analysis and quantification of medicinally important essential oil...
TRANSCRIPT
Analysis and quantification of medicinally important
essential oil by mass spectrometry method and
chemical studies on Opuntia dillenii
A thesis submitted for the partial fulfilment of the degree
of
DOCTOR OF PHILOSOPHY
by
Lubna
Department of Chemistry
University of Karachi,
Karachi -75270,
Pakistan
2017
Dedicated
to,
my beloved parents
and
my siblings
Author Introduction
The author was born in Karachi, Pakistan. She received her early education at Karachi and
passed her matriculation examination from Sir Syed Children’s Academy, Nazimabad, in
1996. She joined Women Degree College, Nazimabad and qualified Intermediate Certificate
in 1998 and got B.Sc. degree in 2000 from Federal Government Urdu Science College. She
got M.Sc. degree in Organic Chemistry from Karachi University in 2005 with 4th position.
Later, she joined International Center for Chemical and Biological Sciences, H.E.J.
Research Institute of Chemistry, University of Karachi, in July 2005, as a research associate
for a research project (H.E.C. project No. 20-161; entitled as “Pesticidal and Medicinal
agents based on Tagetes species”). She got M. Phil. degree (2006-2014) under the
supervision of Prof. Dr. Shaheen FaiziS.I. after that she submitted her Ph. D. studies from
Department of Chemistry, University of Karachi, under the supervision of Prof. Dr. Rahat
Sultana in 2017. During her research studies she earned more than twelve research
publications and submitted one US and one Pakistani patent.
Acknowledgement
To begin with the name of Al-Mighty Allah (SWT), the most gracious and the most merciful
Alhamdullilah. All praises to Allah for the blessing me to complete my Ph. D. research
work. All high opinions for our last Holy Prophet (Peace be upon him), whose teachings and
deeds provide us the clear approach to accomplish our destination successfully.
I wish to express my gratitude to a number of peoples who were involved in this thesis, in
different ways. In this regards, first of all I have great pleasure in expressing my deep sense
of gratitude and indeptedness to my dear supervisor, Prof. Dr. Rahat Sultana, for her
enthusiastic encouragement, active co-operation and expert guidance to elaborate things
clearly and simply. I also pay my sincere thanks to my research co supervisor Dr. Syed
Ghulam Musharraf for providing modern spectroscopic facilities. I am also grateful to Prof.
Dr. Shaheen faiziS.I. for giving me valuable suggestion. I am whole heartedly thankful to
Prof. Dr. Dilshad Waqar, Chairperson, Department of Chemistry, University of Karachi, for
providing excellent and modern research facilities for conducting my research work.
Any sense of gratitude will be incomplete without giving glory and words of thanks to
founding Director of this institute, (Late) Prof. Dr. Salimuzzaman SiddiquiF.R.S.,H.I.,S.I.,T.I.. My
sincere thanks are due to the Chief Parton of the Institute, Prof. Dr. Atta-ur-
RahmanF.R.S,N.I.,H.I.,S.I.,T.I. and the Director, Prof. Dr. M. Iqbal ChoudharyH.I,S.I.,T.I. for providing
opportunities and world-class facilities, equipped with most sophisticated instruments,
where I carried out all of my spectral studies. I have paid my sincere thanks to all my
research group fellows. In particular, Dr. Perveen for their valueable suggestions. I am
grateful to compound bank to cooperate for the evaluation of biological activities. I am also
thankful to Prof. Dr. Ahsana Dar and her student Dr. Faheema Siddiqui for the analysis of
anticancer activity.
Last but not least, my deepest gratitude goes to my parents, specially my beloved father Mr.
Zaheer Ahmed and Abida Begum and my sisters and brothers (Syeda Uzma, Syeda Huma,
Mohammad Humayun and Danish Khan) for their highly valuable contributions and prayers
in compilation of this thesis.
Lubna
Karachi, 2017
CONTENTS
Page No.
Summary………………………………………………………………………….. 01-02
Urdu Summary…………………………………………………………................ 03
Chapter 1
1. General introduction……………………………………………………. 04-08
Chapter 2
2. Biosynthesis……………………………………………………………… 09-37
2.1.Biosynthesis of fatty acids…………………………………………...
2.2.Biosynthesis of terpenes………………………………………………
2.3.Biosynthesis of steroids……………………………………………….
2.4.Biosynthesis of flavonoids…………………………………………….
Part A
Analysis and quantification of medicinally important essential oil by mass
spectrometry method
Chapter 3
3. Introduction……………………………………………………………………
38-58
Chapter 4
4. Results and discussion.…………………………………………………………
59-160
GC and GC-MS studies of leaves, flowers and stalks on different medicinal
plants……………………………………………………………………………..
a. Antibacterial activity……………………………………………….
b. Antifungal activity………………………………………………….
c. Antioxidant activity……………………………………………......
d. Cytotoxic activity..…………………………………………………
e. Immunomodulating activity………………………………………...
Outlook………………………………………………..………………………….. 161-162
Chapter 5
5. Experimental….………………………………………………………………...
163-166
Chapter 6
6. References………………………………………………………………………
167-191
Part B
Chemical studies on Opuntia dillenii
Chapter 7
7. Introduction……………………………………………………………………
192-207
Chapter 8
8. Results and discussion …………………………………………………………
208-252
Chemical investigation on Opuntia dillenii cladode butanol phase…………....….
Outlook………………………………………………..………………………….. 253-254
Chapter 9
9. Experimental…………………………………………………………………… 255-259
Chapter 10
10. References……………………………………………………………………..
260-280
List of publications……………………………………………………………….. 281-287
1
Summary
2
The thesis comprises two parts, A and B. Part A deals with the analysis and quantification of
medicinally important essential oil, by mass spectrometry method while, the part B describes
the chemical studies on cladode of Opuntia dillenii. A short summary on the biosynthesis of
steroids, flavonoids, fatty acids and terpenoids are also included in the thesis.
Part A
This part explains the chemical constituents and their quantification of medicinally important
components of extracts on Ixora fulgens, I. coccinea (yellow and orange color flowers), I.
polyantha, I. chinensis, Ipomoea batata (blackie), I. batata pink frost, and Cassia fistula, by
mass spectrometry method. GC and GC-MS studies were carried out on extracts of different
parts especially flowers, leaves and stalks of the plants which led to the identification of 230
chemical compounds from hexane extracts, 324 chemical compounds were identified from
chloroform extract of Ixora species. Moreover, 26 metabolites were characterized from
methanol extracts of Ixora chinensis flowers extract. From Ipomoea species, 106 chemical
compounds were identified from hexane extracts, while, 72 compounds were identified from
chloroform extract of the plant. Moreover, 57 phytochemicals were identified from hexane
extract, whereas, 51 metabolites were identified from chloroform extracts of C. fistula plant.
Different class of compounds were identified from this extracts including hydrocarbons, long
chain fatty acids and their esters, alcohols, aromatic acids and their esters, terpenes and
vitamins. Antibacterial, antifungal, antioxidant, anti-inflammatory, and anti-cancer activities
have also been carried out from all the above mentioned extracts.
Part B
Total thirteen different fractions of vaccum liquid chromatography (VLC) of antidepressant
butanol phase of Opuntia dillenii were selected for chemical analysis. For this purpose GC
and GC-MS studies were carried out, which led to the identification of 409 chemical
compounds.
3
Urdu summary
4
Chapter 1
General Introduction
5
All the time men have based on environment for their fundamental requirements for the
invention of goods, protections, clothes, medicines etc. Natural World has recommends us a
range of prescriptions to treat a lot of diseases. 2600 BC was the starting period for the
preparation of remedies for diseases. More than 1000 plants derivatives were utilized as oils
of Cedrus species, Commiphora species, Cupressus sempevirens, Glycyrrhiza glabra, and
Papaver somniferum, all of these are instant applied.1
Egyptian medicines (2900 BC), manuscript name is the “Ebers Papyrus” (1500 BC), more
than 700 treatments mostly based on plants and their formula like pills and ointment, with
milk, honey, beer, and wine being collectively used as means of transportations. The Chinese
Materia Medica (dated 1100 BC), and Indian Ayurvedic system (1000 BC), the following
system founded the origin for the initial documentation of Tibetan medicines.
In Western history, the Greeks added significantly to the progress of the application of herbal
medicines. The scientists and philosophers, in his plants records, showed the herbs medicinal
qualities, and modify their qualities through cultivation. Dioscorides, a Greek physician (100
AD), all through his activities among Romans soldiers all through later ‘known world’,
precisely documented the collected works, and application of herbal medicines, and is
believed by a lot of to be the good number of agent of the science of herbal medicines in
‘ancient times’. Galen (130-200 AD), applied, and educated about pharmacy and drugs in
Rome, and published approximately 30 books on the following topics, and famous for his
perceptions (containing dozen ingredients; galenicals) used in drugs.
Throughout the Dark and Middle time periods, from the fifth to the twelfth centuries, Ireland,
England, Germany and France saved the remnants of this Western understanding. On the other
hands, Arabs were accountable for the protection of Greeco-Romans knowledge, and for
expand it to comprise the application of their individual resources, along with Indians and
Chinese plants, which were unidentified to the Greeco-Romans World. In eighteen century,
initially, medicines stores were starts the Arabs, and the Persian pharmacist, philosopher, and
physician Avicenna works a lot in the field of science, pharmaceuticals and medicines, for
example Canon Medicinae, considered as ‘the concluding systematic form of the entire
6
Greeco-Roman medication’. Afterward the complete collection of medicines, known as the
collection of simple text by Ibn al-Baitar, who practiced in Malaga for the time period of
Moorish profession of Spain.
In 1618 of the London Pharmacopoeia were arranged a lot of expertise, at any coast in the UK
by the publication and the idea of ‘pure’ compounds as medicines with the help of isolation
of the active chemical compounds and herbs namely atropine, morphine, strychnine, and
colchinine in the early 1800s. Ayurvedic system of medicines was made on the required rules
of nature and its elements after a vigilant and systematic study of human beings. The
advantage of herbal decoction is that it can maintain the body by maintaining many organ
system.2
Natural products are the active secondary metabolites with boundless varieties produced by
plants, bacteria, fungi and marine organisms. Plants products play a significant task in the
healthcare organizations of the remaining 20 % of the people, commonly live in developed
countries. Data study on dealing dispensed from pharmacies communities in the United State
showed that about 25 % plant extracts have active phytochemicals, which were obtained from
higher plants. Medicinal plants have long been an excellent source of pharmaceutical
representatives. Result oriented objectives of research plan are to discover and design new
chemotherapeutic representatives based on plant derived phytochemicals leads by using
medicinal chemistry approach, which is combination of chemistry and biology.3 Biologically
active natural products and their synthetic compounds, including quassinoids,
naphthoquinones, sesquiterpenes lactones, etc., will be presented with respect to their findings
and preclinical development as potential clinical trial candidates. Research approaches
containing bioactivity or mechanism of action-directed isolation and classification of active
chemical compounds, rational drug design based modification and compounds synthesis, and
structure activity relationship and mechanism of action studies.3-6 Isolation has been
performed as a result of several natural products has been obtained, from plants and other
organisms, and revealed to obtain strong physiological response in humans. Modern
pharmaceutical companies have been developed natural products as antifungal,
immunosuppressive, antibiotic and anticancer agents in addition to other medicines. Even
7
though, several pharmaceuticals companies have ceased to follow up natural and synthetic
chemical constituents.
Pakistan is a developing country of South Asia, there is a need for represent concentration to
health as the perfect situation of psychological, physical, social, and spiritual workings of
human beings, additionally medicinal plants to relief human from diseases. Herbal decoctions
(various herbs) demonstrating positive results for healing are usually used in Ayurveda.
The research work performed for the doctor of philosophy dissertation, comprised of two
parts. One deals with the analysis and quantification of medicinally important essential oils
by gas chromatography-mass spectrometry methods while other describes the chemical
studies on O. dillenii.
8
1 a) Newman, D. J., Cragg, G. M., Snader, K. M. (2000). The influence of natural
products upon drug discovery. J. Nat. Prod., 17, 215-234. b) Cragg, G. M., Grothaus,
P. G., Newman, D. J. (2009). Impact of natural products on developing new anticancer
agents. Chem. Rev., 109(7), 3012-3043. c) Hostettmann, K., Marston, A., (2007).
Plants as a still unexploited source of new drugs. Nat. Prod. Comm., 3(8), 1307-1315.
2 a) Jain, S., Gill, V., Vasudeva, N., Singla, N. (2009). Ayurvedic medicine in treatment
of cancer. J. Chin. Integr. Med., 7(1), 1096-1099. b) http://www.rsc. org/publishing/
journals cb/volume/2008/1/natural_remedies.asp
3 a) Lee, K-H., (2010). Discovery and development of natural product derived
chemotherapeutic agents based on a medicinal chemistry approach. J. Nat. Prod.,
73(3), 500-516. b) Dwight, D., Baker, M. C., Uma, O., Vineet, R. (2007). The value
of natural products to future pharmaceutical discovery. Nat. Prod. Rep., 24, 1225-
1244.
4 Newman, D. J., Cragg, G. M. (2007). Natural products as sources of new drugs over
the last 25 years. J. Nat. Prod., 70(3), 461-477.
5 Ghavami, G., Kazemali, M. R., Sardari, S. (2011). Informatics of drug synergism in
naturally occurring anticancer agents. Rec. patent anti-cancer drug discov., 6, 26-44.
6 Setzer, W. N., Setzer, M. C. (2003). Plant derived triterpenoids as potential
antineoplastic agents. Mini Rev. Med. Chem., 3, 540-556.
9
Chapter 2
Biosynthesis
10
A process in which simple substrates by using chemical energy (adenosine triphosphate; ATP)
and enzymes (catalyst) synthesizes complex products are called biosynthesis. Glucose is the
starting material in this process, while the complex molecules are formed including proteins,
vitamins and antibiotics. In this process one step product is the starting product of another step
whereas, many enzymatic steps are involved in these process. Amino acids, carbohydrates,
fats, proteins and nucleic acids are primary metabolites; while natural products are secondary
metabolites.1
During photosynthesis, solar energy, carbon dioxide and water are consumes in plants and
some bacteria, and glucose is produce. Respiration is a process, where glucose gives ATP,
which works as a fuel and provides energy to all living organisms. Glycolysis is a process in
which glucose, is divided into two compounds of pyruvic acid with the help of enzyme
catalyst. In multistep glycolysis process, several intermediates and amino acids consisting
acetyl coenzyme A (acetyl-CoA), deoxyxylulose-5-phosphate and shikimic acid are formed.
Several secondary metabolites have been synthesized through these intermediates
(originators) (Figure 1).1
The acetate pathway
Different catabolic routes have been used for the formation of pyruvate via glycolysis. In
plants and animals (aerobic organisms), oxidation takes place through elimination of its
carboxyl group as CO2, to acetyl group of acetyl-CoA; the acetyl group then oxidized
completely to CO2 by the citric acid cycle. The acetyl-CoA produced a large number of natural
products with polyketides, including fatty acids, lipids, sterols, terpenes etc. biosynthesis of
these compounds is called “Acetate Pathway” (Figure 2). 1
The polyketides pathway
Natural compounds produce in plants, fungi, and soil bacteria called as polyketides.2 These
secondary compounds are produce completely or partially from poly-β-keto methylenes chain
(CH2CO)n which get through the decarboxylative condensation of 2-, 3- or 4- carbon building
units like acetyl-CoA, propinoyl-CoA or butyryl-CoA.1a,b,2b Many enzymes have been
catalyzed the process of biosynthesis, polyketide synthases (PKSs), are the large multi
11
CO2H2O
O
OH
OH
OH
OH
PO
O
OH
OH
OH
OH
HO
OH
OPO
OH
PO
OHC OH
NH2
CO2H
NH2
CO2H
HO
CO2H
OH
HO OH
PO
HO2C OH
SHIKIMIC ACID
NH
NH2
CO2H
HO2C OP
O OH
OP
OH
HO2C O
CoAS O
HO2C OH
HO
NH2
CO2HHO2C
HO2C
NH2
CO2H
HO2C
O
CO2H
O
CO2HHO2C
NH2
CO2HH2NNH2
CO2HH2N
Figure 1: The building blocks
GLYCOLYSIS
Polysaccharides
Glycosides
Nucleic acids
glucose 6-P
glyceraldehyde 3-P
3-phosphoglyceric acid
phosphoenolpyruvate
pyruvic acid
ACETYL-CoA
KREBS CYCLE
Oxaloacetic acid 2-Oxoglutaric acid
L-glutamic acid
L-ornithine
L-aspartic acid
L-lysine
DEOXYXYLULOSE 5-P
D-glucose
PENTOSE PHOSPHATE
CYCLE
erythose 4-P
PHOTOSYNTHESIS
h
L-phenylalanine
L-tyrosine
L-tryptophan
Phenols
Cinnamic acid derivatives
Lignans
Alkaloids
Phenylpropanoids
Aromatic compounds
MEVALONIC ACID
Isoprenoids
(Terpenes, Steroids,
Carotenoids)
Phenols
Prostaglandins
Macrolide antibiotics
Fatty acids
Polyketides
Isoprenoids
(Terpenes, Steroids,
Carotenoids)
12
enzyme protein compounds, having the typical core of coordinated active sites. The PKSs
structures are similar to synthesis of fatty acid (FASs). These fatty acid synthase catalyzes the
pathways of fatty acids. Polyketides contain many structures, the common of these secondary
metabolites are formed by three large categories of polyketide synthases (PKSs) consisting
multienzyme type I, iterative type II and homodimeric type III PKSs, containing the same
mechanism having condensation reactions, which produces different kinds of aromatic
skeletons for example, chalcones and flavonoids etc. 2
From poly β-keto ester chain, polyketides are formed, it is composed of four acetates parts,
the reaction is start from one acetate unit, whereas remaining three acetates are used in their
activated malonoate structure as chain addition units, thus, the process is known as acetate
polymalonyl formation route (Figure 3). The poly-keto chain is stabilized on the surface of
enzyme, upto certain chain length limit, the cyclization or further reactions initiated,
commonly aldol or Claisen type reactions occour, these reactions depends on the enzyme
nature and the folding pattern of substrate. Several paths of cyclization proceed through β-
keto ester chain i.e. A-D (Figure 4) the aromatic polyketides are produced, orsellinic acid,
floroacetophenone and pyrones containing α- and β-pyrones.1a, b, 2, 3
Penicillium patulum, produces 6-methyl salicylic acid, when the loss of hydroxyl group at C-
4 position was performed, and produced, orsellinic acid. The enzyme 6-methyl salicylic acid
synthase with a cofactor NADPH, ketone moiety has been reduced to alcohol at triketide step,
and dehydration takes place for the loss of oxygen moiety. Later than aldol condensation,
enolization and ejection from enzyme, 6-methyl salicylic acid is produced. Through
enolization and ester formation triacetic acid lactone is formed (Figure 5), known as α-pyrone,
it may be the precursor of several other compounds. It is isolated from Gerbera hybrid.1a, b, 3
Different structure of the substrate moiety leads to the formation of a large number of natural
products e.g, when substrate is p-hydroxy cinnamoyl-CoA, condensation with three malonyl-
CoA units followed by aldol or Claisen type cyclization, flavonoid naringenin and a stilbene,
resveratrol have been produced (Figure 6).1a,b
13
Biosynthesis of fatty acids
Fatty acids biosynthesis are same to polyketides, chain lengthening step in fatty acids pursued
by a fixed sequence of reduction of ketone group, then loss of water and enol reduction, while
in polyketide biosynthesis carbonyl group is not reduced and able to a definite chain length
limit cyclization performs, it provides a large number of secondary metabolites.1a,b,2b Enzyme-
bound thioesters of acetyl-CoA and malonyl-CoA have been used in the fatty acid
biosynthesis, this reaction performed with Claisen condensation i.e. the malonyl ester,
transfered the acyl carrier protein (ACP), giving acetoacetyl-ACP (β-ketoacyl-ACP), which
through NADPH, is stereospecifically reduced to the β-hydroxy ester. Later than dehydration
α,β-unsaturated ester is formed, a saturated fatty acyl-ACP has been produced by the help of
NADPH, in this reaction double bond has been reduction, with the addition of two carbon
atoms in the substrate. This is the lengthening in the fatty acids carbon skeleton are collected
in a reproducing series, till the required carbon skeleton is achieved. Fatty acid synthase
enzyme, is used in fatty acid biosynthesis1a-c,4(Figures 3-9).
Biosynthesis of terpenes
Terpenes are the main biosynthetic building blocks contained by nearly all living organisms.
They are wide class of natural compounds produced by plants and insects. The oxidized or
rearranged forms of terpenes are called as terpenoids (isoprenoids). Essential oils are formed
by terpenes and terpenoids.1a,d Isoprene units are the basic unit of terpenes, dimethylallyl
pyrophosphate (DMAPP), and isopentenyl pyrophosphate (IPP) are joined collectively into
head and tail approach producing simple chains or rings (after rearangement). The active
elements DMAPP and IPP are obtained through two routes the mevalonate independent
pathway and the mevalonate pathway via deoxyxylulose phosphate following the
intermediates mevalonic acid and deoxyxylulose 5-phosphate correspondingly produced
throughout glycolysis (Figures 1, 2). Formation of mevalonic acid (MVA) is the starting point
of mevalonic pathway by using the three units of acetyl-CoA. Through Claisen condensation
of two acetyl-CoA elements followed through stereospecific aldol addition of third acetyl-
CoA entity, a branched chain ester β-hydroxy-α-methylglutaryl-CoA (HMG-CoA) is
produced. The MVA (3R) is formed through the two-step reduction of thioester group of
HMG-CoA to primary alcohol, irreversible transformation and a rate determining
14
2CO2
2 Acetyl-CoA
aerobic condition
4 CO2 + 4 H2O
Mevalonic acid Polyketides
Fatty acids
Lipids
Prostaglandins
Thromboxanes
Leukotrienes
Glucose 2 Pyruvate
Figure 2: Products of acetyl-CoA
Terpenes, sterols
-H2O
R
CO2H
CH2CO SCoA
RCH2 CH2CO
OHH
S ACP
RCH2
CO S ACP
NADPH
NADPH
CO2H
CH2CO S ACP
RCH2COCH2CO-S-ACP
RCH2CH2CH2CO
HSCoA
SCoA
RCH2CO
RCH2CH2CH2CO
CH3CO SCoA
S Enz
H2O
S ACP
RCH2CH2CH2CO2H
malonyl-CoA
acyl carrier protein (ACP)
malonyl-ACP
Claisen
reaction
acetyl-CoA
acyl-enzyme thioester
each turn of the cycle
extends the chain length of
the acyl group by two
carbons -keto acyl-ACPstereospecific
reduction of carbonyl -hydroxy acyl-ACP
E2 elimination of H2O
E
-unsaturated acyl-ACP
reduction of double bond fatty acyl-ACP
fatty acyl-CoA fatty acid
Figure 3: Biosynthesis of saturated fatty acids
15
H3C C
O
SCoA
H3C C
O
CH2 C
O
SCoAH3C C
O
SCoA
CH2 C
O
SCoA
H2C
CO O
C
O
SCoA
H
CO2
CH3CO [CH2-C]n CH2COSCoA
O
Acetyl-CoA
Malonyl-CoA
Claisen
reaction
Acetoacetyl-CoA
Malonyl-CoA
CO2
repeat o f Claisen
reaction
Poly- -keto ester
Figure 3: Biosynthesis of poly-keto chain
16
O
CoAS
CO2H
O
SCoA
O
CH3
OO
SEnz
O
H
O
O
SEnz
OO OH3C CH3
O
SEnz
O
CH3
O O
O
OEnz-S
O
O
CH3
O
SEnz
CH3O
OH
O
OH
SEnz
CH3
O
O O
O
O O
OH
H3C
O
SEnz
CH3O
O O
O
CH3
OO
O
CO2H
CH3HO
OH OH
CH3
OOH
HO
H2O
H2Ofolding
B
Poly- -keto ester (Tetraketide intermediate)
folding
A
3x
Malonyl-CoA
aldol reaction
enolization hydrolysis
Orsellinic acid
claisen reaction
enolization
Phloracetophenone
Figure 4: Biosynthesis of aromatic polyketides
Acetyl-CoA
folding
D
folding
C
Tetraketide -pyrone
-pyrone
17
O
CH3CoAS
CO2H
O
SCoA
Enz-S
O O O
NADPH
O
O
O
SEnzO
H
O
SEnz
enolization then ester formation-H2O
O
O
SEnz
O
O
HO
triacetic acid lactone
Malonyl-CoA
O
O
SEnz
Oaldol
reaction
-H2O
O
SEnz
O enolization hydrolysis OH
CO2H
O
OH
O
poly- -keto ester (Triaketide intermediate)
folding
A
2x
Malonyl-CoA
Figure 5: Biosynthesis of 6-methylsalicylic acid and triacetic acid lactone - the polyketides
Acetyl-CoA
folding
D
6-Methyl salicylic acid
4 6 4
6
18
OH
OH O
HO
OH
SCoA
O O
O
O
OH
HOR
OH
O
OOH
OH
OH
OH
O
SCoA
O
O
HO
OH
OH
R
OH
O
OH O
HO
NADPH
O
OH
HO
OH
OH
R
OH
OH
OH
O
O
HO
OH
R
O
OHO
OH
OH
R
OH
O
OH
HO
OH
OH
R
OH
NADPH
OHO
OH
OH
R
OH
+ 3 Malonyl CoA
(from acetate pathway)
R = H, naringenin
R = OH, ericodictyol
(flavanones)R = H, dihydrokaempferol
R = OH, dihydroquercetin
(dihydroflavonols)
R = H, kaempferol
R = OH, quercetin
(flavonols)
O2
2-oxoglutarate
R = H, apigenin
R = OH, luteolin
(flavones)
O2
2-oxoglutrate
Claisen
chalcone synthase
Naringenin-chalcone
O2
2-oxoglutarate
R = H, leucopelargonidin
R = OH, leucocyanidin
(flavandiols; leucoanthocyanidins)
R = H, afzalechin
R = OH, (+)-catechin
(catechins)
-2 H2O
R = H, pelargonidin
R = OH, cyanidin
(anthocyanidins)
Figure 6: Biosynthetic pathways of flavonoids
RR
R
R
R = H, 4-Hydroxy
cinnamoyl-CoA
19
O
SCo-A
ATP ADPHCO3
NHN
S
O
Co Enz
O
O
NHHN
S
O
Co Enz
O
SCo-AO
O
O
SCoA
SH
CysACP
SH
NADPH
S
ACP
O OHH
SH
CysS
Cys
O
CH3 S
ACP
O O
SH
CysS
ACP
O
O
O
-H2O
S
Cys
O
S
ACP
O
SH
CysS
ACP
O
SH
Cys
SH
ACP
H2O
HO
O
Claisen condensation
( -hydroxyacyl dehydratase)
transfer
Figure 7: Biosynthesis of fatty acid
+
biotin
carboxylase
+Pi
transcarboxylase
acetyl-CoA
Malonyl CoA
+ Biotin enzyme
Malonyl transacylase
(Fatty acid synthase)
acetyl-CoA
-Hydroxy acyl-ACP -keto acyl-ACP
Fatty acyl-ACP
NADPH
enoyl reductase
--Unsaturated acyl-ACP
Biotin enzyme
Acetyl transacylase
( -ketoacyl synthase) -keto acyl
reductase
E
Fatty acid
Fatty acyl-CoA
20
O
SCo A
O
SCo-AHO
O
SH
CysSP
ACP
O
SR
SR
O
O
SRHO
O
SR
O
SR
O
SR
OSR
O
SR
O
SR
O
SR
O
Acetyl-CoA Malonyl CoA
+
R = CoA in animals/fungi
R = ACP in plants
Oleate (18:1, 9)
desaturation
a-Linolenate (18:3, 9,12,15
)
Fatty acid synthase
( )
Palmitate (16:0)
desaturation
( )
Malonoate (elongation)
Stearate (18:0)
desaturation (in plants only)
-Linolenate (18:3, 6,9,12
)
Eicosatrienoate (20:3, 8,11,14
)
Eicosatetraenoate (Arachidonate) (20:4, 5,8,11,14
)
elongation/desaturation
elongation
desaturation
Polyunsaturated fatty acids
EPA (20:5, 5,8,11,14,17
)
DPA (22:5, 7,10,13,16,19
)
DHA (22:6, 4,7,10,13,16,19
)
Figure 8: Biosynthesis of saturated and unsaturated fatty acids
7
Linoleate (18 : 2, 9,12
)
desaturation
Palmitate (16:1, 9)
desaturation (in plants only)
9
11
9 12
9 12 15
9 12
9
9
6
8
11 14
8 5
11 14
21
step. The MVA produced, by phosphorylation in the catalysis of two dissimilar ATP-
dependent enzymes, followed via dehydration/decarboxylation, providing IPP, IPP convert to
DMAPP isomer by an enzyme named as isomerase (Figure 10). The IPP and DMAPP are
appeared through deoxyxylulose 5-phosphate, the route, commonly used in nature known as
mevalonate independent route (Figure 11).1a,b,d Different classes of terpenes are given below:
Hemiterpenes (C5):
Isoprene unit is also called as hemiterpenes whereas, IPP and DMAPP are its active forms.
Monoterpenes (C10):
Two isoperene units join to form monoterpene. IPP and DMAPP combine via an enzyme
prenyl transferase to produce geranyl diphosphate (GPP). These are the intermediate of
monoterpenes geraniol, citronellol etc. (Figure 12).
Sesquiterpenes (C15):
Sesquiterpenes are formed through three isoprene units. C5 units (IPP) combine with GPP in
the presence of catalyst prenyl transferase gives sesquiterpene precursor farensyl diphosphate
(FPP), which directs to the formation of several sesquiterpenes e.g. bisabolene etc. (Figure
12).
Diterpenes (C20):
Diterpenes are formed with the combination of four isoprene units, more addition of C5 IPP
unit in FPP produces geranyl geranyl pyrophosphate (GGPP). Diterpene (phytol), gives the
lipophylic side chain of chlorophyll (Figure 12).
Sesterpenes (C25):
Sesqueterpenes are formed, when five isoprene units combine together. Further addition of
C5 IPP unit in GGPP to forms geranyl farnesyl pyrophosphate (GFPP) (Figure 12).
22
OH
O
H
O
OH
-COOH
OH
O
O
O
OH
Fatty acids
Hydrocarbons
oxidase
Figure 9: Biosynthesis of other fatty compounds via fatty acids
( ) n
Fatty acids 1o
( ) n
Fatty aldehydes
( )n
( )n
Fatty alcohols 2o
( )n
Fatty acid
( )n +
( )n ( )n
( )n
Fatty alcohols
(1o or 2
o)
Fatty ester
reductase
23
HH
SCoA
O O
SCoA
O
HO2C
OHO
SCoA
SCoA
O
SEnz
O
NADPH
HO2C
OH
OHO2C
OH
SCoA HO2C
OH
SCoA
OH
HNADPH
P
O
HO
OH
ADPH
ATP
-CO2
OPPOPP
HSHR
OH
OPP
O
HO
Claisen
reaction
Acetyl-CoA
EnzSH
+ EnzSH
HMG-CoA
Acetoacetyl-CoA
enzyme-bound
acetyl group
HMG-CoA
reductase
Mevalonic acid
(MVA)
1
23
4
56
Mevaldic acidMevaldic acid
hemithioacetal
2 x ATP
isomerase
12
3
4
5
Isopentenyl PP
(IPP)
Dimethylallyl PP
(DMAPP)
Figure 10: Biosynthesis of precursors of terpenes, IPP and DMAPP through mevalonate pathway
Stereo specific
aldol reaction
24
OH
OPP
-H2O
OPP
OH
OH
OP
O
OH
OP
OH
OH
CTP
N SR
1
R2
NADPH
ONCH2
OHHO
N
NH2
OOP
O
OH
OP
O
OH
O
OH
OH
OH
NADPH
OH
OPP
N SR
1
CH3C
R2
O
H OH
OH
OP
-H2O
OH
OP
OH
O
OH OH
OPP
P
O
N P
OH
T
OPP
A
OH
OP
O
OH
NADPH
O
OH
OP
H
OH O
OPP
N SR
CH3C OH
R2
OH
R
OH
ONCH2
OHHO
N
NH2
OOP
O
OH
OP
O
OH
O
OH
O P
O
OH
OH
OH
CO2
O OH
OP
OH
H
P
O
P
OOOHO
O
OHOH
OHH
O
CO2H
Pyruvic acid
Thiamine PP
(TPP)
D-glyceraldehyde
3-P
TPP/pyruvate-derived
enamine
1-Deoxy-D-xylulose 5-P
TPP anion
regenerated
Fosmidomycin
1-Deoxy-D-xylulose 5-P2-C-Methyl-D-erythritol 4-P
-------------------------------------------- steps to be determined --------------------------------------------
isomerase
Dimethyl PP
(DMAPP) Isopentenyl PP
(IPP)
2-Phospho-4-(CDP)-2-C-methyl-D-erythriol4-(CDP)-2-C-methyl-D-erythritol
2-C-methyl-D-erythritol-2,4
-cyclophosphate
Figure 11: Biosynthesis of precursors of terpenes, IPP and DMAPP via mevolonate independent
pathway-deoxyxylulose pathway
Triterpenes (C30):
25
Six isoprene units give triterpenes. C5 IPP unit combine together by tail-to-tail fusion of two
molecules of farensyl pyrophosphate (FPP), forming a (squalene) hydrocarbon, after
selectively oxidation to forms squalene oxide. Intermediates of squalene oxide have been fold
on the enzyme surface pursue through Wagner-Meerwein (W-M) migration of hydrides and
methyl gives many triterpenoids such as, α- and β-amyrins, cycloartenol, and lanosterol, etc.
(Figures 13, 14).
Tetraterpenes (C40):
Eight isoprene units combine together to form tetraterpenes, for example α- and β-carotenes,
and acyclic lycopenes.
Polyterpenes:
Long chain of isoprene units combine together to form polyterpenes e.g. natural rubber having
polyisoprene structure with cis double bond.1a, b, d
Biosynthesis of steroids
Lanosterol and cycloartenol are the examples of triterpenes (vide supra, Figure 13), it is the
starting material of sterols e.g. lanosterol produces the cholesterol (animals) while,
cycloartenol gives phytosterols for example, stigmasterol, β-sitosterol, etc. and also forms
cholesterol (plants) (Figure 15).1
Shikimate pathway
Shikimate pathway is a biosynthetic route of formation of aromatic compounds, particularly
the aromatic amino acids, namely L-tyrosine, L-tryptophan, and L-phenylalanine. Amino acids
play the role of starting material for a number of aromatic secondary metabolites, for example
flavonoids, alkaloids, indole acetate, phytoalexins, and lignins.1a,b,d,5 Shikimic acid is formed
by the aldol-type condensation of two intermediates of glycolysis (Figure 1), the D-erythrose-
4-phosphate and phosphoenol-pyruvate (PEP) giving 3-deoxy-D-arabino- heptulosonic acid 7-
phosphate (DAHP). 3-Dehydroquinic acid is formed by the intramolecular aldol condensation
of DAHP, which followed the routes of dehydration,
26
OPP
DMAPP
OPP
OPP
HSHR
OPP
HSHR
OPP
OPP
OPP
HSHR
OPP
OPP
OPP
OPP
OPP
HR HS
OPP
IPPOPP
OPP
HSHR
IPP
E
Geranyl PP
(GPP)
Setp 1: Monoterpene precursor
Farnesyl PP
(FPP)allylic cation
Geranylgeranyl PP
(GGPP)
Geranyl PP
(GPP) allylic cation
Farnesyl PP
(FPP)
Setp 2: Sesquiterpene precursor
Setp 3: Diterpenes precursor
Geranylfarnesyl PP
(GFPP)
Figure 12: Biosynthesis of precursors of different classes of terpenes
Setp 4: Sesterpenes precursor
Geranylgeranyl PP
(GGPP)
27
FPP
*PPO
FPP*
OPP
HH
*
*
OPP**
*
H*
H
*
H
H
*
H
H
HH
*
*
**
H
H
(NADPH)O2
NADPH
HOH
H
HH
H
O HOH
H
HH
HOH
H
H
HOH
H
sequence of W-M 1,2-hydride
and 1,2-methyl shifts
cyclizations
animals
fungi
plants loss of proton leads
to cyclopropane
Squalene
synthase
Allylic cationElectrophilic addition giving tertiary cation
Allylic cation
PresqualenePP
W-M
1,3 Alkyl shift
Squalene
Lanosterol Cycloartenol
Squalene oxideProtosteryl cation
loss of proton
gives alkene
Figure 13: Biosynthesis of triterpenes
28
H
HO H
H
O
H
H H
H
H
O2
NADPH
H H
**
H
HO H
H
H
HH
HO H
H
H
H
HO H
H
H
H
HO H
H
H
H
HO H
H
H
H
HH
HO H
H
H
cyclizations
Squalene
Squalene oxideDammarenyl cation
Baccharenyl cation
Lupenyl cation
Oleanyl cation
W-M
1,2-alkyl shift
Oleanyl cation
W-M
1,2-methyl shift
Taraxasteryl cation
-Amyrin
-Amyrin
W-M
1,2-hydride shiftsW-M
1,2-hydride shift
Figure 14: Biosynthetic pathway of -and -amyrin triterpenes
W-M
1,2 alkyl shift
29
HO
HOH
H
H
HO
HOH
HH
H
HO
H
H
H
Lanosterol
CycloartenolCholesterol
-Sitosterol Stigmasterol
Figure 15: Biosynthetic routes for steroids
30
reduction, oxidation, and changes into shikimic and aromatic acids (Figure 16). The shikimic
acid is changed into chorismic acid, by more reaction with protonated PEP followed by
elimination of phosphoric acid. Under the process of Claisen rearrangement, chorismic acid
in the presence of catalysis of enzyme chorismate mutase, transforms into prephenic acid,
following many routes to offer aromatic amino acids L-tyrosine (L-Tyr) and L-phenylalanine
(L-Phe) (Figure 17). L-Tryptophan is also attained by chorismic acid via anthranilic acid as
intermediate. The amino acids L-Phe and L-Tyr are the forerunners of large number of aromatic
secondary metabolites (Figure 18).1a,b
Biosynthesis of flavonoids
Flavonoids belong to secondary metabolites, play vital character in plant like flowers color,
auxin transport inhibition, allelopathy, defense, and UV protection. Flavonoids having several
pharmacological activities like antibacterial, anti-inflammatory, antioxidant, antitumor, and
anti-proliferative.6 Polyketide (acetate) and shikimic acid pathways unite to form flavonoids.
Shikimic acid gives cinnamoyl-CoA (vide supra, Figure 18), merges with three malonyl-CoA
units in the presence of enzyme chalcone synthase producing a polyketide which by aldol or
Claisen condensation gives chalcone precursor for many flavonoids (Figure 6).1a,b,d
Glycosylation of natural products
Several secondary metabolites having glucose (O-, N-, S- and C-glycosides) and
polysaccharides are present in nature. All these glycosides are formed through glycosylation
reaction via a compound, uridine diphospho sugar (UDP glucose), which is formed in nature
from UTP and glucose 1-phosphate (Figure 19).1a
31
CO2H
OP
H
O
OP
HO
OH
CO2H
OHHO
OH
H
NADPH
CO2H
OHO
OH
CO2H
OH
H
OH
OH
POO
DAHP
- H2O
NAD
HO
OHO
OH
CO2H
H
CO2H
OH
OH
- H2O
O
OH
OHHO
CO2H
CO2H
OHOH
OH
NADH
HO
OHHO
OH
CO2H
PEP
D-Erythrose 4-P
- HOP
Shikimic acid 3-Dehydroshikimic acid Quinic acid3-Dehyroquinic acid
dehydration and
enolization
- 2H
oxidation and
enolization
Gallic aicdProtocatechuic acid
aldol-type
reaction
Aldol type
reaction
Figure 16: Biosynthetic pathway of shikimic acid and other aromatics
32
CO2H
OHHO
OH
AT P
CO2H
OHPO
OH
H
PO CO2H
CO2H
O
OH
O
CO2H
O
OH
CO2H
OH
O
H
NAD
PLP
-HOP
CO2H
O
OH
CO2H
NH2
OH
CO2HO
HO
CO2H
OPO
OH
HHH
OPCO2H
CO2H
OPO
OH
CO2H
CO2H
NH2
-HOP
CO2H
NH2
OH
NAD
Shikimic acid Shikimic acid 3-P
EPSP synthase
EPSPChorismic acid
Figure 17: Biosynthesis of amino acids L-phenyalanine (L-phe) and L-tyrosine (L-tyr) via skikimic acid
Phenyl pyruvic acid
4-Hydroxyphenyl pyruvic acid
L-phe
L-tyr
L-arogenic acid
PLP
PLP
Prephenic acid
33
NH2
CO2H
O2
NADPH
CO2H
O2
NADPH
O2
NADPHSAM
CO2H
OH
MeO OMe
SAM
CO2H
OH
MeO
CO2H
OH
MeO OH
CO2H
OH
HO
NH2
CO2H
OH
CO2H
OH
CH2OH
OH
MeO OMe
CH2OH
OH
MeO
CH2OH
OH
H2O
NAD+
CO2H
OH
R
HOCHO
OH
R
CO2H
OH
R
reverse
aldol
Figure 18: Biosynthesis of several aromatic compounds through amino acid L-Phenylalanine (L-Phe)
L-Phe
PAL
Cinnamic acid
hydroxylation
L-Tyr p-Coumaric acid
sequence of hydroxylation and methylation reaction
Caffeic acid Ferulic acid Sinapic acid
p-Coumaryl alcoholConiferyl alcohol
Sinapyl alcohol
34
O
OHOH
H2C N
O
OO
OP
O
OP
O
OHOH
OHO
HOOH
OH
N
O
OOH2C
HO OH
OP
O
OH
OP
O
O
OH
O
OH
OH
HOHO
OHO
HO
OPOH
OHUTP
O
OH
OH
O
HO
OHO
HO
OPOH
H
OH
UTP OHO
HO
OHOR
OH
UDP
O
OH
OH
O
HO
OOH
HOOH
HO
-H+
UDP+
UDP glucose
Glucose 1-P
UDP glucose
O- -D - glucoside
Figure 19: Glycosylation in nature
Glucose 1-P
+
ROH SN2 reaction
C-glucosidation
O-glucosidation
Oreintin
(Luteolin-8-C-glucose)
35
References
36
1 (a) Dewick, P. M. (2002). Medicinal Natural Products, A biosynthetic approach, 2nd
Edition, John Wiley and Sons, Chichester, UK. (b) Barton, S. D. (1979).
Comprehensive Organic Chemistry, vol. 5, Pergamon Press, New York. (c) Nelson,
D. L., Cox, M. M. (2003). Lehninger principles of biochemistry, 3rd Edition, Worth
Publishers, New York, (d) http://en.wikipedia.org
2 (a) Jeong, J-C., Srinivasan, A., Grüschow, S., Bach, H., Sherman, D. H., Dordick, J.
S. (2005). Exploiting the reaction flexibility of a type III polyketide synthase through
in vitro pathway manipulation. J. Am. Chem. Soc., 127, 64-65. (b) S-Wörgötter, E.
(2008). Metabolic diversity of lichen-forming ascomycetous fungi: culturing,
polyketide and shikimate metabolite production, and PKS genes, Nat. Prod. Rep., 25,
188-200. (c) Gokhale, R. S., Saxena, P., Chopra, T., Mohanty, D. (2007). Versatile
polyketide enzymatic machinery for the biosynthesis of complex mycobacterial lipids.
Nat. Prod. Rep., 24, 267-277. (d) Thomas, R. (2004). Biogenetic speculation and
biosynthetic advances. Nat. Prod. Rep., 21, 224-248. (e) Rawlings, B. J. (1999).
Biosynthesis of polyketides (other than actionomycete macrolides). Nat. Prod. Rep.,
16, 425-484. (f) Rawlings, B. J. (1997). Biosynthesis of polyketides. Nat. Prod. Rep.,
14, 523-556. (g) Eckermann, S., Schröder, G., Schmidt, J., Strack, D., Edrada, R. A.,
Helariutta, Y., Elomaa, P., Kotilainen, M., Kilpeläinen, I., Proksch, P., Teeri, T. H.,
Schröder, J. (1998). New pathway to polyketides in plants. Nature, 396, 387-390.
3 (a) Goel, A., Ram, V. J. (2009). Natural and synthetic 2H-pyran-2-ones and their
versatality in organic synthesis. Tetrahedron, 65, 7865-7913. (b) Eckermann, C.,
Schröder, G., Eckermann, S., Strack, D., Schmidt, J., Scheider, B., Schröder, J.
(2003). Stilbenecarboxylate biosynthesis: a new function in the family of chalcone
synthase-related proteins. Phytochemistry, 62, 271-286. (c) Eckermann, C., Matthes,
B., Nimtz, M., Reiser, V., Lederer, B., Böger, P., Schröder, J. (2003). Covalent
binding of chloracetamide herbicides to the active site cysteine of plant type III
polyketide synthases. Phytochemistry, 64, 1045-1054.
37
4 (a) Bernard, J. (1998). Biosynthesis of fatty acids and related metabolites. Nat. Prod.
Rep., 275-308. (b) Buist, P. H. (2004). Fatty acid desaturases: Selecting the
dehydrogenation channel. Nat. Prod. Rep., 21, 249-262. (c) Rashotte, A. M., Jenks,
M. A., Feldmann, K. A. (2001). Cuticular waxes on eceriferum mutants of
Arabidopsis thaliana. Phytochemistry, 57, 115-123. (d) E Fahy, S Subramaniam, H A
Brown, C K Glass, A H Merrill, R C Murphy, C R Raetz, D W Russell, Y Seyama,
W Shaw, T Shimizu, F Spener, G Meer, M S VanNieuwenhze, S H White, J L
Witztum, E A Dennis. (2005). A comprehensive classification system for lipids. J.
Lipid Res., 46, 839-861.
5 (a) Herrmann, K. S. (1995). The shikimate pathway as an entry to aromatic secondary
metabolism. Plant Physiol., 107, 7-12. (b) Knaggs, A. R. (2003). The biosynthesis of
shikimate metabolites, Nat. Prod. Rep., 20, 119-136.
6 (a) Buer, C. S., Imin, N., Djordjevic, M. A. (2010). Flavonoids: New roles for old
molecules. J. Integ. Plant Biol., 52(1), 98-111. (b) Crozier, A., Jagnath, I. B., Clifford,
M. N. (2009). Dietary phenolics: Chemistry bioavailability and effects on health. Nat.
Prod. Rep., 26, 1001-1043. (c) Veitch, N. C., Grayer, R. J. (2008). Flavonoids and
their glycosides, including anthocyanins. Nat. Prod. Rep., 25, 555-611.
38
Part A
Analysis and quantification of medicinally important
essential oil by mass spectrometry method
39
Chapter 3
Introduction
40
Ixora chinensis Ixora coccinea
Ixora fulgens Ixora polyantha
Ixora coccinea
41
3.1. Ixora species
3.1.1. Ixora coccinea Linn.
Ixora coccinea Linn. a time-honored medicinal plant commonly known as ‘jungle geranium’
and flame of wood or vethi in ayurvedha. It belongs to member of Rubiaceae family and found
in the tropical and subtropical areas of all over the world. It is cultivated throughout India as
an ornamental plant. There are more or less 400 species of Ixora genus, only some are
cultivated. I. coccinea is a shrub with dense, long multi-branched, twigs compressed, and thick
at nodes, generally 4-6 ft but in able of reaching 12 ft height. Leaves of Ixora are coriaceous,
from 2 cm to 6 inches in length, sessile or sub-sessile, oblong, and obtuse. The flowers appear
in clusters, which are produced at the end of branches. Each cluster may contain upto 60
individual flowers. The flowers is very small in size and tubular, with four petals.1
A. Pharmacological importance
I. coccinea is widely used in ayurvedic purposes, pharmacological studies have shown that
the plant of I. coccinea have been used in the treatment of antimicrobial, antibacterial,
antifungal, antioxidant, analgesic, cytotoxic, sedative, stomachic, fever, CNS depressant,
gonorrhea, antiseptic, dysentery, headache, sores, and chronic ulcer diseases. It has been
reported to possess many medicinal properties like hepatoprotective, chemoprotective,
antinociceptive, antiasthematic and hypolipidemic activities. The plant extracts have been
reported in the treatment of astringent, anti-mitotic, anti-inflammatory, tuberculosis, wound
healing, skin diseases, cardiovascular and antimutagenic properties. Different parts of the
plants have been used in the medication of dysmenorrheal, heamoptysis, catarrhal bronchitis,
blood diseases, anti-leucorrhoea, anti-diarrhea, anti-catarrhal, antigenotoxic and chemo
protective activities.1-16
B. Phytochemical importance
The plant is a rich source of bioactive chemical constituents with potential therapeutic
activities. Phytochemical studies revealed that the plant contains important class of
compounds including alkaloids, amino acids, carbohydrates, fatty acids, flavonoids,
glycosides, proteins, resin, saponins, steroids, tannins and terpenoids (Table 1).1,3e,17-25
42
3.1.2. Ixora chinensis
I. Chinensis Lam. commonly known as Chinese Ixora, belongs to family Rubiaceae, more
than 500 species of this family have been found all over the world. It is a shrub or a small tree
native to China, Thailand and commonly found in Pakistan. Most of the species are usually
utilized as decorative plant in gardens. Leaves have small stalked, obviate, oblong, waxy, 6-
10 cm in length, pointed at both ends and borne on short petioles. Flowers are compactly
organized with 4 pink color petals. The genus Ixora has been reported to possess different
classes of compounds, including amyrins, fatty acids, aromatic acids, flavonoids, oils, tannins,
saponins, carbohydrates, triterpenoids, and sterols which were described in Table 2.26-28
A. Pharmacological importance
I. chinensis plant have been used in several medicinal purpose like antimicrobial, antibacterial,
antiaging, antioxidant, hepatoprotective, chemoprotective, antinociceptive, antimitotic,
antiinflammatory, antitumor and anticancer diseases. It is usually used in traditional system
of medicines, like liver diseases, spasmolytic, antiallergic, antihistaminic, and mental
diseases. Different parts of the I. chinensis have been used in the medication of skin diseases,
dizziness, headache, gout, hemorrhoids, melanosis, juindice, tuberculosis, chronic bronchitis,
facial paralysis, and lowering blood pressure. Moreover, several medicines have been
prepared for the treatment of digestion problems, tonsillitis, stomachache, kidney stones and
gastric ulcer. Furthermore, Chinese medicines have been prepared for the treatment of
dysmenorrhea and trigeminal neuralgia.29-38
B. Other uses
Anthocyanins are natural colorants which have increased a cultivating interest due to their
wide variety of colors and valuable health effects, assessment of anthocyanins have been
performed in red and blue color flowers. Extraction and stability of red pigment from I.
chinensis Lam and the preparation and uses of all kinds of new pigment in food products,
shampoo, and cosmetics etc. have been prepared and reported in literature.39
43
Table 1: Isolated chemical constituents from Ixora coccinea
S.
No.
Compounds Molecular
formula
Molecular
weight
Parts of
plant
References
Alkyl Esters
1 2,3-Dihydroxy propyl eicosanoid acid C23H46O4 386 Leaves 1g
2 Di-n-octyl phthalate C24H38O4 390 Root 25a
Coumarins
3 Scopoletin C10H8O4 192 Leaves 1g
4 Caumarin C9H6O2 146
5 Erythro 1,2-albiflorin C16H18O5 290
Diterpenoids
6 16α-Hydroxy-19-acetoxy-(-)-kauran-
17-oic acid
C22H35O4 363 Leaves 1g
7 16α-Hydroxy-19-ol-(-)-kauran-17-
oic-acid
C20H32O3 320
Fatty Acids
8 Oleic acid C18H34O2 282 Root,
root bark
1d, 4i, 6d,
12b 9 Linoleic acid C18H30O2 278
10 Myristic acid C14H28O2 228
11 9,12-Octadecadienoic acid C18H34O6 276
12 Palmitic acid C16H32O2 256
13 Stearic acid C18H36O2 284
Flavonoids
14 Ixoratanin A-2 C45H36O18 864 Leaves 6d, 16b
15 Procyanidin A-2 C30H24O12 576
16 (+) Catechin C15H14O6 290
17 (-)-Epicatechin C15H14O6 290
18 Kaempferol C15H10O6 286
19 Kaempferol-7-O- α-L-rhamnoside C21H20O10 432
20 Kaempferol-3-O-α -L-rhamnoside C21H20O10 432
21 Kaempferol-3,7-O- α-L-dirhamnoside C27H30O14 578
44
22 Quercetin-3-O-α-L-rhamnopyranoside C21H20O11 448
23 Luteolin C15H10O6 286
24 Kaempferitin C27H30O14 578 Root 6d, 16b
25 Kaempferol-7-O-glucoside C21H20O11 448
26 Quercetin C15H10O7 302
27 Cyanidin-3-glucoside C21H21O12 610 Flowers 1d, 1h
28 Delphinidine-3-glucoside C21H21O12 465
29 Rutin C27H30O16 610
30 Kaempferol-3-rutinoside C27H30O16 595
Peptides
31 Ixora peptide I C30H32O5N2 500 Leaves 6d
32 Ixora peptide II C31H42O4N4 534
Quinones
33 1,4-Dihydroxy-3-methyl-
anthraquinone
C14H8O4 240 Leaves 6d
34 α-Tocopherol C29H50O2 430
Steroids
35 Stigmasterol C29H48O 412 Leaves 6d
36 6 β-Hydroxy-stigmast-4-en-3-one C29H48O2 428
37 β-Sitosterol C29H50O 414 Flower,
Leaves
4h, 6d
Steroidal Glycosides
38 Stigmast-5-en-3-O- β-D-glucoside C35H61O6 577 Flowers 1l, 6d
Sugars
39 D-Mannitol C6H14O6 182 Root,
rootbark,
leaves
4h, 4i
Tannins
40 5-O-Caffeoyl quinic acid C6H14O6 354 Flowers 1l
41 Cinnamtannin B-1 C6H14O6 864 Leaves 5e
Terpenoids
45
42 21,23-Epoxy-tirucall-7-en-3β-ol
(ixoroid)
C30H50O2 442.3 Flowers 1l
43 Ursolic acid C30H48O3 457 6d, 49
44 α- Amyrin C30H50O 426 1d, 5d
45 17β-Dammara-12,20-diene-3β-ol
(ixorene)
C30H50O 354 Leaves 4h, 5d, 6d
46 Lupeol C30H50O 426
47 3-Acetyl betulinic acid C32H50O4 498
48 Betulinic acid C30H48O3 456
49 3-Acetyl ursolic acid C32H50O4 498
50 Oleanic acid C30H48O3 456
51 β - Amyrin C30H50O 426 Roots 12b
46
Table 2: Isolated chemical constituents from Ixora chinensis
S.
No.
Compounds name References
1 Rutin 27a
2 Kaempferol 27a
3 Quercetin 27a
4 Formononetin 27a
5 Catechin 27a
6 Kaempferitrin 27a
7 Rubiothiagepine 27a
8 Oleanolic acid 27a
9 β-sitosterol 28b
11 D-Mannitol 28b
12 Steric acid 28b
13 (10E)-9-Oxooctadec-10-en-12-ynoic acid 28b
14 Azelic acid 28b
15 Dihydromasticadienoic acid 28b
16 Ixorene 27a
17 Ixoric acid 28c, 28d
18 Crepenynic acid 28c, 28d
19 Ixoroside 28e
20 Ixoside (7,8-dehydroforsythide) 28e
47
Ipomoea batata blackie
Ipomoea batata pink frost
48
3.2. Ipomoea batatas (Ornamental sweet potato)
The genus Ipomoea with approximately 500-600 species was considered the large genus
within the Convolvulaceae family, its looks climbing woody, twining, and herbaceous plants
that commonly have funnel-shaped flowers and heart-shaped, leaves. The genus Ipomoea
arises in the warm areas of the world although a number of species were found in moderate
regions. The species are frequently scattered all through central and the south America, and
tropical Africa countries and consumed almost all over the world. Decorative sweet potato
have many types consisting 'Blackie' and 'Black Heart' with reddish-purple-black plants and
'Tricolor' or 'Pink Frost' is spotted with violet-pink and blush in colors with smaller roots.
A number of species of the genus Ipomoea, as well as, of the Convolvulaceae family have the
property of phytotoxicity, which means suppressing the growth of other plants consisting
invasive weeds. The genus Ipomoea has been used traditionally for diverse reasons, for
example, nutritional, medicinal, and agricultural purposes. The plant is used as antimicrobial,
antifungal, nematicidal, antimutagenic, anticarcinogenic, inflammatory and anticancer
diseases. They also possess antioxidant, analgesic, spasmolitic, spasmogenic, hypotensive,
hepatotoxicity and trypsin inhibitor activity. The plant has been used in the dealing of asthama,
diabetes, infections, prostatitis, anaemia, hypertension, allergies, aging, HIV and
cardiovascular diseases.
The phytochemistry of the Ipomoea genus has been examined as a result of different class of
compounds have been identified namely, alkaloids, coumarins, isocoumarins, diterpenes,
benzenoids, flavonoids, phenolics compounds, norisoprenoids, lignins, glycolipids and
triterpenes.40
3.2. 1. I. batata blackie
The plant I. batatas Lam. (blackie), commonly called “sweet potato” belongs to the family
Convolvulaceae, is a famous ornamental plants. This tender tuberous perennial with heart-
formed, lobed, leaves are black in color, flowers are bears trumpet-shaped lavender to pale
purple in color. Plant stems have thin shaped with light green and purple in color. Leaves
appear alternate heart shaped, palmate lobed leafy vegetable. The plant has good appearance,
49
fast growth rate and high size. I. batata blackie plant has several biological and chemical
properties. It is the World’s most significant multipurpose food crop with greater than 133
million ton in annual production. I. batata plant have several medicinal importance, it has
been used in the treatment of cardiovascular diseases, anticancer, anti-bacterial,
anticarcinogenic, fever, tumor, asthama, burn, nausea, and stomach problems. The plant have
been used in the healing of aphrodisiac, laxative, antihyperglycemic, antidiabetes, and
hypertension. Phytochemical studies on I. batata showed that the plant contains important
class of compounds including aromatic compounds, terpenes, vitamins, hydrocarbons, fatty
acids and their esters, terpenes, and anthocyanins. The purple color of sweet potatoes are due
to the presence of anthocyanin. The total antioxidant activity of purple fleshed sweet potatoes
were higher.41-51
3.2. 2. I. batata tri-color pink frost
This sweet potato vine is blessed with leaves that are edged in pink. Use it in pots, like
groundcover, in beds and borders.42a-d
3.3. Cassia fistula
C. fistula is a very common Indian tree belongs to family Fabaceae usually known as “Golden
Shower Indian Laburnum.” It is indigenous plant to India, Sri Lanka, Mexico, China, South
Africa, West Indies and Brazil. It is a semi wild, medium deciduous tree with trunks consisting
of greenish grey smooth bark, when young hard reddish wood, attain a height of 40 feet. The
flowers are bright yellow in color and occur in clusters, the fruit is cylindrical pod and pulpy.
Seeds are described as flattish brown. Whole plant has been reported for its pharmacological
potential, so, it is called “disease killer”.52,53
A.Pharmacological importance
Medicinal plants have great significance for the treatment of diseases. Over the years,
scientists have understood the importance of active chemical constituents, which showed the
pharmacological properties of the plants. C. fistula is usually known for its antimicrobial,
antifungal, antibacterial, antioxidant, analgesic, anticancer, anti-tumor, anti-inflammatory
properties. The plant also possess anti-psoriatic, antipyretic, anti-malarial, hypolipidemic,
anti-diabetic, hepatotoxicity, anticonvulsant, antileishmanial and anthelmintic activities. It
50
Cassia fistula tree
Cassia fistula flower
51
has been used in the treatment of hypoglycemic, constipation, cardiac problems, dyspepsia,
fever, asthama, liver, stomach, kidney and skin diseases. The plant was widely used in the
medication of burns, diahorrea, facial paralysis, and leucoderma. It is also known to treat
intestinal disorders, hepatoprotective, jaundice, anti-cholesterol, antiulcers, antifeedant and
larvicidal activities. Nanoparticles have been synthesized by using the plant extracts and their
pharmacological activities have been studied. 53-79
B. Phytochemical importance
Several class of compounds have been isolated from the entitled plant named as alcohols,
alkaloids, amino acids, anthraquinones, carbohydrates, chromenes, fatty acids, flavonoids,
minerals, lectin, phenolic compounds, steroids, saponins, terpenoids, volatile oils, and
tannins.81-99
Alcohols:
Ethanol, 1-hexacosanol,1-octacosanol, 1, 2, 3-propanetriol 1-acetate were found in the stem
bark of the plant.80
Aldehydes:
Acetaldehydes and 2,4-dihydroxy benzaldehyde have been identified from different parts of
C. fistula.81
Alkaloids:
Fistulatins A and B, fistulalkaloid A (Isoquinoline); and 2-(2-hydroxyethyl)-5-methyl-6-
prenylisoindolin-1-one, (isoindole) and pyrrolidone have been identified as important plant
constituents.54a,82,56f
Amino acids:
Aspartic acid, asparaginine, β-alanine, arginine, leucine, methionine, phenylalanine,
tryptophan, glutamic acid were identified from different parts of the plant.83
52
Anthocyanins:
Cyanidin-3-O-galactoside, and petunidin-3-O-glucoside were isolated from C. fistula.84
Anthraquinones:
Rhein (1), 6-Methylrhein, 1,3,8-trihydroxy-6-methylanthraquinone, N,N'-bis(1-
methylethyl)-6-chloro-4-phenyl-2-propylquinoline, benzoquinoline 2,4-di-methyl-,
chrysophenol (2), physcion (3), 7-methylphyscion, fistulic acid (4), 3-formyl-1-hydroxy-8-
methoxy anthraquinone (5), sennoside A (6) and B (7), 8-O-D-glucopyranosyl-1-hydroxyl-3-
methyl-anthraquinone (8) were identified from various parts of the entitled plant.
Cassiquinone A, aloe-emodin, 1,3-dihydroxy-2-methyl-5,6- dimethoxy anthraquinone, fistula
quinone A, 9,11-dihydroxy-2-(hydroxymethyl)-5-methyl-4H-naphtho [2,3] chromene-4,7,12-
trione, 9, 11-dihydroxy-2,5-dimethyl-4H-naphtho[2,3]chromene-4,7,12-trione, 4-dodecyl-
1,3,8-trihydroxyanthraquinone, hexacosanoyl quinate, 7, 12-dimethyl benzanthracene, 1,8-
dihydroxy-6-methoxy-3-methyl anthraquinone, 1,8-dihydroxy-3-methyl anthraquinone,
bianthraquinone have been isolated from C. fistula. 1,3-Dihydroxy-6,8-dimethoxy-2-
isoprenyl anthraquinone and 3-formyl-1-hydroxy-8-methoxy-anthraquinone have been
identified from the entitled plant.52,56c,80b,d,81b,86
Aromatic compounds:
Isovanillic acid, vanillic acid, salicyl alcohol, and 4-hydroxybenzyl alcohol have been
identified from the plant of C. fistula.81b
Carbohydrate:
Inisitol, galactose, mannose, epimelibiose, galactomannobiose, mannobiose, manno- tetraose,
mannopentaose and mannohexaose have been isolated from the plant.87
Carbonyl Compounds:
2-Hentriacontanone (21), 2-hexadecanone (22), 5-nonatetracontanone (23), heptacosanoic
(24), nonacosanoic (25), and triacontanoic acids (26), 5(2) hydroxyl phenoxy methyl furfural
(27), benzyl-2-hydroxy-3,6-dimethoxy benzoate (28), benzyl 2β-O-D-glucopyranosyl-3,6
53
dimethoxy benzoate (29), heptacosyl eicosanoate, 5-nonatetracontanone and 2-hentriacontan-
one have been identified from the plant.88
Caumaurin:
Isoscopoletin and scopoletin have been identified from the C. fistula.89
Chromones:
Fistucacidin (17), 7-hydroxy-5-hydroxy methyl-2-(2'-hydroxypropyl) chromone (30),
fistulain A (31), and B (32), 8-hydroxy-2-(3-hydroxypropanoyl)-5-(2-oxopropyl)-4H-
chromen-4-one,2-(2-hydroxyethyl )-7-methoxy-5-(2-oxopropyl)-4H-chromen-4-one, 2-(3-
hydroxypropanoyl)-8-methoxy-5-(2-oxopropyl)-4H-chromen-4-one, 6-methoxy-3-methyl-8-
(2-oxopropyl) benzooxepin-5(2H)-one, 5-hydroxy-2,2-dimethyl-7-(2-oxopropyl)-2,3-
dihydrochromen-4-one, 8-methoxy-2,2-dimethyl-7-(2-oxopropyl)-2,3-dihydro chromen-4-
one, 5-methoxy-2,2-dimethyl -7-(2-oxopropyl)-2,3-dihydrochromen-4-one, 5-methoxy-2,2-
dimethyl-8-(2-oxopropyl)-2,3-dihydrochromen-4-one,1-(3,4-dihydro-5-methoxy-2,2-dimeth
yl-2H-chromen-7-yl) propan-2-one, 7-hydroxy-2-(2'-hydroxy propyl)-5-methylchromone,
2,5-dimethyl-7-hydroxychromone, and 2,5-dimethyl-7-methoxy chromone have been isolated
from various parts of the plant of C. fistula.86a,u,89,90
Essential oil:
Eugenol, phytol, camphor, 1,8-cineole, limonene, linalool, nerolidol, and 2-hexadecanone
have been identified from the essential oil of the plant.81a,92
Fatty acids and their esters:
2-Propenoic acid, octanoic, palmitic, stearic, oleic, linoleic, linolenic, myristic, myristoleic,
arachidonic, behenic acids and heptacosyl eicosanate have been identified from different parts
of the C. fistula.54a,86r,u,93
Flavonoids
Kaempferol (9), kaempferol-3-O-glucoside, kaempferol 3-neohesperidoside, (+)
epiafzelechin (10), (-) epiafzelechin (11), epiafzelechin-3-O-glucoside, procyanidin B2 (12),
catechin (13), epicatechin (14), naringenin, flavanol, quercetin, rutin, hesperidin, rhamnetin-
54
3-O-gentiobioside (15), rhamnetin 3-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside,
5,7,3',4'-tetrahydroxy 6,8-dimethoxy flavone-3-O-α-arabinopyranoside, biochannin A, fistula
flavonoids B and C; 5,7,4'-trihydroxy-6,8,3'-trimethoxyflavone-3-O-α-L-rhamnosyl (1→2)-
O-β-D-glucopyranoside; 1,8-dihydroxy-3,7-dimethoxyxanthone-4-O-α-L-rhamnosyl (1→2)-
O-β-D-glucopyranoside, and 3-neohesperidoside have been identified from different parts of
C. fistula plant.56a,d,58f,84,85,94a-e
Glycerols:
Glycerol-1-tetra-eicosanoate, glycerol-1-penta-eicosanoate, tri-myristin, and glyceryl tri-
linoleate have been isolated from the entitled plant. 80c,94g
Heterocyclic compounds:
Ascorbic acid (vitamin C), 2(3H)-furanone, naphtho [1,2] furan, 2,9-dihydroxy-7-methoxy-4-
methyl naphtha [1,2] furan-3(2H)-one, 5-(2-hydroxyphenoxymethyl) furfural (27) and 5,6-
dihydroxyisoaurone-4'-O-β-D-glucoside have been identified from different parts of C.
fistula.53b,54a,58b,86u,95
Hydrocarbons:
Glyceryl-1-tetracosanoate, heptacosanyl-5-hydroxypentadec-2-enoate, and octacosan-5,8-
diol were isolated from the entitled plant.96
Minerals:
Minerals namely K, Na, P have been identified from C. fistula.86l
Phenolic Compounds:
Vanillic acid, cinnamic acid, gallic acid, benzofuran 2,3,dihydro-2-methoxy-4-vinylphenol,
3-(4-hydroxyphenyl)-methyl ester, phenolic amide, 3-(3,4-dihydroxyphenyl)-N-[2-(4-
methoxyphenyl)-2-oxoethyl]-prop-2-enamide, benzyl 2-hydroxy-3,6-dimethoxy benzoate
(28), benzyl 2β-O-D-glucopyranosyl-3,6-dimethoxy benzoate, and dibenzyl 2,2'-dihydroxy-
3,6,3'',6''-tetramethoxy-biphenyl-1,1'-dicarboxylate (I), have been identified from the plant.
86u,87b, 97a-d
55
Steroids:
β-sitosterol (16), β-sitosterol-3-O-β-glucopyanoside, stigmasterol, ergosterol, lupeol, β-
amyrin, friedelin, daucosterol, aurantiamide acetate, fucosterol, and 28-isofucosterol were
isolated from different parts of the C. fistula.54a,69,80c,86r,87b,94d,98
Terpenes:
Lupeol (19), nerolidol (20), 3β-hydroxy-lup-20(29) en-28-oic acid, and 3β-17-norpimar-8(9)-
en-15-one were identified from the plant.56,86r,89,99a,b
3.4.3. Other uses:
The plant has been used for the manufacturing of chemical fertilizer, decolorization of
reactive dyes, and textile application.100
56
O
OOH OH
COOH
O
OOH OH
CH3 O
OOH OH
CH3H3COO
OOH OH
H3CO CH3
COOHH3CO
OH
O
OOCH3
OH
OCH3
Physcion (3)Rhein (1) Chrysophenol (2) Fistulic acid (4)
3-Formyl-1-hydroxy-8-
methoxy anthraquinone (5)
Sennoside A (6) Sennoside B (7)
O
O
HH
HO
O
OH
O
O
O
O
O
OH
OHHO OH
HO OH
OH
HO O
O
H
HO
O
OH
O
O
O
O
O
OH
OHHO OH
HO OH
OH
HO
H
8-O-Glucopyranosyl-1-hydroxyl
-3-methyl anthraquinone (8)
OH
CH3
OO
OH
OH
OHHO
O
O
OH
OH
OH
HO
Kaempferol (9)
O
OH
OH
HO
(+) Epiafzelechin (10)
OH
Figure 1: Chemical constituents identified from C. fistula
57
O
OH
OH
HO
(-) Epiafzelechin (11)
OH
O
OHOH
HO
OH
OH
O
OH
OH
OHHO
OH
O
OH
OH
OH
OH
HO
O
OH
OH
OH
OH
HO
Catechin (13)
Epicatechin (14)
Procynidin B2 (12)
O
O
H3CO
OH
OHOH
O
O
OH
OHOHO
O
OHOHOH
HO
Rhamnetin-3-O-gentiobioside (15)
HO
Sitosterol (16)
HO
O
OH
HO
OH
OH
Fistucacidin (17 )
OH
Hexacosanol (18 ) Lupeol (19)
HO
OH( )23
Nerolidol (20 )
58
2-Hentriacontanone (21 )
O
2
2-Hexadecanone (22) 5-Nonatetracontanoate (23)
( )10
O
2( )25
O
5( )40
OH
O
Triacontanoic acid (26 )Heptacosanoic acid (24)
OHH3CO
OCH3
O
O
HO
OO
H
O
Benzyl 2-hydroxy 3,6 dimethoxy benzoate (28)5(2) Hydroxy phenoxy methyl furfural (27)
Nonacosanoic acid (25)
( )20 OH
O
( )22 OH
O
( )24
O
OH
OHO
HO
H3CO
OCH3
O
O
O
OHOHO
OH
HO
(2'S)-7-Hydroxy-5-hydroxy methyl-
2-(2-hydroxypropyl) chromone (30) Benzyl 2--O-D-glucopyranosyl
3,6-dimethoxy benzoate (29)
Fistulain A (31)
ON
O
CH3
OH3C
O
HO
CH3
O
O
O
HO
OH3C
O
O
OMe
Fistulain B (32)
59
Chapter 4
Results and discussion
60
Present studies
Phytochemical and biological research on medicinally important plants namely, Ixora fulgens
(red flowers), I. polyantha (white flowers), I. chinensis (pink flowers), I. coccinea (orange and
yellow color flowers), Ipomoea batata blackie, I. batata pink frost and Cassia fistula showed
that it has remarkable medicinal significance in folk and traditional medicines. In the present
studies bioassay directed isolation method on flowers, leaves and stalks of all the above
mentioned plants were carried out for the extraction and evaluation of anticancer, antibacterial,
antifungal, antioxidant and immunomodulatory activities. Flowers were extracted with hexane,
chloroform and methanol to understand its biology and chemistry more elaborately.
All extracts showed promising cytotoxic activity, against three human cancer cell lines namely,
HeLa (cervical cancer), MCF-7 (breast cancer), and NCI H-460 (lung cancer) cell lines, which
have been used for the evaluation of cytotoxic effect. Triplicate experiments have been
performed, while doxorubicin was used as a standard drug (Tables 3, 11, 18). The extracts were
studied through GC and GC-MS examination, with the identification of 349 chemical
constituents from hexane extracts, 273 compounds from chloroform extracts, and 26
phytochemicals from I. chinensis methanol extracts (Tables 4-6, 12, 13, 19, 20).
The extracts were screened for antibacterial assay against two Gram positive (Bacillus subtilis
and Staphylococcus aureus) and four Gram negative bacteria (Escherichia coli, Shigella
flexenari, Pseudomonos aeruginosa and Salmonella typhii) and most of the samples were found
to be inactive against all the tested bacteria (Tables 7, 14, 21). Subjected plant extracts have
been examined for antifungal assay against five fungi namely Trichphyton rubrum, Candida
albicans, Aspergillus niger, Microsporum canis, Fusarium lini and Candida glabrata, which
were found to be inactive against all the tested fungi (Tables 8, 15, 22). These plants extracts
also possessed antioxidant and immunomodulatory properties.
61
OH( )n
n
07 = (1)
11 = (2)
12 = (233)
13 = (3)
15 = (338)
16 = (434)
21 = (4)
25 = (5)
20 = (386)
31 = (389)
OH
(7)
OH
(12)
(8)
(E)OH( )5
(9)
HO
(6)
HO
(11) (10)
CH2 O
(232)
OH
HO(E) (E)
(15)
HO
(E)
(E)
(14)
HO(E)
(16)
(17)
OH
CH2
OO
O
HO (Z)
(18)
(13)
HO
( )5( )7
(19)
HO OH
OH
(22)
OH
(20)
Figure 2: Chemical constituents identified from medicinal plants extracts
62
(24)
OH
(E) (E)
(E) (E)
(23)
H
O
( )11H
O
(29)
( )13
(30)
(Z)H
O
( )11
(28) (31)
O
O(E)
(25)
H
O
(E)
(27)
( )6
HON
O
OH
(32)
( )10
OOH
(33) (34)
N
O
O
(Z)(E)
OOH
OCH3
OH
(36)
(26)
H
O
OOH
OH
OCH3
(35)
O
O
O
O
(40)
OH
OH
OCH3
(38)
OH
OCH3
(39)
OO
O
H
OH
OCH3H3CO
(37)
(44) (43)
O
O
O
O
( )3
( )3
(42)
O
O
O
O
(41)
(45)
(48) (49) (47) (46)
63
(50)(51)
(52)
(54) (53) (55)
(56)
CH2
(58) (57)
(Z)
(59)
(60)
(57)
(r)
(r)
(Z) (Z)
(62) (63) (64)
(Z)
(65)
( )3
(67) (68) (66)
(70)(69)
( )6( )6
(71) (72)
64
(73)
(Z)
(E)
(74)
CH2
CH2
(76) (75)
(Z)
OH
O
(77)
(Z)H
O
(78) (80)
(Z)
(79)
(E)
(83)
(E)
(Z)
(87)(86)
O
(Z)
(82)
(89)
H2C
H3C
O
(Z)
(E)
(84)
O
(Z)(E)
(81)
O
H2C
(88)
(85)
(91)
O
(97)
O
O ( )15
O( )7 ( )7
(96)
H3C
O
CH2
(93)
CH3O
(94)
O
(95)
65
(99)
O
(100)
O
(98)
(103)
ON
O
NH
O
O
(101)
OO
CH2
HO
(106) (105)
O
OO
O
( )14
OO
O
O
OO
(104)
( )3
O
(Z)
O
(102)
Cl ( )11
(116)
Cl
O
(E) (E)
(115)
( )7 ( )4
O
(E)
(114)
OF
O
F F
F F
FF
( )17
(119)
OCl
O
(120)
( )13
(S)N
Cl
O
OCH3
O
(117)
(118)
OFF
F
FF
O
(121)
( )18
(122)
Cl
O
( )8
(255)
Cl( )17
(123)
Cl
O
(140)
( )10
(139)
HO H
O O
( )7H OCH3
OO
(E)( )4
8
9
(138)
O
(141)
O
(142)
CH3
O
(E) (E)
(143)(144)
(E)
O
(145)
O
(146)
CH3
H3CO
66
( )n
n
09 = (124)
10 = (125)
11 = (126)
12 = (127)
13 = (128)
14 = (129)
15 = (355)
16 = (426)
17 = (130)
18 = (131)
19 = (133)
21 = (357)
22 = (132)
27 = (358)
28 = (455)
31 = (134)
32 = (135)
33 = (136)
34 = (137)
41 = (427)
OO
( )8
(149)
(147)OH
OH
O
(148)
OO
H3COOH
O
O
H3CO
OCH3O
H3CO OCH3
OO
H3CO
O
(156)
(152) (154)
( )6
(157)
O O
H3CO
HO
(151)
H3CO OH
OO
H3CO H
O O
(159)(158)
( )7
( )7
(160)
H3CO O
O OCH3
( )7H3CO OCH3
OO
(161)
( )8O
O
O
O
(162)
( )9( )8( )9
OH
O
( )n
n
03 = (107)
04 = (108)
08 = (109)
12 = (110)
14 = (111)
15 = (112)
16 = (113)
06 = (344)
07 = (454)
10= (345)
19 = (346)
13 = (418)
OCH3
O
( )n
n
06 = (155)
10 = (164)
12 = (165)
14 = (166)
15 = (172)
16 = (174)
17 = (181)
18 = (191)
19 = (184)
20 = (185)
21 = (186)
22 = (187)
23 = (188)
24 = (189)
26 = (183)
O
O
( )14
(168)
OCH3
O
(163)
( )8 OCH3
O
O
O( )7
( )5
(167) (169)
( )14
O
O
( )7( )5
(173)
67
OCH3
O
OCH3
O
(170) (169)
O
O
(171)
OCH3
O
(E) OCH3
O
(E) (E)
9
10
(175) (177)
9
1012
13
O
O
(E)( )7( )7
(176)
OCH3
O
(178)
( )7 OCH3
O
(E) (E) (E)
(179)
9
1012
1315
16
O
O
(E) (E)
(180)
()7()4
9
1012
13
O
O
( )14
(182)
OCH3
O
(190)
( )19
OCH3
O
(193)
( )8
OCH3
O(E)
(192)
O
POF
(194)
68
(197)
NH(E)
(Z)
O
HS
O
HO(Z)
(200)
O
(198)
O
OH(E)
(E)
(199)
OS
O
O
(196)
( )12
NP
N
O
CH3(Z)
(Z)
(Z)
(Z)
(195)
O O
(203)
(201)
O
OH3C
OO Si(E)
(202)
O
OOCH3
(204)
(209)
OH
(E)
(205)
O
O
O
O
(206)
OCH3
O
O
O
( )7
N
O
O
CH3
OCH3H3CO
(Z)
(207) (208)
OH
(210)
O
(211)
(213)
(E) (E) O
O
(212)
HO (E)
69
(215)
OH
(216)
O
OH
CH3
OH
CH2
(218)(217)
OO
HO
(220)
H2CCH2
(219)
(217)
OH OH3C
O
(221)
(222)
(227)
HO
(223)(224)
(225)
(228)
O
CH3HO
CH3
H3C
(229)
O
CH3HO
CH3
(230)
O
HO
CH3
H3C
70
(235)
OH
(236)
(E)OH
(237)
(Z) OH
(239)
(238)
H3C O(E)
O
H3C O(E)
O
(240)
(E)
OH
(243)
OH
OH
(242)
OH OH
2 63 7
(241)
OH
(234)
HO
(247)
(E) (E) H
O
(248)
H
O
(250)
O
(251)
(E)
O
(249)
O (E) ( )4
O(E)
(244)
( )5
(245) (246)
H
O
(Z)
(E)
H
(Z)
(E)
O
HON
O
HO
HO
OCH3
OH
(252)
( )9
(253)
OHOH
OCH3OCH3
(254) (256)
O
O
O
O
HHH
71
(257) (258)(259) (260)
(261)
H3CO
O
O
(E)
O
HOOCH3
(E)
(262)
( )3
(263) (272)
(273) (274) (275)
(276) (277) (278)
4
5 53 5
3
84
7
5 7
(279) (280)
3
6 2
73
(281)
2 6 10
(282)(283) (284)
(E)
2
7
10 2
5 6
H
72
(285)
5
(286) (287)(288)
(289) (290)
(291)
(E)
O
23 5
2
5
253
7
(292)
(293)
(E)13
4
3 5
(295)
O
(294)
(E)( )13 ( )3
O
(309)
OO
(Z)
(329)
(Z)
(318)
HO
O
(E)
(349)
( )5( )9
O
(343) (347)
OH
OO
(348)
(E) (E) (E) OH
O
(356)
O(E)
O
OH
OH
(350)
HO
OH
73
O
(359)
SO
O
(361)
(369)
OS
O
O
( )10
(370)
SiO
SiO
SiO
Si
OSi
OSi
O
O
Si
Si
(371)
SiO
HS
OSi
OS
O
O
Si
Si
(372)
O
SiO
Si
OSi
OSi
OSi
OSi
OSi
OSi
OSi Si
O
(374)
OS
O
O
( )10
(375)
S
O
O
OH
OH
HO
H3COOH
(376)
(377) (378)
(373)
Si
OSi
O
SiO
SiO
SiO
SiO
SiO
SiO
SiO
(379) (380)
OH
(383)
HO
(381)
O
OHOH O
HO
(382)
74
(385)
OO
OH
3
(390)
(393)
O
O
( )9 ( )2
(391)
HO
OHOH
OH
(392) (394) (395)
H3CO
O
O
(398) (397)
H
O
CH3
OOHHO
(399)
(400)
O
O
O
O
(396)
HOO
(402)
(403) (406)
CH2
HO
O
O
(408)
(407)
O
(404)(405)
(401)
O
O
O
O 7
7
O
OHO
(411)
(414)
H3CO
H3CO
O
(413)OH
H3COO
(415)
75
O
(416)
( )5
(419)
O
Cl
O
O
O
O
(417)
OF
FF
O
(420)
( )9
ClOH
Cl
Cl
(421)
(422)
Cl
Cl O
(428)
(424)
Cl Cl
ClCl
(425)
(426)
ClCl
Cl
Cl
Cl
(423)
O
O
(431)
( )14 ( )5
HO
OH
O
(433)
H3CO
O
(432)
( )14
O
H3COO
O
O
(429) (430)
(436)
OH
5
68
911
12
(437)
OH( )10( )3
O
OH
O
OCH3
H3CO
O
OH
HO
(438) (439) (440)
O
OH
(442)
76
(460)
OS
O
O
( )14
H
O
(465)
O
O
(457)
(461)
O OH
(462)
HO
(463)
(464)
( ) 6
( )7
(460)
OS
O( )13( )4
OCH3
H3CO
HN
NHO
O OCH3
OCH3
(441)
(443)
3 5
64
(467)
H
O
( )5( )3
(469)
OH
(470) (468)
(475)
(471)
(472) (474)
O
O
(473)
O
O
CH3OH
OH
O
( )7
(476)
O
OCl
O
O
(479)
OH
O
(478) (480)
Br
( )10
77
O
OH
OCH3
(485)
(E)
(482)
( )5( )5
O
(481)
( )9
(484)
2
(486)
S
(490)
HON
O
O
( )9
O
OO
O
(488)
(493)
2
3 5
OHO
OH
(492) (491)
OH
151153
(514)
O
O
(513)
HSi
(Z)
(Z)
Si
OSi
O
SiO
SiO
SiO
SiO
SiO
SiO
SiO
(512)
(506)
(E) O
O
(505)
OH
O17
78
A) Bioassay guided isolation studies on Ixora species
a) Cytotoxic activity
World has facing a cruel health disease known as cancer, extensive types of cancers, namely
stomach, liver, lung, cervical, and breast cancer; lung cancer (men) and breast cancer (women)
have been recognized the major widespread cancer. Cancer deaths (>70 %) happened in poor
countries. Conventional treatments of cancer consist of involvements such as psychosocial
support, radiotherapy, chemotherapy and surgery. On the other hand, death rate increase
related with cancer and harmful results of cancer radiation therapy and chemotherapy,
isolation of anticancer agents from plants, is currently under investigation. Numerous natural
products, with diverse structures, were identified as anticancer compounds. A number of
compounds such as vincristine, podophyllotoxin, camptothecin, taxol, combretastatins,
vinblastine, etc. were identified from plants and modified to produce enhanced analogues for
toxicity, activity or solubility. More than a few flourishing molecules also known as potential
drugs and several are forthcoming.1d,101a,b
Uncrushed, dried flowers, leaves and stalks of Ixora species were subjected for the chemical
and biological studies named as including I. fulgens (red), I. polyantha (white), I. chinensis
(pink), I. coccinea (orange and yellow). All the above mentioned plants were extracted
through soxhlet extraction method with hexane (H) followed by chloroform (C), and methanol
(M) to give respective extracts, which were evaporated through rotary evaporator
(Experimental). Total twelve extracts were obtained from hexane (H1-H12), twelve from
chloroform (C1-C12) and twelve from methanol (M1-M12), which were examined for
phytochemical and pharmacological studies.
All the extracts were tested for anticancer activity (Tables 3a-c). All the non-polar (hexane)
and moderately polar (chloroform) extracts and methanol extract of I. chinensis flowers were
examined to GC-FID and GC-MS evaluation, which demonstrated the occurrence of alcohols,
aldehydes, amide group, aromatic compounds, benzene derivatives, cyclic and bicyclic
compounds, hydrocarbons, branched chain hydrocarbons, heterocyclic compounds, fatty
acids, and their esters, halogen containing compounds, phenols, terpenes, tocopherols, and
miscellaneous compounds (Tables 4-6)(Figure 2).101c
79
All extracts of I. fulgens, I. polyantha, I. chinensis, and I. coccinea plants (orange and yellow
color flowers) (10-250 µg/mL) have been evaluated for growth inhibitory and cytotoxic
activity. Among I. fulgens flowers, chloroform extracts was found to be most potent in growth
inhibition and cytotoxic effect (GI50: 10 ± 2.9 µg/mL and LC50: 165 ± 3.1 µg/mL) in
opposition to HeLa cell line. It was ¬ 4.8 times less potent than MCF-7 and H-460 cell lines.
While leaves extracts of I. fulgens showed less potent growth inhibitory effect (Tables 3a-c;
Figure 3).
Chloroform extract of I. polyantha leaves was consistently most active against NCI H-460 in
producing growth inhibitory effect (GI50: 50 ± 4.5 µg/mL) and equipotent effect against both
HeLa and MCF-7 cell lines (GI50: 128 ± 3.5 µg/mL) (Table 3). However, flowers extracts
were inactive against all aforementioned cell lines. Among I. coccinea flowers again
chloroform extract was equipotent (GI50: 15 ± 3.8 µg/mL) against HeLa, MCF-7 and NCI H-
460 cancer cell lines, while its leaves and stalks demonstrated non significant growth
inhibitory effects. Both leaves and flower extracts of I. chinensis displayed weaker growth
inhibitory consequences not in favor of tested cell lines. These results are in agreement with
previously reported work that I. coccinea flowers exhibited cytotoxic effect, extract of the
flower was reported to include flavonoids, phenols, terpenoids and tannins. Ethanol extract
fractionate by using petroleum ether to give lupeol that has potential of cytotoxicity.
Kaempferol has been isolated earlier from the plant showed anti-platelet aggregation activity.
In addition, ixora peptide I, a peptide isolated from I. coccinea revealed cytotoxicity against
the Hep3B liver tumor cell line.4,6,5d,20
The chemical constituents of the flowers, leaves and stalks of Ixora species were presented in
Tables 4-6. Total twelve different plant extracts have been studied for GC-MS analysis
including five extracts from different (species and colors) flowers, five extracts from leaves
and two extracts from stalks. Total 233 chemical constituents have been identified from
hexane extracts of Ixora species, 225 from chloroform extracts and 26 chemical constituents
were identified from methanol extract of I. chinensis flowers. The extracts were dominated by
different class of compounds. Total 55 phytochemicals have been identified from hexane
extract of I. fulgens leaves, 56 from hexane extract of I. polyantha leaves, 51 from hexane
extract of I. chinensis leaves, 33 from chloroform extract of I. fulgens flowers, 72 from I.
80
fulgens leaves, and 17 from I. coccinea flowers (yellow)(Figure 2). Bis (2-ethylhexyl) 1,2-
benzene dicarboxylate (40), 2,6-dimethyl-2,6-octadiene (64), 3-(4-methyl-3-pentenyl)-3-
cyclohexene-1-carboxaldehyde (77), n-tetradecanoic acid (110), n-tridecane (126), n-
hexadecane (129), n-eicosane (131), methyl tetracosanoate (187), 4,8,12-trimethyltridecan-
4-olide (203), 3,7,11-trimethyl-1,6,10-dodecatrien-3-ol (218), α-tocopherol (228) and β-
tocopherol (229) were the identical compounds present in hexane extracts of I. fulgens, I.
polyantha and I. chinensis leaves. Whereas, bis (2-ethyl hexyl)-1,2-benzenedicarboxylate
(40), 6,10,14-trimethyl-2-pentadecanone (295), n-tetradecanoic acid (110), n-hexadecane
(129), n-eicosane (131), methyl octadecanoate (174), methyl hexadecanoate (166), were
identified as alike phytochemicals present in chloroform extracts of I. fulgens flowers, I.
fulgens leaves, and I. coccinea flowers (yellow color). Some of compounds present in extracts
have been reported to exhibit cytotoxic effect. Bis (2-ethylhexyl)-1,2-benzene dicarboxylate
(40)102a,b n-tetradecanoic acid (110),102c n-tridecane (126), n-hexadecane (129), n-eicosane
(131),103a have been reported to have anticancer potential. Methyl tetracosanoate (187),
methyl hexadecanoate (166), methyl octadecanoate (174) showed good anticancer activity
against MCF-7 cancer cell line.103b 1,6,10-Dodecatrien-3-ol, 3,7,11-trimethyl- (15), exhibited
significant cytotoxic potential on colon and breast cancer cells.103c α-Tocopherol (228), and β-
tocopherol (229) possesess strong anticancer activity against colon cancer cell line.103d
whereas, 6,10,14-trimethyl-2-pentadecanone (295), has been identified in Viscum album L.
extract on ehrlich tumour cells line.103e Strong cytotoxic effect of hexane extracts of I. fulgens,
I. polyantha, and I. chinensis leaves and chloroform extracts of I. fulgens flowers, leaves, and
I. coccinea flowers (yellow color), were found to be active against cervical (HeLa), breast
(MCF-7) and lung cancer cell lines (NCI H-460), because of the synergistic behaviour of
chemical compounds identified through GC-MS analysis (Table 3). The structures of chemical
constituents were identified through GC and GC-MS studies and verified through mass library
search software were given in Figure 2.101c
These studies showed that the Ixora species flowers, leaves and root extracts contained 33
chemical constituents have been identified from I. fulgens flowers while 17 chemical
constituents were present from I. coccinea flowers (yellow color) generally accountable for
its growth inhibitory and cytotoxic potential, which is favorable in the direction of cervical
81
cancer cell line. Furthermore, all the non-polar and moderately polar extracts, were
investigated for their identifications of phytochemicals by using GC-MS technique, which
revealed the presence of alcohols, aldehydes, amide group, aromatic compounds, benzene
derivatives, bicyclic compounds, branched chain hydrocarbons, cyclic and heterocyclic
compounds, fatty acids and their esters, halogen containing compounds, hydrocarbons,
phenols, terpenes, tocopherols, and miscellaneous compounds (Tables 4-6). The identification
of phytochemicals were made by GC and GC-MS spectra and Mass hunter library search data
base.101c Present investigation showed that chloroform extracts of I. fulgens and I. coccinea
flowers were found to be highly active extracts against HeLa cell lines.
Clustering of chloroform extracts of plants metabolites
Hierarchical clustering was presented by using Euclidean distance metric, whole linkage to
create a dendrogram for clustering of different flower species extracts using normalized
intensities of important chemical compounds. Vertical lines length in the dendrogram is a
determination of difference, wheras, small lines showed close linkage of the groups. This
method clustered the five different plant species. The two plant species metabolites namely I.
coccinea flowers (orange color) and I. fulgens flowers were the most similar from other
species. While, the most dissimilar species were examined of dissimilarity index according to
plant metabolite, named as I. coccinea flowers (orange color) and I. chinensis flowers Figure
4.
b) Antibacterial activity
Plants having medicinal properties commonly known for their disease protecting character,
are used as a source of active phytochemicals.104 A number of plant derived compounds are
well recognized as antimicrobial agents.105 So, the current work clearly documented the
significant chemical compounds which act as an antibacterial agents.
Hexane (H) and methanol (M) extracts of Ixora species namely, I. coccinea (orange and
yellow flowers), I. fulgens, I. polyantha, and I. chinensis were evaluated for antibacterial
82
Table 3a. Growth inhibitory and cytotoxic effects of Ixora species extracts against Hela cell lines (Cervical).
S.
No.
Plant name (Codes)
Do
ses
(µg
/mL
)
Cervical cancer cell line
(HeLa)
% Cell growth
inhibition/
kill
(µg/mL)
GI50 LC50
1 I. fulgens flowers (H1) 250 +39 ± 2.8 >250 >250
2
I. fulgens flowers (C1)
10 +51 ± 2.9***
10 ± 2.9b 165 ± 3.1b
50 +62 ± 1.5***
100 +92 ± 2.8***
200 -80 ± 1.6***
250 -87 ± 2.3***
3 I. fulgens flowers (M1) 250 10 ± 1.9 >250 >250
4
I. fulgens leaves (H2)
10 +00 ± 1.5
130 ± 2.1 >250
50 +03 ± 1.9
100 +30 ± 2.5
200 +93 ± 2.4
250 -14 ± 2.5
5
I. fulgens leaves (C2)
10 +08 ± 2.1
52 ± 2.7
>250
50 +48 ± 1.6
100 +98 ± 0.9
200 -13 ± 3.1
250 -23 ± 2.8
6
I. fulgens leaves (M2)
10 +49 ± 3.4
150 ± 3.2 >250
50 +56 ± 2.7
100 +59 ± 3.9
200 +74 ± 3.4
250 +88 ± 1.9
7
I. polyantha flowers (C3)
10 +18 ± 1.5*
140 ± 3.7c >250
50 +32 ±2.4**
100 +43 ± 3.1***
200 +61 ± 2.7***
250 +69 ± 3.4***
8 I. polyantha flowers (M3) 250 +00 ± 1.4 >250 >250
83
9 I. polyantha leaves (H4)
10 +01 ± 2.4
14 ± 1.0 22 ± 0.8
50 +16 ± 2.6
100 +19 ± 1.6
200 +51 ± 3.7
250 +65 ± 2.4
10 I. polyantha leaves (C4)
10 +03 ± 1.4
128 ± 3.5 >250
50 +16 ± 2.9
100 +26 ± 3.4
200 +90 ± 4.1
250 -47 ± 2.7
11 I. polyantha leaves (M4) 250 +35 ± 2.8 >250 >250
12 I. chinensis flowers (C5) 250 +26 ± 2.4* >250 >250
13 I. chinensis flowers (M5) 250 00 ± 2.4 >250 >250
14 I. chinensis leaves (H6)
10 +11 ± 1.6
200 ± 3.0 >250
50 +11 ± 1.5
100 +12 ± 2.4
200 +36 ± 3.5
250 +61 ± 2.1
15
I. chinensis leaves (C6)
10 +03 ± 1.3
110 ± 3.1 >250
50 +08 ± 2.6
100 +48 ± 3.4
200 -39 ± 2.1
250 -41 ± 0.9
16 I. chinensis leaves (M6) 250 +15 ± 2.9 >250 >250
17
I. coccinea flowers (Y)(H7)
10 +00 ± 2.6
40 ± 1.5 170 ± 1.8
50 +60 ± 1.4
100 -16 ± 2.9
200 -61 ± 3.8
250 -72 ± 2.4
18
I. coccinea flowers (Y) (C7)
10 +45 ± 2.2***
15 ± 3.8b 230 ± 3.9c
50 +99 ± 3.1***
100 -13 ± 2.4***
200 -36 ± 1.9***
250 -54 ± 2.5***
84
19
I. coccinea leaves (Y) (H8)
10 +00 ± 1.9 90 ± 2.9 >100
50 +07 ± 1.4
100 +58 ± 1.9
200 +64 ± 1.8
250 +88 ± 2.7
20 I. coccinea leaves (Y) (C8)
10 +00 ± 1.5
120 ± 3.1 >250
50 +20 ± 1.3
100 +37 ± 3.1
200 -62 ± 2.9
250 -65 ± 2.1
21 I. coccinea leaves (Y)(M8)
10 +00 ± 2.6
110 ± 3.0 >250
50 +21 ± 1.4
100 +43 ± 2.9
200 +84 ± 3.8
250 +93 ± 2.4
22 I.coccinea stalks (Y)(H9) 250 +00 ± 2.8 >250 >250
23 I. coccinea stalk (Y) (M9)
10 -
>250 >250
50 -
100 -
200 -
250 +00 ± 2.5
24 I .coccinea flowers (O)(H10) 250 +01 ± 3.7 >250 >250
25 I. coccinea flowers (O) (C10)
10 +13 ± 1.4
60 ± 2.7c >250
50 +49 ± 2.4***
100 +68 ± 3.5***
200 +89 ± 3.4***
250 -31 ± 4.1***
26 I. coccinea flowers (O)(M10) 250 +00 ± 2.5 >250 >250
27
I. coccinea leaves (O) (H11)
10 +00 ± 3.7
230 ± 3.7 >250
50 +05 ± 1.4
100 +13 ± 3.7
200 +26 ± 2.1
250
-52 ± 1.9
85
28 I. coccinea leaves(O)(M11) 10 +03 ± 1.4 190 ± 2.6 >250
50 +06 ± 2.9
100 +13 ± 3.4
200 +52 ± 4.1
250 +64 ± 2.7
29 I.coccinea stalks (O)(H12)
10 +61 ± 4.2
120 ± 2.4 240 ± 3.5
50 +35 ± 1.4
100 +42 ± 2.1
200 +75 ± 2.4
250 -61 ± 1.4
30 I. coccinea stalks (O)(M12) 250 00 ± 2.7 >250 >250
31 Doxorubicin µg/mL (µM)
(standard)
0.006 +5.0 ± 3.0
0.5±0.02a
(0.88 ±0.04)
5.8±0.1a
(10±0.1)
0.06 +7.0 ± 3.0
0.6 +60 ± 3.0***
6.0 -52 ± 7.0***
0.006 +5.0 ± 3.0
H = Hexane extract; C = chloroform extract; M = methanol extract; O = orange color flowers containing plant
extracts; Y = yellow color flowers containing plant extracts
Control absorbance (515 nm): MCF-7 cell line (2.9 ± 0.6), NCI H-460 cell line (2.7 ± 0.5), HeLa cell line (2.4
± 0.9)
Each value represents mean ± SEM of three independent experiments
Growth inhibition = (+); cytotoxicity = (–)
Concentration causing 50 % of cell growth inhibition = GI50
Concentration of drug that killed 50% cells = LC50
Values within parentheses are expressed in µM.
Asterisk indicates significant (*p < 0.05, **p < 0.01 and ***p < 0.001) growth inhibition and
cytotoxicity as compared to respective controls
In columns, dissimilar superscript alphabets (a-d) represent significant
(p < 0.05) and similar alphabets non-significant GI50 and LC50 values.
86
Table 3b. Growth inhibitory and cytotoxic effects of Ixora species extracts against MCF-7 cell lines (Breast
cancer cell lines).
S.
No.
Plant name (Codes)
Do
ses
(µg
/mL
)
Breast cancer cell line
(MCF-7)
% Cell growth
inhibition/
kill
(µg/mL)
GI50 LC50
1 I. fulgens flowers (H1) 250 +16 ± 1.5 >250 >250
2
I. fulgens flowers (C1)
10 +04 ± 1.2
48 ± 2.2c >250
50 +53 ±2.2***
100 +71 ± 1.8***
200 +80 ± 1.5***
250 +87± 2.7***
3 I. fulgens flowers (M1) 250 +00 ± 2.9 >250 >250
4
I. fulgens leaves (H2)
10 +00 ± 1.3
110 ± 2.6 >250
50 +36 ± 1.9
100 +41 ± 2.5
200 -04 ± 3.4
250 -25 ± 2.9
5
I. fulgens leaves (C2)
10 +12 ± 2.2
38 ± 2.5
200 ± 2.2
50 +68 ± 2.6
100 +87 ± 1.4
200 -49 ± 3.2
250 -63 ± 1.0
6
I. fulgens leaves (M2)
10 -
>250 >250
50 -
100 -
200 -
250 +32 ± 3.0
7 I. polyantha flowers (C3)
10 +01 ± 0.4
220 ± 4.3d >250
50 +09 ± 1.1
100 +37 ± 2.5**
200 +43 ± 3.5***
250 +55 ± 4.1***
87
8 I. polyantha flowers (M3)
250 +18 ± 2.1 >250 >250
9 I. polyantha leaves (H4)
10 +00 ± 1.6
160 ± 3.5 >250
50 +02 ± 1.9
100 +12 ± 2.2
200 +68 ± 3.1
250 +86 ± 2.1
10
I.polyantha leaves (C4)
10 +00 ± 1.4
125 ± 3.2 >250
50 +11 ± 2.9
100 +37 ± 3.4
200 +74 ± 4.1
250 +95 ± 2.7
11 I. polyantha leaves (M4)
250 +07 ± 2.4 >250 >250
12 I. chinensis flowers (C5) 250 +00 ± 2.4
>250
>250
13 I. chinensis flowers (M5)
250 +02 ± 2.4
>250 >250
14
I. chinensis leaves (H6)
10 +00 ± 2.6
190 ± 2.2 >250
50 +00 ± 1.4
100 +04 ± 2.9
200 +58 ± 3.8
250 +83 ± 2.4
15
I. chinensis leaves (C6)
10 +00 ± 1.5
150 ± 3.5 >250
50 +11 ± 2.2
100 +30 ± 2.4
200 +68 ± 2.5
250 +84 ± 1.0
16 I. chinensis leaves (M6)
250 +04 ± 2.6 >250 >250
17 I. coccinea flowers (Y)(H7)
10 +02 ± 2.6
68 ± 2.9 230 ± 3.3
50 +26 ± 1.4
100 +95 ± 2.9
200 -42 ± 3.8
250 -52 ± 2.4
10 +45± 1.3*** 12 ± 1.4b 205 ± 4.5b
88
18 I. coccinea flowers (Y) (C7) 50 +92 ± 1.5***
100 -26 ± 1.9***
200 -49 ± 2.1***
250 -64 ± 0.8***
19
I.coccinea leaves (Y) (H8)
10 +00 ± 0.9
178 ± 3.2 >250
50 +00 ± 1.2
100 +02 ± 0.8
200 +59 ± 1.4
250 +67 ± 2.5
20
I. coccinea leaves (Y) (C8)
10 +00 ± 0.6
210 ± 2.1 >250
50 +22 ± 1.4
100 +34 ± 2.0
200 +46 ± 2.1
250 +71 ± 2.5
21
I. coccinea leaves (Y)(M8)
10 +02 ± 2.1
150 ± 3.5
>250
50 +16 ± 1.9
100 +23 ± 2.9
200 +73 ± 3.0
250 +84 ± 2.9
22 I.coccinea stalks (Y)(H9)
250 +00 ± 2.8 >250 >250
23
I.coccinea stalk (Y) (M9)
10 -
>250 >250
50 -
100 -
200 -
250 +36 ± 2.2
24 I.coccinea flowers (O) (H10)
250 +45 ± 3.2 >250 >250
25 I. coccinea flowers (O) (C10)
10 +24 ± 1.2*
58 ± 3.7c >250
50 +41 ± 2.4***
100 +72 ± 3.1***
200 +89 ± 2.7***
250 -38 ± 4.5***
26
I.coccinea flowers (O) (M10)
250 +00 ± 4.2 >250 >250
89
27 I. coccinea leaves (O)(H11)
10 +00 ± 4.7
150 ± 2.3 >250
50 +00 ± 2.4
100 +24 ± 3.7
200 +77 ± 2.9
250 +86 ± 1.8
28
I.coccinea leaves(O)(M11)
10 -
>250 >250
50 -
100 -
200 -
250 +44 ± 2.5
29
I.coccinea stalks (O)(H12)
10 +00 ± 2.2
100 ± 3.1 250 ± 3.3
50 +25 ± 3.1
100 +51 ± 2.9
200 +98 ± 1.9
250 -03 ± 3.2
30 I.coccinea stalks (O)(M12)
250
+18 ± 1.9 >250 >250
31 Doxorubicin µg/mL
(µM) (standard)
0.1 +37±5.0**
0.17±0.03a
(0.3±0.05)
5.8±0.01 a
(10±0.02)
0.5 +71±2.0***
5.0 -18±3.0***
10.0 -50±2.0***
0.1 +37±5.0**
H = Hexane extract; C = chloroform extract; M = methanol extract; O = orange color flowers containing plant
extracts; Y = yellow color flowers containing plant extracts
Control absorbance (515 nm): MCF-7 (2.9 ± 0.6), NCI-H460 (2.7 ± 0.5) and HeLa (2.4 ± 0.9).
Each value represents mean ± SEM of three independent experiments
Growth inhibition = (+) and cytotoxicity = (–)
Concentration causing 50 % of cell growth inhibition = GI50
Concentration of drug that killed 50% cells = LC50
Values within parentheses are expressed in µM.
Asterisk indicates significant (*p < 0.05, **p < 0.01 and ***p < 0.001) growth inhibition and cytotoxicity as
compared to respective controls
In columns, dissimilar superscript alphabets (a-d) represent significant
(p < 0.05) and similar alphabets non-significant GI50 and LC50 values.
90
Table 3c. Growth inhibitory and cytotoxic effects of Ixora species extracts against NCI H-460 (Lung cancer
cell line).
S.
No.
Plant name (Codes)
Do
ses
(µg
/mL
)
Lung cancer cell line
(NCI H-460)
% Cell growth
inhibition/
kill
(µg/mL)
GI50 LC50
1 I. fulgens flowers (H1) 250 +42 ± 4.3 >250 >250
2
I. fulgens flowers (C1)
10 +07 ± 1.9
46 ± 1.9c
>250
50 +56 ± 1.7***
100 +63 ± 1.4***
200 +94 ± 2.8***
250 -12 ± 2.1***
3 I. fulgens flowers (M1) 250 +00 ± 2.9 >250 >250
4 I. fulgens leaves (H2)
10 +00 ± 1.7
46 ± 1.9
>250
50 +34 ± 1.7
100 +63 ± 2.9
200 -14 ± 1.5
250 -66 ± 1.9
5 I. fulgens leaves (C2)
10 +45 ± 1.1
40 ± 2.7
190
50 +53 ± 1.9
100 -14 ± 1.9
200 -52 ± 2.1
250 -70 ± 1.8
6 I. fulgens leaves (M2)
10 +00 ± 1.7
120 ± 1.1 190 ± 1.9
50 +08 ± 2.5
100 +38 ± 1.9
200 -65 ± 3.0
250 -70 ± 2.9
7 I. polyantha flowers (M3) 250 +00 ± 4.4 >250 >250
8 I. polyantha leaves (H4)
10 +01 ± 3.5
40 ± 2.7
190
50 +05 ± 3.4
100 +15 ± 3.9
200 +66 ± 4.1
250 +71 ± 3.8
91
9 I.polyantha leaves (C4)
10 +00 ± 1.9
50 ± 4.5 250 ± 3.1
50 +54 ± 2.4
100 +88 ± 2.4
200 -24 ± 3.1
250 - 46 ± 4.7
10 I. polyantha leaves (M4) 250 +01 ± 2.4 >250 >250
11 I. chinensis flowers (C5) 250 +01 ± 3.8 >250 >250
12 I. chinensis flowers (M5)
250 +00 ± 2.4 >250 >250
13 I. chinensis leaves (H6)
10 +05 ± 2.6
50 ± 4.5 250 ± 3.1
50 +10 ± 1.4
100 +16 ± 2.9
200 +60 ± 3.8
250 +65 ± 2.4
14 I. chinensis leaves (C6)
10 00 ± 1.3
200 ± 3.5 >250
50 +00 ± 1.9
100 +04 ± 2.2
200 +48 ± 1.0
250 +91 ± 0.9
15 I. chinensis leaves (M6)
250 +00 ± 2.4 >250 >250
16 I. coccinea flowers (Y)(H7) 10 +00 ± 2.9
200 ± 3.5 >250
50 +29 ± 1.9
100 +88 ± 2.1
200 -34 ± 2.7
250 -40 ± 2.8
17 I. coccinea flowers (Y) (C7) 10 +40 ± 2.4***
12 ± 3.1b 200 ± 4.7b
50 +98 ± 2.9***
100 -31 ± 3.9***
200 -51± 4.2***
250 -62 ± 4.6***
18 I.coccinea leaves (Y) (H8)
10 -
>250
>250
50 -
100 -
200 -
92
250 +47 ± 2.1
19 I. coccinea leaves (Y) (C8)
10 +7.0 ± 1.6
120 ± 1.1 >250
50 +20 ± 2.1
100 +32 ± 2.1
200 +90 ± 1.9
250 +96 ± 1.7
20 I. coccinea leaves (Y)(M8)
10 -
>250 >250
50 -
100 -
200 -
250 +00 ± 2.6
21 I.coccinea stalks (Y)(H9) 250 +00 ± 2.8
>250
>250
22 I.coccinea stalk (Y) (M9)
10 +01 ± 1.9
250 ± 3.1 >250
50 +02 ± 3.1
100 +20 ± 2.8
200 +44 ± 3.4
250 +50 ± 2.7
23 I.coccinea flowers (O )(H10)
250 +02 ± 2.5
>250 >250
24
I. coccinea flowers (O) (C10)
10 +20 ± 1.6*
60 ± 3.1c
>250
50 +46 ± 2.7***
100 +58 ± 2.9***
200 +78 ± 3.0***
250 +88 ± 3.9***
25 I.coccinea flowers (O) (M10)
250 +01 ± 2.1 >250 >250
26
I. coccinea leaves (O) (H11)
10 +01 ± 2.2
220 ± 1.9 >250
50 +02 ± 1.3
100 +10 ± 2.8
200 +43 ± 2.0
250 +52 ± 1.9
27 I.coccinea leaves(O)(M11) 10 -
>250 >250
50 -
100 -
200 -
250 +01 ± 2.9
93
H = Hexane extract; C = chloroform extract; M = methanol extract; O = orange color flowers containing plant
extracts; Y = yellow color flowers containing plant extracts
Control absorbance (515 nm): MCF-7cell line (2.9 ± 0.6), NCI H-460 cell line (2.7 ± 0.5), HeLa cell line (2.4 ±
0.9)
Each value represents mean ± SEM of three independent experiments
Growth inhibition = (+) and cytotoxicity = (–)
Concentration causing 50 % of cell growth inhibition = GI50
Concentration of drug that killed 50% cells = LC50
Values within parentheses are expressed in µM
Asterisk indicates significant (*p < 0.05, **p < 0.01 and ***p < 0.001) growth inhibition
and cytotoxicity as compared to respective controls
In columns, dissimilar superscript alphabets (a-d) represent significant (p < 0.05) and similar alphabets non-
significant GI50 and LC50 values.
28 I.coccinea stalks (O)(H12)
10 +00 ± 2.2
135 2.1 >250
50 +06 ± 2.6
100 +21 ± 3.2
200 +98 ± 3.1
250 +99 ± 2.8
29 I.coccinea stalks (O) (M12) 250 00 ± 1.8 >250 >250
30 Doxorubicin µg/mL
(µM) (standard)
0.1 +42±5.0**
0.17±0.05a
(0.26 ±0.08)
5.4 ± 1.2a
(9.3 ± 1.2)
0.5 +76±2.0***
5.0 -18±3.0***
10.0 -53±2.0***
0.1 +42±5.0**
94
aa) 50 % Cell growth inhibition (GI50) against hexane series
Treatments
ab) 50 % Cell growth inhibition (GI50) against chloroform series
Treatment
Figure 3: The bar graph represents concentration values of (aa, ab, ac) GI50 (50 % cell growth inhibition)
and (ba, bb, bc) LC50 (50 % cell kill) of Ixora species hexane, chloroform and methanol extracts against
HeLa, MCF-7 and NCI H-460 cancer cell lines.
0
50
100
150
200
250
300
Co
nce
ntr
ati
on
(µ
g/m
L)
HeLa
MCF-7
NCI H-460
0
50
100
150
200
250
300
Co
nce
ntr
ati
on
(µ
g/m
L)
HeLa
MCF-7
NCI H-460
95
ac) 50 % Cell growth inhibition (GI50) against methanol series
Treatments
ba) 50 % Cell kill (LC50) against hexane series
Treatments
0
50
100
150
200
250
300
Co
nce
ntr
atio
n (
µg/
mL)
HeLa
MCF-7
NCI H-460
0
50
100
150
200
250
300
Co
nce
ntr
ati
on
(µ
g/m
L)
HeLa
MCF-7
NCI H-460
96
bb) 50 % Cell kill (LC50) against chloroform series
Treatments
bc ) 50 % Cell kill (LC50) against methanol series
Treatments
0
50
100
150
200
250
300
Co
nce
ntr
ati
on
(µ
g/m
L)
HeLa
MCF-7
NCI H-460
0
50
100
150
200
250
300
Co
nce
ntr
ati
on
(µ
g/m
L)
HeLa
MCF-7
NCI H-460
97
I. coccin
ea (O
)
I. fulg
ens
I. coccin
ea (Y
)
I. polya
nth
us
I. chin
ensis
Methyl tetradecanoate
Methyl tetracosanoate
2-Decenal
Methyl octanoate
2-Heptenal
Methyl 9-oxo nonanoate
n-Hexadecanoic acid
n-Octanoic acid
9-Oxononanoic acid
Methyl octadecanoate
Dibutyl 1,2 benzenedicarboxylate
n-Hexadecane
n-Eicosane
Bis (2ethyl hexyl)-1,2-benzenedicarboxylate
n-Tridecane
Methyl eicosanoate
Methyl hexadecanoate
Methyl docosanoate
6,10,14-Trimethyl 2-pentadecanone
N,N−Bis(2-hydroxyethyl) dodecanamide
2,6,10,14-Tetramethyl hexadecane
n-Tricosane
n-Tetradecanoic acid
9,12-Octadecadienoate
4,7-Dimethyl undecane
n-Octadecanoic acid
1500
1000
500
0
−500
−1000
Figure 4: Hierarchical clustering of significant metabolites (p value < 0.05) in five different species of
flowers chloroform extracts by using normalized intensity.
98
Table 4: Identification of chemical compounds through GC and GC-MS studies of hexane extracts on Ixora species.
Hexane extracts
S.
No.
Compounds name
I. f
ulg
ens
flo
wer
s
I.fu
lgen
s le
av
es
I. p
oly
an
tha
flo
wer
s
I. p
oly
an
tha
lea
ves
I. c
hin
ensi
s fl
ow
ers
I. c
hin
ensi
s le
av
es
I. c
occ
inea
flo
wer
s (Y
)
I.co
ccin
ea l
eav
es (
Y)
I. c
occ
inea
s ta
lks
(Y)
I.
cocc
inea
flo
wer
s (O
)
I. c
occ
inea
lea
ves
(O
)
I. c
occ
inea
sta
lks
(O)
1 1-Octanol (1) - - - + - - - - - - - -
2 1-Dodecanol (2) - - - - - - - - + - - -
3 1-Tetradecanol (3) - - + - - - - + + - - -
4 1-Docosanol (4) - - - - - - + - - - - -
5 1-Hexacosanol (5) - - - - - - - - + - - -
6 2-Butyl 1-octanol (6) - + - + - - - - - - - +
7 4-Ethylcyclohexanol (7) - + - - - - - - - - - -
8 2-Decen-1-ol (8) - - - + - - - - - - - -
9 cis-Myrtanol (9) - + - - - - - - - - - -
10 2-Methylene-3-(1-methylethenyl) cyclohexyl acetate (10) - - - + - - - + - - - -
11 3,7,11,15-Tetramethyl-1-hexadecanol (11) - - - + - - - + - - - -
12 14-Methyl-8-hexa decyn-1-ol (12) - + - - - - - - - - - -
13 2,3-Dimethyl-undec-1-en-3-ol (13) - - - - - - - + - - - -
14 7,11-Dimethyldodeca-2,6,10-trien-1-ol(14) - - - + - - - - - - - -
99
15 cis-Farnesol or [3,7,11-trimethyl 2,6,10-dodecatrien-1-ol] (15) - - - + - + - - - - - -
16 Phytol or [3,7,11,15-Tetramethyl 2-hexadecen-1-ol] (16) - + - + - - + + + - - -
17 5-(tert-Butoxy carbonyloxy)- or 2-(1-buten-3-yl) phenol(17) - + - - - - - - - - - -
18 4-Methyl- 4-hexen-1-ol (18) - - + - - - - - - - - -
19 2-Hexyl-1-decanol (19) - - - - - - - - - - + -
20 5-Methyl-1-heptanol (20) - - - - - - - - - - - +
21 Pluchidiol (21) - - - - - - - - + - - -
22 1,2,3-Propanetriol (22) - - - - - - - - + - - -
23 2,6,10,15, 19,23-Hexamethyl- 1,6,10,14,18,22-tetracosahexaen-3-ol (23) - - - - - + - - - - - -
24 Cycloheptane methanol (24) - - - - + - - - - - - -
25 2-Heptenal (25) - - - - - - - - - - + +
26 n-Nonanal (26) - - - - - - - - - - + +
27 2-Decenal (27) - - - - - - - - - - + +
28 Tetradecanal (28) + - - + - + - - - - - -
29 Pentadecanal (29) - + - - + + + - - + + -
30 13-Octadecenal (30) - - - - + - + - - - - -
31 3-Methyl-2-butenal (31) - - - - - - - + - - - -
32 N,N-Bis(2-hydroxyethyl) dodecanamide (32) + - + + - + + - + + + +
33 Benzoic acid (33) - - - - - - - - + - - +
34 3-Pyridinyl benzoate (34) - - - - - - - + - - - -
35 4-Hydroxy 3-methoxy benzoic acid or (Vanillic acid) (35) - - - - - - - - + - - -
36 3-Hydroxy-4-methoxybenzaldehyde (36) - - - - - - - - + - - -
37 4-Hydroxy-3,5-dimethoxy benzaldehyde (Syringaldehyde) (37) - - - - - - - - + - - -
38 4-Hydroxy 3-methoxy benzaldehyde or (Vanillin) (38) - - - - - - - - - - - +
39 Ethyl homovanillate or (Ethyl 4-hydroxy-3-methoxy phenyl acetate) (39) - - + - - - - - - - - -
100
40 Bis (2-ethylhexyl) 1,2-benzene dicarboxylate (40) + + + + + + + + + + + +
41 Butyl 2-methylpropyl 1,2-benzenedicarboxylate (41) - - - - - - - - - - - +
42 Dibutyl 1,2-benzenedicarboxylate (42) - + - - - + - - - - - -
43 (1-Propyloctyl) benzene (43) - - + - - + - - - - - -
44 (1-Propyl nonyl) benzene (44) - - + - - - - - - - - -
45 (1-Propyl decyl) benzene (45) - - + - - - - - - - - -
46 1-Ethyl-4-methyl benzene (46) - - + - - - - - - - - -
47 (1-Ethyl nonyl) benzene (47) - - + - - + - - - - - -
48 (1-Ethyl decyl) benzene (48) - - + - - - - - - - - -
49 (1-Ethyl undecyl) benzene (49) - - + - - - + - - - - -
50 (1-Butylheptyl) benzene (50) - - + - - + - - - - - -
51 (1-Butyl octyl) benzene (51) - - + - - - - - - - - +
52 (1-Butyl nonyl) benzene (52) - - + - - - - - - - - -
53 (1-Methyl decyl) benzene (53) - - + - - - - - - - - -
54 (1-Methyl undecyl) benzene (54) - - + - - + - - - - - +
55 (1-Pentyl heptyl) benzene (55) - - + - - - - - - - - -
56 (1-Pentyl octyl) benzene (56) - - + - - - - - - - - -
57 Hexamethyl benzene (57) - - - - - - - - - + - -
58 2-Methylene bicyclo[4.3.0] nonane (58) + - - - - - - - - - - -
59 Bicyclo[4.3.1]dec-1(9)-ene (59) - - - - - - - - - + - -
60 7-Methylene-2,4,4-trimethyl-2-vinyl-bicyclo[4.3.0] nonane (60) - - - - - - + - - - - -
61 1,2,3,6-Tetramethyl bicyclo[2.2.2]octa-2,5-diene (61) + - - - - - - - - - - -
62 4-Methylene-2,8,8-trimethyl-2-vinyl- bicyclo[5.2.0] nonane (62) - + - - - - - - - - - -
63 2,6,10,14 Tetramethyl hexadecane (Phytane) (63) - - + - + - - - - - - +
64 2,6-Dimethyl-2,6-octadiene (64) - + - + - + - - - - - -
101
65 3-Methyl-5-propyl nonane (65) + - - - - - - - - - - -
66 2-Methyl-5,7-dimethylene-(1,8-nonadiene) (66) - - + - - - - - - - - -
67 3,8-Dimethyl decane (67) - - - - - - - + - - - -
68 5,9-Dimethylpentadecane (68) - - + - - - - - - - - -
69 8-Heptyl pentadecane (69) - - + - - - - - - - - -
70 Tetradeca-6,8-diyne (70) - - + - - - - - - - - -
71 4-Methyl- 1-undecene (71) - - - + - - - - - - - -
72 2,6,11-Trimethyl dodecane (72) - - - - - - - + - - - -
73 6-Methyl tridecane (73) - - + - - - - - - - - -
74 3-(1-Hexenyl) cyclohexene (74) - + - - - - - - - - + +
75 4-Hydroxy-2-cyclo hexen-1-one (75) - - - + - - - - - - - -
76 1,1,4,4-Tetramethyl-2,5-dimethylene-cyclohexane (76) + - - - - - - - - - - -
77 3-(4-Methyl-3-pentenyl)- 3-cyclohexene-1-carboxaldehyde (77) - + - + - + - - - - - -
78 1,2,4-Trivinyl cyclohexane (78) - + - - - - - - - - - -
79 3-Ethyl cyclohexene (79) - - - - - - - - - + + -
80 2,4-Diethenyl-1-methyl cyclohexane (80) - - - - - - - - - - + -
81 4-(3-Hydroxy-1-butenyl) 3,5,5-tri methyl 2-cyclohexen-1-one(81) - - - - - - - + + - - -
82 Tricyclo[3.3.0.0(2,8)]octan-3-one, 8-methyl-(82) - + - - - - - - - - + +
83 1-(1-Methylethyl) cyclopentene (83) - - - - - + - - - - - -
84 3-Methyl-2-(2-pentenyl), 2-cyclopenten-1-one (84) - - - - - - - - - + - +
85 1,3-Dimethyl-2-(1-methyl ethenyl) cyclopentane (85) - - - - - - - - - - - +
86 3-t-Pentylcyclopentanone (86) - - - - - + - - - - - -
87 1,5-Cyclodecadiene (87) - - - - - + - - - - - -
88 Octahydro-1-methylene 4(1H)-azulenone (88) - - - - - - - - - - + -
89 1,5-Dimethyl-6-methylene spiro [2.4] heptane (89) - + - - - - - - - - - -
102
90 7-Octylidene bicycle [4.1.0] heptane (90) - - + - - - - - - - - -
91 1,5-Dimethyl 7-oxa bicycle [4.1.0] heptane (91) - - - + - - - - - - - -
92 2,3,4,5-Tetra methyl- tricyclo [3.2.1.02,7]oct-3-ene (92) - - - + - - - - - - - -
93 Dihydrocarvone (cyclohexanone, 2-methyl-5-(1-methyl ethenyl))(93) - - - - - - - - + - - -
94 3-Cyclohexylidene-4-ethyl- 2-hexanone (94) - - - - - - - - - - + -
95 Pulegone or (cyclohexanone, 5-methyl-2-(1-methylethylidene)) (95) - - - - - - - - - - - +
96 1-Octyl ether (96) - - - - - + + - - - - -
97 Oxirane, [(hexadecyloxy) methyl] (97) - - - + - - - - - - - -
98 2-Hexadecyloxirane (98) - - - - - + - - - - - -
99 Decahydro-1,6-dimethyl naphthalene (99) + - - - - - - - - - - -
100 N-Methyl-2-pyrrolidone (100) - + - - - + - - - - - -
101 2,5-Pyrrolidinedione (101) - - + - - - - - - - - -
102 5,6,7,7-Tetra hydro-4,4,7-trimethyl 2(4H)-benzofuranone (102) - + - + - - + - - - + -
103 (1'S,5S)-5-Aminomethyl-3-(1'-phenyl ethyl)-1,3-oxazolidin-2-one (103) - - + - - + - - - - - -
104 Phthalic acid, butyl ester, ester with butyl glycolate (104) - - - - - - + - + - - -
105 (2,2-Dimethyl-1,3-dioxolan-4-yl) methyl hexadecanoate (105) - - - - - - - - - - - +
106 4-(Hydroxyl methyl)-4-methyl-2-methylene butanolide (106) - - - - - - - - + - - -
107 n- Pentanoic acid (107) - + - - - - - - - - - -
108 n-Hexanoic acid (108) - - - - - - - - - - - +
109 n-Decanoic acid (109) - - - - - - - - + - - -
110 n-Tetradecanoic acid(110) + + + + + + + + + + + +
111 n-Hexadecanoic acid (111) - - + - + + + + + + + +
112 n-Heptadecanoic acid (112) - - - - - - - - - - + +
113 n -Octadecanoic acid (113) - - - + + + + + + - + +
114 5-Dodecenoic acid (114) - - - - - - - + - - - -
103
115 9,12-Octadecadienoyl chloride (115) + - - - - - - - + + - -
116 1-Chloro dodecane (116) - - - - - + - - - - - -
117 3-Chloro-4-oxo-1-(1-phenyl ethyl) azetidine-2-carboxylic acid methyl ester isomer
(117)
- - - - - - - - - - + -
118 5β-Iodo methyl-1β-iso propenyl-4α, 6β-bicyclo[4.3.0]nonane (118) - - - - - - - + - - - -
119 n-Octadecyl heptafluorobutyrate (119) - - + - - - - - - - - -
120 Tetra decyl chloro acetate (120) - - - - - - - - - + - -
121 Nona decyl penta fluoro propionate (121) - - - - - - - - - + - -
122 10-Undecenoyl chloride (122) - - - - - - - - - - + +
123 1-Chloro octadecane (123) - - - + - - - - - - - -
124 n-Undecane (124) - - + - - - - - - - - -
125 n-Dodecane (125) - + - - - - - - - - - -
126 n-Tridecane (126) - + - + + + - - - - - +
127 n-Tetradecane (127) - - - - - + - - - - - +
128 n-Pentadecane (128) - - - - - + - - - - + -
129 n-Hexadecane (129) + + + + + + + + + + + +
130 n-Nonadecane (130) - + - - - + - - - - + -
131 n-Eicosane (131) + + + + + + + + - + + +
132 n-Tetracosane (132) - - - + - - - - - - - -
133 n-Heneicosane (133) + + - - + - - - - - - -
134 n-Tritriacontane (134) - - - - - - - - - - - +
135 n-Tetratriacontane (135) - - + - - - + - - - - -
136 n-Pentatriacontane (136) - - - - - - - - - - + -
137 n-Hexatriacontane (137) - - - - - + - - - - + -
138 Methyl 10-oxo-8-decenoate (138) + - - - - - - - - + - -
104
139 9-Oxo nonanoic acid (139) - - - + - - - - - + + +
140 2-Tridecanone (140) - + - - - - - - - - - -
141 5-Hexen-2-one (141) - - - - - + - - - - - -
142 3-Penten-2-one, 4-methyl-(142) - - - - - + - - - - - -
143 6,10-Dimethyl 5,9-dodecadien-2-one (143) - - - + - - - - - - + -
144 6,10-Dimethyl- 5,9-undecadien-2-one (144) - - - + - - - - - - - -
145 6-Methyl-5-hepten-2-one (145) - - - + - - - - - - - -
146 1-(4-Methylphenyl) ethanone (146) - - - + - - - - - - - -
147 1-(3',5'-Dihydroxy phenyl) heptan-2-one (147) - + - - - - - - - - - -
148 Pent-2-en-1,4-dione (148) - - - - - - - + - - - -
149 2,11-Dodecanedione (149) - - + - - - - - - - - -
150 8-Methyl-5-(1-methylethyl) 6,8-nonadien-2-one (150) - - - - - + - - - - - -
151 7-Hydroxy-6-methoxy-2H-1-benzopyran-2-one (151) - - - - - - - - + - - -
152 Methyl methoxy acetate (152) - - - - - - - - + - - -
153 4-Pentadecyl valerate (153) - - - - - - - + - - - -
154 Butanedioic acid, monomethyl ester (154) - - + - - - - - - - - -
155 Methyl octanoate (155) - + - - - - - - - - + -
156 Dimethyl octanedioate (156) - + - - - - - - - - - -
157 Methyl 6-methyl octanoate (157) - - - - - - - - - + - -
158 Nonanedioic acid, mono methyl ester (158) + - + + - - - - - + + +
159 Dimethyl nonanedioate (159) - + + - - - - - - - - -
160 Methyl 9-oxo-, nonanoate (160) + + - + - - - - - + + +
161 Dimethyl decanedioate (161) - + - - - - - - - - - -
162 Didecyl decanedioate (162) - - - - - - - - - - + -
163 Methyl 10-undecenoate (163) - + - - - - - - - - - -
105
164 Methyl dodecanoate (164) - + - - - - - - - - - -
165 Methyl tetradecanoate (165) + + + + - - - - - + + -
166 Methyl Hexadecanoate (166) - - + + + + + + + + + +
167 Methyl 9-hexadecenoate (167) + - + - - - - - - + - -
168 Ethyl hexadecanoate (168) + - - - - - - - - + - -
169 Ethyl 1-methyl hexadecanoate (169) - - + - - - + - + - - -
170 15-Methyl, hexadecanoate (170) + - - - - - - - - - - -
171 2,6,10,14-Tetramethyl-15-hexadecen-1-yl acetate (171) - - - - - - - - - - + -
172 Methyl heptadecanoate (172) - - - + - - - - - - - -
173 Methyl10-heptadecenoate (173) + - - - - - - - - - - -
174 Methyl octadecanoate (174) + + + + - - + + + + + +
175 Methyl 9-octadecenoate (175) - - + - - - - + - + - +
176 Ethyl 9-octadecenoate (176) + - - - - - - - - - - -
177 Methyl 9,12-octadecadienoate (177) + - + + + + + - - + + -
178 Methyl 9,12-octadecadiynoate (178) + - + + - - - - - + - +
179 Methyl 9,12,15-octadecatrienoate (179) + - + - + - + - - + + +
180 Ethyl linoleate (180) + - - - - - - - - + - -
181 Methyl nonadecanoate (181) - + - - - - - - - - - -
182 Isopropyl palmitate (182) - - - - + - - + - + - +
183 Methyl octacosanoate (183) + - - - - - - - - + - -
184 Methyl heneicosanoate (184) + - - - - + - - - + - -
185 Methyl docosanoate (185) - + - - - + - - - - + -
186 Methyl tricosanoate (186) + + - + - - - - - + - -
187 Methyl tetracosanoate (187) + + + + - + - - - + + -
188 Methyl n-pentacosanoate (188) + + - - - - - - - - - -
106
189 Methyl hexacosanoate (189) + - - - - - - - - - - -
190 2,4,6-Trimethyl, 1-methyl hexacosanoate (190) - - - - + - - - - - - -
191 Methyl eicosanoate (191) - + + - - + - - - - - -
192 Methyl 11-eicosenoate (192) - - - - - - - - - + - -
193 Methyl 10,12-octadecadiynoate (193) - - - + - - - - - - - -
194 2-Tertbutyl cyclohexyl propyl phosphono fluoridate (194) - - + - - - - - - - - -
195 1,3-Dihydro-2-methyl-1,3,2-naphtho [1,8]diaza phosphole-2-oxide (195) - - - - - - - + - - - -
196 2-Propyl tridecyl ester sulfurous acid (196) - - - - - - - - + - - -
197 3-Mercapto-2(1H)-pyridinone (197) - - - + - - - - - - - -
198 2-Octylfuran (198) - - - - - - - - - - + -
199 3,4-Di[1-butenyl]- tetrahydrofuran-2-ol (199) - - - - - - - - - - + -
200 3,4-Bis(1,3-benzo dioxol-5-ylmethyl) dihydro, 2(3H)-furanone (200) - - + - - - - - - - - -
201 3-Acetoxy 2,4-dimethylfuran (201) - - - - - - - - - - - +
202 1-Ethoxy 4-trimethylsilyl 1,2-butadiene (202) - - - - - - - + - - - -
203 4,8,12-Trimethyltridecan-4-olide (203) + - + + + + + + + + + +
204 2,2-Diphenyl-1-(4-methoxyphenyl)-1,2-dihydroazeto[2,1]quinazolin-8-one (204) - - + - - - - - - - - -
205 Diisopropylidene mannitol (205) - - - - - - - - - - - +
206 Methyl octyl Phthallate (206) - - - - - - - - - - - +
207 7,8-Dimethoxy-3-(3',4'-methylene dioxyphenyl)-2-methyl-1,2,3,4-tetrahydro isoq...
(207)
- - - - - - - - - - + -
208 Cyclohexanol (208) - + - - - - - - - - - -
209 4-(1-Methylethyl) cyclohexanol (209) - - - + - - - - - - - -
210 Isomyocorene (2,6-Dimethyl-1,3,7-octatriene) (210) - + - - - - - - - - - -
211 6,10-Dimethyl 5,9-undecadien-2-one (211) - + - - - - - - - - - -
212 Farnesol isomer a (212) - - - + - - - - - - - -
107
213 Farnesol, acetate (213) - + - - - - - - - - - -
214 Geranyl linalool isomer B (214) - + - + - + - - - - - -
215 Geranyl geraniol (215) - + - - - - - - - - - -
216 7-Methyltocol (216) - + - - - - - - - - - -
217 Nerolidol 1(217) - - - + - - - - - - - -
218 3,7,11-Trimethyl- 1,6,10-dodecatrien-3-ol or (E-farnesol) (218) - + - + - + - - - - - -
219 Neophytadiene (7,11,15-trimethyl,3-methylene-1-hexadecene) (219) - + - + - + + + + - - -
220 (-)-Loliolide (220) - - - + - + - + + - + +
221 Isomyrcenyl acetate or [ 2-(2-methyl-1-propenyl)-5-hexenyl acetate] (221) - - - + - - - - - - - -
222 2,6,10,15,19,23-Hexamethyl- 2,6,10,14,18,22-tetracosa hexaene (221) - - - + - + + - - - - -
223 Elemene (223) - - - - - + - - - - - -
224 Elemol (224) - - - - - + - - - - - -
225 Norolean-12-ene (225) - - - - - + - - + - - -
226 Noruns-12-ene (226) - - - - - - - - + - - -
227 Ergost-5-en-3-ol (227) - - - + - - - - - - - -
228 α-Tocopherol (228) - + - + + + + + + - + +
229 β-Tocopherol (229) + + + + - + + + - - + -
230 δ- Tocopherol (230) - - - + + - - - + + - -
Ixora = I.; + = present; - = absent
108
Table 5: Identification of chemical compounds through GC and GC-MS studies of chloroform extract on Ixora species.
S.
No.
Compounds name
I. f
ulg
ens
flo
wer
s
I. f
ulg
ens
lea
ves
I. p
oly
an
tha
flo
wer
s
I. p
oly
an
tha
lea
ves
I. c
hin
ensi
s fl
ow
ers
I. c
hin
ensi
s l
eav
es
I. c
occ
inea
f l
ow
ers
(Y)
I. c
occ
inea
lea
ves
(Y)
I. c
occ
inea
sta
lks
(Y)
I. c
occ
inea
flo
wer
s (O
)
I. c
occ
inea
lea
ves
(O
)
I. c
occ
inea
sta
lks
(O)
1 1-Tetradecanol (3)
- - - - + - - - - - - -
2 1-Hexacosanol (5)
- - - + - - - - - - - -
3 3,7,11,15-Tetramethyl-1-hexadecanol (11)
- - - + - - - + - - + -
4 3,7,11,15-Tetramethyl 2-hexadecen-1-ol (16)
- + - - - + - + - - + -
5 Pluchidiol (21)
+ - - - - - - - - - - -
6 2-Heptenal (25)
+ + - - - + - + + + + -
7 n-Nonanal (26) - - - + - - - - - + + -
8 2-Decenal (27)
+ + - + - - - - + + + +
9 n-Pentadecanal (29)
- - - + - - - - - - - -
10 13-Octadecenal (30)
- - - + - - + - + - - -
11 Benzoic acid (33)
- + - - - + - - - - - -
109
12 Syringaldehyde (37)
- - - - - + - - + - - -
13 Vanillin (38) - - - - + + - - + + - -
14 Dibutyl 1,2-benzenedicarboxylate (42)
- - - - + + - + - + - -
15 Hexamethyl benzene (57)
- + - - - - - - - - - -
16 3,8 Dimethyl decane (67)
- - + - - + - - - - - -
17 2,6,11 Trimethyl dodecane (72)
- - - - - - - + - - - -
18 3-(1-Hexenyl) cyclohexene (74)
- - - - - - - - - - + -
19 4-Hydroxy 2-cyclohexen-1-one (75)
- + - - - - - - - - - -
20 1,1,4,4-Tetramethyl 2,5-dimethylene cyclohexane (76)
- - - - - - - - - + - -
21 1-(1-Methylethyl) cyclopentene (83)
- - - - - - - + - - - -
22 Octahydro-1-methylene 4(1H) azulenone (88)
- - - - - - - - - - - +
23 3 Cyclohexylidene-4-ethyl- 2-hexanone (94)
- + - - - - - - - - - -
24 Phthalic acid, butyl ester, ester with butyl glycolate (104)
- - + + - - - - + - - -
25 n-Decanoic acid (109)
- + - - - - - - + - - -
26 n-Tetradecanoic acid (110)
+ + + + + + + + + + + +
27 n-Hexadecanoic acid (111)
+ - - + - + + + - + + +
28 n-Octadecanoic acid (113)
- + + + + + - + - + + -
110
29 10-Undecenoyl chloride (122) - + - - - - - - - + - -
30 n-Dodecane (125)
- - - - - - - - - - + -
31 n-Tridecane (126) - - + + + + - + + - + -
32 n-Tetradecane (127)
- - - - - - - + - + - -
33 n-Pentadecane (128)
- - - - + + - + - - - +
34 n-Hexadecane (129)
+ + + + + + + + + + + -
35 n-Nonadecane (130)
- - - - + - - - - - - -
36 n-Eicosane (131)
+ + + - + + + + + + - -
37 n-Tritriacontane (134)
- - - - + - - - - - - -
38 n-Pentatriacontane (136)
- - - + - - - + - - - -
39 n-Hexatriacontane (137)
- - - - - - - - + - - -
40 6,10-Dimethyl- 5,9-dodecadien-2-one (143)
- - - - - - - - + - + -
41 Pent-2-en-1,4-dione (148)
- + - - - - - - - - - -
42 Methyl octanoate (155)
+ + - - - - - - - + + -
43 Nonanedioic acid, mono methyl ester (158)
- + - - - - - - - + + -
44 Dimethyl nonanedioate (159)
- + - - - - - - - - - -
45 Methyl 9-oxo, nonanoate (160)
+ + - + - - - - + + + -
111
46 Methyl tetradecanoate (165)
+ + - - - - - - - + + -
47 Methyl hexadecanoate (166)
+ + + + - + + + + + + +
48 Ethyl hexadecanoate (168)
+ - - + - - - + - - - -
49 Ethyl 1-methyl hexadecanoate (169)
- - - - - - - - - + - -
50 Methyl 15-methyl, hexadecanoate (170)
- - - - - - - - - - - +
51 Methyl 9-octadecenoate (175)
+ - - - - - - + - - - +
52 Methyl heptadecanoate (176)
- + - - - - - - - - - -
53 Methyl 9,12-octadecadienoate (177)
+ + + - + + - + + - + -
54 Methyl octadecanoate (178)
+ + + - + + + + + + + +
55 Methyl 9,12,15-octadecatrienoate (179)
+ - - - - + - - - - + +
56 Isopropyl palmitate (182)
- - + + - + - + + - - -
57 Methyl octacosanoate (183)
- - - + - - - - - - + -
58 Methyl heneicosanoate (184)
- + - - - - - - - - - -
59 Methyl docosanoate (185)
+ + + + - - - + + + + -
60 Methyl tricosanoate (186)
+ + - - - - - - + - + -
61 Methyl tetracosanoate (187)
+ + - + - - - - - + - -
62 Methyl pentacosanoate (188)
- + - - - - - - - - - -
112
63 Methyl eicosanoate (191)
+ - + - - - + + + + - -
64 3-Mercapto-2(1H)-pyridinone (197)
- - - - - - - - + - - -
65 4,8,12-Trimethyltridecan-4-olide (203)
- - - + - - - + - - - -
66 4-(1-Methylethyl) cyclohexanol (209)
- + - - - - - - - - - -
67 7,11,15-Trimethyl, 3-methylene-1-hexadecene (219)
- + - - - + - + - - + -
68 (-)-Loliolide (220)
+ + - + - + - + + - + -
69 Norolean-12-ene (225)
- - - - - - - + - - - -
70 α-Tocopherol (228)
- + - + - + - + + - + -
71 β-Tocopherol (229)
- + - + - + - + - - + -
72 δ-Tocopherol (230)
- + - - - - - + - - - -
73 3-Pentanol (232)
- + - - - - - - - - + -
74 1-Tridecanol (233) - + - - - - - - - - - -
75 (2,4,6-Trimethylcyclohexyl) methanol (234)
- + - - - - - - - - + -
76 5-Methyl-5-hexen-3-yn-2-ol (235)
- + - - - + - - - - + -
77 2,5,5-Trimethyl-3,6-heptadien-2-ol (236)
- - - - - - - - - - + -
78 1-Cyclopenten-4-ol (237)
- - - + - - - + - - + -
79 2-Hexen-1-ol, acetate (238) - - - - - - - + - - + -
113
80 3-Hexen-1-ol, acetate (239) - - - - - - - + - - - -
81 3,3,6-Trimethyl-1,4-heptadien-6-ol (240) - + - - - - - + - - - -
82 5-Methyl-2-(1-methylethyl) 1-hexanol (241)
- - - + - - - - - - - -
83 2,6-Dimethyl 3,7-octadiene-2,6-diol (242)
- - - - - - - - + - - -
84 2,6-Dimethyl- 1,7-octadiene-3,6-diol (243)
- - - - - - - - + - - -
85 2-Nonenal (244)
- - - - - - - - + - - -
86 2,4-Heptadienal (245)
- + - - - - - - - - + -
87 2,4-Decadienal (246)
- + - - - - - - - - + -
88 2,4-Dodecadienal (247)
- + - - - - - - - - - -
89 2 Ethyl butanal (248) - - - + - - - - - - - -
90 n-Octenal (249)
- - - - - - + - - - - -
91 n-Undecenal (250)
- - - - - - - - - - - -
92 14-Methyl 8 hexadecenal (251)
- - - + - - - - - - - -
93 N,N-Bis(2-hydroxy ethyl) dodecanamide (252)
- + + + - - - + + + - -
94 Isovanillic acid (253)
- - + - - - - - - - - -
95 3,4-Dimethoxybenzene1,2-diol (254)
- - - - - - - - + - - -
96 Bis (2-ethyl hexyl) 1,2-benzenedicarboxylate (255)
+ + + + + + + + + + + +
114
97 Bis (2-methyl propyl) 1,2-benzenedicarboxylate (256)
- - - + - - - - - - - -
98 1 Methyldecyl benzene (257)
- - - - + - - - - - - -
99 1 Methylundecyl benzene (258)
- - - - + - - - - - - -
100 1,4 Dimethyl benzene (259)
- - - + - - - - - - - -
101 1,3,5-Trimethyl benzene (260)
- - - + - - - - - - - -
102 1-Methyl-4-(1-methylethyl) benzene (261)
- + - + - + - - - - + -
103 3-(4-Methoxy phenyl), 2-ethyl hexyl 2-Propenoate (262)
- - - + - - - - - - - -
104 3-(4-Hydroxy-3-methoxy phenyl)- 2-propenal (263)
- - - - - - - - + - - -
105 Bicyclo[[7.1.0]dec-2-ene (264)
- - - - - - - - - - + -
106 Bicyclo[3.1.1]hept-2-en-4-ol, 2,6,6-trimethyl-, acetate (265)
- - - - - - - - - - + -
107 Bicyclo[4.3.0]non-3-ene, 3,4,5-trimethyl-(266) - + - - - - - - - - - -
108 5,7,7-Trimethylbicyclo[3.3.0]oct-8-en-2-one (267)
+ - - - - - - - - - - -
109 1,5-Dimethyl- 7-oxabicyclo [4.1.0] heptane (268)
- - - - - - - + - - - -
110 1,6,8-Trimethyl-10-methylene-5-phenyl-1,2,3-triazo[4,5-β]tricyclo[3... (269)
+ - - - - - - - - - - -
111 Methyl-4-isopropyl-bicyclo [2.2.2] octa-5-ene-2,3-dicarboxylic anhydride
(270)
- - + - - - - - - - - -
112 Hexahydro 2,5-methano-1H-inden-7(4H)-one (271)
- + - - - - - - - - - -
113 4 Methyl undecane (272)
+ - - - - - - - - - - -
115
114 5 Methyl undecane (273)
- - + - - - - - - - - -
115 5 Ethyl undecane (274)
- - - - - - - - - - + -
116 3,5 Dimethyl undecane (275)
- - - - - + - - - - - -
117 3,8 Dimethyl undecane (276)
- - - + - - - - - - - -
118 4,7 Dimethyl undecane (277)
- - + + + - - - - - - -
119 5,7 Dimethyl undecane (278)
- - - - - - + - - - - -
120 3,3,6 Trimethyl decane (279)
- + - - + - - - - - - -
121 2,3,7 Trimethyl decane (280)
- - - - - + - - - - - -
122 2,6,10 Trimethyl dodecane (281)
- + - - - - + + + - + -
123 2,7,10 Trimethyl dodecane (282)
- - - - - - - - - - + -
124 2,5 Dimethyl tridecane (283)
- - + - - - - - - - - -
125 7 Methyl 6-tridecene (284)
- - - - - - - - + - - -
126 5-Propyl tridecane (285)
- - - - - - - - + - - -
127 2,5-Dimethyl tetradecane (286)
- - + - - - - - - - - -
128 3,5-Dimethyl 2-octanone (287)
- - - - - - - - - - + -
129 2,3,5-Trimethyl hexane (288) - - - - - - - - - - + -
130 2,6,10,14-Tetramethyl hexadecane (289)
- - + + - - + + - - - +
116
131 2,6,10,15-Tetramethyl heptadecane (290)
- - + - - - - - - - - -
132 7-Hexadecene (291)
- - - + - - - - - - - -
133 3-Methyl 1,4-heptadiene (292)
- + - - - - - - - - - -
134 3 Methyl-5-propyl nonane (293)
- - + - - - - + - - - -
135 5-Eicosene (294)
- + - - - - - - - - - -
136 6,10,14-Trimethyl 2-pentadecanone (295)
+ + + + - + + + + + + +
137 5,7-Dimethyl 1,6 octadiene (296)
- + - - - - - - - - - -
138 Branched nonadecane (297)
- - - - + - - - - - - -
139 3,13-Dimethyl heptadecane (298)
- - - - - + - - - - - -
140 2,3-Dimethyl 2-pentene (299)
- - - - - - - + - - - -
141 3 Hydroxy 3,5-dimethyl-2-hexanone (300)
- - - - - - - + - - - -
142 Acetate, 4-hydroxy-3-methyl-2-butenyl (301) - - - - - - - + - - - -
143 4-(3-Hydroxy-1-butenyl)-3,5,5-trimethyl-2-cyclohexen-1-one (302)
- + - - - - - + + - - -
144 1-Methyl cyclohexene epoxide (303)
- - - + - - - - - - - -
145 1,5-Diethenyl-2,3-dimethyl cyclohexane (304) - - - - - - - - - + - -
146 3-Isopropenyl-1,2-dimethyl cyclopentane (305)
- + - - - + - - - - - -
147 1,1,3 Trimethyl cyclopentane (306)
- - - + - - - - - - - -
117
148 2,4,4-Trimethylcyclopentanol (307)
- - - - - - - + - - - -
149 2-Ethyl-1,1-dimethyl cyclopentane (308)
- - - - - - - - - - + -
150 8-Methyl tricyclo [3.3.0.0(2,8)]octan-3-one (309)
- + - - - - - - - + + -
151 Tricyclo[4.3.1.0(2,5)]decane (310)
- + - - - - - - - + - -
152 1-(2-Methylene-3-butenyl)-1-(1-methylene propyl) cyclopropane (311)
- + + - - - - - - - - -
153 3-Cyclopropyl-2-butanone (312)
- - - - - - - + - - - -
154 2-(1,1-Dimethyl ethyl) anthracene (313)
- - + - - - - - - - - -
155 1-(1-Cyclohexen-1-yl) ethanone (314)
- - - + - - - + - - + -
156 1 Cyclopropyl ethanone (315)
- - - - - - - + - - - -
157 α-Pinene, 2,6,6-Trimethyl- (-)-bicyclo[3.1.1]hept-2-ene (316)
- - - + - - - - - - - -
158 1-Methyl-1,3-cyclononadiene (317)
- - - + - - - - - - - -
159 4,8,12,16-Tetramethylhepta decan -4-olide (319)
- - - - - - + - + - - -
160 2-Isopropenyl-5-methyl cyclohexyl acetate (320)
- - - - - - - + - - - -
161 Cyclopentylcyclopentanol (321)
- - - - - - - + - - - -
162 3-(2-Butenyl)-2-cyclohepten-1-one (322)
- - - - - - - - + - - -
163 1-[(1,2-epoxy-3-hydroxy) propyl] cyclohexane (323)
- - - - - - - - - - + -
164 1,5-Dimethyl- 7-oxabicyclo[4.1.0]heptanes (324)
- - - - - - - - - - + -
118
165 2-Methyl[1,3,4] oxadiazole (325)
- + - - - - - - - - - -
166 3-Ethyl-4-methyl- 1H-pyrrole-2,5-dione (326)
- + - - - + - - - - + -
167 1H-Pyrrole-2,5-dione, 3-ethyl-4-methyl- 2-decenal (327)
- - - - - - - + - - - -
168 2-2,4-Ethanopentaleno[1,2]oxirene, hexahydro (328) - + - - - - - - - - - -
169 5,6,7,7-Tetrahydro-4,4,7-trimethyl 2(4H) benzofuranone (329)
- + - - - + - + + - + -
170 7-Hydroxy-6-methoxy-2H-1-benzopyran-2-one (330)
- - + - - + - - + - - +
171 Methyl 8-(2-furyl) octanoate (331)
- + - - - - - - - - - -
172 Dihydro-5-(hydroxymethyl)-2(3H)-furanone (332)
- + - - - - - - - - - -
173 2-Pentyl furan (333)
- - - + - - - - + - - -
174 Methyl-2,3,4,5-tetrahydro-4-methyl-1,5-dioxo-1H-benz[c]azepine-3-c.. (334)
- - - - - + - - - - - -
175 Dihydro methyl furanone (335)
- - - - - - - + - - - -
176 1 Methyl 1H-pyrazole (336)
- - - - - - - - - - + -
177 3 Methyl 1H-pyrazole (337)
- - - - - - - + - - - -
178 Tetramethyl 1-benzoyloxy-4,5-dihydro pyrido(1,2)azepine-2,3,4,5-
tetracarbox... (338)
- - - - - - - - + - + -
179 2-Hydroxy-3-methyl-5-methoxy-p-benzo quinone (339)
- - - - - - - - + - - -
180 6,7-Dimethoxy-1,4-dihydro-2,3-quinoxalinedione (340)
- - - - - - - - + - - -
181 6,7-Dimethyl-1,2,3,5,8,8-hexahydro naphthalene (341)
- - - - - - - - + - - -
119
182 2-Methyl[1,3,4] oxadiazole (342)
- - - - - - - - - - + -
183 1,2 Epoxy dodecane (343)
- - - - - - - - - - + -
184 n-Octanoic acid (344)
+ - - - - - - - - + - +
185 n-Dodecanoic acid (345)
+ - - - - - - - - - - -
186 n-Eicosanoic acid (346)
- - - - - - - - - - + -
187 9-Oxononanoic acid (347)
+ + - + - + - - + + - +
188 6,9,12-Octadecatrienoic acid (348)
- + - - - - - - - - - -
189 cis-Vaccenic acid (349)
- - - - - - - + - - - -
190 Kaempferol (350)
+ - - - - - - - - - - -
191 2-Fluoro-3,3-dimethylbutanal (351)
- - + - - - - - - - - -
192 3,7-Dimethyl-1-octyl methyl phosphono fluoridate (352)
- + - - - - - - - - - -
193 1-Chloroheptylacetate (353)
- - - - - - - + - - - -
194 1,2-Dicyclohexyl-1,1,2,2-tetrafluoro ethane (354)
- - - - - - - - - - - +
195 n-Heptadecane (355)
- - - - - - - + - - - -
196 1-Nonadecene (356)
- - - - + - - - - - - -
197 n-Tricosane (357)
- - + - - - + - - - - -
198 n-Nonacosane (358)
- - + - - - - - - - - -
120
199 2-Pentadecanone (359)
- - - - - - - - + - - -
200 5-Methyl- 3-hexen-2-one (360)
- + - - - - - - - - + -
201 Hexadecanoic acid, trimethylsilyl ester (361)
- - - - - - + - - - - -
202 Methyl 10-oxo-8-decenoate (362)
+ - - - - - - - - - - -
203 Methyl 8-oxo octanoate (363)
- + - - - - - - - - - -
204 Methyl 9-octadecenoate (364)
- - - - - - - - - + - -
205 Methyl 6-octadecenoate (365)
- - - - - - - - - - + -
206 Methyl 4,7,10,13-hexadecatetraenoate (366)
- + - - - - - - - - - -
207 Acrylic acid octadecanyl ester (367)
- - - + - - - - - - - -
208 Neopentyl 2,2-dimethylbutanoate (368)
- - - - - - - - - - + -
209 2-Ethylhexyl tridecyl sulfuate (369)
- - + - - + - - - - - -
210 3-Isopropoxy-1,1,1,7,7,7-hexa methyl-3,5,5-tris (trimethyl siloxy)
tetrasiloxane (370)
- - - + - - - - - - - -
211 1,1,1,5,7,7,7-Heptamethyl-3,3-bis (trimethyl siloxy)tetrasiloxane (371)
- - - + - - - - - - - -
212 Eicosa methyl cyclodecasiloxane (372)
- - - + - - - - - - - -
213 Octadeca methyl cyclonona siloxane (373)
- - - + - - - - - - - -
214 2-Propyl undecyl sulfate (374)
- - - - - - - + - - - -
215 1,1-Dioxide 2,3-dimethylthiirane (375)
- - - - + - - - - - - -
121
216 4-Vinylphenol (376)
+ - - - - - - - - - - -
217 2,4-Bis(1,1-dimethyl ethyl) phenol (377)
- - - - + - - - - - - -
218 4-3-Hydroxy-1-propenyl)-2-methoxy phenol (378)
- - - - - - - - + - - -
219 α-Amyrin (379)
- - - - - - - - + - - -
220 β- Amyrin (380)
- - - - - - - - + - - -
221 Vomifoliol (381)
- + - - - - - - + - - -
222 γ-Tocopheryl methyl ether (382)
- + - - - - - - - - - -
223 Stigmasterol (383) - - - - - + - - - - - -
224 9-Octadecenoic acid (478)
+ - - - - - - - - - - -
I = Ixora; + = present; - = absent; O = orange color flowers containing plant; Y = yellow color flowers containing plant
122
Table 6: Identification of chemical compounds of I. chinensis flowers methanol
extracts through GC and GC-MS studies.
S.
No.
List of Compounds
1 2-Heptenal (25)
2 2-Decenal (27)
3 N,N-Bis(2-hydroxyethyl) dodecanamide (32)
4 Bis (2-ethylhexyl) 1,2-benzenedicarboxylate (40)
5 (1-Propyloctyl) benzene (43)
6 Decanoic acid (109)
7 Tetradecanoic acid (110)
8 Hexadecanoic acid (111)
9 Heptadecanoic acid (112)
10 Octadecanoic acid (113)
11 n-Hexadecane (129)
12 Methyl hexadecanoate (166)
13 Methyl 9-octadecenoate (175)
14 Methyl 9,12-octadecadienoate (177)
15 Methyl octadecanoate (178)
16 Isopropyl palmitate (182)
17 Methyl docosanoate (185)
18 Methyl eicosanoate (191)
19 α-Tocopherol (228)
20 1-Tridecanol (233)
21 Undecenal (250)
22 6,10,14-Trimethyl- 2-pentadecanone (295)
23 5,9-Dimethyl 4,8-decadienal (384)
24 4,8,12,16-Tetramethylheptadecan-4-olide (385)
25 1-Eicosanol (386)
26 (3β)- Cholesta-4,6-dien-3-ol, benzoate (387)
123
activity against two Gram positive and four Gram negative bacteria (Table 7). B. subtilis and
S. aureus were the name of Gram positive bacteria while, E. coli, S. flexenari, P. aeruginosa
and S. typhii were the name of Gram negative bacteria. I. fulgens flowers (H1), I. fulgens
leaves (H2), I. polyantha flowers (H4), I. chinensis flowers (H7), I. chinensis leaves (H8), I.
coccinea flowers (yellow, H10), I. coccinea leaves of yellow flowers (H11), and stalks (H12),
I. coccinea flowers (orange, H13), I. coccinea leaves of orange flowers (H14), and stalks
(H15), were the hexane extracts. Whereas, I. fulgens flowers (M1), I. fulgens leaves (M2), I.
polyantha flowers (M4), I. polyantha leaves (M5), I. chinensis flowers (M7), I. chinensis
leaves (M8), I. coccinea flowers (yellow, M10), I. coccinea leaves of yellow flowers (M11),
and stalks (M12), I. coccinea flowers (orange, M13), I. coccinea leaves of orange flowers
(M14), and stalks (M15), were the extracts of methanol solvent, which were used for the
evaluation of antibacterial activity. Hexane extracts showed inactive behavior against all the
examined organisms except few, which showed non significant activity. H1 (11.87 %), H7
(15.36 %), H8 (8.47 %), H11 (20.74) and H15 (13.66 %) showed low % of inhibition of
compounds against E. coli. All the tested methanol extracts were exhibited insignificant
antibacterial potential against B. subtilis, S. flexenari and S. aureus. Methanol extracts of
different parts of the plants exhibited, M1 (13.28 %), M5 (12.58 %), M7 (16.17 %), M8 (31.66
%), M10 (24.44 %), M11 (24.98 %), M12 (14.15 %), M13 (38.80 %), and M15 (6.57 %)
inhibition against B. subtilis. Moreover, only four extracts were exhibited activity against S.
aureus, M1 (3.78 %), M4 (23.69 %), M8 (29.96 %) and M13 (22.32 %) inhibition. M1 (6.91
%), M4 (16.39 %), M5 (6.13 %), M7 (6.72 %), M10 (12.51 %), M11 (2.42 %), M12 (7.03 %),
M13 (10.07 %), and M14 (13.59 %) showed low activity against S. flexnari. Whereas, only
M4 showed (5.98 %) inhibition against P. aeruginosa and M10 exhibited (3.47 %) inhibition
against S. typhii. All the tested methanol extracts were inactive agaist E. coli. Amount of
samples were used 06mg, while the concentration of compounds used 150 µg/mL. As
mentioned earlier different plant extracts have earlier been reported in literature having as
antibacterial agents.1d-g,3a-c,4a-c
c) Antifungal activity
Hexane (H) and methanol (M) extracts of Ixora species namely, I. coccinea (orange and
yellow flowers), I. fulgens, I. polyantha, and I. chinensis were examined for antifungal activity
against A. niger, C. albicans, C. glabrata, F. lini, M. canis, and T. rubrum (Table 8). Ixora
124
fulgens flowers (H1), I. fulgens leaves (H2), I. polyantha flowers (H4), I. chinensis flowers
(H7), I. chinensis leaves (H8), I. coccinea flowers (yellow, H10), I. coccinea leaves of yellow
color flowers (H11), and stalks (H12), I. coccinea flowers (orange, H13), I. coccinea leaves
of orange flowers (H14), and stalks (H15), were the hexane extracts. Whereas, I. fulgens
flowers (M1), I. fulgens leaves (M2), I. polyantha flowers (M4), I. polyantha leaves (M5), I.
chinensis flowers (M7), I. chinensis leaves (M8), I. coccinea flowers (yellow, M10), I.
coccinea leaves of yellow flowers (M11), and stalks (M12), I. coccinea flowers (orange,
M13), I. coccinea leaves of orange flowers (M14), and stalks (M15), were the extracts of
methanol solvent, which were used for the evaluation of antifungal assay and found to be
inactive against all the tested fungi. Concentration of compounds were used 400 µg/mL, while
standard drug were named as micrnazole and amphotericin B. Antifungal activity of Ixora
species have been cited previously in literatue. 1d-g,3a-c,4a-c
d) Antioxidant activity
Antioxidant assay can be defined as, the activity of a chemical constituents to decrease
oxidative degradation, similar to lipid peroxidation. Phenolic class is the major example of
antioxidant element of food material. Although antioxidant assay of polyphenols are
distributed with various mechanisms, the supporting activity of phenolics towards of active
free radicals is considered as the important mechanism. Antioxidant reaction ability provides
the knowledge about the time period of antioxidative reaction, the reactivity tells the first
dynamics of antioxidation at a exact concentration of an antioxidant. The valuable influence
of a lot of food stuff consisting fruits, vegetables, tea, and coffee on man physical state to
initiate from the chain-breaking antioxidant action of natural polyphenols, a significant factor
of the above mentioned products. The dietary worth of products is created to a large amount
by their antioxidant potential.106 DPPH estimation is the method for determining antioxidant
assay. The DPPH test is situated on the potential of stable free radical 2,2-diphenyl-1-
picrylhydrazyl to react with H-donors consisting phenolics. As DPPH shows a very intensive
absorption in the visible region, it can be easily concluded by the UV–Vis spectroscopy, while
ESR method in addition is appropriate.106
The antioxidant potential of the flowers, leaves and stalks extracts of Ixora species were
125
estimated by using DPPH assay. Methanol extact (M9) of I. coccinea stalk showed maximum
inhibition whereas, I. coccinea leaves (M8) and I. chinensis leaves (M6) sowed significant
activity as compared to standard compound (Table 9).
Furthermore, I. fulgens flowers, and leaves, I. coccinea leaves (orange color flowers) and I
chinensis flowers and I. polyantha leaves showed good activity. The order of antioxidant
potency trend of methanol extracts of the plants were as follows: I. coccinea stalk > I.
chinensis leaves > I. coccinea leaves (yellow color flowers) > I. fulgens flowers > I. chinensis
flowers = I. coccinea leaves (orange color flowers) > I. polyantha leaves > I. fulgens leaves >
I. coccinea stalk (orange color flowers) (Table 9). All the hexane and chloroform extracts of
the plants were inactive on DPPH assays. Antioxidant activity of plant extracts and isolated
compounds were reported in literature as antioxidant agent.1a-g,5a-c
e) Immunomodulatory activity
Immunomodulation is a process in which immune system of an organism can be transform
with its function, it results in increase of immune responses,is known as an immunostimulative
behavior which mainly indicates support of non specific system, like, macrophages,
granulocytes, complement, and T-lymphocytes. Immunosuppression implies usually to reduce
opposition against stress, infections, and chemotherapeutic issues. Both processes equally
required to be tackled in order to control the normal immunological tasks. Natural and
synthetic compounds are used as immunosuppressive and immunostimulative representatives.
Both types of immunomodulating agents have their own position for productive compounds
for appling these activities in the field of main impact in the world. However there are
restrictions to the general use of these compounds for example increased chances of infection
and complete results all over the immune system.107
In these studies, immunomodulatory assay of hexane, chloroform and methanol extracts of
Ixora species (flowers, leaves, and stalks) have been determined. Effects of these extracts on
extra and intracellular eactive oxygen species (ROS) production were studied. Intracellular
effects was determined by using oxidative brust assay chemiluminesence technique (whole
blood phagytes, isolated neutophils and from mice peritoneum macrophages). These studies
126
showed that, all the extracts except only one methanol extract were found to be non active
against ROS. I. chinensis leaves methanol extract showedsignificant activity on extracellular
ROS production from whole blood, macrophages and neutrophil assay. It showed a very
potent inhibitory effect of chemluminesence, with IC50 value of 19.5 ± 0.4 µg/mL. For
immunomodulating assay Ibuprofen has been used as standard compound (Table 10). This is
the first time investigation of immunomodulatory assay on this plant extracts.
B) Bioassay guided isolation studies on Ipomoea species:
a) Cytotoxic activity
Medicinally important phytochemicals have been isolated and identified from medicinal
plants. These chemical constituents have been recognized for their healing properties. A large
number of plant derived natural chemical constituents have been isolated from the plants are
well known and well documented in literature a anticancer agent.40a,42,51 Uncrushed, dried
leaves of two Ipomoea species plant were subjected for the chemical and biological studies
namely Ipomea batata blackie, and I. batata pink frost. Both plants were extracted with hexane
(H) followed by chloroform (C), and methanol (M) through soxhlet extraction method. All
the above mentioned extracts were evaporated through rotary evaporator to give respective
residue (Experimental). Total two extracts were obtained from hexane (H1-H2), two from
chloroform (C1-C2) and two from methanol (M1-M2). Hexane and chloroform extracts were
evaluated through GC and GC-MS examination for the identification of chemical compounds.
The cytotoxic activity of extracts of flowers of Ipomoea species Linn. (Convolvulaceae),
reported for the first time against HeLa, NCI H-460 and MCF-7 human cancer cell lines and
identification of chemical constituents via GC-MS analysis of all the subjected extracts.
Hexane extract, of I. batata pink frost was found to be most active against HeLa cell line,
exhibiting both growth inhibitory (GI50: 85 ± 3.2 µg/mL) and cytotoxic properties (LC50: 20
± 3.5µg/mL) against HeLa cancer cell line (HeLa). While, chloroform extract of I. batata
(blackie) exhibited good effects against HeLa cell line showed growth inhibition (GI50: 80 ±
3.9 µg/mL). Moreover, methanol extract of I. batata pink frost was most potent against
127
Table 7: In vitro antibacterial activity of extracts of different parts of Ixora species.
S.
No.
Plants name (parts)
Pla
nt
co
de
Gram positive
bacteria
Gram negative bacteria
Ba
cill
us
sub
tili
s
Sta
ph
ylo
cocc
us
au
reu
s
Esc
heri
chia
co
li
Sh
igel
la f
lex
ena
ri
Pse
ud
om
on
os
aer
ug
ino
sa
Sa
lmo
nel
la t
yph
ii
Percent (%) inhibition of drugs
1 I. fulgens (flowers) H1 - - 11.87 - - -
2 I. fulgens (leaves) H2 - - - - - -
3 I. polyantha (flowers) H3 - - - - - -
4 I. polyantha (leaves) H4 - - - - - -
5 I. chinensis (flowers) H5 - - 15.36 - - -
6 I. chinensis (leaves) H6 - - 8.47 - - -
7 I. coccinea (flowers)(Y) H7 - - - - - -
8 I. coccinea (leaves)(Y) H8 - - 20.74 - - -
9 I. coccinea (stalk) (Y) H9 - - - - - -
10 I. coccinea (flowers) (O) H10 - - - - - -
11 I. coccinea (leaves)(O) H11 - - - - - -
12 I. coccinea (stalk) (O) H12 - - 13.66 - - -
13 I. fulgens (flowers) C1 20.28 25.37 23.35 - - -
14 I. fulgens (leaves) C2 - - - - - -
15 I. polyantha (flowers) C3 - 10.98 - - - -
16 I. polyantha (leaves) C4 - - - - - -
128
Amount of extracts = 6 mg; Concentration of extracts =150 µg/mL; - = Inactive; Y = yellow color flowers
containing plant; O = orange color flowers containing plant; H = hexane extract; C = chloroform extract; M =
methanol extract
17 I. chinensis (flowers) C5 - - - - - -
18 I. chinensis (leaves) C6 - 38.57 - - - -
19 I. coccinea (flowers) (Y) C7 - 2.86 12.89 - - -
20 I. coccinea leaves (Y) C8 - 19.27 - - - -
21 I. coccinea (stalk) (Y) C9 - - - - - -
22 I. coccinea (flowers) (O) C10 32.59 7.60 - - - -
23 I. coccinea (leaves) (O) C11 - - - - - -
24 I. coccinea (stalk) (O) C12 - - 6.48 - 5.11 -
25 I. fulgens (flowers) M1 13.28 3.78 - 6.91 - -
26 I. fulgens (leaves) M2 - - - - - -
27 I. polyantha (flowers) M3 - 23.69 - 16.39 5.98 -
28 I. polyantha (leaves) M4 12.58 - - 6.13 - -
29 I. chinensis (flowers) M5 16.17 - - 6.72 - -
30 I. chinensis (leaves) M6 31.66 29.96 - - - -
31 I. coccinea (flowers) (Y) M7 24.44 - - 12.51 - 3.47
32 I. coccinea (leaves) (Y) M8 24.98 - - 2.42 - -
33 I. coccinea (stalk) (Y) M9 14.15 - - 7.03 - -
34 I. coccinea (flowers) (O) M10 38.80 22.32 - 10.07 - -
35 I. coccinea (leaves)(O) M11 - - - 13.59 - -
36 I. coccinea (stalk) (O) M12 6.57 - - - - -
37 Ampicillin (standar drug) 94.97 92.45 93.12 90.64 93.97 90.23
129
Table 8: In vitro antifungal activity of different parts of Ixora species.
S.
No.
Plants name (part)
Pla
nt
co
de
Tri
chp
hyt
on
ru
bru
m
Ca
nd
ida
alb
ica
ns
Asp
erg
illu
s n
iger
Mic
rosp
oru
m c
an
is
Fu
sari
um
lin
i
Ca
nd
ida
gla
bra
ta
Percent (%) inhibition of drugs
1 I. fulgens (flowers) H1 - - - - - -
2 I. fulgens leaves H2 - - - - - -
3 I. polyantha (flowers) H3 - - - - - -
4 I. polyantha leaves H4 - - - - - -
5 I. chinensis (flowers) H5 - - - - - -
6 I. chinensis leaves H6 - - - - - -
7 I. coccinea (flowers) (Y) H7 - - - - - -
8 I. coccinea leaves (Y) H8 - - - - - -
9 I. coccinea stalk (Y) H9 - - - - - -
10 I.coccinea (flowers) (O) H10 - - - - - -
11 I.coccinea leaves (O) H11 - - - - - -
12 I. coccinea stalk (O) H12 - - - - - -
13 I. fulgens (flowers) M1 - - - - - -
14 I. fulgens leaves M2 - - - - - -
15 I. polyantha (flowers) M3 - - - - - -
16 I. polyantha leaves M4 - - - - - -
17 I. chinensis (flowers) M5 - - - - - -
18 I. chinensis leaves M6 - - - - - -
19 I. coccinea (flowers) (Y) M7 - - - - - -
130
Concentration of extracts = 400 µg/mL; - = Inactive; Y = yellow color flowers containing plant; O = orange
color flowers containing plant; H = hexane extract; M = methanol extract
20 I. coccinea leaves (Y) M8 - - - - - -
21 I. coccinea stalk (Y) M9 - - - - - -
22 I. coccinea (flowers) (O) M10 - - - - - -
23 I. coccinea leaves (O) M11 - - - - - -
24 I. coccinea stalk (O) M12 - - - - - -
25 Micronazole (standard drug) - - - - - - -
26 Amphotericin B (standard drug) - - - - - - -
131
Table 9: Antioxidant (DPPH) activity of extracts on Ixora species.
S. No. Plants name (parts) Sample
codes
% RSA
(Radical Scavenging
Activity)
(µg/mL)
IC50 ± SEM
µg/mL
1 I. fulgens (flowers) H1 16.084 -
2 I. fulgens (leaves) H2 15.611 -
3 I. polyantha (flowers) H3 36.097 -
4 I. polyantha (leaves) H4 24.471 -
5 I. coccinea (leaves) (O) H11 37.680 -
6 I. coccinea (stalk) (O) H12 35.418 -
7 I. fulgens (leaves) C2 21.467 -
8 I. polyantha (flowers) C3 29.745 -
9 I. polyantha (leaves) C4 29.616 -
10 I. chinensis (flowers) C5 14.852 -
11 I. coccinea (stalk) (Y) C9 58.747 Insoluble
12 I. coccinea (flowers) (O) C10 20.414 -
13 I. coccinea (leaves) (O) C11 17.105 -
14 I. coccinea (stalk) (O) C12 36.779 -
15 I. fulgens (flowers) M1 90.377 116.107± 1.19
16 I. fulgens (leaves) M2 88.173 116.536 ±2.13
17 I. polyantha (flowers) M3 54.169 390.839±4.2
18 I. polyantha (leaves) M4 89.240 86.367±1.55
19 I. chinensis (flowers) M5 89.811 105.134±0.27
20 I. chinensis (leaves) M6 91.435 54.261±0.94
21 I. coccinea (flowers) (Y) M7 20.218 -
132
IC50 = 50% effective conc. of antioxidant activity; - = Inactive; Y = yellow color flowers containing plant;
O = orange color flowers containing plant; H = hexane extract; C = chloroform extract; M = methanol
extract
22 I. coccinea (leaves) (Y) M8 90.725 25.501±0.132
23 I. coccinea (stalk) (Y) M9 92.145 30.110±0.24
24 I. coccinea (flowers) (O) M10 59.403 389.60±2.47
25 I. coccinea (leaves) (O) M11 89.811 120.52±0.5
26 I. coccinea (stalk) (O) M12 82.452 193.57±1.6
27 Gallic acid (standard drug) - 93.93 23.436±0.43
28 N-acetyl cysteine
(standard drug)
- 95.95 111.44±0.7
133
Table 10: Immunomodulatory activity of extracts on Ixora species.
S. No. Plants name (parts) Sample
codes
% Inhibition
/stimulation
IC50 ± SD
1 I. fulgens (flowers) H1 -25.8 -
2 I. fulgens (leaves) H2 20.2 -
3 I. polyantha (flowers) H3 - -
4 I. polyantha (leaves) H4 -31.4 -
5 I. chinensis (flowers) H5 - -
6 I. chinensis (leaves) H6 - -
7 I. coccinea (flowers)(Y) H7 - -
8 I. coccinea (leaves) (Y) H8 - -
9 I. coccinea (stalk) (Y) H9 30.6 -
10 I. coccinea (flowers)(O) H10 - -
11 I. coccinea (leaves) (O) H11 - -
12 I. coccinea (stalk) (O) H12 - -
13 I. fulgens (flowers) C1 12.8 -
14 I. fulgens (leaves) C2 1.8 -
15 I. polyantha (flowers) C3 22.2 -
16 I. polyantha (leaves) C4 19.2 -
17 I. chinensis (flowers) C5 -3.0 -
18 I. chinensis (leaves) C6 -7.4 -
19 I. coccinea (flowers)(Y) C7 -2.5 -
20 I. coccinea (leaves) (Y) C8 - -
21 I. coccinea (stalk) (Y) C9 36.3 -
22 I. coccinea (flowers) (O) C10 - -
23 I. coccinea (leaves) (O) C11 - -
134
Concentration of extracts = 25 µg/mL; * = 250, 50, 10 µg/mL; - = Inactive; Y = yellow color flowers
containing plant; O = orange color flowers containing plant; H = hexane extract; C = chloroform
extract; M = methanol extract
24 I. coccinea (stalk) (O) C12 - -
25 I. fulgens (flowers) M1 15.2 -
26 I. fulgens (leaves) M2 - -
27 I. polyantha (flowers) M3 - -
28 I. polyantha (leaves) M4 16.5 -
29 I. chinensis (flowers) M5 3.5 -
30 I. chinensis (leaves)* M6 - 19.5 ± 0.4
31 I. coccinea (flowers) (Y) M7 - -
32 I. coccinea (leaves) (Y) M8 34.3 -
33 I. coccinea (stalk) (Y) M9 - -
34 I. coccinea (flowers) (O) M10 -16.9 -
35 I. coccinea (leaves) (O) M11 13.2 -
36 I. coccinea (stalk) (O) M12 25.0 -
37 Ibuprofen (drug) - 73.2 11.2 ± 1.9
135
HeLa cell line showing both growth inhibitory (GI50: 12 ± 3.4 µg/mL) and cytotoxic properties
(LC50: 70 ± 4.7 µg/mL). All the extracts were tested for anticancer activity (Table 11) (Figure
5). Moreover, 41 chemical constituents have been identified from hexane extract of I. batata
blackie leaves extract and 49 Phytochemicals were identified from chloroform extract of I.
batata blackie. Whereas, 77 chemical constituents have been identified from hexane extract
of I. batata pink frost leaves while, 37 Phytochemicals were identified from chloroform
extract of I.batata pink frost leaves including hydrocarbons, fatty acids, esters of fatty acids,
alcohol, aromatic acids, their esters, terpenes and tocopherols through GC and GC-MS studies
(Tables 12,13).
In conclusion, the cytotoxic activity was examined for the first time of extracts of Ipomoea
species. These studies revealed that the leaves of non-polar and moderately polar extracts of
Ipomoea species contained medicinal chemical constituents, which were analyzed by GC and
GC-MS technique (Tables 12, 13). The identification of compounds were made through GC
and GC-MS spectra and mass hunter data base.101c
b) Antibacterial activity:
Hexane (H) and methanol (M) leaves extracts of Ipomoea species namely, I. batata (Blackie)
and I. batata pink frost were screened for antibacterial assay against Gram positive (B. subtilis
and S. aureus) and Gram negative bacteria (E. coli, P. aeruginosa, S. flexenari, and S. typhii)
and found to be non significant against all the examined bacteria (Table 14). The antibacterial
activity of Ipomoea species have been previously reported in literature.40a
c) Antifungal activity:
Hexane (H) and methanol (M) leaves extracts of Ipomoea species namely, I. batata (Blackie)
and I. batata pink frost were examined for antifungal activity against T. rubrum, C. albicans, A.
niger, M. canis, F. lini and C. glabrata. I. batata blackie (H16, M16), and I. batata pink frost
leaves (H17, M17), were the extracts, which have been used for the evaluation of antifungal
assay and found to be inactive against all the tested fungi. Concentration of compounds were
used 400 µg/mL, while standard drug were named as micrnazole and amphotericin B (Table
15).
136
d) Antioxidant activity:
Leaves extracts of I. batata (blackie) and I. batata pink frost were screened for antioxidant
activity, by using DPPH radical scavenging assay. Methanol extract of I. batata blackie
(91.187 % RSA) and I. batata pink frost (85.609 % RSA) showed significant activity.
Whereas, hexane and chloroform extracts showed non-significant antioxidant assay (Table
16). The antioxidant activity of I. batata have been previously reported in literature.40a,c,41b,42
e) Immunomodulatory activity
The immune system is suggested to maintain the host from invading pathogens and to decrease
disease. Establishment of the system by alloantigen or auto antigen is commonly considered
to need practicing of the antigen from beginning to end the phagocytic cells like macrophages,
monocytes, or related cells. Changes of the immune reaction by medicinal agents is facilitate
in treatment is start than disclosure to the antigen has a possibility to create a initial reaction.
An autoimmune sickness, like nephritis, thyroiditis, uveitis, diabetes mellitus and rheumatoid
arthritis, demonstrate to involve response to auto antigen, a likely role for immunosuppressive
drug has been reported. The drug which effects the immune system is known as
immunomodulatory. Some suppress the system, like, preventing rejection of transplanted
organs and other are stimulating and can be used to help combat viral infection such as AIDS
or help in the medication of cancer.108
In the current studies, immunomodulatory activity of I. batata blackie and I. batata pink frost
leaves have been determined, which exhibited non-active results against ROS (reactive
oxygen species) production from wholeblood, macrophages and neutrophil assays (Table 17).
The concentration of compounds were used 25µg/mL. This is the first report for the
determination of immunomodulatory activity of Ipomoea species.
C) Bioassay guided isolation studies on C. fistula:
a)Cytotoxic activity:
Uncrushed, dried flowers, and leaves with stalks of C. fistula plant were subjected for the
chemical and biological studies. Plant material were sequentially extracted with different
polarity solvents, first non polar solvent hexane (H) then moderately polar solvent,
137
aa) 50 % Cell growth inhibition (GI50)
Treatment
ab) 50 % Cell kill (LC50)
Treatment
Figure 5: The bar graph represents concentration values of (aa) GI50 (50 % cell growth inhibition) and
(ab) LC50 (50 % cell kill) of Ipomoea species hexane, chloroform and methanol extracts against HeLa,
MCF-7 and NCI H-460 cancer cell lines.
0
50
100
150
200
250
300
Co
nce
ntr
ati
on
(µ
g/m
L)
HeLa
MCF-7
NCI H-460
0
50
100
150
200
250
300
I. batata
blackie
hexane
I. batata
blackie
chloroform
I. batata
blackie
methanol
I. batata
pink frost
hexane
I. batata
pink frost
methanol
Co
nce
ntr
ati
on
(µ
g/m
L)
HeLa
MCF-7
NCI H-460
138
Table 12: Identification of chemical compounds of Ipomoea batata species hexane extracts
through GC and GC-MS studies.
S.
No.
Compounds name
I. b
ata
ta b
lack
ie
I. b
ata
ta p
ink
fro
st
1 1-Dodecanol (2) - +
2 1-Tetradecanol (3) - +
3 3,7,11,15-Tetramethyl- -+1-hexadecanol (11) + -
4 3,7,11,15-Tetramethyl -2-hexadecen-1-ol (16) + +
5 2-Hexyl-1-decanol (19) + -
6 N,N-Bis (2-hydroxyethyl) dodecanamide (32) + -
7 Bis (2-ethylhexyl) 1,2-benzene dicarboxylate (40) + +
8 Dibutyl 1,2-benzenedicarboxylate (42) + -
9 (1-Propyloctyl) benzene (43) - +
10 (1-Propyl nonyl) benzene (44) - +
11 (1-Propyl decyl) benzene (45) - +
12 (1-Ethyl decyl) benzene (48) - +
13 (1-Ethyl undecyl) benzene (49) - +
14 (1-Butylheptyl) benzene (50) - +
15 (1-Butyl octyl) benzene (51) - +
16 (1-Butyl nonyl) benzene (52) - +
17 (1-Methyl decyl) benzene (53) - +
18 (1-Pentyl heptyl) benzene (55) - +
19 (1-Pentyl octyl) benzene (56) - +
20 3,8-Dimethyldecane (67) - +
21 Decanoic acid (109) + -
22 n-Tetradecanoic acid (110) + -
23 n-Heptadecanoic acid (112) + -
24 n -Octadecanoic acid (113) + -
25 n-Octyl chloride (123) - +
26 n-Undecane (124) - +
139
27 n-Dodecane (125) - +
28 n-Tridecane (126) - +
29 n-Tetradecane (127) + -
30 n-Pentadecane (128) - +
31 n-Hexadecane (129) + +
32 n-Nonadecane (130) - +
33 n-Eicosane (131) + +
34 n-Tritriacontane (134) - +
35 n-Pentatriacontane (136) - +
36 Nonanedioic acid, monomethyl ester (158) + -
37 Methyl 9-oxo, nonanoate (160) + -
38 Methyl hexadecanoate (166) + +
39 Methyl octadecanoate (174) + -
40 Methyl 9-octadecenoate (175) + -
41 Methyl 9,12-octadecadiynoate (178) + -
42 Methyl 9,12,15-octadecatrienoate (179) + -
43 Isopropyl palmitate (182) + +
44 Methyl hexacosanoate (189) - +
45 3-Acetoxy-2,4-dimethyl-furan (201) + -
46 4,8,12-Trimethyltridecan-4-olide (203) + +
47 Diisopropylidene mannitol (205) + -
48 Methyl octyl phthallate (206) + -
49 (-)-Loliolide (220) + +
50 Norolean-12-ene (225) - +
51 α-Tocopherol (228) + +
52 1-Tridecanol (233) + -
53 2-Nonenal (244) + -
54 2,6,10-Trimethyl dodecane (281) + +
55 1-Dodecene oxide (343) + -
56 Vaccenic acid (349) + -
57 n-Heptadecane (355) - +
58 1-Nonadecene (356) + +
59 n-Tricosane (357) + -
60 1-Eicosanol (386) + +
61 1-Hexadecanol (388) + -
140
62 1-Dotriacontanol (389) - +
63 3-Hexanol (390) - +
64 2-Ethyl-2-methyl tridecanol (391) - +
65 3,7,11-Trimethyl 1-dodecanol (392) + -
66 11-Hexadecen-1-ol, acetate (393) - +
67 3-Methyl-6-(1-methyl ethyl) 2-cyclohexen-1-ol (394) + -
68 3,4,4-Trimethylcyclohexa-2-en-1-ol (395) + -
69 5-Methoxy-2-methyl-2-pentanol (396) - +
70 Benzaldehyde (397) - +
71 4-Hydroxy cinnamic acid (398) - +
72 2,3-Dihydroxy 5-methyl acetophenone (399) - +
73 Bis(1-methylheptyl) 1,2-benzenedicarboxylate (400) - +
74 Diisodecyl 1,2-benzenedicarboxylate (401) - +
75 (1-Ethyloctyl) benzene (402) - +
76 4,5-Dimethyl nonane (403) - +
77 2,3,3,4-Tetramethyl pentane(404) - +
78 4,8-Dimethyl undecane (405) - +
79 1-Tert-butyl-3-methylene-1-cyclobutanol (406) - +
80 Cyclohexanone (407) - +
81 Cyclohexyl propionate (408) - +
82 3-Cyclohexyl undecane (409) - +
83 Di(2-ethylhexyl) adipate (410) - +
84 2-Benzyloxy phenyl acetic acid (411) - +
85 3,5-Di-tert-butyl-4-hydroxy phenyl propionic acid (412) - +
86 3,4,4,5,6,7-Hexahydro-9,10,11-trimethoxy-2H-dibenzocyclohepten-2-one (413) - +
87 Isopropyl 2-phenylheptanoate (414) - +
88 Methyl-3-(3,5-ditertbutyl-4-hydroxyphenyl) propionate (415) - +
89 1-Methoxy hexane (416) - +
90 4-Hydroxy-5-oxohexanoic acid lactone (417) - +
91 n-Pentadecanoic acid (418) + -
92 4-Chloropentylacetate (419) - +
93 Decyl trifluoroacetate (420) + -
94 2,2,2-Trichloroethanol (421) - +
95 3,4-Dichloro 3-buten-2-one (422) - +
96 Pentachloro ethane (423) - +
97 1,1,1,5-Tetrachloropentane (424) - +
141
+ = present; - = absent
98 1-Hexadecene (425) - +
99 n-Octadecane (426) - +
100 n-Triatetracontane (427) - +
101 (trans)-2-Nonadecene (428) - +
102 Methyl 9-oxo-8-oxabicyclo[4.3.0]nona-1(6),2-diene-2-carboxylate (429) - +
103 Friedelan-3-one (430) - +
104 Hexadecyl hexadecanoate (431) - +
105 Methyl 16-methyl, heptadecanoate (432) - +
106 3-(4-Methoxyphenyl), 2-ethylhexyl 2-propenoate (433) - +
142
Table 13: Identification of chemical compounds of Ipomoea batata species chloroform extracts through
GC and GC-MS studies.
S.
No.
Compounds name
I. b
ata
ta b
lack
ie
I. b
ata
ta p
ink
fro
st
1 3,7,11,15-Tetramethyl 2-hexadecen-1-ol (16) + +
2 2-Heptenal (25) + +
3 2-Decenal (27) - +
4 N,N-Bis(2-hydroxyethyl) dodecanamide (32) + +
5 Benzoic acid (33) + -
6 Phthalic acid, butyl ester, ester with butyl glycolate (104) + +
7 n-Decanoic acid (109) - +
8 n-Tetradecanoic acid (110) + +
9 n-Hexadecanoic acid (111) + +
10 n-Heptadecanoic acid (112) + -
11 n-Octadecanoic acid (113) - +
12 n-Dodecane (125) - +
13 n-Tridecane (126) - +
143
14 n-Hexadecane (129) + -
15 n-Nonadecane (130) - +
16 n-Eicosane (131) + -
17 n-Tetracosane (132) + -
18 6,10-Dimethyl, 5,9-dodecadien-2-one (143) - +
19 Methyl hexadecanoate (166) + +
20 Methyl 9,12-octadecadienoate (177) + -
21 Methyl octadecanoate (178) + +
22 Methyl 9,12,15-octadecatrienoate (179) + -
23 Isopropyl palmitate (182) - +
24 Methyl tetracosanoate (187) + -
25 Methyl eicosanoate (191) + -
26 7,11,15-Trimethyl,3-methylene-1-hexadecene (219) + +
27 Loliolide (220) + -
28 α-Tocopherol (228) + -
29 1-Tridecanol (233) + -
30 2,4-Decadienal (246) + -
31 n-Undecenal (250) - +
32 Bis (2-ethyl hexyl) 1,2-benzenedicarboxylate (255) + +
144
33 4,7-Dimethyl undecane (277) - +
34 2,6,10-Trimethyl dodecane (281) + +
35 6,10,14-Trimethyl 2-pentadecanone (295) + +
36 n-Octanoic acid (344) - +
37 cis-Vaccenic acid (349) - +
38 n-Heptadecane (355) - +
39 1-Nonadecene (356) - +
40 4,8,12,16-Tetramethylheptadecan-4-olide (385) + -
41 1-Hexadecanol (388) - +
42 1-Octadecanol (434) - +
43 n-Eicosanol (435) + +
44 5,8,11-Heptadecatrien-1-ol (436) + -
45 11-Hexadecyn-1-ol (437) + -
46 Cinnamic acid (438) + -
47 2-Ethyl hexyl 4-methoxy cinnamate (439) - +
48 p-Coumaric acid (440) + -
49 2-(2, 3-Dimethoxy phenyl) carbonyl amino-N-(2,3-dimethoxy benzyl)-3-methylb...
(441)
+ -
50 Benzene acetic acid (442) + -
51 3-Heptadecen-5-yne (443) + -
52 2-Methyl-5-propyl- nonane (444) - +
53 2-Methyl nonadecane (445) + +
145
54 Tetracyclo[6.3.2.0(2,5).0(1,8)]tridecan-9-ol, 4,4-dimethyl (446) + -
55 1,2,3,4,4α, 5,8,9,12,12α-Decahydro-1,4-methanobenzocyclodecene (447) + -
56 1-[1-Methoxy-3,3-dimethyl-2-(3-methyl buta-1,3-dienyl) cyclopentyl] ethanone (448) + -
57 3-Methyl-4-(1,3,3-tri methyl-7-oxabicyclo[4.1.0] heptan-1-yl) 3-buten-2-one (449) + -
58 Di(2-ethylbutyl) ether (450) - +
59 Methyl octyl ether (451) - +
60 5,6,7,7-Tetrahydro-4,4,7-trimethyl 2(4H) benzofuranone (452) + -
61 Octahydro1,4,9,9-tetramethyl-1H-3α,7-methanoazulene (453) + -
62 n-Nonoic acid (454) - +
63 n-Triacontane (455) + -
64 5,9-Dimethyl 2-decanone (456) + -
65 1,1-Dimethoxyhexan-2-one (457) + -
66 2-Methylpropyl 3-phenyl-2-propenoate (458) - +
67 2-Ethyl hexyl 3-(4-hydroxyphenyl) propenoate (459) + -
68 Hexyl tetradecyl Sulfate (460) - +
69 Linalool oxide (461) + -
70 Lanceol (462) + -
71 Ledane (463) + -
72 Octahydro-1-(2-octyldecyl) pentalene (464) + -
I = Ipomoea; + = present; - = absent
146
Table 14: In vitro antibacterial activity of extracts of leaves of Ipomoea species.
Amount of extracts = 6 mg; Concentration of extracts = 150 µg/mL; - = Inactive; H = hexane extract; C =
chloroform extract; M = methanol extract
S.
No.
Plants name P
lan
t c
od
e
Gram positive
bacteria
Gram negative bacteria
Ba
cill
us
sub
tili
s
Sta
ph
ylo
cocc
us
au
reu
s
Esc
heri
chia
co
li
Sh
igel
la f
lex
ena
ri
Pse
ud
om
on
os
aer
ug
ino
sa
Sa
lmo
nel
la t
yph
ii
Percent (%) inhibition of drugs
1 I. batata blackie H16 - - - - - -
2 I. batata blackie C16 - 11.6 - - - -
3 I. batata blackie M16 27.74 - - 18.66 - -
4 I. batata pink frost H17 - - - - - -
5 I. batata pink frost C17 - - 7.93 - - -
6 I. batata pink frost M17 3.56 6.85
7 Ampicillin (standard drug) - 94.97 92.45 93.12 90.64 93.97 90.23
147
Table 15: In vitro antifungal activity of extracts of leaves on Ipomoea species.
Concentration of extracts = 400 µg/mL; - = Inactive; H = hexane extract; M = methanol extract
S.
No.
Plants name P
lan
t c
od
e
Tri
chp
hyt
on
ru
bru
m
Ca
nd
ida
alb
ica
ns
Asp
erg
illu
s n
iger
Mic
rosp
oru
m c
an
is
Fu
sari
um
lin
i
Ca
nd
ida
gla
bra
ta
Percent (%) inhibition of drugs
1 I. batata blackie H16 - - - - - -
2 I. batata blackie M16 - - - - - -
3 I. batata pink frost H17 - - - - - -
4 I. batata pink frost M17 - - - - - -
5 Micronazole (standard drug) - 97.8 113.1 - 98.1 73.50 -
6 Amphotericin B
(standard drug)
- - - 20.70 - - -
148
Table 16: Antioxidant (DPPH) activity of leaves extracts on Ipomoea species.
IC50 = 50% effective conc. of antioxidant activity; - = Inactive; H = hexane extract;
C = chloroform extract; M = methanol extract
Table 17: Immunomodulatory activity of leaves extracts on Ipomoea species.
Concentration of extracts = 25 µg/mL; - = Inactive; H = hexane extract;
C = chloroform extract; M = methanol extract
S. No.
Plants name
Sample
codes
% RSA
(Radical
Scavenging
Activity)
(µg/mL)
IC50 ± SEM
µg/mL
1 I. batata blackie H16 - Insoluble
2 I. batata blackie C16 33.751 -
3 I. batata blackie M16 91.187 73.227± 1.1
4 I. batata pink frost H17 38.940 -
5 I. batata pink frost C17 - Insoluble
6 I. batata pink frost M17 85.609 177.15± 5.93
S. No. Plants name Sample
codes
% Inhibition
/stimulation
IC50 ± SEM
µg/mL
1 I. batata blackie H16 3.2 -
2 I. batata blackie C16 7.0 -
3 I. batata blackie M16 9.5 -
4 I. batata pink frost H17 10.0 -
5 I. batata pink frost C17 -
6 I. batata pink frost M17 - 4.1 -
7 Ibuprofen (drug) - 73.2 -
149
chloroform (C), and in the last with polar solvent methanol (M). Soxhlet extraction method
was used for the process of extraction. All the extracts were collected and evaporated through
rotary evaporator to give respective residue (Experimental). Two extracts were obtained from
hexane, two from chloroform and two from methanol. The hexane and chloroform extracts
were examined by GC-FID and GC-MS spectral studies for the identification of chemical
constituents. All the hexane and chloroform extracts of the plants were subjected to mass
spectral studies, resulting 22 phytochemicals from hexane extract of leaves, 41 metabolites
from chloroform extract of leaves, 45 chemical compounds from hexane extract of flowers,
whereas, 08 metabolites from chloroform extracts of flowers of C. fistula. These studies
disclosed the presence of different class of compounds, which have been listed in Tables 18-
19.
The cytotoxic potential of the C. fistula flowers and leaves extracts were determined by the
Sulforhodamine-B assay using MCF (breast), Hela (cervical) and NCI H-460 (lung) cancer
cell lines. Triplicate experiments were performed. The extracts showed non significant
activity against all the examined cell lines (Table 20a-c). The extracts and compounds isolated
from the entitled plants have been previously reported in literature as anticancer agent.52,56
b)Antibacterial activity:
Hexane (H) and methanol (M) extracts of C. fistula flowers and leaves were examined for
antibacterial assay against Gram positive (B. subtilis and S. aureus) and Gram negative
bacteria (E. coli, P. aeruginosa, S. flexenari and S. typhii) and found to be insignificant against
all the examined bacteria (Table 21). The extracts and compounds isolated from the entitled
plants have been previously reported in literature as antibacterial agents.52,54
c)Antifungal activity:
Hexane (H) and methanol (M) leaves extracts of C. fistula flowers and leaves were examined
for antifungal activity against A.niger, C. albicans, F. lini, T. rubrum, M. canis, and C.
glabrata and were found to be inactive. 400 µg/mL Concentration of extracts were used for
150
the evaluation of antifungal activity, while standard drug which were used in experiment
named as micrnazole and amphotericin B (Table 22). The extracts and compounds isolated
from the plants have been previously reported as antifungal agents in literature.52,54
d)Antioxidant activity:
Extraction on different parts of plants were performed by using different polarity solvents to
give respective extracts. These extracts were analyzed by in vitro antioxidant activity by using
DPPH assay. The methanol extract of C. fistula flowers and leaves were examined. The flowers
extract of methanol exhibited 64.159 % RSA while, the leaves of the plant showed 65.770 %
RSA. Both extracts showed the antioxidant activity with IC50 value of >> 200 µg/mL (Table
23). Gallic acid and N-acetyl cysteine were used as standard compounds. The extracts and
compounds isolated from the entitled plants have been previously reported in literature as
antioxidant agents.52,55
e) Immunomodulatory activity
In the current investigation, immunomodulatory potential of hexane, chloroform and methanol
extracts of C. fistula flowers and leaves have been determined, which were found to be non
active against the ROS chemiluminesence technique (Table 24). Ibuprofen was applyied as a
standard drug in this assay. Extracts and compounds isolated from the entitled plants have been
previously reported in literature as immunomodulating agents.75
151
Table 18: Identification of compounds of C. fistula hexane extracts through GC and
GC-MS studies.
S.
No.
Compounds name
C.
fist
ula
lea
ves
C.
fis
tula
flo
wer
s
1 1-Dodecanol (2)
+ +
2 3,7,11,15-Tetramethyl-(2-hexadecenol) (16)
+ -
3 Pentadecanal (29)
- +
4 N,N-Bis(2-hydroxyethyl) dodecanamide (32)
- +
5 Benzoic acid (33)
- +
6 Bis (2-ethylhexyl) 1,2-benzene dicarboxylate (40)
+ +
7 2-Hexadecyloxirane (98)
- +
8 Decanoic acid (109)
- +
9 n-Tetradecanoic acid (110)
- +
10 n-Hexadecanoic acid (111)
+ -
11 n -Octadecanoic acid (113)
+ -
12 n-Tetradecane (127)
- +
13 n-Pentadecane (128)
- +
14 n-Hexadecane (129)
- +
15 n-Eicosane (131)
- +
16 n-Heneicosane (133)
- +
17 2,11-Dodecanedione (149)
- +
18 Methyl hexadecanoate(166)
+ +
19 Ethyl hexadecanoate (168)
+ -
20 Ethyl 1-methyl hexadecanoate (169)
+ -
21 Methyl octadecanoate (178)
+ +
22 Methyl 9-octadecanoate (175)
+ -
152
23 Methyl 9,12-octadecadienoate (177)
+ +
24 Methyl 9,12,15-octadecatrienoate (179)
- +
25 4,8,12-Trimethyltridecan-4-olide (203)
+ +
26 7,11,15-Trimethyl,3-methylene-1-hexadecene (219)
+ -
27 (-)-Loliolide (220)
+ -
28 α-Tocopherol (228)
+ -
29 Octenal (249)
- +
30 2,6,10,15-Tetramethyl heptadecane (290)
- +
31 Stigmast-5-en-3-ol (318)
- +
32 n-Tricosane (357)
+ -
33 n-Triacontane (455)
- +
34 Decanal (465) - +
35 7,10 Hexadecadienal (467)
+ -
36 (1-Methyldodecyl) benzene (468)
- +
37 5-(2-Methylpropyl) nonane (469)
- +
38 1-Ethynyl-1-cyclopentanol(470)
- +
39 2-Ethyl-1,1-dimethyl cyclopentane (471)
+ -
40 Anthracene (472)
- +
41 1,8-Dihydroxy-3-methyl 9,10-anthracenedione (473)
- +
42 4-Methyldecahydronaphthalen-1-yl Acetate (474)
- +
43 8-Methyl-exo-tricyclo [5.2.1.0 (2.6)] decane (475)
+ -
44 Nonanoic acid (476)
- +
45 9-Octadecenoic acid (478)
- +
46 Butyl 2-chloroethyl Phthallate (479)
- +
47 2-Bromotetradecane (480)
- +
48 2-Methyl tetradecane (481)
- +
49 7-Tetradecene (482)
+ -
50 5-Eicosene (483)
- +
153
+ = present; - = absent
Table 19: Identification of chemical compounds of C. fistula chloroform extracts through GC and
GC-MS studies.
S.
No.
Compounds name C. fistula
leaves
C. fistula
flowers
1 3,7,11,15-Tetramethyl 1-hexadecanol (11) + -
2 3,7,11,15-Tetramethyl 2-hexadecenol (16) + -
3 n-Pentadecanal (29) + -
4 N,N-Bis(2-hydroxyethyl) dodecanamide (32) + -
5 Dibutyl 1,2-benzenedicarboxylate (42) + -
6 2,6-Dimethyl 2,6-octadiene (64) + -
7 n-Decanoic acid (109) + -
8 n-Tetradecanoic acid (110) + -
9 n-Hexadecanoic acid (111) + -
10 n-Octadecanoic acid (113) + -
11 n-Hexadecane (129) + -
12 n-Heneicosane (133) + -
13 Methyl hexadecanoate (166) + -
14 Ethyl hexadecanoate (168) + -
15 Methyl 9,12-octadecadienoate (177) + -
16 Methyl octadecanoate (178) + -
17 Methyl 9,12,15-octadecatrienoate (179) + -
18 Isopropyl palmitate (182) + -
19 4,8,12-Trimethyltridecan-4-olide (203) + -
51 2-Dodecanone (484)
- +
52 5-Hydroxy-6-methoxy-1-indanone (485)
- +
53 1-Methyldibenzothiophene (486)
- +
54 Thioxanthene (487)
- +
55 2-[1,3-Dioxan-2-yl]-1,3-dioxane (488)
- +
56 Mome inositol (489)
+ -
57 N-Methyl-N-(1-oxododecyl) glycine (490)
+ -
154
20 7,11,15-Trimethyl,3-methylene-1-hexadecene (219) + -
21 (-)-Loliolide (220) + -
22 α-Tocopherol (228) + -
23 6,10,14-Trimethyl- 2-pentadecanone (295) + -
24 Bis (2-ethyl hexyl) 1,2-benzenedicarboxylate (255) + +
25 4,8-Dimethyl undecane (405) + -
26 3,5,11,15-Tetramethyl 1-hexadecen-3-ol (491) + -
27 4-Hydroxy benzoic acid (492) - +
28 α-Isopropoxymethoxy-2-isopropoxy methoxy benzyl (493) - +
29 2,3,5 Trimethyl decane (493) + -
30 β-Decalone (494) + -
31 2,6,10, 15,19,23-Hexamethyl 2,6,10,14,18,22-tetracosa hexaene
(495)
+ -
32 2,6,10-Trimethyl 1,5,9-undecatriene (496) + -
33 1-(1-Methylethyl) cyclopentene (497) + -
34 1-Cyclopropyl ethanone (498) + -
35 1-Acetyl-2-methylcyclopropane (499) + -
36 2(4H)-Benzofuranone, 5,6,7,7-tetrahydro-4,4,7-trimethyl (500) + -
37 n-Cetyl thiocyanate (501) + -
38 2-(1-oxy-2,2,6,6-tetramethylpiperidin-4-yl) 6-hydrogen
naphthalene dicarboxylate (502)
+ -
39 5-Methyl- 2(3H)-furanone (503) - +
40 Adenine (504) - +
41 17-Octadecynoic acid (505) + -
42 3,7-Dimethyl-2,6-octadienyl butanoate (506) + -
43 5-Chloro-6,6,6-trifluoro-2,2-dimethyl-4-hexen-3-one (507) - +
44 Ethyl 2-dichloromethyl hexanoate (508) - +
45 1-Butenyl methyl ketone (509) + -
46 Farnesol, acetate (510) + -
47 Isopulegol acetate (511) + -
48 Octadeca methyl cyclononasiloxane (512) - +
49 1-Silacyclo-2,4-hexadiene (513) + -
50 Limonene dioxide 1 (514) + -
51 α -Tocopheryl methyl ether (515) + -
+ = present; - = absent
155
Table 20a: Growth inhibitory and cytotoxic effects of C. fistula plant extracts
against Hela cell lines (Cervical).
S.
No.
Plant name
(Codes)
Doses
(µg/mL)
Cervical cancer cell line
(HeLa)
% Cell
growth
inhibitio
n/
kill
(µg/mL)
GI50 LC50
1 C. fistula leaves (H18) 250 +00 ± 3.5 >250 >250
2 C. fistula flowers (H19) 250 +20 ± 3.2 >250 >250
3 C. fistula leaves (C18) 250 +00 ± 1.8 >250 >250
4
C. fistula leaves (M18)
10 +04 ± 0.7
250 ± 3.9 >250
50 +05 ± 1.4
100 +15 ± 2.8
200 +44 ± 1.1
250 +51 ±
2.4
5 C. fistula flowers (M19) 250
+09 ±
2.5 >250 >250
H = hexane extract; C = chloroform extract; M = methanol extract
156
Table 20b: Growth inhibitory and cytotoxic effects of C. fistula plant extracts
against MCF cell lines (Breast cell lines).
S.
No.
Plant name (Codes)
Doses
(µg/mL)
Breast cancer cell line
(MCF-7)
% Cell growth
inhibition/
kill
(µg/mL)
GI50 LC50
1 C. fistula leaves (H18) 250 +10 ± 2.8 >250 >250
2 C. fistula flowers (H19) 250 +29 ± 1.9 >250 >250
3 C. fistula leaves (C18) 250 +00 ± 2.9 >250 >250
4
C. fistula leaves (M18)
10 -
>250
>250
50 -
100 -
200 -
250 +02 ± 3.3
5 C. fistula flowers (M19) 250 +00 ± 2.5 >250 >250
H = hexane extract; C = chloroform extract; M = methanol extract
157
Table 20c: Growth inhibitory and cytotoxic effects of medicinally important
plant extracts against NCI H-460 (Lung cancer cell line).
S.
No. Plant name (Codes)
Doses
(µg/m
L)
Lung cancer cell line
(NCI H-460)
% Cell growth
inhibition/
kill
(µg/mL)
GI50 LC50
1 C. fistula leaves (H18) 250 +00 ± 2.5 >250 >250
2 C. fistula flowers (H19) 250 +34 ± 0.9 >250 >250
3 C. fistula leaves (C18) 250 +01 ± 2.3 >250 >250
4 C. fistula leaves (M18)
10 -
>250
>250
50 -
100 -
200 -
250 +01 ± 1.9
5 C. fistula flowers (M19) 250 +00 ± 2.3 >250 >250
H = hexane extract; C = chloroform extract; M = methanol extract
158
Table 21: In vitro antibacterial activity of extracts of flowers and leaves on C. fistula.
Amount of extract = 6 mg; Concentration of extract =150 µg/mL; - = Inactive ; H = hexane extract; C =
chloroform extract; M = methanol extract
S.
No.
Plants name (parts)
Pla
nt
co
de
Gram positive
bacteria
Gram negative bacteria
Ba
cill
us
sub
tili
s
Sta
ph
ylo
cocc
us
au
reu
s
Esc
heri
chia
co
li
Sh
igel
la f
lex
ena
ri
Pse
ud
om
on
os
aer
ug
ino
sa
Sa
lmo
nel
la t
yph
ii
Percent (%) inhibition of drugs
1 C. fistula (flowers) H19 - - - - - -
2 C19 - - - - - -
3 M19 18.97 - - 6.58 - -
4 C. fistula (leaves) H18 - - - - - -
5 C18 - 18.3 - - - -
6 M18 33.64 22.32 - - - -
7 Ampicillin (standard drug) - 94.97 92.45 93.12 90.64 93.97 90.23
159
Table 22: In vitro antifungal activity of extracts of flowers and leaves on C. fistula.
Concentration of extracts = 400 µg/mL; - = Inactive; H = hexane extract; M = methanol extract
Table 23: Antioxidant (DPPH) activity of methanol extracts on C. fistula.
IC50 = 50% effective conc. of antioxidant activity; M = methanol extract
S.
No.
Plants name
Pla
nt
co
de
Tri
chp
hyt
on
ru
bru
m
Ca
nd
ida
alb
ica
ns
Asp
erg
illu
s n
iger
Mic
rosp
oru
m c
an
is
Fu
sari
um
lin
i
Ca
nd
ida
gla
bra
ta
Percent (%) inhibition of drugs
1 C. fistula flowers H19 - - - - - -
2 M19 - - - - - -
3 C. fistula leaves H18 - - - - - -
4 M18 - - - - - -
5 Micronazole (standard drug) - - - - - - -
6 Amphotericin B (standard drug) - - - - - - -
S. No.
Plants name
Sample
codes
% RSA
(Radical Scavenging Activity)
(µg/mL)
IC50 ± SEM
µg/mL
1 C. fistula flowers M19 64.159 243.670±1.86
2 C. fistula leaves M18 65.770 305.163±8.9
160
Table 24: Immunomodulatory activity of extracts of C. fistula.
S. No. Plants name (parts) Sample
codes
% Inhibition
/stimulation
IC50 ± SEM
µg/mL
1 C. fistula (flowers) H19 8.7 -
2 C. fistula (flowers) M19 -12.1 -
3 C. fistula (leaves) H18 5.2 -
4 C. fistula (leaves) C18 11.2 -
5 C. fistula (leaves) M18 6.1 -
6 Ibuprofen (drug) - 73.2 -
Concentration of extracts = 25 µg/mL; - = Inactive; H = hexane extract;
C = chloroform extract; M = methanol extract
161
Outlook
162
1 The phytochemical and biological studies of medicinally important plants namely, I.
fulgens, I. polyantha, I. chinensis, I. coccinea (yellow, orange color flowers), I. batata
blackie, I. batata pink frost and C. fistula showed that all these plants have tremendous
medicinal importance.
2 The phytochemistry and pharmacology were carried out on flowers, leaves and stalks
of all the above mentioned plants. All these parts were extracted sequentially with
different polarity solvents like non polar solvent, hexane, moderately polar solvent,
chloroform and polar solvent, methanol.
3 Total forty eight (48) extracts of different medicinal plants were studied.
4 The chemistry of hexane and chloroform extracts were determined through mass
spectroscopic technique, GC-FID and GC-MS studies. All the structures of chemical
compounds were determined through GC-MS chromatogram and verified through MS
data base.
5 The identification of 382 chemical compounds from hexane extracts, 323 compounds
from chloroform extract and 26 metabolites from methanol extracts of all the
subjected plant were carried out, including hydrocarbons, fatty acids and their esters,
alcohol, aromatic acids, aromatic esters, terpenes and vitamins.
6 All the extracts were subjected to biological assays including antibacterial, antifungal,
antioxidant, anti-inflammatory, and anti cancer activities.
7 I. fulgens, I. coccinea yellow color flowers and I. batata blackie hexane, chloroform
and methanol extracts were found to be active against HeLa (cervical), MCF-7 and
NCI H-460 (lung) cancer cell lines.
8 All the subjected extracts were found to be inactive against antibacterial and anti-
fungal examinations.
9 Most of the methanol extracts of all subjected plants were found to be active.
10 Only one extract showed activity against ROS, immunomodulatoy assay.
11 Polar extracts of all the subjected medicinal plants will be examined through ESI-MS
studies, for the identification of chemical constituents found in polar solvent extracts.
163
Chapter 5
Experimental
164
Material and methods
Plant material
Ixora species:
Five species of Ixora plant namely, Ixora coccinea (yellow color flowers), I. coccinea (orange
flowers), I. fulgens (red flowers), I. chinensis (pink flowers) and I. polyantha (white flowers),
belongs to family Rubiaceae were collected during May, 2015 from HEJRIC (ICCBS) gardens
of Karachi University. They were submitted in the herbarium of the department, with
following voucher specimen number.
Ixora coccinea plant (yellow flowers), the voucher specimen (KUH GH 91566).
I. coccinea plant (orange flowers), voucher specimen (KUH GH 91565).
I. fulgens plant (red flowers), the voucher specimen (KUH GH 91567).
I. chinensis plant (pink flowers), the voucher specimen (KUH GH 91566).
I. polyantha plant (white flowers), the voucher specimen (KUH GH 91562).
Ipomoea batata (blackie) and I. batata pink frost (Tricolor):
Both species of Ipomoea were collected during May, 2015 from HEJRIC (ICCBS) gardens of
University of Karachi, Karachi.
Cassia fistula:
C. fistula plant was collected during May, 2015 from HEJRIC (ICCBS) gardens of Karachi
University, Karachi. All the above mentioned plants were identified by plant taxonomist Miss
Muneeba Khan under the guidance of Dr. Anjum perveen of Botany Department, University
of Karachi, Karachi.
Soxhlet extraction
Different parts of all the subjected plants were soaked with hexane followed by chloroform
and then methanol, for 8 hrs. in a conventional soxhlet apparatus to gave respective extracts.
165
The liquid extracts were collected, filtred and then evaporated to complete dryness in a
vacuum by using rotary evaporator equipment. At last, the dried extracts were placed at room
temperature for pharmacological and phytochemical studies.
Analysis of plant constituents
The chemical constituents of extracts were identified by mass spectrometric methods. The
identification and quantification of the phytochemicals were evaluated by gas chromatography
mass spectrometry (GC-MS) methods.
GC-MS studies of plant extracts
The non polar extracts were examined by gas chromatographic flame ionization detection
(GC-FID) and (gas chromatography mass spectrometry) GC-MS method. GC-FID spectra
were operated on a Shimadzu GC-17A [FID mode; column, fused silica capillary column OV-
1, DB-1 (30 m x 0.53 mm, 0.5mm film thickness). Carrier gas was nitrogen, at 75 oC and
programmed to 75 oC at 240 oC min-1 and 3-5 min hold. Injector and detector were at 240 and
250 oC correspondingly. 2 mL of each extract sample were triplicate injected and quantities
characterized as relative area % as obtained from integrator.
GC-MS system was functioned on a JSM 600H (Jeol, Japan) with Agilent 6890N USA [EI
mode; ionization potential, 70ev; column DS-6 (0.32 mm x 30 m); column temperature 50-
250 oC (rate of temperature increases 5 oC/min); carrier gas, He; flow rate, 1.8 mL/min; split
ratio, 1:30]. Sample injection was carried out by means of a split ratio of 35:1 at a temperature
of 25oC. Data processing together with peak integration and deconvolution of spectrum were
presented using the Agilent Mass Hunter Qualitative Analysis (version B.04.00).
Antibacterial, antifungal, antioxidant, cytotoxic and anti-inflammatory activities
Antibacterial, antifungal, antioxidant, immunomodulatory and anticancer assays were
performed at ICCBS, HEJRIC, Karachi University, Karachi. The antibacterial, antifungal,
166
antioxidant, cytotoxic, and antiinflammatory assays were performed by methods as described
in the literature.105-113
Cytotoxic activity samples preparation
The stock solutions of plants extract (hexane, chloroform and methanol), I. fulgens flowers
(H1, C1, M1); I. fulgens leaves (H2, C2, M2); I. polyantha leaves (H5, C5, M5); I. chinensis
leaves (H8,C8,M8); I. coccinea flowers (Y)(H10, C10, M10); I. coccinea leaves (Y)(H11,
C11, M11); I. coccinea stalks (Y)(H12, C12, M12); I. coccinea flowers (O)(H13, C13,M13);
I. coccinea leaves (O)(H14, C14, M14); I. coccinea stalks (O)(H15, C15, M15); I. batata
blackie (H16, C16,M16); I. batata pink frost (H17, C17, M17); C. fistula leaves (H18, C18,
M18) and flowers (H19, C19, M19) (40 mg/mL) were made in DMSO (100%) with a final
concentration of DMSO not increasing 0.5 % (v/v) whereas, doxorubicin (20 mM) in sterile
(D/W). Additional dilutions were made in RPMI-1640 medium.109a
167
Chapter 6
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168
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192
Chemical studies on Opuntia dillenii
193
Opuntia dillenii cladode
Opuntia dillenii flower Opuntia dillenii fruit
194
Introduction
195
Opuntia, a large genus belongs to family Cactaceae, comprising 300 species, it is native of
Mexico, however generally found in the warmer regions of all over the world and has been
naturalized all over Indo-Pakistan subcontinent. They are usually identified as prickly pears.1-
3 It has rapid growth, having good adaptation to poor soils and low requirement for water, it
multiply naturally by just sinking its pads on the ground. This specie have been reported to
have many phytochemical and pharmacological significance, a number of chemical
constituents have been reported to have medicinal properties such as antioxidant, anti-
inflammatory, antimicrobial, anti-viral, anti-ulcer, anti-rush, anti-dysentery and anticancer.
The plant have been used in several diseases like ulcers, sores, diabetes, gout, rheumatism,
skin problem, hemorrhoids, earaches, asthmatic, anthelmintic, purgative, refrigerant, laxative,
hemostatics, gastrointestinal, and diuretic.1-7
Opuntia dillenii (Ker Gawl.) Haw. a cactus plant, generally called as Nagphani and Prickly
Pear. The plant is cultivated all over the World and also found in abundance in the Himalayas
up to 1500 meters. The leaves of the plant are modified into spines by glochids. Flowers of
the cactus are yellow in color, due to the presence of the flavonoids. The fruits are pear shaped,
purple in color and edible, sugars contents are present in the fruits, while the seeds of the fruit
are rounded in shape. O. dillenii have great pharmacological significance; it is used as folk
medicine in all over the world.1,8-10
Chemistry of O. dillenii
Chemical constituents (Table 1) isolated from the entitled plant have been described in the
following text.
Alcohols:
Methanol (1) and heptadecanol (2) have been isolated from O. dillenii.1,11b-e
Aldehydes:
Hexanal (3), nonanal (4), and 2-decenal (5) were identified from cladode of O. dillenii.1,11e,12
196
Alkaloid:
Auraniamide acetate (6), was identified from cladode of O. dillenii.10b
Amino acids:
Amino acids including glycine (7), alanine (8), valine (9), leucine (10), isoleucine (11),
serine (12), threonine (13), glutamate (14), asparagine (15), lysine (16), arginine (17),
methionine (18), phenylalanine (19), tyrosine (20), histidine (21), tryptophan (22), and proline
(23), have been isolated from the entitled plant.1,13a
Aromatic acids and esters:
Different parts of fruits (peel, pulp and seeds) of O. dillenii have been extracted and
chemically studied, they have large amounts of aromatic acids. Benzoic acid (24), 4-hydroxy
benzoic acid (25) vanillic acid (26), 3, 4-dihydroxy benzoic acid (27), gallic acid (28), methyl
4-hydroxy benzoate (29), ethyl 3,4-dihydroxy benzoate (30), 4-hydroxy acetophenone (31),
piceine (32), p-coumaric acid (33), ferulic acid (34), sinapic acid (35), methyl 4-hydroxy-
cinnamate (36), 3, 5-dimethoxy-4-O-β-D-glucopyranosyl cinnamic acid (37), octadecyl
caffeate (38), isobutyl phthalate (39), and butyl phthalate (40), have been isolated and
identified as plant chemical constituents from different parts of O. dillenii cladode.1,10b,11b-e,13d-
g,19h
Betanins:
Betanin (41), betanidin (42), isobetanin (43), and betalamic acid (44), have been identified
from the cactus.1,19h,21a-d
Carbohydrates:
Different saccharides including polysaccharides, tetrasaccharides, trisaccharides, D-glucose
(45), L-galactose (46), fructose (47), rhamnose (48), xylose (49), L-arabinose (50), and ethyl
α-L-rhamnopyranoside (51) have been identified from the entitled plant.1,13a,h,14d,15,16,20
Fatty acids and their ester:
Palmitic (52), stearic (53), heptadecenoic (54), dodecanoic (55), palmitoleic (56),
197
heptadecanoic (57), linolenic (58), linoleic (59), and eucomic (60) acids and esters namely,
methyl eucomate (61), methyl linoleate (62) have been identified from the different parts of
the plant by GC-MS analysis.1,17,18b
Flavonoids:
Flavonoids have been isolated from different parts of the entitled plant named as:
aromadendrin, isorhamnetin (63), isorhamnetin-3-O-glucoside (64), isorhamnetin-3-O-
rutinoside (65), isorhamnetin-3-O-glactoside (66), 3-O-methyl isorhamnetin (67), quercetin
(68), quercetin-3-O-β-glucopyranoside (69), 3-O-methyl quercetin (70), 3, 3-dimethyl
quercetin (71), 3-O-methyl quercetin-7-O-β-D-glucopyranoside (72), quercetin-3-O-
rhamnoside (73), quercetin-3-O-β-galactoside (74), rutin (75), morin (76), kaempferol (77),
kaempferide (78), kaempferol 7-O-β-D-glucopyranosyl-(14)-β-D-glucopyranoside (79),
kaempferol-7-O-β-D-glucopyranoside (80), kaempferol-3-O-β-arabinoside (81), catechin
(82), epicatechin (83), orientin (84), vitexin (85), and manghaslin (86) have been identified
from the cactus, O. dillenii.1,11a-e,13a,c,d,h,14c,18b,19b-h,l, 29a
Glycerols:
Isolation of 9, 12, 13 trihydroxyl-octadeca 10,15-dienoic acid (87), and 9, 12, 13, trihydroxy-
10-octadecenoic acid (88) have been reported from the cladodes.1,20
Heterocyclic compounds:
Heterocyclic components have been isolated from the plants named as flavin (89), opuntiol
(90), opuntioside I (91), opuntioside II (92), opuntioside III (93), 4-ethoxyl-6-hydroxymethyl-
α-pyrone (94), opuntiaester (95), guaiacylglycerol-β-ferulate (96), friedlin (97), 5-(hydroxyl
methyl)-2-furaldehyde (98), 3-hydroxy-3-methyl glutarate (99), ethyl tetrahydro-5-oxo-2-
furan, carboxylate (100), androsine (101), equisetin (102), neoechinulin A (103), and
echinulin (104), are listed in Table 1. Several derivatives of opuntiol have been synthesize and
reported in literature.1,6b,10b,11b-e,13c,e-g,18b,19f,20, 57-59
Hydrocarbons:
Tricosane (105), tetracosane (106), pentacosane (107), hexacosane (108), heptacosane (109),
198
octacosane (110), nonacosane (111), triacontane (112), hentriacontane (113), dotriacontane
(114), tritriacontane (115), tatratriacontane (116), pentatriacontane (117) and hexatriacontane
(118) were identified from stem and cladode of O. dillenii cladode.1,23
Lignans:
Secoisolarici resinol (119) and liriodendrin (120) were identified from cactus of the
plant.10b
Mineral elements:
Mineral elements including K, Ca, Na, Mg, P, Fe, Cu, Zn, B, Mn, Se, Pb, As, Hg have been
identified in wild cactus.1,11a,24
Organic acids:
Malic acid (121), succinic acid (122), cyclodopaglucoside hexaacetate (123), and butyl malate
(124) have been isolated from the stems and fruit of O. dillenii cladode.1,11b-d,13g,21a, 25
Steroids:
β-Sitosterol (125), β-sitosterol-3-O-β-D-glucoside (126), 7-oxo-sitosterol (127), 6β-
hydroxylstigmast-4-ene-3-one (128), daucosterol (129), opuntisterol I (130), and
opuntisteroide II (131) have been isolated from different parts of O. dillenii.1, 13f,18b,19c,e
Terpenes:
Taxerol (132), and 2-hydroxy diplopterol (133), have been identified from stem and cladode
of the plant.1,10b,18b
Vitamins:
Ascorbic acid (134) has been isolated from fruit of the plant.1,19h
Pharmacological significance:
O. dillenii cladodes have been reported for pharmacological purpose. It has been used for the
treatment of antibacterial, antifungal, antimicrobial, antidiabetic, antioxidant, antidepressant,
199
hypotensive, liver problems, immunomodulatory, antituberculosis, anticancer, anti-
inflammatory, antiviral, anti-aging, analgesic, anti-obesity diseases. Cladode of the plant has
been reported for wound healing, skin problems, baldness, local hyperthermia, entrokinesia,
stomach diseases, ulcer, expectorant, cholagogue, spleen-strengthening, and diarrhea. Cladde
of the plant has been used in the treatment of blood and heart diseases, angiogenesis,
hypoglycemic, hypolipidemic, chronic, paralysis, and antispermatogenic. Cladode was also
reported to treat in bartholinitis, synovitis, respiratory infection, hepatitis B, cough, asthama,
whooping, endometritis, rhinitis, antiaging, urinary tract infection, breast hyperplasia and
dyspepsia disease. Extract of the cladode have several medicinal properties like stomatocace,
tonsillitis, pneumonia, tinea favosa, neurasthenia, women diseases. The plant has also been
used for the treatment of skin diseases, mosquito bite, parotitis, anti-hyperlipidemic,
thrombocythemia, bartholinitis, rheumatoid, arthritis, gout, eye diseases, and obesity.1, 6i,8-
10,13a,e,h,19d,m,n,29,30c,40-53
Nutraceutical Importance
Opuntia plant has great nutritional importance. The prickly pears and the cladodes have been
used as food, like vegetables, salads and nutrients food supplements, such as nutraceutical
milk products, yoghurts, soft candies, steamed bread premix flour, noodles, glutinous rice
cake, vinegar beverages, mixed grain, juices, nectars, marmalades, jellies, jams and natural
sweetners etc. A herbal tea is composed of O. dillenii, can effectively remove fat, reduce
weight and improved the human immune system. 54-71
Other Properties
O. dillenii is a multipurpose plant many compositions have been used in preparation of
toothpastes, cosmetics, detergents, soap, bath powder, natural colorants, natural emulsion.1,72-
83 The chemical constituents of the plant have already been described elaborately in the Ph.D.
dissertations of Dr. Lubna Abidi and various M.Sc theses including Ms Safina and Ms
Javeria.1
200
Table 1: Chemical constituents isolated from various parts of O. dillenii.
S.
No. Class of compounds and names of compounds
Molecular
Formula/
Molecular
weight
Parts References
Alcohols
1 Methanol (1) CH4O (32) Cladode 11e
2 Heptadecanol (2) C17H36O (256) Stem/cladode 11b-e
Aldehydes
3 Hexanal (3) C6H12O (100) Cladode 11e, 12
4 Nonanal (4) C9H18O (142) Cladode 11e, 12
5 2-Decenal(5) C10H18O (154) Cladode 12
Alkaloid
6 Aurantiamide acetate (6) C28H30N2O4 (458) Cladode 10b
Amino Acids
7 Glycine (7) C2H5 NO2 (75) Stem/cladode 13a
8 Alanine (8) C3H7 NO2 (89) Stem/cladode 13a
9 Valine (9) C5H11 NO2 (117) Stem/cladode 13a
10 Leucine (10) C6H13NO2 (131) Stem/cladode 13a
11 Isoleucine (11) C6H13NO2 (131) Stem/cladode 13a
12 Serine (12) C3H7NO3(117) Stem/cladode 13a
13 Threonine (13) C4H9NO3 (119) Stem/cladode 13a
14 Glutamate (14) C5H9NO4 (147) Stem/cladode 13a
15 Asparagine (15) C4H8N2O3 (132) Stem/cladode 13a
16 Lysine (16) C6H14N2O2 (146) Stem/cladode 13a
17 Arginine (17) C6H14N2O2 (174) Stem/cladode 13a
18 Methionine (18) C5H5NO2 (149) Stem/cladode 13a
19 Phenyl alanine (19) C9H11NO2 (165) Stem/cladode 13a
20 Tyrosine (20) C9H11NO3 (181) Stem/cladode 13a
21 Histidine (21) C6H9N3O2 (155) Stem/cladode 13a
22 Tryptophan (22) C11H12 N2O2
(204)
Stem/cladode 13a
23 Proline (23) C5H9NO2 (115) Stem/cladode 13a
Aromatic compounds
24 Benzoic acid (24) C7H6O2 (122) Stem/cladode 13e
25 4-Hydroxy benzoic acid (25) C7H6O3 (138) Stem/cladode 10b,11b-e
26 Vanillic acid (26) C8H8O4 (168) Stem/cladode 11b-e
27 3,4-Dihydroxy benzoic acid (27) C7H6O4 (154) Stem/cladode 11b-d,13d
28 Gallic acid (28) C6H7O5 (170) Seeds 19h
29 Methyl 4-hydroxy benzoate (29) C8H8O3 (152) Stem/cladode 10b, 13e
30 Ethyl 3,4-dihydroxybenzoate (30) C9H10O4 (182) Stem/cladode 11b-d
31 4-Hydroxy acetophenone (31) C8H8O2 (136) Stem/cladode 13g
32 Piceine (32) C14H18O7 (298) Stem/cladode 13g
33 p-Coumaric acid (33) C9H8O3 (164) Seeds 19h
34 Ferulic acid (34) C10H10O4 (194) Stem/cladode 11b-d,13f, 19h
201
35 Sinapic acid (35) C11H12O5 (224) Seeds 19h
36 Methyl 4-hydroxy cinnamate (36) C10H10O3 (178) Stem/cladode 13e
37 3,5-Dimethoxy-4-O- β-D-glucopyranosyl cinnamic
acid (37) C17H22O4 (370) Stem/cladode 13g
38 Octadecyl caffeate (38) C27H44O4 (432) Cactus 10b
39 Iso-butyl phthalate (39) C12H14O4 (222) Cladode 11e
40 Butyl phthalate (40) C12H14O4 (222) Cladode 11e
Betanin
41 Betanin (41) C24H26N2O13
(550) Fruit 19h, 21a,b,d
42 Betanidin (42) C18H16N2O8 (468) Fruit 21a
43 Isobetanin (43) C24H26 N2 O13
(550) Fruit 19h, 21b,d
44 Betalamic acid (44) C9H9NO5 (211) Fruit 21a
Carbohydrates
45 D-Glucose (45) C8H16O6 (208) Stem/cladode 13a,h,14d-16
46 L-Galactose (46) C6H12O6 (180) Pods 14a, 14d-16
47 Fructose (47) C6H12O6(180) Stem/cladode 13a,h
48 Rhamnose (48) C6H13O5 (165) Stem/cladode 14d,15
49 Xylose (49) C5H10O5 (150) Pods 16
50 L-Arabinose (50) C5H10O5 (150) Pods 14a, 16
51 Ethyl α-L-rhamno pyranoside (51) C8H16O5 (192) Stem/cladode 20
Fatty acids and their methyl esters
52 Palmitic acid (52) C16H32O2 (256) Seeds 17
53 Stearic acid (53) C18H36O2 (284) Seeds 17
54 Heptadecenoic acid (54) C19H38O2 (270) Seeds 17
55 Dodecanoic acid (55) C18H40O2 (216) Seeds 17
56 Palmitoleic acid (56) C16H30O2 (254) Seeds 17
57 Heptadecanoic acid (57) C19H36O2 (282) Seeds 17
58 Linolenic acid (58) C18H30O2(278) Seeds 17
59 Linoleic acid (59) C18H32O2 (280) Seeds 17
60 Eucomic acid (60) C11H12O6 (240) Stem/cladode 18b
61 Methyl eucomate (61) C12H15O6 (255) Stem/cladode 18b
62 Methyl linoleate (62) C19H34O2 (294) Stem/cladode 18b
Flavonoids
63 Isorhamnetin (63) C16H12O7 (316) Stem/cladode 14, 11a, 19c
64 Isorhamnetin-3-O-glucoside (64) C22H22O12 (478) Flowers 14c, 11a, 19d,g,
29a
65 Isorhamnetin-3-O-rutinoside (65) C28H34O16 (626) Stem/cladode 11,19f,g
66 Isorhamnetin-3-O-galactoside (66) C22H22O12 (478) Stem/cladode 19d, 29a
67 3-O-Methyl isorhamnetin (67) C17H14O7 (330) Stem/cladode 11e, 18b, 19f
68 Quercetin (68) C15H10O7 (302) Flowers/seeds 14c,11a, 19c,e,h
69 Quercetin-3-O-β-D-glucopyranoside (69) C21H20O12 (465) Stem/cladode
/flowers 11a, 14c,19b,e
70 3-O-Methyl quercetin (70) C16H12O7 (316) Stem/cladode 19c,e,f
71 3,3'-Dimethyl quercetin (71) C17H14O7 (330) Stem/cladode 11b-d
72 3-O-Methyl quercetin 7-O-β-D-glucopyranoside (72) C22H22O12 (462) Stem/cladode 11b-d, 13d, 19f
73 Quercetin-3-O-rhamnoside (73) C21H20O11(448) Stem/cladode 11a, 14c, 19d,
29a
202
74 Quercetin-3-O-β-galactoside (74) C21H20O12 (464) Stem/cladode 19c,e
75 Rutin (75) C27H30O16 (610) Stem/cladode 13a,h, 11b-e,
19h
76 Morin (76) C15H10O7 (302) Stem/cladode 13a,h
77 Kaempferol (77) C15H10O6(286) Stem/cladode 19c,f
78 Kaempferide (78) C16H12O6(300) Stem/cladode 19c,f
79 Kaempferol-7-O-β-D-glucopyranosyl-(1→4)-β-D-
glucopyranoside (79) C27H28O16 (639) Stem/cladode
11b-d, 13c,d,
19l
80 Kaempferol-7-O-β-D-glucopyranoside (80) C21H20O11 (448) Stem/cladode 11b-d, 13d, 19f
81 Kaempferol-3-O-α-arabinoside (81) C20H19O10 (419) Stem/cladode 19g
82 Catechin (82) C15H14O6 (290) Seeds 19h
83 Epicatechin (83) C15H14O6 (290) Seeds 19h
84 Orientin(84) C21H20O11 (448) Stem/cladode 19d,29a
85 Vitexin (85) C21H20O10 (432) Stem/cladode 19d,29a
86 Manghaslin (86) C33H40O20 (756) Stem/cladode 11b-d, 13d
Glycerols
87 9,12,13-Trihydroxy octadeca-10, 15-dienoic
acid (87) C18H32O5(328) Stem/cladode 20
88 9, 12,13-Trihydroxy 10-octadecenoic acid (88) C18H34O5(330) Stem/cladode 20
Heterocyclic Compounds
89 Flavin (89) - Stem/cladode 57-59
90 Opuntiol (90) C7H8O4(156) Stem/cladode 11b-d, 13c, 19f
91 Opuntioside I (91) C13H18O9(318) Stem/cladode 11b-d, 13c,f
92 Opuntioside II (92) C13H18O9(319) Stem/cladode 20
93 Opuntioside III (93) C11H14O7(258) Stem/cladode 20
94 4-Ethoxy-6-hydroxy methyl-α-pyrone (94) C8H10O4 (170) Stem/cladode 11b-e, 13c
95 Opuntio ester (95) C12H18O6 (258) Stem/cladode 13e
96 Guiacylglycerol-β-ferulic acid ester (96) C20H24O8 (392) Stem/cladode 13e
97 Friedlin (97) C30H50O (426) Stem/cladode 18b
98 5-(Hydroxymethyl)-2-furaldehyde (98) C6H6O3 (126) Stem/cladode 20
99 3-Hydroxy-3- methyl glutarate (99) C6H10O4 (162) Stem/cladode 20
100 Ethyl tetrahydro-5-oxo-2-furan carboxylate (100) C7H10O4 (158) Stem/cladode 20
101 Androsine (101) C15H20O8 (328) Stem/cladode 13g
102 Equisetin (102) C22H31NO4(373) Fungus 6b
103 Neoechinulin A (103) C19H21N3O2 (323) Cactus 10b
104 Echinulin (104) C29H39N3O2 (461) Cactus 10b
Hydrocarbons
105 Tricosane (105) C23H48(324) Stem/cladode 23
106 Tetracosane (106) C24H50(338) Stem/cladode 23
107 Pentacosane (107) C25H52(352) Stem/cladode 23
108 Hexacosane (108) C26H54(366) Stem/cladode 23
109 Heptacosane (109) C27H56(380) Stem/cladode 23
110 Octacosane (110) C28H58(394) Stem/cladode 23
111 Nonacosane (111) C29H63(408) Stem/cladode 23
203
112 Triacontane (112) C30H62(422) Stem/cladode 23
113 Hentriacontane (113) C31H64(436) Stem/cladode 23
114 Dotriacontane (114) C32H66(450) Stem/cladode 23
115 Tritriacontane (115) C33H68(464) Stem/cladode 23
116 Tetratriacontane (116) C34H70(478) Stem/cladode 23
117 Pentatriacontane(117) C35H72(492) Stem/cladode 23
118 Hexatriacontane (118) C36H74(506) Stem/cladode 23
Lignans
119 Secoisolarici resinol (119) C20H26O6 (362) Cactus 10b
120 Liriodendrin (120) C34H46O18 (742) Cactus 10b
Organic Acids
121 Malic acid (121) C4H6O5 (134) Stem/cladode 25, 11b-d, 13g
122 Succinic acid (122) C4H6O4 (118) Stem/cladode 25
123 Hexaacetate of cyclodopaglucoside (123) C27H32O12 (548) Fruit 21a
124 Butyl (L) malate (124) C8H14O5(190) Stem/cladode 13g
Steroids
125 β-Sitosterol (125) C29H50O(414) Stem/cladode 18b,19c
126 β-Sitosterol-3-O-β-galactoside (126) C35H63O6(576) Stem/cladode 13f, 19e
127 7-Oxositosterol (127) C29H48O2(428) Stem/cladode 18b
128 6-β-Hydroxy stigmast-4-ene-3-one (128) C29H48O2(428) Stem/cladode 18b
129 Daucosterol (129) C35H60O6(576) Stem/cladode 18b
130 Opuntisterol I (130) C29H75O2 (430) Stem/cladode 18b
131 Opuntisteroside II (131) C35H63O7 (592) Stem/cladode 18b
Terpenes
132 Taxerol (132) C30H50O(426) Stem/cladode 18b
133 2-Hydroxydiplopterol (133) C30H50O2(442) Cactus 10b
Vitamin
134 Ascorbic acid (134) C6H8O6(176) Fruits 19h
204
R3
R2
R1
O R
R R1 R2 R3
Benzoic acid (24) OH H H H
4-Hydroxy benzoic acid (25) OH H OH H
Vanillic acid (26) OH OCH3 OH H
3,4-Dihydroxy benzoic acid (27) OH OH OH H
Gallic acid (28) OH OH OH OH
Methyl 4-hydroxy benzoate (29) OCH3 H OH H
Ethyl 3,4 dihydroxy benzoate (30) OC2H5 OH OH H
4-Hydroxyacetophenone (31) CH3 H OH H
Piceine (32) OCH3 H O-Glu H
R3
R2
R1
O OR
(E)
Figure 1:Compounds isolated from Opuntia dillenii
R R1 R2 R3
p-Coumaric acid (33) H H OH H
Ferulic acid (34) H H OH OCH3
Sinapic acid (35) H OCH3 OH OCH3
Methyl 4-hydroxy cinnamate (36) OCH3 H OH H
3,5-Dimethoxy-4-O-β-D-glucopyranosyl cinnamic acid (37) H OCH3 O-D-glu OCH3
205
(S)
OHO HO
OH
O
OR
O
OH
O
O
O
O
O
O
NH
N
N
NH
O
O
(E)
(E)
OH
O
HO
O-
O
HO
OH
OH
OH
NHO
OO
N
H
O
(E)
(Z)
(E)
NOHHO
OO
N
HO
HOOH
O
H
(Z)
(E)
(E)
OH
NOHHO
OO
N
O
HO
O
H
O
HO
OH
OH
OH
(Z)
(E)
(E)
O OR
OR1
(Z)
(E)
R
(60) H Eucomic acid
(61) CH3 Methyl eucomate
(39) Isobutyl phthalate (40) Butyl phthalate
(89) Flavin
(41) Betanin (42) Betanidin
(43) Isobetanin
R R1
(90) Opuntiol OH CH3
(91) Opuntioside I O-D-glu CH3
(94) 4-Ethoxy-6- hydroxy- methyl OH C2H5
-a-pyrone
NOHHO
OO
O
(Z)
(E)
(44) Betalamic acid
(S)(R)
O
(R)
(S)
O
HO
HO
O
OH
OH
OH
OH
HO
OH
(Z)
(S)
(R)
O
(R)O
(S)
O
OH
OH
HO
HO
OH
HO
OH
(Z)
OH
O
OO
O
O
O
CH3
OHOH
OH
HO
OH
CH3OHOH
HO
O
O
OH
OH
HO(E)
(84) Orientin
(86) Manghaslin(85) Vitexin
O
OO
OO OH
HO
HO
O
OCH3
(E)
O
H3C
HO O
O
HO
HOO
(E)
(Z)
(92) Opuntioside II (93) Opuntioside III
206
O
O
A
B
C
R1
R2
R3
R4
R5
R6
R7
1
3
4 5
7
Flavonoids R1 R2 R3 R4 R5 R6 R7
Isorhamnetin (63) O-Glu OH OH H OH OCH3 H
Isorhamnetin-3-O-glucoside (64) OH OH OH H H OCH3 H
Isorhamnetin 3-O-rutinoside (65)
O-rutinoside H OH H OH OCH3 H
Isorhamnetin-3-O-galactoside (66) O-Gal H OH H OH OCH3 H
3-O-Methyl isorhamnetin (67) OCH3 H OH H OH OCH3 H
Quercetin (68) OH OH OH H OH OH H
Quercetin-3-O-β-D- glucopyranoside (69) O-Galp H OH H OH OH H
3-O-methyl quercetin (70) OCH3 OH OH H OH OH H
3,3’-Di methyl quercetin (71) OCH3 H OH H OH OCH3 H
3-O-Methyl quercetin-7-O-β-D-
glucopyranoside (72)
OCH3 H O-β-D-Glcp H OH OH H
Quercetin-3-O-rhamnoside (73) O-Rha H OH H OH OH H
Quercetin-3-galactoside (74) O-Gal OH OH H OH OH H
Rutin (75) O-rutinoside OH OH H OH OH H
Morin (76) OH H OH H OH H OH
Kaempferol (77) OH OH OH H OH H H
Kaempferide (78) OH OH OH H OCH3 H H
Kaempferol-7-O-β-D-
glucopyranosyl(1→4) β-D-
glucopyranoside (79)
OH H H O-β-D-Glcp
(1→4) β-D-
Glcp
OH H H
Kaempferol-7-O-β-D- glucopyranoside
(80)
OH H O-β-D-Glcp H OH H H
Kaempferol 3-O-α- arabinoside (81) O-Ara H OH H OH H H
207
(97) Friedlin
n
(105) 21
(106) 22
(107) 23
(108) 24
(109) 25
(110) 26
(111) 27
(112) 28
(113) 29
(114) 30
(115) 31
(116) 32
(117) 33
(118) 34
( ) n
(S)
(R)
(R)
OHO
(R)
(Z)
RO (Z)
(R)
(R) Pr-i
O
OH
H
(R)(S)
(S)
(E)
O
OCOCH3OCOCH3
H3COCO
O
H3COCO N
OCH3
O
H3COCO
(128) 6-b-Hydroxy stigmast-4-ene-3-one
(123) Hexaacetate of cyclodopaglucoside
(127) 7-Oxositosterol R
(125) H beta-Sitosterol
(126) O-glu beta-Sitosterol-3-O-galactoside
(R)(S)
(R)
(S)
(R)(S)
HOR
H
OH(R) (R)
(E)
R
(130) Opuntiosterol I OH
(131) Opuntiosteroside II beta-D-Glc
(R) (R)
(S) (R)
OH3C
O (E)
(132) Taxerol
(R)(S)
(R)(R) (R)(S)
(R)(R)
(R)
O
208
Results and discussion
209
Present studies
Earlier phytochemical research on O. dillenii, cladodes showed that it has remarkable
medicinal significance in folk and traditional medicines. In the current studies isolation and
identification of chemical constituents on butanol phase of cladode, and its vaccum liquid
chromatography (VLC) fractions were carried out.
Cladodes were extracted with different solvents sequentially to understand its chemistry more
elaborately. Different extracts, fractions, phases were obtained through solvent solvent
separation technique. Butanol phase was subjected to vaccum liquid chromatography (VLC)
to obtain (1-43) fractions. These VLC fractions were studied through GC and GC-MS
analysis, with the identification of chemical constituents (1-409) including long chain
acetophenone, acetylenic compound, alcohols and phenols, aromatic aldehyde, alkyl benzene,
amide, alkaloids, benzoic acid and its derivatives, derivatives of benzyl groups, carbohydrates,
cinnamic acid and their derivatives, cyano group containing compounds, cyclic compounds,
flavonoid, heterocyclic compounds, hydrocarbons, long chain fatty acids, long chain
aldehyde, long chain amide, sesquiterpenes, sulphur containing compounds, terpenes and
miscellaneous compounds (Table 2)(Figure 2).
210
CH3
O
R2
R3
H
R1
R4
R3
CH3
O
R1
R2
R1 R2 R3 R4
(1) H H H H
(2) H H OH H
(3) H OCH3 OH H
(4) H OCH3 OCH3 H
(5) OCH3 H OCH3 H
(6) H OCH3 H OCH3
R1 R2 R3
(7) CH3 H OH
(8) H CH3 OH
O
OCH3
O
(9)
H3C
O
CH3
CH3
(10)
O
H3CO CH3
(12)
HO
HO
(15) (14)
HO
(13)
HO
(16)
( )15
( )10
( )13
OH
(17)
9 ( )7( )7
Figure 2: Compounds identified through GC and GC-MS analysis on butanol phase of O. dillenii
cladode
211
HOOH
(E)
HO
OH
(19)
( )12
OH(E) (E)
3
(18)
OH(E)(E)
(20)
OH
OH
(E)
(E) (E)
(21)
NH
NH
O
O
NH
OCH3
O
O
O
NH
O
O
NH
O
O
O
O
(40) (41)
N
O
O
OH
(43)
NH
NH
O
NH
O
(48)
N
NH
O
O
(45)
N O
( )16
(47)
OH
CH3
OH3C
O
(30)
(22)
(42) (44)
(50)
212
R1
H
R3
R2
R4
R5
R1 R2 R3 R4 R5
(23) OH OH H H H
(24) OH OH H H C(CH3)3
(25) OH OCH3 H OH H
(26) OCH3 OH OCH3 H H
HO
R1
NH2
R2
R1 R2
(27) CH3 H
(28) OCH3 H
(29) H OH
R2
R1
R4
R3
HO
R1 R2 R3 R4
(33) H OH H H
(34) OCH3 OH H H
(35) OH OCH3 H H
(36) OCH2CH3 OH H H
(37) OCH3 OH OCH3 H
(38) H H H CH3
(39) OCH3 CH3 H OCH3
213
HN
NH
O
O
(52)
O
O
HN
N
(53)
NH
CN
O
O(Z)
O NH
HN O
(55)
(54)
NH
H
H3CO
OOH3COOC
NH
(56)
N
HN
O OCH3
OCH3
OCH3
(51)
HN O
(63)
HN
O
(E) (E)
(66)
R3
H
R2
R1R5
R4
R3
R4 R2
R1R5
RO
R1 R2 R3 R4 R5
(57) CH3 H H H H
(58) H H CH(CH3)2 H H
(59) CH3 H CH2CH3 H H
(60) H H CH3 CH3 CH3
(61) CH3 CH3 H H CH3
(62) CH3 H CH3 H CH3
R R1 R2 R3 R4 R5
(68) OH H H H H H
(69) OH H OH H H H
(70) OCH3 OH H H H H
(71) OCH3 H H OH H H
(72) OCH3 H H OCOCH3 H H
(73) OH H H OH OCH3 H
(74) OCH3 OH H H H OH
(75) OH H OCH3 OH OCH3 H
(76) OCH3 H OCH3 OH OCH3 H
214
H2N
O
(E)( )n1
( )n2
n1 n2
(64) 7 7
(65) 11 7
OH
OO
H
(78) (77)
OCH3
HOO
HO OH
OO
OH
O
O
OH
OCH3H3CO
OCH3
H3CO
OCH3
H3CO
H3CO
H3CO OCH3
OH
O
CH3
OHO
O (E)
O
O
OH
OH
OH
HO
HO
O
(82)
(83)
(84)
(81)
(80) (79)
O
OH
OH
HO
HOHO
HO
O
OH
OH
HO
OH
O
OH
OH
OH
HO
HO
(86)
(87)
(85)
( )2
( )5
OHO
OH
OH
OCH3
HO
(88)
OHO
OH
OH
OCH3
HO
O
OCH3
OHOH
OH
(90) (89)
(91) (92) (93)
(94)
215
(S)
(S)HO
OH
OH
O(S)
O
OHO
HO
O
OH
OHOH
HOO
HO
HO
HO
OCH3
(R)
(S)
(S)
(S)O
HO
HO
OH
OH
O
OH
OH
OH
OH
O
OH
OH
OH
OH
O
OHO
OH
HOOH
OOH
(98) (95) (96) (97)
(99)
O
OH
OH
OH
OH
S
(101)
O
OH
OH
OH
OH
S
(100)
OCH3
OH
OH
O
O
O
(102)
O O
OHHO
O
OH
OHOH
OH
OH
OH
(103)
HO
H3CO
OH(E)
(113)
OOCH3
O
(E)
O
OH(E)
(114)
(115)
( )10
( )11 ( )11
N CO
(116)
NCS
(117)
( )7
CN
(118)
OH
O
(E)
O
(Z)
(120) (122)(121)
O
O
(123)
OH
O
(124)
O (E)H
O
216
O
RO
R3
R2
R4
(E)
R1
R R1 R2 R3 R4
(105) H H H H H
(106) CH3 H H OH H
(107) CH3 H H OH OCH3
(108) CH3 H H OCOCH3 OCH3
(109) H H OCH3 OH OCH3
(110) H H OCH3 OCH3 OCH3
(111) H OCH3 H OCH3 OCH3
(112) H H OCH3 OCH3 OCH3
(125)
(Z)
H
O
O
OOH
(Z)
(E)
(126)
O OCH3
O
(127)
(E)
(E)
(128)
O
OCH3
O
O
O OH
(129) (130)
O
HO
(Z)
(131)
OH3C
O
O CH3
O
(132)
O
O
O
O
O
NO2
OCH3
O
(133) (135)(134)
H3CO
O
H3CO
O
(136)
217
O
(E)
HO
OH
OCH3
O
OOH
OH
(E)
O
HN
O
HN
NC
O
O
(Z)
NH
OON NH2
OCH3
NH2N
N
NNH
NH2
NH2
OO
O
N
N
S
CH3
HN
NH2
O
NH
O S
CH3
N
HO
CH3
H3CO
N
(146)
(151)
(144)
(148) (147)
(142)
(139)
(141) (143)
(145)
(137) (138)
( )17
O
O
O
O
CH3CH3
Cl
H3C
OH
O
CH3
NOH
(E)
(E)
(E)
(Z)
(E)
(150)
O
OH
(Z)
OHO
HO
(153)
(152)
( )4
( )4O
O
(154) (156)
OOHO
(155)
O
(E)
OO
( )14 ( )4
218
O
O
(158) (149)
O
(E)
(160) (161)
O
OH
(E)
(E)
OOO
O
O
(162)
OO
(163)
( )6
( )8
( )11
OOO
( )15 O
(164)
O
O
(Z)
(E)
(165)
N
N
S
NS
NH
O (E)
(Z)
(166)
( )5
NH
N
H3CO
OO
CH3(Z) (E)
(167)
NH
H3C CH3
O
S
O
H3C
(Z)
(168)
(169)
S
NH2N (Z)
OO
OCH3H3CO
(Z) (Z)
(170) (171)
NH
S
CH3
OO OHOH2C
OCH3
(Z)
(E)
OH3C
HO OH
O
(E)
H3C
OCH3
OO
(Z)
(E)
O
O
CH3
H3C
H3C (Z)
O CH3
O
(E)
(173) (176) (178)
(172)
(157)
(174)
219
OH3CO CH3
CH3
(Z)
O
OO
O
O
OH
O O
OCH3
O
(E)
OH3CO CH3
CH3
H3CO(Z)
(179) (180) (181)
( )12
(182)N
N
SHN
NH
N
O
(E) (E)
(183) (184)
NH
O
O
ON
OH
O
O H3C
HO OH
(185)
7
4
(186) (187)
( )16
O
H3CO(E)
S
O
O
O
H3CO
OH
N N(E)
O
CN
(189)(188) (190)
(194) (193)
( )7
O
H3C
OO
O
NH
Si
(192)
(191)
( )13
(Z)(E)
O
OH
O
(195)
220
Cl( )n
n
8 = (197)
9 = (198)
10 = (199)
11 = (200)
12 = (201)
13 = (202)
14 = (203)
34 = (204)
(207) (205)(208)
(206)
(209)
( )17
( )17
HO
O(Z)
(217)
( )4
( )6
( )13
( )5
HO
O
( )n
n
07 = (210)
09 = (211)
10 = (212)
12 = (213)
13 = (214)
14 = (215)
16 = (216)
(218)
HO
O
HO
O
(E)
(219)
HOOH
O
O
(224)
(222)
OH
OH
O
OHO
OHO
(225)
OH
O
(223)
(221)
HO
O
(E) HO
O
(E)
(220)
HO (E) (E)
O
( )7
O
O
O
(E)
(227)
( )4
( )11
( )3( )3
( )14
OCH3
O
O
10
(226)
O
O
O
(E)
(228)
OO
(229)
( )8
221
OCH3
OOH
OH
OCH3
O
H3C OH
H3COOCH3
O H3C OH
O
10
(230) (232)
(233)
(231)
( )7
( )10
O
O OH
(E)
(236)
OCH3
O
O
OH
(E) (234)
(235)
( )8 ( )5 ( )8
OH
O
O
O
O(E)
H3COO
O
O
H3CO
O
O
O O O
OO
O O
O O
OO
O
O
OO
O
O
(E)
(237)
(243)
(244)
(242)
(238)
(241)
(240)
( )7
( )5
H3C O
O
O CH3
O
(245)
OO
O
O
OO
O
O
(246)
(247)
H3CO
O
O
O
(248)
222
CH3
O
O
OO
(E) (E)
(249)
OCH3
O
O
O
(250)
O
O
OO(E) O
O
O
O
(E)
(251)
(252)
( )16
( ) 12 O
O
(253)
( )9
H3CO
O
O
O
H3CO
O
(256) (254) (255)
10
11
O CH3
O
(E)
(257)
( )14 ( )6( )10
H3CO
O
(258)
O
O
O
(E)
(262)
(259)
O
OO
O
O
O
O
O
(264) (263)
(261) (260)
O
( )6
( )11
( )9
( )9( )6
OCH3
O
O
O
O
O
(E)
(267)(265)
( )8( )7 ( )18
(266)
223
OCH3
O
(269)
371115
O
O
(E)
O
O
(E) (E)
(270)
(268)
O
HOOH
O
(271)
O
(E)H3CO CH3
O O(272) (273)
O
O
(274)
74
H3COCN
O
CN
(276)
(277)
OO
O
O
(E) (E)
(275)
9
1410
15
O
O
O
O
O
O
5
(278)
5
(279) (280)
( )6 O
O
O
O
(281)
(282)
O
O
(283)
( )9 ( )13 O
O11
12
(285)
( )8
O CH3
O
O
O
(E)
(284)
136
14
15
(286)
11
12
224
H3COOH
O
O
HOO
OHO
(288)
(287)
O
H3C O
H3C
O
O
O CH3
( )17
(289)
H3CO OCH3
OCH3
OCH3
OCH3
(290)
O
H
(E)
(295)
HN
O
O2N
O
O P OCH3H2C
OCH3
O
(300)
(301) (302)
OH
OCH3
OHOCH3H3CO
HN
NH2
O
O2NO
O
O OH
O
O
H3CO
OCH3
NH2
(303)
(306) (308)
(304)
(305) (307)
( )6
Cl
Cl Cl O O
O
(297) (298) (299)
O
H(E)
(296)
NH
OCH3
O
OCH3
O
HN
O
(309)(311)
OH
O
NH
O
(312) (310)
OHN O
O O
225
S
NH
OO
(313)
HO N
OH
(314)
OHN
O
O
O
(315)
(S)O
OH
OCH3
OOH
O
O
O
HOHO
OH
OHOHO
OH
(316)
OH
O
(E)
O
O
O (E)O
HOOH
HO
HO
S
(318)
(319) (320)
OH
(321)
OH
OH
OHHO
O
(323)
(324)
(327)
HO COOH
OCH3H3CO
OCH3 OCH3
(325)
(Z)
(E)
(E) (E) (E) O
O
( )14
(317)
226
(Z)
(331) (332) (333)
OH
(Z)
OH
O
OH
(Z)
(334)
O
(335)
(330)
OOH
(E)
O
(Z)
(E)
(337) (336)
OH
O
(Z)
(338)
O
(Z)(E)
(339)
O(Z)
(Z)
(340)
O
O CH3
O(Z)
O
OH
O CH3
CH2 O
(342)(341)
(343)
OH
O
OCH3
O
(Z)(E) (E)
(344)
(Z)
(345)
OH
OH
HO
(346)
OO
(347)
(Z)
(Z)
(349)
(Z)
(348)
227
O
(350)
O
O
(352)
OO
O
(Z)
(353) (351)
O
OH
(Z)
(356)
O
(354)
O
(Z)
(355)
H3C CH2
CH3
H3C
SO O
(372)
S CH3
CH3 CH3O
(E)
(371)
S
OCH3
O
S S
H
O
O
H
(373)
(375)
H3CS
CH3
OS
OCH3
O
S
O
(377)(374) (376)
(Z)
(Z)O
OCOCH3
H3CO
O
HOO
O
OHOH
O
(379) (380)(378)
228
O
OO
(Z)
O
O
O
O
O
(382) (381)
(383)
(Z) (Z)
(Z)
(384)
O
HO H3CO
HO
O
N
HO
O
O
O
O
Cl
(E)
(385) (386) (387)
OH
HO
HO
O
O
NH
CN
O
(389)(388)
229
O
O
O
(Z)(Z)
(390) (391)
O
O
HOO
(Z) (E)O
O
O O
(393) (392)
O
O
OH
O
HO OH
OH
O
OH
O
HOO
OO
OH
(Z)
(Z)
(394) (395)
(396) (397)
230
Fresh and undried O. dillenii cladodes (45 kg) were cut into small cubes and extracted three
times with methanol at room temperature to give methanol extracts. The extract was
partitioned with ethyl acetate and water. The aqueous phase was then shaken five times with
butanol to give butanol and aqueous phases. The butanol phase was evaporated under vacuum
affording a gummy residue OM-But (18 g) (Schemes 1). The TLC of OM-But showed number
of spots, but the two major spots were purified through VLC, which were identified as known
chemical constituents opuntiol (172) and opuntioside (172a).77a,b
The butanol fraction was analyzed by antidepressant potential, through two behavioral assays
viz a) forced swimming and b) tail suspension tests in mice and rats, respectively.84 It
considerably less the period of immobility interval of rodents in a dose dependant mode in
both behavioral stress models, showing antidepressant analysis. The result was equivalent to
standard drug fluoxetine. Butanol fraction was not produced great change in the locomotor
assay recommending that it was not significant. Yohimbine treated mice showed four times
better activity in the presence of butanol fraction as contrast to control animals implies that
butanol fraction was same interfering with the alpha-2 feedback mechanism of
norepinephrine. Therefore the fraction of O. dillenii possessed antidepressant potential.84a
Therefore the aim of current studies was to identified the chemical constituents present in
butanol phase of O. dillenii cladode. Fractionation was performed for determination of
phytochemicals present in butanol phase. For this purpose the butanol phase was subjected to
vacuum liquid chromaography (VLC)84b and eluated with petroleum ether, ethyl acetate,
methanol, acetone and water with increasing polarity, yielding 45 fractions.
Out of 45 fractions initial thirteen fractions encoded (09, 10, 11, 12, 13, 14, 15, 16-17, 20, 21,
24, 25, 26) were subjected to GC-FID and GC-MS analysis for the identification of chemical
constituents present in the above mentioned VLC fractions (Scheme 1).These studies on the
13 non polar to moderately polar VLC fractions of O. dillenii cladode was performed through
GC-MS analysis resulted in the detection of 409 metabolites (Table 2)(Figure 2). To the best
of our knowledge 382 of these chemical compounds were identified for the first time from O.
dillenii cladode. These phytochemicals belongs to different classes of compounds including
acetophenone, acetylene, aromatic aldehydes, alcohols and phenols, amides, alkaloids, alkyl
231
benzene, benzoic acid and its derivatives, benzyl derivatives, carbohydrates, cinnamic acid
and their derivatives, cyano group including compounds, cyclic compounds, flavonoid,
hydrocarbons, heterocyclic compounds, long chain fatty acids, long chain aldehydes, long
chain amides, sulphur containing compounds, sesquiterpenes, terpenes and miscellaneous
compounds (Table 2). Besides identification of chemical constituents from the VLC fractions
of butanol phase through GC-MS analysis, two pure compounds were also obtained from VLC
fractions 16-22, and 27, which were identified as opuntiol (172), and opuntioside (172a)
through spectroscopic studies. 77a,b
Isolation of opuntiol (172):
Fraction No: 16-22 eluting with P.E: EtOAc (50:150) to EtOAc: MeOH (180:20) yielded
needle like crystals, which were washed with P.E, CHCl3 and EtOAc. TLC chromatogram of
the crystals showed a single spot (Rf = 0.42, CHCl3:MeOH (9.5:0.5), silica gel 60 GF254, the
spectral studies EI-MS, 1H-NMR of which revealed its structure as a known compound
opuntiol (172).77a,b
Isolation of opuntioside (172a):
Fraction No: 27-33 eluting with EtOAc:MeOH (150:50) to EtOAc:MeOH (190:10), showed
a single spot on TLC (Rf = 0.428, CHCl3:MeOH (8:2), silica gel 60 GF254), the spectral studies
EI-MS, 1H-NMR of this pure fraction revealed its structure as a known compound opuntioside
(172a).77a,b
Fraction number 9 furnished promising results in GC-MS analysis, with the identification of
18 chemical constituents. These metabolites include an alkaloid, 7-isobutyl-1H-indole-2,3-
dione (41)(0.47%), a carbohydrate, 1-O-hexyl-D-mannitol (88)(0.53%), two heterocyclic
compounds, tert-butyl (5-cyano-4,4,5-trimethyl-2-pyrrolidinylidene)-,1,1-dimethylethyl ester
(142)(1.45%), and 3-benzylidene-5-phenyl-2(3H)-furanone (165)(2.68%); six long chain
fatty acids and their methyl esters, n-dodecanoic acid (212) (0.83%), n-tetradecanoic acid
(213)(1.59%), n-pentadecanoic acid (214)(5.79%), palmitic acid (215)(40.0%), stearic acid
(216)(1.57%), and oleic acid (217)(2.0%); seven miscellaneous compounds, N-butyl benzene
sulfonamide (313)(6.87%), methyl bis (3,5-dimethoxyphenyl)-hydroxyacetate (325)(0.68%),
232
trans-α-lonone (336)(0.6%), 1,2-dihydrothujopsene (348)(0.08%), and bisabolol oxide A
(356)(0.33%), and one steroid, β-sitosterol (381)(1.39 %). Eighteen of these compounds 41,
88, 136, 165, 213, 214, 313, 336, 348, 356, 381 and 399 have been identified for the first time
from O. dillenii.
Fraction number 10 provided the most promising results. Out of 54, 53 phytochemicals have
been identified. These included one benzoyl derivative, methyl 4-benzoyl butyrate (9), four
alcohols, 2-hexadecanol (15), 2-methyl hexadecanol (16), 12-methyl-2,13-octadecadien-1-ol
(18) and 2-methyl-3,13-octadecadien-1-ol (21)(0.61%); one aromatic aldehyde, 4-hydroxy-3-
ethyl vanillin (36), one alkyl benzene, 1,2,4-trimethyl benzene (61)(0.09%); three benzoic
acid derivatives, 3-hydroxy benzoic acid (69) (0.3%), methyl 4-hydroxy benzoate (71), methyl
4-acetoxy benzoate (72) and 2-napthyl benzoate (87)(1.59%) three derivatives of benzoyl
group, methyl 4-hydroxy phenyl acetate (79), 4-butoxyphenyl acetic acid (80); five cinnamic
acid derivatives, trans-cinnamic acid (105), methyl 4-hydroxy cinnamate (106)(0.22%),
methyl ferulate (107)(0.15%), methyl, 4-hydroxy-3-methoxy, cinnamate (108) and sinapic
acid (109) (4.96%); three heterocyclic compounds, 4-methoxy octahydro-1(2H)-
naphthalenone (129)(0.26%), 5-acetyl-2,2-dimethyl-7-(2-oxopropyl) cycloheptanone (133),
7-isobutyl-1H-indole-2,3-dione (143), N-butyl benzene sulfonamide (147), and p-
octyloxybenzonitrile (194)(0.08%); nine hydrocarbons, decane (179)(0.15%), undecane (198)
(0.24%), dodecane (199)(0.22%), tridecane (200)(0.22%), tetradecane (201)(0.01%),
pentadecane (202)(0.05%), hexadecane (203)(0.24%), 2,6-dimethyl undecane (205)(0.02%),
and 3,4-dimethyl undecane (206)(0.04%), fourteen long chain fatty acids and their derivatives,
n-nonanoic acid (0.03%), n-dodecanoic acid (212), n-tetradecanoic acid (213)(0.73%), n-
pentadecanoic acid (214)(1.72%), palmitic acid (215), stearic acid (216), 11-hexadecenoic
acid (219), 9,12-octadecadienoic acid (220), methyl 3-hydroxy undecanoate (230), butyl
fumarate (237), adipic acid, decyl methyl ester (240)(0.3%), 8-dodecenyl acetate (257), nonyl
2-ethyl hexanoate (260), and isopropyl palmitate (263)(0.31%), one long chain aldehyde, 4-
undecenal (295)(0.07%) one sesquiterpene, and three steroids, 5α-pregnan-20-
one,5,6α:16α,17-diepoxy-3β-hydroxy, acetate (383)(0.59%), 4,6,8(14)-cholestatriene
(384)(0.16%). Out of fifty four compounds, 42 metabolites are being reported for the first time
from O. dillenii.
233
GC-MS studies on fraction 11, 12, 13, 14, 15, 16-17 resulted in identification of 34, 72, 38,
48, 27, 32 phytochemicals of VLC fractions respectively. These included seven acetophenone
derivatives, one acetylenic compound, six alcohols, four phenols, six aromatic aldehydes, six
alkaloids, seven alkyl benzene, five amides, two aromatic compounds, six derivatives of
benzyl group, four cinnamic acid derivatives, three cyano group containing compounds.
Fourteen cyclic compounds, twenty three heterocyclic compounds, three hydrocarbons, about
fifty compounds of fatty acids and their methyl esters, one long chain aldehyde, about fifty
miscellaneous compounds, two sulphur containing compounds, two terpenes, seven steroids,
have been identified from the cladode of O. dillenii through GC-MS studies. Less than ten
metabolites were previously reported from the O. dillenii cactus, while rest of the
phytochemicals are being identified for the first time from the subjected fractions.
GC-MS analysis on VLC fractions numbers 20, 21, 24, 25 and 26 resulted in the identification
of 21, 19, 37, 31, and 51 metabolites from which the total chemical constituents were 21, 24,
38, 34 and 51 of VLC fractions respectively. These fractions resulted in the identifications of
three acetophenone, six alcohols, and phenols, eight alkaloids, four benzoic acid derivatives,
two derivatives of benzyl group, sixteen derivatives of carbohydrates, three cinnamic acid
derivatives, six cyclic compounds, one flavonoid, about twenty miscellaneous compounds,
three hydrocarbons, more than twenty five fatty acids and their methyl esters, four
miscellaneous compounds, nine sesquiterpenes, five sulphur containing compounds, and eight
steroids (Table 2). Out of more than one hundred and fifty compounds only eight compounds
have been reported earlier in literature. While rest of the compounds have been identified for
the first time from O. dillenii.
Acetophenone (1), 4-hydroxy acetophenone (2), 4-hydroxy-3-methoxy acetophenone (3), and
acetosyringone (76), have been isolated and identified from different plants namely Phellinus
sp., Tanacetum sinaicum, Cynanchum paniculatum, Haloxylon griffithii, Apocynin
cannabinum, Formoson ficus septica, Penicillium baarnense, having several medicinal
properties.85a-d,86a,b,87a Moreover, 8-hydroxylinalool (20), 1, 2-benzenediol (23), 4-hydroxy
benzaldehyde (33), vanillin (34), isovanillin (35), syringaldehyde (37) and 9, 10-
234
octadecenoamide (67), have been identified in the present studies. All these compounds have
previously isolated from other medicinal plants namely Ilex kudingcha, Lannea
coromandelica, Pyrola calliantha H. Andres II, Daphne genkwa, Opuntia milpa,
Dendropanax proteus, and Gynostemma pentaphyllum, reported to have a number of
pharmacological properties.88a-i Benzoic acid (68), methyl 4-hydroxy benzoate (71), 3-
methoxy-4-hydroxy benzoic acid (vanillic acid)(73), syringic acid (75) have been reported in
Cynanchum paniculatum, Annona cherimola, Smilax riparia, and O. dillenii.10b,85c,89a-b
Furthermore, galacto heptulose (89), methyl β-D-glucopyranoside (92) and methyl β-D-
galactopyranoside (93) have been reported in fed L-sorbose, Coriaria japonica and O. dillenii
to have many medicinal properties.89c,90a-c Additionally, trans-cinnamic acid (105), methyl 4-
hydroxy cinnamate (106), methyl ferulate (107), methyl 4-hydroxy-3-methoxy cinnamate
(108) and sinapic acid (109) have been reported in Cynanchum paniculatum, O. dillenii,
Smilax riparia, and Haloxylon griffithii. 85c,89b,91a-d
Cyclic compounds including 1,2-cyclohexadione (122), opuntiol (172), and hydrocarbons
namely, decane (197), undecane (198), dodecane (199), tridecane (200), tetradecane (201),
pentadecane (202), and hexadecane (203) have been identified by GC-MS analysis from the
current studies. These phytochemicals have also been isolated and showed several therapeutic
importance. 92a-i In addition, long chain fatty acids and their methyl esters have also been
identified through the spectral studies namely, nonanoic acid (210), dodecanoic acid (212),
tetradecanoic acid (213), palmitic acid (215), stearic acid (216), oleic acid (217), methyl
pentadecanoate (258) and diethylene glycol (322) have been identified through GC-MS
studies. All these chemical compounds have many therapeutic properties.93a-i Only a
flavonoid, isorhamnetin (141) has been identified in the current study. 141 has been previously
isolated and reported in the literature from the O. dillenii cactus, O. monacantha, exhibited
antiradical activity, antidepressant, antioxidant and inhibitory activity on Farnesyl Protein
Transferase (FPTase). Total eighteen steroids have been identified in the current studies. β-
Sitosterol (381) has been isolated previously from flowers of O. ficus indica and O.
dillenii.88b,g,93d,94a-d
235
To summarize, 403 compounds were isolated from the VLC fractions, most of these
compounds were recognized first time from the cladode. Only 22 of these were known and
have been previously reported from the plant cladode, whereas, 382 phytochemicals are
reported as a new phytochemicals from the plant. Most of the compounds reappeared in
different VLC fractions. Structures of all chemical compounds were determined through mass
library search.95 Strong antidepressant potential of the butanol extracts may be due to the
synergistic effect of the phytochemicals present in the plant.
236
Table 2: Identification of compounds through GC and GC-MS studies of VLC fractions of butanol phase of O.
dillenii cladodes and their confirmation through mass spectral library search.
S.
No.
Compound Name
Molecular
formula
(Molecular
Weight)
%
Fra
ctio
n n
um
ber
Ret
enti
on
Tim
e
1 Acetophenone (1) C8H8O (120) 0.05 12 0.091
2 4-Hydroxy acetophenone (2) C8H8O2 (136) 0.40 12 17.572
3 4-Hydroxy 3-methoxy acetophenone (3) C9H10O3 (166) 0.27 11 28.078
0.05 12 18.288
4 3,4-Dimethoxyacetophenone (4) C10H12O3 (180) 0.10 21 29.676
1.99 26 29.687
5 2,4-Dimethoxy acetophenone (5) C10H12O3 (180) 0.65 12 19.634
0.51 20 18.184
6 3,5-Dimethoxy acetophenone (6) C10H12O3 (180) 1.13 11 29.659
1.22 24 29.666
4.91 26 29.727
7 4-Hydroxy 2-methyl acetophenone (7) C9H10O2 (150) 0.02 21 24.271
8 4-Hydroxy 3-methyl acetophenone (8) C9H10O2(150) 0.87 15 24.234
9 Methyl 4-benzoyl butyrate (9) C12H14O3(206) - 10 45.975
10 2,2,4,6-Trimethylphenyl isobutyl ketone (10) C14H20O (204) 1.12 11 30.610
11 1-(4-Methoxyphenyl)-1-heptanone (11) C14H20O2 (220) 6.0 26 24.302
12 Methyl 2,5-octadecadiynoate (12) C19H30O2 (290) - 14 26.668
13 Hexadecanol (13) C16H34O (242) 0.99 12 21.661
14 2-Ethyl 4-methyl pentanol (14) C8H18O (130) 0.11 12 7.063
15 2-Hexadecanol (15) C16H34O (242) 0.99 12 21.661
- 10 20.237
16 2-Methyl hexadecanol (16) C17H36O (256) 0.09 10 27.481
17 9-Heptadecanol (17) C17H36O (256) 8.15 14 21.075
18 12-Methyl-2,13-octadecadien-1-ol (18) C19H36O (280) - 10 45.489
19
13-Methyl pentadec-14-ene-1,13-diol (19) C16H32O2 (256) 2.09 12 20.710
0.19 14 15.381
20 8-Hydroxylinalool (20) C10H18O2 (170) 0.76 14 15.509
237
21 2-Methyl-3,13-octadecadien-1-ol (21) C19H36O (280) 0.61 10 34.050
22 2,6,10,15,19,23-Hexamethyl-tetracosa-2,10,14,18,22-pentaene-
6,7-diol (22)
C30H52O2 (444) 1.34 25 56.991
23 1,2-Benzenediol (23) C6H6O2(110)
C8H10O3(154)
0.46/
0.12
20 11.523/
14.272 24 2,6-Dimethoxy phenol (26)
25 1,2-Dihydroxy 4-tert-butylbenzene (24) C10H14O2(166) 0.55 26 28.153
26 2-Methoxyhydroquinone (25) C7H8O3(140) 0.34 26 26.452
27 2-Hydroxy 5-methyl aniline (27) C7H9ON(123) 0.56 26 27.242
28 2-Amino 4-methoxy phenol (28) C7H9NO2(139) 0.03 12 15.512
29 2-Methoxy 4-vinyl phenol (31) C9H10O2(150) 0.31 13 24.230
0.84 14 14.946
4.62 16,17 24.264
0.07 20 13.576
1.17 24 24.251
30 4-Aminoresorcinol (29) C6H7 NO2(125) 0.62 16,17 28.393
31
2-Methyl resorcinol, acetate (30) C9H10O3(166) 3.13 13 34.438-
34.478
32 2,4-Diethyl 1-methyl cyclohexane (32) C11H22(154) 0.03 16,17 31.163
33
4-Hydroxy benzaldehyde (33) C7H6O2(122) 0.13 11 25.523-
25.546
1.39 12 16.078
34 Vanillin (34) C8H8O3(152) 0.78 12 16.654
35 Isovanillin (35) C8H8O3(152) 0.28 11 26.209
0.35 14 16.647
36 4-Hydroxy 3-ethyl vanillin (36) C9H10O3(166) - 10 26.389
37 Syringaldehyde (37) C9H10O4(182) 4.4 12 21.302
15.5 13 31.548
13.1 14 21.322
38 2-Methyl benzaldehyde (38) C8H8O (120) 0.55/
0.03
16,17 21.967/
29.683 39 2,5-Dimethoxy 4-methyl benzaldehyde (39) C10H12O3(180)
40 Octahydro-3-methyl-1H-indole (40) C9H17N (139) 0.39 12 15.474
41 7-Isobutyl-1H-indole-2,3-dione (41) C12H13NO2
(203)
0.47 09 20.482
42 Methyl 3-(3-methyl-2,4-dioxo pyrrolidin-3-yl) propionate (42) C9H13NO4(199) 3.33 13 31.246
43 3-Methyl 3-propyl-2,5-pyrrolidineone (43) C8H13NO2(155) 0.63 14 14.675
0.76 15 23.936
44 1-(Butyryloxy)-2,5-pyrrolidinedione (44) C8H11NO4(185) 0.68 15 26.317
238
45 Hydroxy succinimide (45) C4H5NO3(115) 3.16 15 27.284-
27.325
46 2-Ethyl 4-methyl succinimide (46) C7H11ON2(141) 0.18 12 13.663
47 5-Ethyl 6-imino-5-(1-methyl-butyl)-dihydro-pyrimidine-2,4-
dione (47)
C11H19N3O2
(255)
4.01 15 39.796
48 3-Isobutyl hexa hydro pyrrolo [1,2]pyrazine-1,4-dione (48) C11H18O2N2
(210)
0.2/
0.54
21 38.370/
39.140
3.90 24 34.997
49 3-Benzyl 6-isopropyl-2,5-piperazinedione (49) C14H18O2 N2
(246)
0.96 24 52.056
50 1-Stearoylpyrrolidine (50) C22H43ON (337) 2.31 24 35.915
51 1,2-Dihydro-3-benzyl-2-(2,4,5-trimethoxy phenyl) quinazolin-
4(3H)-one (51)
C24H24O4N2
(404)
- 25 67.531
52 3-Isobutyl-2,5-piperazinedione (52) C14H18N2O2
(170)
0.32 20 15.759
53 3-Benzyl hexa hydro pyrrolo[1,2] pyrazine-1,4-dione (53) C14H16O2N2
(244)
3.57 25 53.963
0.47 26 54.176
- 24 53.845
54
3-Benzyl-6-isobutyl-2,5-piperazinedione (54) C15H20O2N2
(263)
2.62 25 53.702
3.45 25 53.833
1.26 24 53.590
55
Tert-butyl-(5-cyano-4,4,5-trimethyl-2-pyrrolidinylidene)
ethanoate (55)
C14H22O2N2
(250)
0.26 26 49.280
56 Malonic acid,2-formamido-2-[4-(4-hydroxy-3-methyl-2-
butenyl) indol-3-yl] methyl, dimethyl ester (56)
C20H24O6N2
(388)
3.33 13, 14 31.246/
55.946
57 Toulene (57) C7H8(93) 0.7 11 3.747
58 Isopropyl benzene (58) C9H12(120) 0.21 14 7.893
59 1-Ethyl-3-methyl-benzene (59) C9H12(120) 0.15 14 6.681
60 1,2,3-Trimethyl benzene (60) C9H12(120) 0.33 13 14.147
61 1,2,4-Trimethyl benzene (61) C9H12 (120) 0.03 16,17 29.683
0.09 10 7.648
0.61 11 14.137
0.25 15 14.131
62 1,3,5-Trimethyl benzene (62)
C9H12(120) 0.12 11 12.942-
12.965
1.19 14 7.069
63 N-(1-methylpropyl) acetamide (63) C6H13ON(115) 1.00 14 16.369
239
64 Oleic acid amide (64) C18H35NO(281) 0.82 13 53.682
65 13-Docosenamide (65) C22H43NO(337) 4.37 11 57.991
0.05 12 54.958
2.94 13 58.012
- 14 54.948
1.43 15 58.008
66 N-Isopentyl-2,4-undecadiene-8,10-diynamide (66) C16H21NO(243) 2.22 12 23.007
67 9,10-Octadecenoamide (67) C18H35NO (281) 1.00 14 16.369
68 Benzoic acid (68) C7H6O2(122) 0.78 24 58.018
69 3-Hydroxy benzoic acid (69) C7H6O3(138) 0.3 10 29.485
70 Methyl 2-hydroxy benzoate (70) C8H8O3(152) 1.35 25 21.164
71 Methyl 4-hydroxy benzoate (71) C8H8O3(152) - 10 17.795
72 Methyl 4-acetoxybenzoate (72) C10H10O4(194) - 10 16.647
73 3-Methoxy-4-hydroxy benzoic acid (vanillic acid) (73) C8H8O4(168) 0.08 16,17 29.941
74 Methyl 2,6-dihydroxybenzoate (74) C8H8O4(168) 0.42 26 29.051-
29.084
75 Syringic acid (75) C7H6O5(170) 0.95 20 26.191
76 Acetosyringone (76) C10H12O4(196) 4.72 13 33.018
22.3 14 22.746
77 Benzene acetaldehyde (77) C8H8O(120) - 12 8.455
78 Benzene acetic acid (78) C8H8O2(136) - 14 44.478
12.62 15 57.590
0.14 16,17 22.891
79 Methyl 4-hydroxy phenyl acetate (79) C9H10O3(166) - 10 18.311
80 4-Butoxyphenyl acetic acid (80) C12H16O3(208) - 10 22.899
81 3-Phenylpentanedioic acid (81) C11H12O4(208) 3.01 12 32.455
82 2,4-Dimethoxybenzyl alcohol (82) C9H12O3(168) 0.43 25 67.531
83 3,4,5-Trimethoxybenzyl methyl ether (83) C11H16O4(212) 2.11 11 33.851
84
Methyl (3,4-dimethoxy phenyl)(hydroxyl) acetate (84) C11H14O5(226) 0.07 20 13.576
1.71 24 45.014
85 2-(3-Isopropylphenyl)-1-propanol (85) C12H18O(178) 0.17 12 18.663
86 2-Hexenyl benzoate (86) C13H16O2(204) 1.25 11 51.858-
51.808
87 2-Napthyl benzoate (87) C17H12O2(248) 1.59 11 51.925
- 10 46.216
88 1-O-Hexyl-D-mannitol (88) C12H26O6(266) 0.53 09 18.174
89 Galacto heptulose (89) C7H14O7(210) 8.16 26 26.851
240
90 1,5-Anhydrohexitol (90) C6H12O5(164) 2.93 24 25.684
91 β-D-Glucopyranose (91) C6H12O6(180) 2.12 25 36.127
92 Methyl β-D-glucopyranoside (92)
C7H14O6(194) 6.06 26 32.413
15.06 26 37.651
93 Methyl β-D-galactopyranoside (93) C7H14O6(194) 23.21 26 40.636
94 Methyl 6-deoxy hexopyranoside (94) C7H14O5(178) 1.89 24 25.141
95 Methyl d-lyxofuranoside (95) C6H12O5(164) 4.02 25 26.556
96 1-Gala-1-ido-octonic lactone (96) C8H14O8(238) 3.06 25 47.188-
47.403
97 1-O-Dodecyl-α-D-riboside (97) C17H34O5(318) 1.33 26 33.221
98 4-O-Hexapyranosyl hexose (98) C12H22O11(342) 1.4 24 37.556
99 6-O-Hexopyranosyl hexose (99) C12H22O11(342) 10.06 26 32.256-
32.330
100 1-Deoxy-1-dodecylthio-α, β-D-glucopyranoside (100) C18H36SO5(364) 5.33 24 57.499
101 1-Dodecyl thio-1-deoxy-galactopyranoside (101) C18H36O5S(364) 0.5 25 53.270
102 4,6-O-(undec-10-enylidene)-methyl-α-D-glucopyranoside
(102)
C18H32O6(344) - 24 53.717
103 4-O-β-D-Galactopyranosyl-β-D-glucopyranose (103) C12H22O11 (342) 3.45 26 48.540-
48.727
104 1-Octylthio-1-deoxy- galacto pyranoside (104) C14H28O5S
(308)
0.20 26 53.610
105 Trans-Cinnamic acid (105) C9H8O2(148) - 10 17.390
0.87 12 17.252
0.74 13 29.662
106 Methyl 4-hydroxy cinnamate (106) C10H10O3(178) 0.22 10 29.997
107 Methyl ferulate (107)
C11H12O4(208)
0.15 10 22.189
8.29 11 35.663
- 10 25.451
108 Methyl 4-hydroxy-3-methoxy cinnamate (108) C13H14O5(250) - 10 40.944
109 Sinapic acid (109) C11H12O5(224) 4.96 10 49.294
110 3,4,5-Trimethoxy cinnamic acid (110) C12H14O5(238) 0.56 26 35.853
5.0 11 47.394
111 2,4,5-Trimethoxy cinnamic acid (111)
C12H14O5(238) 0.03 16,17 28.765
0.78 26 47.652
0.05 12 29.926
112 2,3,4-Trimethoxycinnamic acid (112) C12H14O5(238) 31.9 12 37.727
113 4-(3-Hydroxy-1-propenyl)-2-methoxyphenol (113) C10H12O3(180) 0.29 24 33.098
241
114 4-Phenyl-3-butenoic acid (114) C10H10O2(162) 0.03 16,17 28.765
115 3-Phenyl-2-propenyl methoxy acetate (115) C12H14O3(206) 0.06 16,17 28.805
116 Cyclohexyl isocyanate (116) C7H11ON(125) 0.08 12 7.173
117 1-Isothiocyanato-3-methyl butane (117) C6H11NS(129) 0.93 13 28.410
118 1-Hydroxy-4,4-dimethyl cyclohexane carbonitrile (118) C9H15ON(153) 0.12 14 12.974
119 1,3,5-Trimethyl-2-octadecyl cyclohexane (119) C27H54(378) - 14 44.478
120 2-Methyl-2-cyclopentenone (120) C6H8O(96) 0.04 21 24.897-
24.917
121 Methyl cyclopentenolone (121) C6H8O2(112) 0.13 16,17 19.321
0.03 - 19.455
122 1,2-Cyclohexadione (122) C6H8O2(112) 0.03 16,17 13.592
0.02 20 6.262-
6.299
123 1-Ethylcyclohexane carboxylic acid (123) C9H16O2(156) 0.05 16,17 27.757
124 5,5-Dimethyl-3-oxo-1-cyclohexene-1-carbaldehyde (124) C9H12O2(152) 0.02 12 11.074
125 1,3,4-Trimethyl-3-cyclo hexen-1-carboxaldehyde (125) C10H16O(152) 0.03 12 12.932
126 4-Hydroxy-3,5,5-trimethyl-4-[3-oxo-1-butenyl]-2-cyclohexen-
1-one (126)
C13H18O3(222) - 14 24.246
0.1 16,17 34.686
127 Methyl 3-(1,4,4-trimethyl-2-olcyclohexyl) propanoate (127) C13H24O3(228) 4.31 26 46.093
128 Cyclododeca-4,8-diene-1-one (128)
C12H18O(178) 0.03 21, 25 28.738-
28.762
129 4-Methoxyoctahydro-1(2H)-naphthalenone (129) C11H18O2(182) 0.26 10 25.262
130 3-Oxooctahydro-4-(2H)-naphthalene carboxylic acid (130) C11H16O3(196) 3.25 13 32.104
131 3-Hydroxymethylene-1,7,7-trimethyl bicyclo[2.2.1] heptan-2-
one (131)
C11H16O2(180) 0.8 13 29.659
132 Bicyclo[2.2.2]octane-1,4-diacetate (132) C12H18O4(226) 0.13 13 34.458
133 5-Acetyl-2,2-dimethyl-7-(2-oxopropyl) cycloheptanone (133) C14H22O3(238) - 10 23.629
134 Methyl 1-methyl cyclooctane carboxylate (134) C11H20O2(184) 0.03 16,17 26.207
135 2-(4-Nitrobutanoyl) cyclo octane (135) C12H19O4N
(241)
0.42 26 52.887
136 2-Carboxy-3-dimethyl-, dimethyl cyclopentane acetate (136) C12H20O4(228) 0.42 13 29.468
137 2,4-Diethyl-1-methylcyclohexane (137) C11H22(154) 0.03 16,17 31.163
138 1,3,5-Trimethyl-2-octadecyl cyclohexane (138) C27H54(378) 0.1 16,17 26.980
139 17-(1,5-Dimethylhexyl)-10,13-dimethyl-3-styrylhexadeca
hydrocyclo pentaphenanthren-2-one (139)
C35H52O(488) 0.10 26 53.985
140 1,2-Dimethyl-1-cyclooctene (140) C10H18(138) - 12 12.283
141 Isorhamnetin (141) C16H12O7(316) 1.06 25 53.408
242
142 Tert-butyl-(5-cyano-4,4,5-trimethyl-2-pyrrolidinylidene)-1,1-
dimethyl ethyl ester (142)
C14H22O2N2
(250)
1.45 09 40.945
143 7-Isobutyl-1H-indole-2,3-dione (143) C12H13NO2(203
)
- 10 20.508
144 6-Methoxy-4,5-pyrimidinediamine (144) C5H8ON4(140) 17.55 14 22.073
145 3-Methyl-4-propyl- 2,5-pyrrolidinedione (145) C8H13O2N (155) 0.63 14 14.675
146 4-Amino-1H-1,2,3-triazole-5-carboxamide (146) C3H5ON5(127) 0.13 11 25.385
147 N-Butyl benzene sulfonamide (147) C10H15NO2S
(213)
- 10 20.588
148 4-Hydrazino-6,6-dimethyl-2-(methyl sulfanyl)-5,8-dihydro-
6H-pyrano [4',3':4,5] furano [2,3] pyrimidine (148)
C12H16 N4O2 S
(280)
0.20 12 49.248
149 4,5-Dipropenyldihydro-furan-2-one (149) C10H14O2(166) 0.15 24 22.586-
22.630
150 Milbemycin b. 13-chloro-5-demethoxy-28-deoxy-6,28-epoxy-
5-(hydroxyl imino-25-(1-methyl ethyl) (150)
C33H46NO7Cl
(633)
- 12 3048.25
151 1,6-Dimethyl-10-methoxy ergoline-8β-methanol (151) C18H124N2O2
(300)
- 12 3018.75
152 6,6-Dimethyl-9-methylene-3-pentyl-6,7,8, 9,10,10-hexa hydro-
6H-benzo chromene-1,8-diol (152)
C21H30O3(330) - 12 3103.8
153 Tetrahydrocannabinol (153) C21H30O2(314) - 12 3106.95
154 4-Ethyl-4-methyl-2-pentadecyl-1,3-dioxolane (154) C21H42O2(326) 1.42 14 17.612
155 5-Hydroxy methyl furfurole (155) C6H6O3(126) 0.26 14 13.097
156 n- Nonenyl succinic anhydride (156) C13H20O3(224) 2.44 14 21.225
157 4,5-Dihydro-2-thiazolamine (157) C3H6N2S(102) 0.03 16,17 21.357
158 4-Hydroxyundecanoic acid lactone (158) C11H20O2(184) 0.04 20 10.994
159 Trans-Linalool oxide (159) C10H18O2(170) 1.96 26 17.464
160 Cis-Linalool oxide (160) C10H18O2(170) 0.95 26 18.006
161 n-Dodecenyl succinic anhydride (161) C16H24O3(264) 0.99 13 33.798
162 Dodecenyl succinic anhydride (162) C16H26O3(266) 0.5 26 33.090
163 3-(16-Methtylheptadecyl) dihydro-2,5-furandione (163) C22H40O3352 0.10 25 56.506
164 2,3-Dihydro benzofuran (164) C8H8O(120) 0.81 26 22.122
165 3-Benzylidene-5-phenyl-2(3H)-furanone (165) C17H12O2(248) 2.68 9 51.053
166 2-Heptyl-5-imino-6-thiophen-2-ylmethylene-5,6-dihydro-
[1,3,4]thiadiazolo [3,2]pyrimidin-7-one (166)
C17H20ON4S2
(363)
0.09 16,17 54.555
167 2-(4-Methylphenyl)-6-methyl-7,7-tetramethylene-6,7-dihydro-
1H-pyrrolo[3,4]pyridine-4,5-dione (167)
C19H20O3N2
(324)
0.09 16,17 57.633
168 1-(6-Methyl-2-piperidinyl) acetone (168) C9H17NO(155) 0.34 20 14.121
243
169 5-Methyl-2(5H)-thiophenone (169) C5H6OS (114) 0.23 20 15.036
170
2-Acetyl-2,3,5,6-tetrahydro-1,4-thiazine (170) C6H11ONS
(145)
0.03 16,17 21.347-
21.401
171
8,8-Dimethoxy-1,4-dioxaspiro[4,5]deca-6,9-diene (171) C10H14O4 (198) 0.25 16,17 34.281-
34.338
172 Opuntiol (172) C7H8O4 (156) 87.45 16,17 33.484
90.17 20 19.409-
21.941
0.29 21 27.713
95.61 21 34.314
14.47 25 31.090
32.82 24 31.012
173 Methyl triacetolactone (173) C7H8O3(140) 0.75 21 19.271
174 3,5-Dihydroxy-6-methyl-2,3-dihydro-4H-pyran-4-one (174) C6H8O4(144) 0.37 26 20.153
175 2-Diethyl amino methyl-3-hydroxy-6-hydroxy methyl pyran-4-
one (175)
C11H17O4N
(227)
0.33 20 26.965-
27.092
176 2,6,6-Trimethyl-2-cyclohexene-1,4-dione (176) C9H12O2(152) 0.2 14 11.091
177 2-Hydroxy-3,5,5-trimethyl-2-cyclohexen-1-one (177) C9H14O2(154) 6.37 25 35.269
178 3-Ethoxy-2-cyclohexenone (178) C8H12O2(140) 0.43 14 16.453
179 7-Methoxy-2,2-dimethyl chromene (179) C12H14O2(190) 7.4 25 27.634
180 6,7-Dimethoxy-2,2-dimethyl-2H-chromene (180) C13H16O3(220) 2.83 25 31.502
181 2-Phenyl-1,3-dioxan-5-yl palmitate (181) C26H42O4(418) 3.1 25 51.271
182 Methyl 4-(7-Allyl-7-hydroxy-2,2-dimethyl tetra hydro [1,3]
dioxolo[4,5]pyran-4-yl)-3-methylbut-2-enoate (182)
C17H26O6(326) 2.84 25 59.698
183 4,5,6-Trimethyl-2-pyrimidinethiol (183) C7H10N2S (154) 1.53 26 25.159-
25.186
184 Crotonaldehyde isonicotinic acid hydrazone (184) C10H11ON3(189
)
1.00 26 27.293
185 Benzyl N-(1-hydroxy-4-oxo-1-phenylper hydroquinolizin-3-yl)
carbamate (185)
C23H26O4N2
(394)
0.2 26 54.076
186 Ethyl 2-(2-methyl nona decyl) acrylate (186) - 0.87 26 33.659
187 5-[1-(Hydroxymethyl) vinyl]-2-methyl cyclohexanol (187) C10H18O2(170) 0.15 14 11.554
188 2-Ethyl-3-methoxy-2-cyclo pentenone (188) C8H12O2(140) 0.43 14 13.744
189 2-Ethyl-cyclohexanethiol, acetate (189) C10H18OS(186) 0.51 14 14.303
190 1-(4-Methylphenyl) anthrax-9,10-quinone (190) C21H14O2(298) - 14 54.322
191 1-Ethyl-1-tetradecyloxy-1-silacyclopentane (191) C20H42OSi(326) 0.93 13 58.766
192 Bencarbate (192) C11H13O4N 4.8 14 21.872
244
(223)
193 2-(4-Diethyl amino phenyl imino methyl)-6- methoxy phenol
(193)
C18H22O2N2
(298)
12.62 15 57.590
194
p- Octyloxybenzonitrile (194) C15H21NO(231) 0.08 10 9.168
195
15-Hydroxy-4-methylpicrasa-1,13-diene-3, 16-dione (195) C21H28O4(344) 0.46 26 59.827
196 3-Methyl-5-phenyl-1,2,4-oxadiazole (196) C9H8N2O(163) - 12 14.302
197 Decane (197) C10H22(142) 0.15 10 7.892
198 Undecane (198) C11H24 (156) 0.24 10 13.431
199 Dodecane (199) C12H26(170) 0.22 10 17.218
200 Tridecane (200)
C13H28(184) 0.22 10 20.148
0.01 21 23.855-
23.872
201 Tetradecane (201) C14H30(198) 0.3 10 22.637
0.03 21 26.163-
26.190
202 Pentadecane (202) C15H32(212) 0.05 10 18.442
203 Hexadecane (203) C16H34(226) 0.24 10 26.883
0.05 21 30.282
4.44 24 41.363
204 Hexatriacontane (204) C36H74(506) 0.06 21 28.286
205 2,6-Dimethyl undecane (205) C13H28(184) 0.02 10 17.667
206 3,4-Dimethyl undecane (206) C13H28(184) 0.04 10 15.662
207 1-Chloro tetradecane (207)
C14H29Cl(232) 0.02 12 13.053
0.19 14 15.438
208 1,4-Dimethyl-3-n-octadecylcyclohexane (208) C26H52(364) 1.72 13 53.859
209 1,3,5-Trimethyl-4-octadecylcyclohexane (209) C27H54(378) 1.4 15 51.049
210 Nonanoic acid (210) C9H18O2(158) 0.03 10 19.515
211 Undecanoic acid (211) C11H22O2(186) 3.5 26 32.511
212 Dodecanoic acid (212) C12H24O2(200) - 10 19.634
1.52 11 29.578
0.83 09 19.718
213 Tetradecanoic acid (213) C14H28O2(228) 1.59 09 23.422
0.73 10 23.505
1.84 11 33.513
214 Pentadecanoic acid (214) C15H30O2(242) 5.79 09 26.215
245
0.29 10 32.131
1.43 10 33.065
215 Palmitic acid (215) C16H32O2(256) 40.0 09 30.275
- 10 31.707
15.22 11 40.278
5.07 13 40.272
- 14 29.910
1.97 20 29.956
216 Stearic acid (216)
C18H36O2(284) 1.57 09 43.585
10 44.036
217 Oleic acid (217)
C18H34O2(282) 2.0 09 42.124
0.31 13 29.632
218
Decenoic acid (218) C10H18O2(170) - - -
219 11-Hexadecenoic acid (219) C16H30O2(254) - 10 18.187
220 9,12-Octadecadienoic acid (220) C18H32O2(280) - 10 42.766
3.47 11 49.494
221 13-Octadecanoic acid (221) C18H34O2(282) 2.34 20 38.680
222 Myristoleic acid (222) C14H26O2(226) 3.33 13 31.246
223 2,2-Diethyl-3-methyl-3-butenoic acid (223) C9H16O2(156) 0.61 21 26.635
224 2,4-Dimethyl hexanedioic acid (224)
C8H14O4(174) 0.49 13 28.537
0.85 15 28.115-
28.145
225 Agaricic acid (225) C22H40O7(416) - 12 -
226 Methyl 10-ketostearate (226) C19H36O(312) 1.95 13 55.192
227 Ethyl 4-oxo-2-hexenoate (227) C8H12O3(156) 6.42 12 14.179
228 Fumaric acid, cyclo butyl ethyl ester (228) C10H14O4(198) 0.79 11 29.823
229 3,4-Anhydro-1,5-dideoxy-4-(4-methyl-3-pentenyl) pent-2-
ulose (229)
C11H18O2(182) 0.10 16,17 30.172
230 Methyl 3-hydroxy undecanoate (230) C12H24O3(216) - 10 16.225
231 Dimethyl 2-hydroxy-2-methyl succinate (231) C7H12O5(176) 2.84 26 24.654-
24.704
232 Methyl 7-hydroxystearate (232) C19H38O3(314) 12 51.661
233 Bis(1-methyl propyl) octanedioate (233) C16H30O4(286) 5.19 13 33.313
234 Methyl 10-hydroxy-11-dodecanoate (234) C13H24O3(228) 0.37 13 28.932
235 8-t-Butyl-8-hydroxy-2,2-dimethyl-dodeca-5,11-dien-3-one
(235)
C18H32O2(280) 2.12 24 42.458-
42.492
246
236 Isopropyl 5-hydroxy-3,7,11-trimethyl-2,10-dodecadienoate
(236)
C18H32O3(296) 0.10 25 56.422
237 Butyl fumarate (237)
C12H20O4(228) - 10 19.861
0.48 11 29.927-
29.947
0.75 13 29.933
2.12 15 29.930
238 Di isobutyl fumarate (238) C12H20O4(228) 6.08 14 19.842
239 Dibutyl malate (239) C12H22O5(246) 31.35 09 20.985
240
Adipic acid, decyl methyl ester (240) C17H32O4(300) 0.3 10 22.454
241 Adipic acid, heptyl methyl ester (241) C14H26O4(258) 1.06 11 32.709-
32.730
242 Dibutyl nonanedioate (242) C17H32O4(300) 3.23 12 25.676
243 Diethyl glutarate (243) C9H16O4(188) 0.40 13 28.815
244 Di(sec-butyl)-2-methylsuccinate (244) C13H24O4(244) 0.7 14 16.724
245 7-(Acetyloxy) heptyl acetate (245) C11H20O4(216) 0.26 24 24.880-
24.927
246 Di butyl succinate (246) C12H22O4(230) 17.95 15 25.399-
25.513
4.11 16,17 25.764
247 4-Pentadecyl acetoxyacetate (247) C19H36O4(328) 0.05 20 14.620
248 Di(sec-butyl) hexanedioate (248) C14H26O4(258) 1.72 15 29.384
249 Ethyl tetradec-3-enyl fumarate (249) C20H34O4(338) 0.16 21 51.983
250 Ethyl 4-methyl succinate (250) C7H12O4(163) 0.87 15 25.935
251 2-Butyl heptadecyl fumarate (251) C25H46O4(410) 0.38 24 56.501
252 Isobutyl tridecyl fumarate (252) C21H38O4(354) 1.00 24 56.414
253 2-(Acetyloxy)-1-[(octadecyloxy) methyl] ethyl acetate (253) C25H48O5(428) 1.08 25 55.119
254 Pentadecyl isovalerate (254) C20H40O2(312) 0.41 26 29.935
255 Methyl 8-octadecynoate (255) C19H34O2(294) 1.32 26 30.303
256 Methyl 2,5-octadecadiynoate (256) C19H30O2(290) 0.41 26 26.275
257 8-Dodecenyl acetate (257) C14H26O2(226) - 10 20.431
258 Methyl pentadecanoate (258) C16H32O2(256) 2.72 12 24.350
259 2-Hexenyl propanoate (259) C9H16O2(156) 0.23 12 15.407
260 Nonyl 2-ethyl hexanoate (260) C17H34O2(270) - 10 20.930
261 Allyl 2-ethyl butyrate (261) C9H16O2(156) 0.12 12 16.466
262 4-Tetradecyl hexanoate (262) C20H40O2(312) 2.7 12 22.073
247
263 Isopropyl palmitate (263) C19H38O2(298) 0.31 10 28.129
264 Tridec-2-ynyl octanoate (264) C21H38O2(322) 0.19 11 31.032-
31.045
265 Ethyl dodecanoate (265) C12H24O2(200) 4.41 13 29.468
266 Oleyl arachidate (266) C38H74O2(562) 12 -
267 Methyl 8-methyl-decanoate (267) C12H24O2(200) 0.55 13 28.534
268 Ethyl 5,9-dimethyl-2,4-decadienoate (268) C14H24O2(224) 6.97 15 33.946
269 Methyl 3,7,11,15-tetramethyl hexadecanoate (269) C21H42O2(326) 4.0 14 20.056
270 i- Propyl 9-hexadecenoate (270) C19H36O2(296) - 14 49.519
271 2-Isopropenyl 2-methyl-butanedioic acid (271) C8H12O4(172) 4.81 15 34.153-
34.183
272 3-Nonen-2-one (272) C9H16O(140) 9.8 15 28.383
273 Methyl-9-oxodecanoate (273) C11H20O3(200) 0.74 15 29.632
274 Ethyl 2-(2-methylnonadecyl) acrylate (274) C25H48O2(380) 0.87 26 33.659
275 Butyl undec-2-en-1-yl fumarate (275) C19H32O4(324) 1.12 26 43.667
276 Methyl 13-tetradecynoate (276) C15H26O2(238) 1.94 15 30.111
277 2,6-Dimethyl tridecane nitrile (277) C15H29N(223) 0.21 26 28.813
278 i-Propyl-16-methyl heptadecanoate (278) C21H42O2(326) 1.32 15 56.756
279 Octyl acetate (279) C10H20O2(172) 0.05 20 8.984-
9.048
280 Octyl pentanoate (280) C13H26O2(214) 0.29 24 19.582-
19.612
281 Vinyl decanoate (281) C12H22O2(198) 0.17 20 17.574
282 Hexadecyl isovalerate (282) C21H42O2(326) 0.44 20 18.338
283 Pentadecyl butyrate (283) C19H38O2(298) 0.67 24 27.516-
27.566
284 Butyl-9-tetradecenoate (284) C18H34O2 (282) 0.57 24 54.762
285 Tridec-2-ynyl octanoate (285) C21H38O2(322) 3.1 24 41.708
286 11,13-Dimethyl-12-tetradecen-1-ol acetate (286) C18H34O2(282) 3.38 25 38.354
287 Mono methyl succinate (287) C5H8O4(132) 1.12 25 19.091
288 2-Hexadecanoyl glycerol (288) C19H38O4(330) - 12 3022.04
289 Tridecyl 2-methyl propanoate (289) C17H34O2(270) 0.02 26 28.006
290 D-(+)-Arabitol, pentamethyl ether (290) C10H22O5 (222) 0.3 16,17 36.548
291 Ethyl 2-oxo tetradecanoate (291) C16H30O3(270) 2.31 12 20.880
292 Dodecyl hexanoate (292) C18H36O2(284) 0.04 12 9.259
293 Methyl 10-oxostearate (293) C19H36O3(312) 0.06 12 51.076
294 Methyl 7-hydroxystearate (294) C19H38O3(314) 1.00 24 51.966
248
295 4-Undecenal (295) C11H20O (168) 0.07 10 21.267
296 2-Isopropyl-4-methyl-2-hexenal (296) C10H18O (154) 0.02 16,17 25.088
297
Trichloromethane (297) CHCl3(118) 0.13 11 3.586-
3.639
298 2,2,6,6-Tetramethyl-3,5-heptandione (298) C11H20O2 (184) 0.42 11 23.283
299 2-Butyl-2-ethyl-5-methyl-3,4-hexadienal (299) C13H22O (194) 7.52 11 30.496
300 N-(2,2-Diphenylethyl)-2,2-dimethylpropionamide (300) C19H23ON (281) 1.06 11 50.291
301 7,9-Dimethyl-8-nitro bicycle [4.3.1]decan-10-one (301) C12H19 N O3
(225)
0.33 12 18.486
302 1-Phenylethenyl dimethyl ester phosphoric acid (302) C10H13O4P(228) 2.66 12 44.231
303 4-Hydroxy-3-methoxy styrene (303)
C9H10O2 (150) 0.03 12 14.938
3.96 25 24.255
304 3-(1-methyl-3-phenylpropoxy) butanoic acid (304) C14H20O3 (236) 0.02 12 9.701
305 2-Phenylpropenal (305) C9H8O (132) 0.03 12 11.409
306
4-Hydroxy-3,5-dimethoxybenzohydrazide (306) C9H12N2O4
(212)
2.47 12 23.586
307 4-Ethyl-2,5-dimethoxyphenethylamine (307) C12H19
NO2(209)
0.20 16,17 29.686
308
4-Nitrophenyl octanoate (308) C14H19O4N
(265)
0.14 14 14.082
309 Methyl 2-(acetyl amino)-3-methyl butanoate (309) C8H15NO3(173) 0.22 12 18.553
310
N-Acetyl-nor-leucine (310) C8H15 NO3
(173)
1.21 13 29.880
311 Methyl 4-methyl-2-[(3-methyl butanoyl) amino] pentanoate
(311)
C12H23NO3
(229)
1.12 12 19.845
312 1-Phenylalanine,N-propoxycarbonyl-, butyl ester (312) C9H12N2O4
(307)
2.05 12 29.675
313 N-Butyl benzene sulfonamide (313) C10H15NO2 S
(213)
6.87 09 24.125
314
1-Dimethylamino 2,3-propanediol (314) C15H13O2N
(119)
0.12 15 -
- 16,17 19.063
315 1-Phenylalanine, N-isobutoxy carbonyl-,butyl ester (315) C18H27 O4(321) 3.71 13 39.920-
39.974
316 Vitamin p (316) C27H30O16(610) - 14 56.673
317 Vitamin A palmitate (317) C36H63O2 (524) 0.04 25 56.428
318 α-D-Mannothio furanoside, S-decyl- (318) C16H32O5S(336) 0.44 20 18.318
319 Methyl (3-oxo-2-[2-pentyl] cyclopentyl) acetate (319) C13H20O3(224) 0.55 21 41.672
249
320 2-Hydroxy-1-methyl cyclopenten-3-one (320) C6H8O2 (112) 0.07 21 13.632
321 p-Cumylphenol (321) C15H16O(212) 3.52 11 31.099
322 Di ethylene glycol (322) C4H10O3 (106) 5.56 25 13.214
6.17 26 15.441
323 2,4-Dihydroxy diphenyl dimethyl methane (323) C15H16O2(228) 0.73 26 50.794
324 4-Phenyl propiophenone (324) C15H14O(210) 1.09 12 25.006
325 Methyl bis(3,5-dimethoxyphenyl) hydroxyacetate (325) C19H22O7(362) 0.68 09 26.383
326 a)Syringol (2,6-dimethoxy phenol) (326)
b)2,2-Di(4-hydroxyphenyl) propane (327)
C8H10O3(154)
C15H16O2(228)
0.13 21 25.121
327 Nonenyl succinic anhydride (328) C13H20O3 (224) 1.89 13 31.437
2.82 15 33.004
328
4-Hydroxy-10-methyl-3,4,7,8,9,10-hexahydro-2H-oxecin-2-
one (329)
C10H16O3 (184) 0.23 24 53.393
329 4-Isopropenyl-1-methyl-1-cyclohexene (330) C10H16 (136) 0.19 25 15.688
330 1,6-Dihydrocarveol (331) C10H18O (154) 0.08 12 8.064
331
5-Isopropenyl-2-methyl cyclohexanol (332) C10H18O (154) - 10 8.063
332 4-Isopropyl-1-methyl-2-cyclohexenol (333) C10H18O (154) 0.19 12 17.394
333 2-Hydroxymethylene-6-isopropyl-3-methyl cyclohexanone
(334)
C11H18O2 (182) 5.03 13 32.291-
32.335
334 Eucalyptol (335) C10H18O (154) 1.56 15 15.748
335
Trans-α-lonone (336) C13H20O (192) 0.6 09 22.638
336 2-Hydroxy-2,4,4-trimethyl-3-(3-methylbuta-1,3-dienyl)
cyclohexanone (337)
C14H22O2 (222) 0.33 11 27.884
1.62 12 21.487
337
4-(2,2,6-Trimethyl-1-cyclohexen-1-yl) butanoic acid (338) C13H22O2 (210) - 14 23.563
338 1-(2,6,6-trimethyl-1cyclohexen-1-yl)-1-penten-3-one (339) C14H22O (206) 0.15 24 26.240
339 3-Methyl-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one
(340)
C14H22O (206) 4.63 13 36.963
340 2-(2,2,6-Trimethyl-7-oxa bicycle [4.1.0] hept-1-yl)-1-propenyl
acetate (341)
C14H22O3 (238) - 14 23.737
341
2,6,10,10-Tetramethyl-1-oxaspiro[4,5] decan-6-ol (342) C13H24O2(212) - 14 23.895
342 1-[2-(2,2,6-Trimethyl bicycle [4.1.0]hept-1-yl)ethyl]vinyl
acetate (343)
C16H26O2(250) 0.63 26 31.506
250
343 Methyl 2-[7-hydroxy-3-methyl-1,3-octadienyl]1,3-dimethyl-4-
oxo-2-cyclohexene-1-carboxylate (344)
C19H28O4(320) 0.10 26 53.721
344 Caryophyllene (345) C15H24(204) 2.15 25 26.730
345 (6,8)-Bis-hydroxymethyl-4-isopropyl-7-methylene-bicyclo
[3,2,1]oct-1-yl) methanol (346)
C15H26O3(254) - 14 28.372
346
3-Isobutyl hexahydro pyrrolo [1,2] pyrazine-1,4-dione (347) C11H18O2 N2
(210)
3.90 24 34.997
347 1,2-Dihydrothujopsene-(l1) (348) C15H26(206) 0.08 09 19.427
348 1-Isopropyl-4,7-dimethyl-1,2,4,5,6,8-hexahydro naphthalene
(349)
C15H24 (204) 0.47 25 28.052
349 7-Isopropyl-4-methyl octahydro-2(1H)-napthalenone (350) C14H24O(208) 2.84 11 34.535
350 3-Isopropyl-6,10-dimethyl-6-cyclodecene-1,4-dione (351) C15H24O2 (236) 0.12 26 50.378
351
2-Isopropyl-5,9-dimethyl-1,4-cyclodecane dione (352) C15H26O2 (238) 4.67 11 38.014
10.5 12 27.954
352 9-Isopropyl-2,6-dimethyl cyclo decanone (353) C15H28O(224) 0.25 26 31.616
353 Cyclopentadecanone (354) C15H28O (224) 1.79 13 31.065
354 3-Methyl-2-cyclopentenone (355) C6H8O (96) - 12 15.481
355 Bisabolol oxide A (356) C15H26O2 (238) 0.33 09 19.283
356 Limonene (357) C10H16 (136) 3.86 11 15.678
357 5-Methyl-2-isopropyl cyclohexanone (358) C10H18O (154) - 12 15.685
358 2-Isopropyl-5-methyl cyclohexenone (359) C10H18O (154) 0.01 16,17 25.108
359 Neo dihydrocarveol (360) C10H18O (154) 0.1 12 13.191
360 2,2,4-Trimethyl-3-(3-oxobutyl)-2-cyclohexen-1-one (361) C13H20O2 (208) 2.29 12 20.405
361 4-(3-hydroxy-6,6-dimethyl-2-methylenecyclo hexyl)-3-buten-
2- one (362)
C13H20O2 (208) 1.06 12 20.485
362 Menthone (363) C10H18O (154) 0.13 12 15.685
363 D-Isomenthone (364) C10H18O (154) 0.13 12 15.685
364 2,2,6,6-Tetramethyl-3,5-heptanedione (365) C11H20O2 (184) 0.83 13 23.276
365 4-(1,5-Dihydroxy-2,6,6-trimethylcyclohex-2-enyl)but-3-en-2-
one (366)
C13H20O3 (224) 3.38 13 30.911
366 4-Isopropyl-1-methyl-2-cyclo hexenol (367) C10H18O (154) 1.67 14 17.394
367 7-Methyl-oxa-cyclododeca-6,10-dien-2-one (368) C12H18O2 (194) 8.17 15 32.305
368 4-(2,2,6-Trimethyl-1-cyclohexen-1-yl)-butyric acid (369) C13H22O2(210) 4.87 15 33.785-
33.815
369 4-Isopropyl-1-methyl-1,2,3-cyclohexanetriol (370) C10H20O3 (188) 0.16 20 17.340
370 2,2-Dimethyl-3-[3-methyl-5-(phenyl thio)pent-3-enyl] oxirane
(371)
C16H22OS(262) 1.1 26 53.074
251
371 Bicyclo[4,3.0]nonane, 1-isopropenyl-4,5-dimethyl-5-phenyl
sulfonyl methyl (372)
C21H30O2 S
(346)
- 14 63.140
372 Methyl 2-methyl-2-thiophene carboxylate (373) C7H8SO2 (156) 2.81 26 31.332
373 Methyl 3-methyl-2-thiophenecarboxylate (374) C7H8O2S (156) 0.21 21 30.858
374 2,2-Bis(5-formylthien-2-yl)- butane (375) C14H14O2 S2
(278)
- 14 52.285
375 Di methyl sulfoxide (376) C2H6OS (78) 5.61 25 5.562
376 2-Methyl-5-propionyl thiophene (377) C9H10OS (154) 0.21 21 26.953
377 Gibberillic acid (378) C19H22O6 (346) 1.05 11 54.571
378 4,4-Dimethyl androsta-5,7-diene (379) C21H32 (284) - 12 -
379 17-Acetoxy-3-methoxy-4,4,7-trimethyl-3, 19:7,8-diepoxy
androstane (380)
C25H38O5(418) 5.99 24 65.218
380 β-Sitosterol (381) C29H50O2(414) 1.39 09 64.557
381 3-Isopropyl-6,10-dimethyl-8-(2-oxo-2-phenyl-ethyl)-
dodecahydro-benzo chromen-7-one (382)
C26H36O3(396) 0.28 10 51.039
382 5α-Pregnan-20-one,5,6α:16α,17-diepoxy -3β-hydroxy acetate
(383)
C23H32O5(388) 0.59 10 52.717
383 4,6,8(14)-Cholestatriene (384) C27H42(366) 0.16 10 54.780
384 13-Ethyl-3-hydroxy- gona-1,3,5,7,9-pentaen-17-one (385) C19H20O2(280) 10.73 11 53.948
385 3,9-Epoxypregnane-11β-20-diol,3α-methoxy-18[N-methyl-N-
(2',14-epoxy ethyl)amino] (386)
C25H41 NO5
(435)
1.00 12 19.429
386 4-Chlorotestosterone acetate (387) C21H29O3Cl
(364)
- 12 3046.72
387 3-O-acetyl-20-[N-{3-cyano-2-methyl propyl} carbamoyl]-
allopregane-3-ol (388)
C29H46N2O3
(470)
- 12 3066.78
388 Androstane-3,11,17-triol (389) C19H32O3(308) - 14 46.116
389 3-Acetoxy-17-acetyl-5,7,9(10)-estratriene (390) C22H28O3 (340) 0.3 16,17 55.928
390 Stigmastan-3,5-diene (391) C29H48 (396) 1.11 20 52.633
391 Methyl-3,7-diketo-5α-cholanoate (392) C25H38O4 (402) 0.59 24 66.890
392 4-Norlanosta-17(20),24-diene-11,16-diol-21-oic acid,3-oxo-
16,21-lactone (393)
C29H42O4(454)
(418)
0.68 24 67.476-
67.499
393 9,19-Cyclocholestene-3,7-diol 4,14-dimethyl-3-acetate (394) C31H52O3 (472) 0.81 24 59.391
394 Ethyl iso-allocholate (395) C26H44O5 (436) 1.59 24 59.548-
59.639
395 3-Deoxy-D-3,17β-estradiol (396) C18H24O (256) 0.14 26 54.967
396 Pregna-1,4-diene-3,20-dione,11β,17,21-trihydrox y-,21-acetate
(397)
C23H30O6 (402) 0.54 25 66.888
252
397 5α-Androstan-17β-ol (398) C19H32O (276) 0.26 16,17 53.701
398 4-Butoxyphenylacetic acid (399) C12H16O3(208) 1.21 09 22.893
399 Methyl salicylate (400) C8H8O3(152) 0.15 24 21.166
400 3-Phenylpentanedioic acid (401) C11H12O4(208) 3.01 12 32.455
401 3-[4-(Acetyloxy)-3-methoxy phenyl], methyl-2-propenoate
(402)
C13H14O5(250) 1.47 13 66.590-
66.667
402 1-n-Dodecyloxy-1-methyl-1-silicacyclo hexane (403) C18H38OSi
(298)
0.47 26 57.723
403 Unidentified (288) - 10 24.359
404 Unidentified (328) 4.19 11 56.286
405 Unidentified (340) - 12 3022.04
406 Unidentified (152) 8.43 14 15.907
407 Unidentified (503) 0.05 21 58.704
408 Unidentified (503) 0.04 21 56.028
409 Unidentified (503) 0.09 21 57.304
410 Unidentified (576) 0.05 21 52.666-
52.689
411 Unidentified (503) 0.03 21 54.571
412 Unidentified (418) 6.12 24 65.932
413 Unidentified (284) 0.19 25 27.466
414 Unidentified (502) 2.27 25 41.713
415 Unidentified (389) 1.21 25 57.557
253
Outlook
254
1 The present studies carried out on O. dillenii Linn. Keeping in mind the remarkable
properties of plants, which have been shown in the following fact.
2 O. dillenii is a rich source of medicinally important photochemicals like opuntiol and
its glucoside, opuntioside, which were isolated previously from this plant. Both
compounds have been reported as anti-inflammatory, hypotensive and analgesic
agents.
3 The cladodes of O. dillenii were extracted with methanol to obtained methanol
extract. This extract was treated with ethyl acetate and water to give ethyl acetate and
aqueous phases. The aqueous phase was treated with butanol to give butanol and
aqueous phases. Chemical study on this butanol phase has been carried out after the
purification and isolation of chemical compounds through vacuum liquid
chromatography (VLC).
4 The vaccum liquid chromatography (VLC) fractions were subjected to GC and GC-
MS studies which led to the detection of 409 chemical compounds, which belongs to
different class of cmpounds.
5 Two known compounds opuntiol (1) and opuntioside (2), have been isolated from
the current working. Structures of these pure compounds have been determined
through TLC chromatogram and EI-MS and 1H-NMR studies.
6 The chemical investigation has proved that O. dillenii comprising important class of
compounds, which possess valuable medicinal importance. There is a need to be
further work on this plant for new and novel medicinally important chemical
components and will be submitted for biological studies.
7 In order to isolate the pure bioactive chemical constituents of the plant, several
chromatographic techniques such as high performance liquid chromatography
(HPLC) vaccum liquid chromatography (VLC), and flash column chromatography
(FCC) will be used.
255
Experimental
256
Materials and methods
General methods
EI-MS and HREI-MS spectra were run on MAT 312 (Finnigan, Germany), and JMS HX110
spectrometers (Jeol, Japan). 1H-NMR spectra were recorded on a Bruker AV-300 and AV-
400 devices functioning at 300 and 400 MHz. The chemical shifts (δ) and coupling constant
(J) were recorded in ppm and Hz. For vacuum liquid chromatography (VLC) silica gel 60
GF254 (Merck) was used. Chemicals and solvents utilized were of analytical grade.
GC and GC-MS studies
The non polar extracts were examined by GC-FID and GC-MS analysis. GC-FID spectra were
run on a 17-A Shimadzu [FID mode; column, fused silica capillary column OV-1, DB-1 (30
m x 0.53 mm, 0.5mm film thickness) carrier gas N2], at 75 oC and programmed to 75 oC at
240 oC min-1 and 3-5 min hold. Injector and detector were at 240 and 250 oC respectively. 2
mL of each sample were triplicate injected and quantities represented as relative area % as
derived from integrator. GC-MS system was functioned on a JSM 630H (Jeol, Japan) with
Agilent 6890N USA [EI mode; ionization potential, 70ev; column DS-6 (0.32 mm x 30 m);
column temperature 50-250 oC (temperature rate increases 5 oC/min); carrier gas, He; flow
rate, 1.8 mL/min; split ratio, 1:30]. Sample injection was carried out with a split ratio of 35:1
at a temperature of 25 oC.
The recognition and verification of chemical compounds were completed by assessment of
their GC-MS spectra, with Mass library search, Agilent Mass Hunter Qualitative Analysis
(version B.04.00) database.99
Plant material
O. dillenii cladodes were collected during 2011 from HEJRIC (ICCBS) gardens, University
of Karachi. Plant taxonomist Dr. Rubina Dawar was recognized this plant, she affiliated with
Botany department, University of Karachi and the voucher specimen (KUH. G.H. 67280) was
placed in the herbarium of the similar department.
Extraction and isolation of pure compounds from O. dillenii
257
Plant cladodes 45 kg were extracted thrice with methanol at room temperature. The extracts
were filtered and mixed collectively followed by evaporation under vacuum, affording a thick
gummy residue OM (1071 g), of which 871 g was treated with ethyl acetate and water and the
two layers were separated into ethyl acetate and aqueous phases. The aqueous phase was
treated six times with butanol to give aqueous phase, the later phase was evaporated under
vacuum affording a gummy residue OM-But (18 g) (Scheme 1). The TLC of OM-But showed
a number of spots, alongwith the spots of two known compounds namely opuntiol (1) and
opuntioside (2) as major chemical constituents.
Butanol phase obtained from O. dillenii possessed antidepressant potential.84 Therefore
further fractionation of butanol phase was performed by subjecting it to VLC (Silica gel; 60
GF254) and eluated with petroleum ether (PE), ethyl acetate (EtOAc), methanol (MeOH), with
increasing polarity, yielding 45 fractions.
Out of 45 fractions, thirteen fractions encoded (09, (eluted with PE:EtOAc: 130:70), 10
(PE:EtOAc: 120:80), 11(PE:EtOAc: 110:90), 12 (PE:EtOAc: 100:100), 13 (PE:EtOAc:
90:110), 14 (PE:EtOAc: 80:120), 15 (PE:EtOAc: 70:130), 16-17 (PE:EtOAc: 63:140), 20,
(PE:EtOAc: 10:190), 21 (EtOAc: 100%), 24 (EtOAc: MeOH: 180:20), 25 (EtOAc: MeOH:
170:30), 26 (EtOAc: MeOH: 163:40)), were subjected to GC-FID and GC-MS studies for the
recognition of chemical constituents, which were present in the above mentioned fractions.
Isolation of opuntiol (172):
On evaporation at room temperature of VLC fractions 16-22 (P.E:EtOAc (50:150) to
EtOAc:MeOH (180:20)), white needle like crystals was deposited. These crystals were
washed with P.E, CHCl3 and EtOAc, after washing these crystals demonstrated a single spot
on TLC chromatogram (Rf = 0.42, CHCl3:MeOH (9.5:0.5), silica gel 60 GF254). The spectral
studies, EI-MS and 1H NMR revealed its structure as a known compound opuntiol (172).77a,b
Isolation of opuntioside (172a):
Fraction No: 27-33 eluting with EtOAc:MeOH (150:50) to EtOAc:MeOH (190:10), showed
a single spot on TLC (Rf = 0.43, CHCl3:MeOH (8:2), silica gel 60 GF254), the spectral studies
EI-MS, 1H-NMR of this pure fraction revealed its structure as a known compound opuntioside
(172a).77a,b
258
Opuntia dillenni cladodes,fresh,undried
45kg, MeOH´ 3
Continue on next page....
Extract Marc
Residue
(OM,1071 gm)
Evaporated
871gm
E.Aphase Aq.phase
EtOAc
phase
Aqueous
washing
Aqueous phase
(OM-Aq)
Butanol phase
E.A:H2O
(1:1 )
+ H20Butanol x 5
Aqueous
washingButanol phase
Residue
(OM-But, 18 gm)
VLC, (silica gel,
PE, EtOAc, MeOH
1A-8
PE
100%
to
PE : EtOAc
40:60
+ H20
Evaporated
09
PE : EtOAc
130 :70
10
PE : EtOAc
120 :80
11
PE : EtOAc
110 :90
12
PE : EtOAc
1 :1
13
PE : EtOAc
90 :110
Scheme 1: Vacuum liquid chromatography on butanol phase of O. dillenii cladodes
259
15
PE : EtOAc
60 :140
16
PE : EtOAc
50 :150
17
PE : EtOAc
40 :160
18
PE : EtOAc
30 :170
19
PE : EtOAc
20 :180
20
PE : EtOAc
10 :190
21-22
EtOAc (100%)
23
EtOAc: MeOH
190: 10
24
EtOAc: MeOH
180: 20
25
EtOAc: MeOH
170: 30
26
EtOAc: MeOH
160: 40
28
EtOAc: MeOH
140 : 60
29
EtOAc: MeOH
130 : 70
30
EtOAc: MeOH
120 : 80
Opuntiol,
needle like crystals
27
EtOAc: MeOH
150: 50
14
PE : EtOAc
70 :130
31
EtOAc : MeOH
110 : 90
32-41
EtOAc : MeOH
1 : 1
to
EtOAc : MeOH
10 : 190
434445
Acetone : H20
1:1
MeOH : Acetone
150 : 50
MeOH : Acetone
1 : 1
42
MeOH
260
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List of publications
282
1.Effect of cream containing Melia azedarach flowers on skin diseases in children, R. Saleem,
R. Rani, M. Ahmed, F. Sadaf, S. I. Ahmad, N. ul Zafar, S. S. Khan, B. S. Siddiqui, Lubna,
F. Ansari, S. A. khan, S. Faizi, Phytomedicine, 15(4), 2008, 231-236.
2. Antibacterial and antifungal activities of different parts of Tagetes patula: preparation of
patuletin derivatives, S. Faizi, H. Siddiqui, S. Bano, A. Naz, Lubna, K. Mazhar, S. Nasim,
T. Riaz, S. kamal, A. Ahmed and S. Ahmed, Pharmaceutical Biology, 46(5), 2008, 309-312.
3. Limited effectiveness of Melia azedarach cream in bacterial skin infection in children, R.
Saleem, R. Rani, M. Ahmed, F. Sadaf, S.I.Ahmad, N. ul Zafar, S. S. Khan, B. S. Siddiqui,
Lubna, F. Ansari, S. A. khan, S. Faizi, Focus on Alternative and Complementary Therapies
(FACT), 13(4),262- 263, 2008.
4. Specific deuteration in patuletin and related flavonoids via keto-enol tautomerism: solvent
and temperature dependent 1H-NMR studies, S. Faizi, H. Siddiqi, A. Naz, S. Bano and
Lubna. Helvetica Chimica Acta, 93, 2010, 466- 481.
5. Bioassay guided isolation of antioxidant agents with analgesic properties from flowers of
Tagetes patula, S. Faizi, A. Dar, H. Siddiqi, S. Naqvi, A. Naz, S. Bano and Lubna.
Pharmaceutical Biology, 49(5), 2011, 516-525.
6. Isolation of phytonematicidal compounds from Tagetes Patula L. yellow flowers: Structure
activity relationship studies against cyst nematode Heterodera zeae infective stage larvae. S.
Faizi, S. Fayyaz, S. Bano, E. Y. Iqbal, Lubna, H. Siddiqi and A. Naz, Journal of Agriculture
and Food Chemistry, 59 (17), 2011, 9080–9093.
7. Green synthesis and molecular recognition ability of patuletin coated gold nanoparticles.
Muhammad Ateeq, Muhammad Raza Shah, Noor ul Ain, Samina Bano, Itrat Anis, Lubna,
Shaheen Faizi, Massimo F. Bertino, Syeda Sohaila Naz. Biosensors and Bioelectronics,
63(1), 2015, 499-505.
8. Cytotoxic and antioxidant properties of phenolic compounds from Tagetes patula flower.
M. Kashif, S. Bano, S. Naqvi, S. Faizi, Lubna, M. A. Mesaik,K. S. Azeemi, A. Dar.
Pharmaceutical Biology, 2015, 53(5), 672-681.
9. Antibacterial activity of flower of Melia azedarach Linn. And identification of its
metabolites. Munira Taj Muhammad, Lubna, Nida Fayyaz, Saima Tauseef, Ummarah Razaq,
Muhammad Ali Versiani, Aqeel Ahmad, Shaheen Faizi, Munawwer Rasheed. Journal of the
Korean Society for Applied Biological Chemistry, 2015, 58(2), 219-227.
283
10. Analgesic potential of Opuntia dillenii and its compounds opuntiol and opuntioside
against models in mice. Faheema Siddiqui, Lubna Abidi, Lubna,Ching Fidelis Poh, Sabira
Naqvi, Shaheen Faizi, Ahsana Farooq. Record Natural Product, 2016, 10(6), 721-734.
11. Opuntia dillenii cladode: opuntioside and opuntiol attenuates PGE2 and LTB4 mediated
inflammation. Faheema Siddiqui, Sabira Naqvi, Lubna Abidi, Shaheen Faizi, Lubna, Lubna
Avesi, Talat Mirza, Ahsana Farooq, Journal of Ethnopharmacology, 2016 182, 221–234.
12. Anti TNF-α and Anti-arthritic effect of Patuletin: A rare Flavonoid from Tagetes patula.
Almas Jabeen, Lubna, Samina Bano, Shabana U. Simjee, Shaheen Faizi and M. Ahmed
Mesaik. International Immunopharmacology, 2016, 36, 232-240.
13. Hypotensive and toxicological effect of methanolic extract of french marigold flowers its
fractions and pure constituents. F. Sadaf, R. Saleem, U. Ahmed, S. I. Ahmad, Lubna, S. Bano
and S. Faizi (In process, 2017).
US Patent
M. A. Mesaik, A. Jabeen, S. Faizi, S. U. Samjhee, S. Bano, Lubna. Patuletin, a potent Anti -
α and anti-arthritic compound from Tagetes patula. (United State Patent Office, 2013).
Pakistani Patent
S. Faizi, A.Dar. M. Kashif, Lubna, S. Bano, Anticancer activity of Tagetes patula flowers
extract, fractions and pure compounds. (Submitted 2015)
Proceedings
Presented at 1st International symposium and workshop-Cum-Training Course on molecular
Medicine and Drug Research held at the Dr. Panjwani Center for Molecular Medicine and
Drug Research (ICCBS, University of Karachi, Karachi-75270) held at Karachi, January 16-
30, 2007.
S. Bano, S. Tauseef, A. Ahmed, Lubna, S. Firdous, A. Tahir, S. A. Khan, H. Siddiqi, A. Naz
and S. Faizi. Evaluation of antimicrobial activity of flowers of Tagetes erecta: Isolation of
quercetagetin as the antibacterial principle (First prize winning poster).
284
Presented at Sixth International Biennial Conference of Pakistan Society for Microbiology
held at Islamabad, 18-21 March, 2007.
S. Tauseef, S. Bano, S. A. Khan, S. K. Khan, M. Sohail, A. Amyn, A. Naz, H. Siddiqi, Lubna,
A. Tahir, S. Firdous, S. Faizi and A. Ahmed. Studies on antimicrobial activity of extracts and
fractions of Tagetes erecta (First prize winning poster).
Presented at 1st International symposium and workshop-Cum-Training Course on Molecular
Medicine and Drug Research held at the Dr. Panjwani Center for Molecular Medicine and
Drug Research (ICCBS, University of Karachi, Karachi-75270) held at Karachi, January 16-
30, 2007.
M. Kashif, N. Kabir, S. Bano, Lubna, H. Siddiqi, A. Naz, S. Naqvi, S. Faizi and A. Dar, As
in vitro anti-cancer activity against skin melanoma by Tagetes patula flower.
Presented at 13th Annual Congress, held at Karachi, 16-18 February, 2007.
M. Kashif, N. Kabir, S. Bano, Lubna, H. Siddiqi, A. Naz, S. Naqvi, S. Faizi and A. Dar. An
in vitro anti-tublin activity against skin melanoma by Tagetes patula flower: A promising
source for anticancer agent(s).
Presented at 11th International symposium on Natural Product Chemistry (ICCBS, University
of Karachi, Karachi-75270) held at Karachi, October 29-November 01, 2008.
H. Siddiqi, S. Bano, Lubna, A. Naz and S. Faizi. Specific deuteration in patuletin and related
flavanoids via keto-enol tautomerism: solvent and temperature dependent 1H-NMR studies.
M. A. Khan, A. Dar, S. Faizi, L. Abidi, Lubna and N. I. Kabir. Chronic treatment of Opuntia
dillenii exhibits anti-depressant like properties in mice in a modified tail suspension test.
285
S. Bano, S. Fayyaz, Lubna, E. Iqbal, H. Siddiqi, A. Naz and S. Faizi. Bioassay on
yellowflowers of Tagetes patula: structure-activity relationship studies against cyst nematode
Heterodera zeae.
M. Kashif, S. Bano, Lubna, H. Siddiqi, A. Naz, S. Naqvi, S. Faizi, N. Kabir and A. Dar.
Anticancer activity of methanolic extract of Tagetes patula flower against non-small cell lung
cancer cell line (NCI-H460).
Presented at 2nd International symposium Cum-Training Course on Molecular Medicine and
Drug Research held at the Dr. Panjwani Center for Molecular Medicine and Drug Research
(ICCBS, University of Karachi, Karachi-75270) held at Karachi, January 12-15, 2009.
1. S. Faizi, Lubna, S. S. Khan, S. K. Khan, R. Saleem, A. Ahmed, F. Ansari, R. Rani, M.
Ahmed, F. Sadaf, S.I.Ahmad, N. ul Zafar, B. S. Siddiqui, S. A. khan. Effective treatment of
bacterial skin infections by herbal cream containing extract of Melia azedarach flowers.
2.
Presented at “Development of network of women neuroscientists in Pakistan” jointly
organized by: Dr. Panjwani Center for Molecular Medicine and Drug Research, ICCBS,
University of Karachi, Karachi-75270 and Women in world neuroscience (WWN) held at
Karachi, July 1st and 2nd , 2010.
H. Ishaq, R. Saleem, Lubna, S. S. Khan, S. I. Ahmed, S. Faizi. Neuropharmacological
studies on methanolic extracts of different part of Melia azedarach.
Presented at 12th International symposium on Natural Product Chemistry (ICCBS, University
of Karachi, Karachi-75270) held at Karachi, November 22-25, 2010.
3. H. Ishaq, R. Saleem, S. I. Ahmad, Lubna, S. Faizi. Anti-depressant and Anxiolytic activity of
Melia azedarach Linn.
4.
286
5. N. Noureen, Lubna, S. Bano, H. Siddiqi, A. Naz, S. Tauseef, A. Irshad, A. Ahmed, E. Iqbal,
S. Fayyaz and S. Faizi. Bioassay-guided Isolation of Antioxidant and Nematicidal
Constituents from the Extract of Yellow Flowers of Tagetes erecta.
6.
Lubna, S. Sumbul, R. Saleem, N. Noureen, A. Sana and S. Faizi. Nasimizine: A Rare and
New Fluorinated Pyrazine from Root of Moringa oleifera.
F. Sadaf, R. Saleem, U. Ahmed, S. I. Ahmad, Lubna, S. Bano and S. Faizi. Hypotensive and
Toxicological Effect of Methanolic Extract of French Marigold Fowers its Fractions and
Pure Constituents.
Presented at 3rd International symposium Cum-Training Course on Molecular Medicine and
Drug Research held at the Dr.Panjwani Center for Molecular Medicine and Drug Research
(ICCBS, University of Karachi, Karachi-75270) held at Karachi, January 3-6, 2011.
S. Tauseef, M. A. Mesaik, S. Faizi, S. Bano, Lubna, H. Siddiqi, B. Fatima, S. A. Khan and
A. Ahmad. Immunomodulatory Studies on potential of some natural flavonoids of Tagetes
patula.
Presented at 13th International symposium on Natural Product Chemistry (ICCBS University
of Karachi, Karachi-75270) held at Karachi, September 21-24, 2011.
S. Z. Ur Rehman, S. Fayyaz, Lubna, S. Bano, E. Y. iqbal and S. Faizi. Nematicidal Activity
of flavonoids against the root-knot nematode Meloidogyne incognita: Structure Activity
Relationship Studies.
Presented at 14th Asian symposium on Medicinal plants, Species and Other Natural Products
(ICCBS, University of Karachi, Karachi-75270) held at Karachi, December 07-10, 2013.
A. Jabeen, M. A. Mesaik, S. Faizi, S. Bano, Lubna and S. U. Simjee. Anti TNF-α and Anti
arthritic effect of Patuletin: a Flavonoid from Tagetes Patula.
287
T. Roome, S. Khan, A. Razzak, A. Noorani, R. Khanani, A. Dar, S. Faizi, L. Abidi, Lubna,
and M. Cathcart. Opuntiol and Opuntioside-I an anti-atherogenic agent interfering with
Chemotaxis via iPLA2β- dependent F-actin polymerization in human monocyte.
F. Siddiqui, S Naqvi, L Abidi, Lubna and A Dar. Anti-inflammatory activity of
Opuntiadillenii via inhibition of inflammatory mediators PGE2 and LTB4.
Mudassar, A. Dar Farooq, S. Haque, Lubna, S. Bano, S. Faizi. Evaluation of T. patula
methanol extract and pure compound, patuletin for their genotoxic actions.
P. Ali, S. Aziz, A. Razzak, T. Roome, S. Khan, F. Siddiqui, A. Dar, S. Faizi, L. Abidi, Lubna.
Inhibitory effect of Opuntiol/ Opuntioside-I against the expression of cytokine and chemokine
in carrageenan and zymosan induced mouse inflammatory model.
P. Ali, S. Aziz, A. Razzak, T. Roome, S. Khan, F. Siddiqui, A. Dar, S. Faizi, L. Abidi, Lubna.
Inhibitory effect of Opuntiol/ Opuntioside-I against the expression of cytokine and chemokine
in carrageenan and zymosan induced mouse inflammatory model.
A. Jabeen, M. A. Mesaik, S. Faizi, S. Bano, Lubna and S. U. Simjee. Patuletin: A flavonoid
from Tagetes patula as TNF-a and rheumatoid arthritis inhibitor.
Presented at 4th Pak-France Bi-national Workshop on Drug Discovery and Molecular
Medicine October 12 -14, 2015, Karachi, Pakistan.
Faheema Siddiqui, Sabira Naqvi, Lubna Abidi, Lubna, Shaheen Faizi and Ahsana Dar
Farooq.Anti-inflammatory and Antinociceptive Properties of Opuntia dillenii and its Pure
Compounds: Opuntiol and Opuntioside.