part iv - shodhganga : a reservoir of indian theses @...

Part IV

Identification of the Bioactive Molecule in the Fractions

ofT. cordifolia

Chapter 1

Introduction

174 Part IV: Chapter 1

4.1.1 Secondary metabolites: an introduction

Plants survive with the use of two important metabolic pathways

which are classified as the primary and the secondary metabolic pathways.

The primary metabolism involves a series of chemical reactions that are

primary for the survival and growth of the plant. The metabolites of the

primary metabolism include the carbohydrates, lipids, proteins and nucleic

acids. The secondary metabolites are derived from the primary metabolites

and are not significant to the plant in terms of its growth and development or

survival. These secondary metabolites serve additional benefits of plant

defence and ecological considerations. In plant defence, the secondary

metabolites can function to prevent attacks of pests, pathogens and

herbivores by acting as anti-feedant.

Secondary metabolites are also important in the regulation of the

primary metabolic pathway and the signalling involved in this regulatory

mechanism. Plant characteristics like the colour of flowers, fruits, etc. are

also imparted by the presence of secondary metabolites and further enhance

the fertilization or pollination processes of the plant. Plant hormones are also

classified as part of secondary metabolites. There are certain types of

primary metabolites that can be specific to a particular plant species or

family with rare occurrence like some fatty acids and sugars and are thus

also considered to be secondary in nature. One such example is the presence

of the apiose sugar specifically in the parsley family.

4.1.2 Classification of Plant Secondary Metabolites

Plant secondary metabolites are broadly classified on the basis of

composition into the nitrogen containing secondary metabolites and the

secondary metabolites that do not contain nitrogen. The nitrogen containing

Introduction 175

secondary metabolites include alkaloids, nonprotein amino acids, cyanogenic

glycosides, glucosinolates, alkamides and lectins. The major groups of

secondary metabolites that do not contain nitrogen are the terpenes, steroids,

saponins, flavonoids, tannins, phenylpropanoids, polyacetylenes and

polyketides. These groups of secondary metabolites are briefly described

below.

4.1.3 Nitrogen containing secondary metabolites

4.1.3.1 Alkaloid

Alkaloids are bitter tasting secondary metabolites that has basic

nitrogen atom and functions mostly as defence compounds. Alkaloids are

often divided into the following major groups (Hesse, 2002):

1. True alkaloids that originate from amino acids.

2. Protoalkaloids that originate from amino acids.

3. Pseudoalkaloids that are not from amino acids

Many of these alkaloids are toxic to other organisms but they also

have pharmacological effects. These compounds can be used as medications

for a wide range of disorders like the analgesic morphine, antibacterial

berberine, anticancer vincristine, anti-hypertension reserpine, anti-asthma

ephedrine or as recreational drugs like stimulant cocaine (Hesse, 2002),

psychedelic psilocin and stimulant caffeine (Veselovskaya, 2000) or nicotine

(Siegmund et al., 1999). Nicotine and anabasine alkaloids are used as

insecticides but was restricted due to their highly toxic nature (Matolcsy et

al., 2002) while liriodenine of tulip tree protects from mushrooms that are

parasitic.


4.1.3.2 Non-protein amino acids

Non-protein amino acids are not found normally in proteins but have

been found to be present in fabaceae, leguminosae and cucurbitaceae

families of plants. The three major possibilities of synthesis are (Ambrogelly

et al., 2007):

a) modification of existing amino acid,

b) modification of an existing pathway and

c) novel pathway

Several non-proteinogenic amino acids are important as biosynthetic

intermediates like ornithine and citrulline in the urea cycle (Curis et al.,

2005), for physiological functions as neurotransmitters or toxins which

provide resistance to insect herbivores (Huang et al., 2011), and as

pharmacologically active compounds (Rosenthal, 1982). Non-protein amino

acids are toxic to herbivores by obstructing with primary metabolism. An

example being mimosine that interferes with tyrosine metabolism,

misincorporating proteins, and interference of insect neurology (Bell, 2003).

Putrescine, spermidine and spermine are found ubiquitous in all plants

(Bagni and Pistocchi, 1992) and are found increased in environmental stress,

salinity stress and acid stress (Flores et al, 1989). Polyamines are important

to regulate DNA replication, and morphogenesis (Galston and Sawnhey,

1990). Polyamines can be precursors of certain alkaloids which may play

important roles in plant defense against herbivores.

4.1.3.3 Cyanogenic glucosides

Cyanogenic glucosides are the secondary metabolites with the

primary function of defence. The release of cyanide from these compounds is

not toxic to the plant and can be detoxified by humans and animals naturally.

Introduction 177

The toxic effect of these compounds is particularly specific for parasites and

herbivores. This effect is caused due to the inhibition of sodium-iodide

symporter and subsequent blocking of the transport of iodide in cells

(Venturi et al., 2000). Plant pesticides that contain cyanogenic glucosides

can liberate cyanide, thereby blocking cytochrome c oxidase and the sodium-

iodide symporter proving toxic to parasites and herbivores (Venturi,

2011). Positive benefits of cyanogenic glucosides include the increase in

growth rates of the southern armyworm larvae when its diet includes plants

with these compounds. Cyanogenic glucosides that are stored in the seeds of

the plant are a source of nitrogen for the growth of the seedling and are

utilized during germination.

4.1.3.4 Glucosinolates

Glucosinolates are sulphur containing secondary metabolites which

are derived from glucose and amino acid with a pungent taste, examples

being mustard and horseradish. A central carbon atom bound to a thioglucose

using a suphur atom and a nitrogen atom is the standard structure for these

glucosinolates. The side chains bound to the central group are responsible for

the differential biologic effects of the glucosinolates. Glucosinolates are

commonly found in plants belonging to the Cruciferae, Capparidaceae,

Caricaceae and Euphorbiaceae families (Rodman et al., 1996). They serve

the main function of defending the plants against pests which is perfectly

achieved by the bitterness that forms once the glucosinolates are released on

damage or chewing by the pests. The toxicity to humans and animals remain

plausible at high concentrations resulting in goitrogenic effects (Van Doorn

et al., 1998) but continual use at below toxic doses of these Brassica

vegetables results in a lower risk of cancer (Hayes et al., 2008). The

anticancer effect of these vegetables are as a result of the induction of the


Phase I and Phase II enzymes, protection against damage from oxidation and

modification of the metabolism of steroid hormones (Das et al., 2000).

Indole and isothiocyanate metabolites of glucosinolates can inhibit the

growth of several types of cancers of the stomach, liver, lungs, breast and

colon (Hecht, 2000; Murillo and Mehta, 2001).

4.1.3.5 Alkamides

Alkamides are a group of amides from unsaturated aliphatic fatty

acids that are joined by an amide bond to amines and include compounds

that promote plant growth. These compounds are usually found in a large

number of plant families like asteraceae, rutaceae, piperaceae, solanaceae

among others and are known for the pungency (Ramírez-Chávez et al.,

2004). Alkamides have demonstrated a significant impact on the

development of roots and provides key references for improvement in plant

production.

4.1.3.6 Lectins, peptides and polypeptides

Lectins are specific proteins that bind to carbohydrates and are better

known as phytohemagglutinins due to the specific binding of other

glycoconjugates. The importance of lectins is underlined by the emphasis on

biological recognition of cells or carbohydrates and proteins (Brudner et al.,

2013) that explains the use of lectins in human blood typing (Komath et al.,

2006). This recognized binding of carbohydrates can be hindered by specific

monosaccharides and oligosaccharides which prevent attachment to cell

membranes (Van Damme et al., 1998). BanLec, the lectin isolated from

bananas have been found to inhibit HIV-1 under in vitro conditions

(Swanson et al., 2010).

Introduction 179

4.1.4 Secondary metabolites without nitrogen

4.1.4.1 Terpenes

Terpenes are secondary metabolites consisting of isoprene units and

can be further classified into hemiterpenes with a single isoprene,

monoterpenes with two, sesquiterpenes with three, diterpenes with four,

sesterterpenes with five, triterpenes with six, sesquarterpenes with seven,

tetraterpenes with eight and polyterpenes with even more units. ß-Myrcene

from cannabis is prominently used for the management and treatment of pain

and inflammation, muscle spasms as well as psychosis. Limonenes found in

citrus fruit peels are used pharmacologically as an antimicrobial, antitumour,

immunostimulant, antidepressant and an insect repellent. Phytols formed by

decomposing chlorophyll can reduce itching and remains beneficial for slow-

healing wounds.

4.1.4.2 Steroids

Phytosterols are those secondary metabolites in plants that include

plant sterols and stanols, which are the counterparts to animal cholesterol.

Stanols have no double bonds in the sterol ring and are thus saturated sterols

(Akhisa and Kokke, 1991). Brassicasterol and beta-sitosterol are the most

common steroids which are important for plant growth promotion. Other

important functions of phytosterols include the hypocholesterolaemic effect in

humans due to the competition with cholesterol for intestinal absorption

(Katan et al., 2003). A supplement of 1.6 g/day of plant sterols was able to

reduce elevated levels of triglycerides by 14% when administered for six

weeks causing a subsequent reduction in risk for cardiovascular disease (Plana

et al, 2008). The supplement was given as phytosterol fortified fermented milk

beverage (Plat and Mensink, 2009) and was further explained by the

mechanism that was proposed by Theuwissen et al. (2009) with a


corresponding decrease in production of triglyceride concentrated VLD-

Lipoprotein particles. Phytosterols were also studied and found to be effective

in breast, ovarian, lung and stomach cancers (Woyengo et al., 2009) with

supportive modulation of antioxidant enzymes, superoxide dismutase,

glutathione peroxidise and catalase activity (Carange et al., 2011).

4.1.4.3 Saponins

Saponins are glycosides that are synthesized as plant secondary

metabolites and consist of varying numbers of saccharide units usually glucose

or galactose linked to a lipophilic aglycone called sapogenin (Hostettmann and

Marston, 2005). The sapogenins are polycyclic organic structures which is

either a triterpene skeleton with 30 carbons or a steroidal skeleton with 27

carbons. The steroidal saponins are more commonly known as saraponins

(Haridas et al., 2001). In plants, saponins function as anti-feedants (Haridas et

al., 2001) protecting the plant against microbial or fungal attacks while other

saponins (oats and spinach) function to enhance absorption of nutrients. Like

most other secondary metabolites that function in defence, saponins have a

bitter taste which can reduce palate of attackers and even cause fatal toxicity

(Klita et al., 1996). Saponins that have been isolated from Gypsophila

paniculata have demonstrated to augment cytotoxic immunotoxins directed

against human cancer cells significantly. Other beneficial effects of saponin

rich plants are the hemolytic (Waller and Yamasaki, 1996), cholesterol

lowering (Sauvaire et al., 1996) (Sowmya and Rajyalakshmi, 1999) and

anticancer properties (Kerwin, 2004). Antitumour effects in human colon

cancer were implemented by down-regulation of alpha-fetoprotein marker,

induction of apoptosis and modulation of the NF-κB signalling pathway by

Astragalus saponins (Auyeung et al., 2009). These saponins could also be used

Introduction 181

for supplementation with conventional chemotherapeutics by its ability to

target NSAID activated gene (Tin et al., 2007).

4.1.4.4 Flavonoids

Flavonoids are secondary plant metabolites which are polyphenolic

compounds mostly used in plants to produce pigments that provides colour

to the plant parts. The flavanoids have demonstrated to be functional as

effective antioxidant, anti-inflammatory, anti-allergic natural supplements

with the ability to modulate normal cell signalling pathways. The anticancer

properties attributed to these compounds are caused by a mechanism where it

activates the natural enzymes that fight against carcinogens. Flavonoids are

found abundant in citrus fruits, red wine, chocolate, berries and tea.

A flavonoid has a basic structure of a diphenylpropane with two

benzene rings that is linked with a closed pyran ring. This structural

character allows the flavonoid group of compounds to remain as aglycones,

glycosides or methylated derivatives. Flavonoids are synthesized by the phenyl

propanoid metabolic pathway where the amino acid phenylalanine produces

chalcones (Crozier and Ashihara, 2006). Chalcones are aromatic ketones with

two phenyl rings that are the precursors of flavonoids. On the basis of the

molecular structure of the flavonoids they can be further subdivided into 6

sub classes as Anthocyanidins, Flavanols, Flavanones, Flavonols, Flavones

and Isoflavones

Flavonoids have been studied to have strong antioxidant properties

(Hertog et al., 1997) with inhibition of excessive free radical generation

leading to the prevention of the toxic effect of reactive oxygen species

(Halliwel, 1991). Studies conducted with Myricetin, Quercetrin and Rutin

have revealed the inhibition of superoxide radical generation (De Groot,


1994; Grace, 1994) while regular consumption of green tea containing

catechins can prevent cardiovascular disorders (Nakagawa et al., 1999). The

antiviral activity of flavonoids was studied by Wang et al. (1998) with the

ability to affect the replicative and infective properties of both RNA and

DNA viruses (Kaul et al., 1985). The use of Flavonoids for the treatment of

osteoporosis, cardiovascular diseases and hormone dependent cancer have

been studied (Wiseman et al., 2000) extensively which has further led to the

study where the loss of bone in postmenopausal women was prevented by

the use of Genistein (Metzner et al., 2009). Flavonoids also exhibit an

important role in cancer prevention and therapeutics with its significant

steroid hormone like activity (Rosenberg et al., 2002)

Studies have associated flavonoids with reduced risk of coronary

heart disease (Zern and Fernandez, 2005) while other studies have shown the

reduction in injury after an incident of myocardial ischemia by

Dihydroxyflavonol with both vasorelaxation and antioxidation functions

(Qin et al., 2008). Diets rich in citrus foods have been known to reduce the

risk associated with cardiovascular disorders (Liu et al., 2008). Anti-

inflammatory activity of quercetin has been demonstrated by Park et al.

(2009) while the role of dietary flavonoids with a reduced risk of cancer

(Bach et al., 2010; Appelt et al., 1999; Kamaraj et al., 2009) has also been

studied. Tangeritin, a flavonoid from citrus products can effectively inhibit

proliferation of cancer cells (Arafa et al., 2009). The hepatoprotectivity of

Flavonoids isolated from German chamomile have shown to produce effects

on the metabolism of sphingolipids in aged liver and in turn regulate the key

enzyme levels in liver (Babenko et al., 2008).

Introduction 183

4.1.4.5 Tannins

Tannins are oligomeric secondary metabolites that are polyphenols and

can precipitate proteins. Tannins are commonly found in dark coloured fruits

(grapes, blueberry, etc.), legumes, chocolate and tea (Waterman and Mole,

1994). Tannins are synthesized through the phenyl propanoid pathway

resulting in two categories of hydrolyzable tannins and condensed tannins

(also known as proanthocyanidins) distinguished with the ability to resist

hydrolytic degradation (Reed, 1995). Hydrolyzable tannins have a central core

of polyol (usually D-glucose) with esterified phenol groups, gallic acid

(gallotannins) or ellagic acid (ellagitannins). Proanthocyanidins are complex

oligomers or polymers of flavan-3-ol (2 to 50 monomer units) that are linked

with carbon-carbon bonds and are resistant to hydrolytic cleavage. When

proanthocyanidins are heated in acidic alcohol solutions red anthocyanidins

are produced and has given rise to its name (Giner-Chavez, 1996).

Condensed tannins can defend the plant from herbivore attacks by

havocking the digestion of the herbivores. These tannins bind to plant

proteins that are consumed by the herbivores and interfere in the digestion

and absorption of the proteins. The high content of condensed tannins in red

wines and red grape juice can effectively inactivate enteric viruses, herpes

simplex virus and polio virus (Bajaj, 1988). Studies on the effect of tannins

in in vitro cells have revealed anti-parasitic (Kolodziej and Kiderlen, 2005),

anti-HIV (Lu et al., 2004) and antibacterial activities (Akiyama et al., 2001).

Tannin enriched fractions from the bark of Myracrodruon urundeuva have

been found to produce effective neuroprotection in 6-hydroxydopamine-

induced toxicity (Nobre-Junior et al., 2008) while demonstrating strong anti-

inflammatory and anti-ulcer properties in rodent models with formalin and


carrageenan induced inflammation and indomethacin induced gastric ulcer

(Souza et al., 2007).

4.1.4.6 Phenyl propanoids, lignins, coumarins and lignans

Phenylpropanoids are a class of plant polyphenols derived from

shikimic acid and gives rise to lignins, coumarins, and lignans as secondary

products of its metabolism.

Lignins are unusual heterogenous biopolymers that lack a definitive

primary structure. The most important function of these compounds being

the strengthening of wood in trees (Wardrop, 1969), lignins can fill the cell

wall spaces particularly in the xylem. Extensive crosslinking among plant

polysaccharides and lignins confers mechanical strength to both the cell wall

and the plant (Chabannes et al., 2001). Lignins are also crucial in helping

water conduction through the xylem with the cross linked polysaccharide

being unable to absorb water (Sarkanen and Ludwig, 1971).

Coumarin with a pleasant odour and bitter taste suppresses appetite in

grazing animals who avoid such plants (Link, 1959). Coumarins are used in

the treatment of asthma (Liu, 2011), as an anticoagulant and for

lymphoedema (Farinola and Piller, 2005). The use of these coumarins as

rodenticides is also widespread and works by blocking the regeneration of

vitamin K. Coumarin has also been studied for its anti-tumor (Weber et al.,

1998) and anti-fungal (Montagner et al., 2008) activities.

Plant lignans are derived by the dimerization of monolignol to a

dibenzylbutane skeleton (Heinonen et al., 2001) and are classified as

phytoestrogens (Axelson et al., 1982). Lignans are important as the plants

defence system against both biotic and abiotic factors. They have

demonstrated anti-inflammatory (Korkina et al, 2011) and antioxidant

activity (Korkina, 2007) in different studies that were conducted.

Introduction 185

Anticarcinogenic activities of lignin include studies on reduced risk of breast

cancer (Boccardo et al., 2006) with dietary exposure and inhibition of breast

cancer in experimental models by enterolactone (Lindahl et al., 2011).

4.1.4.7 Polyacetylenes, and unusual fatty acids

Polyacetylenes are natural plant secondary metabolites with carbon-

carbon triple bonds or alkynyl functional groups and are as a consequence

highly reactive interacting with proteins and biomembranes (Lie et al., 1996).

These compounds are also highly toxic with neurotoxicity. A diynoic acid

compound isolated from the Indonesian plant (Mitrephora celebica) has

shown potent antimicrobial activity (Zgoda J et al., 2001), while a triynoic

acid isolated from Orophea enneandra (Cavin et al., 1998) and Mitrephora

celebica (Zgoda J et al., 2001) displayed significant antifungal activity. Other

significant studies include the cytotoxic effects of polyacetylenes from carrot

and ginseng (Zidorn et al., 2005). The difference between chemoprevention of

pure carotene and that in carrots was better understood by the involvement of

polyacetylenes in carrots. Further investigations into the anticancer properties

of falcarinol, an allergen from carrots have revealed that the induction of

apoptosis by it causes a delay in tumour formation (Kobaek-Larsen et al.,

2005). The content of falcarinol in carrots were significantly reduced up to

70% on boiling for 12 minutes (Hansen et al., 2003) while its content was

increased significantly after 4 months storage at 1°C (Kidmose et al., 2004).

4.1.4.8 Polyketides

Polyketides are a group of structurally diverse secondary metabolites

that are not restricted to plants but are widely prevalent in fungi and microbes.

These compounds are further subdivided into three classes where type I

polyketides are often macrolides, type II polyketides are often aromatic

molecules and type III polyketides are often small aromatic molecules by


fungus. Biologically active polyketides are the macrolide antibiotics derived

from bacteria, cholesterol lowering lovastatin from Aspergillus sp. and

immunosuppressant rapamycin from Streptomyces sp.

4.1.4.9 Complex carbohydrates and organic acids

Complex carbohydrates are a major component of plant systems with

cellulose being the main structural component of plant cell walls. Some

complex carbohydrates are also used to provide immediate energy to plant cells

and for the storage of energy (starch) in seeds, roots, or fruits for later use. The

carbohydrates produced by plants are an important source of energy for animals.

4.1.5 Secondary metabolites in T. cordifolia

The secondary metabolites in T. cordifolia have been extensively

studied. The pharmacological activity of T. cordifolia has been attributed to

the classes of secondary metabolites like alkaloids (Kumar et al., 2000),

steroids (Pathak et al., 1995), glycosides (Gangan et al., 1994),

sesquiterpenoids (Maurya and Handa, 1998), diterpenoid lactones (Gangan et

al., 1994), and aliphatic compounds (Dixit and Khosa, 1971). The study by

Sao et al. (2014) has reported the presence of flavonoids, glycosides,

alkaloids, steroids, and carbohydrates in the phytochemical screening of root

extracts of T. cordifolia. Kiem et al. (2010) have reported the presence of two

aporphine alkaloids in T. cordifolia. Tembetarine, Choline, Tinosporin,

Isocolumbin, Palmatine, Tetrahydropalmatine and Magnoflorine were also

isolated from root extract of T. cordifolia (Singh et al., 2003). Pradhan et al.

(2013) has studied T. cordifolia extracts prepared in methanol, petroleum

ether, water, chloroform and ethyl acetate were screened for the presence or

absence of phytochemicals. Tannins was absent in all the extracts, while the

aqueous and methanol extracts contained saponins. Flavonoids, steroids,

alkaloids, proteins, cardiac glycosides and carbohydrates, were present in all the

Introduction 187

extracts. These results correlate with the studies by Tanwar et al. (2012),

Sivakumar and Dhanarajan, (2010) and Nasreen et al. (2010) who have reported

on the presence of phytochemicals in T. cordifolia. Three major groups of

compounds, protoberberine alkaloids, terpenoids and polysaccharides have been

reported to be responsible for the observed therapeutics activity of T. cordifolia

(Bisset and Nwaiwu, 1983; Chintalwar et al. 1999).

4.1.6 Objectives of Part 4

Mass spectrometry has routinely been applied to the efficient

identification of unknown compounds that are present in trace levels utilizing

the electron ionization (EI) mass spectrum (Nyiredy, 2004). The EI mass

spectrum when compared to the spectra from other molecular analytical

methods is unusually high in information (McLafferty et al., 1998) making it

useful for computer predictions of even global unknowns (Demuth et al.,

2004). The information provided by mass spectroscopy can be used in three

ways for the identification of unknown compounds and includes the

determination of molecular weight, molecular formula, and molecular

structures of the compounds (Henneberg et al., 1993). The identification of

the compounds in the bioactive fractions of T. cordifolia extracts were dealt

with in this chapter.

The detailed objectives for Part IV are as follows:

1. HPLC analysis of active fractions.

2. UPLC/Q-Tof Analysis of Active fractions to identify

bioactive molecules in fractions of T. cordifolia.

Chapter 2

Review of Literature


4.2.1 Structure Elucidation of Bioactive Molecules

Structure elucidation of bioactive molecules has been the most

pursued avenue of the scientific community and still remains the most

difficult analysis thereby resulting in newer and improved methods. The

approaches in structure elucidation of the plant bioactive molecules is to use

different combination of methods of purification, such as separation of

compounds by HPLC/UPLC and other chromatographic methods followed

by determination of the structure by Mass spectroscopic analysis, Nuclear

magnetic resonance (NMR) and X-ray crystallography forming hyphenated

instruments. High performance liquid chromatography is an experimental

technique that can be used for the separation of components in a mixture of

compounds. The separation is based on the interaction of the separating

molecule to its environment, the stationary phase and the mobile phase. In a

reversed phase HPLC, the more polar molecules elute readily in a non-polar

stationary phase with a longer retention time required for non-polar

compounds. HPLC technique can be used for the separation of compounds

for research or for industrial use. The separation of compounds include the

use of pump to force the pass of solvent containing sample through the

stationary phase.

The first two phases in elucidation of the structure of molecules are

the identification of the exact molecular mass of the compounds and the

determination of the molecular formula. The main objective of spectroscopic

analysis is to identify functional groups in the molecule and molecular

fragmentation leading to structural correlations by the examination of the

spectra of already known compounds.

Review of Literature 191

4.2.2 Mass Spectrometry

Mass spectrometry is useful to calculate the accurate mass of small

molecules and to identify the chemical entities in the structure of the

molecule based on its fragmentation pattern (Adams, 1992). Tandem mass

spectrometry combines two mass spectrometers for this analysis (MS/MS)

usually the transmission quadrupole or the time-of-flight elements

(Strathmann and Hoofnagle, 2011). This creates a highly specific and

sensitive environment for the detection of the molecules. The use of ultra-

performance liquid chromatography mass spectrometric (UPLC-MS/MS)

techniques has increased greatly over the past few years for applications in

structure elucidation of plant and microbial metabolites. Time-of-flight

(ToF)-MS instruments have high resolving power and high acquisition speed

with accurate mass measurements (<2ppm) (deHoffman and Stroobant,

2003). It is preferred in combination with the quadrupole mass analyzers

because the latter has issues of lack of sensitivity in full scan spectrum

analysis. The added advantage of the QToF MS/MS system is the high

accuracy of measurements without compromise on sensitivity of the analysis

(Grebe and Singh, 2011).

4.2.3 Phytochemical investigations based on Mass Spectrometry

Several studies have been reported based on the application of the

QToF MS/MS analyzers in plant metabolite profiling particularly identifying

new unknown compounds (Wewer et al., 2011). These analyzers can also be

used for the standardization of medicinal plants by its metabolite profile as

shown in the study by Rogachev and Aharoni (2012). Madala et al (2014)

have reported on the use of the QToF analyzer with in-source collision

induced dissociation for the study of the identification of the isomers of

chlorogenic acid in the Momordica species for the first time. The presence of


environmental contaminants in economically and medicinally important

plants can be detected as part of stringent quality control measures

(Macherius et al., 2014).

4.2.4 T. cordifolia and mass spectrometry

A large fraction of the constituents in the plant parts of T. cordifolia

have been successfully elucidated but very few studies have been conducted

using the LCMS/MS system. The storage of T. cordifolia stem juice in the

cold can affect the phytochemical profiles of the sample. The study was

conducted by Shirolkar et al (2013) in an UPLC-QToF MS/MS analyzer to

identify menisperine, jatrorrhizine, berberine magnoflorine, columbamine, and

tinosporoside in the sample, 99% of which degrades in 30 days. Another study

by Pushp et al (2013) has reported on the presence of epicatechin in T.

cordifolia stem based on the data from QToF MS for the first time. More

compounds form the stems of T. cordifolia were elucidated by the study

conducted by Sharma et al (2012). This study deals with the elucidation of the

structure of seven compounds in T. cordifolia extracts which are as follows:

Compound 1: N-formylannonain, Compound 2: 11-hydroxymustakone,

Compound 3: N-methyl-2-pyrrolidone, Compound 4: cordifolioside A,

Compound 5: magnoflorine, Compound 6: tinocordiside, Compound 7:

syringin. Compounds 4 and compound 7 have been reported to possess

immunomodulatory activity while the compounds 1, 2, 3, 5, 6 demonstrate

enhanced phagocytic activity.

Review of Literature 193

4.2.5 Other Compounds in T. cordifolia

The details of the different constituents isolated and characterized in

T. cordifolia are as given below

Table 4.1: Name and reference of compounds in T. cordifolia

Sl. No. Name of compound Bioactivity, if any Reference

A: Terpenoids

1 Tinosporide Nil Hanuman et al., 1986a

2 Furanolactone diterpene Nil Hanuman et al., 1986a

3 Furanolactone clerodane diterpene

Nil Bhatt et al., 1988;

Hanuman et al., 1988

4 Furanoid diterpene Nil Bhatt and Sabata, 1989

5 Tinosporaside Nil Khan et al., 1989

6 Ecdysterone, makisterone, several glucosides

Nil Gangan et al., 1994

7 Phenylpropene, disaccharides cordifolioside A, B and C

Nil

Immunostimulation

Gangan et al., 1994;

Maurya et al., 1997

8 Cordifoliside D and E Nil Gangan et al., 1994

9 Tinocordioside, cordioside Nil Wazir et al., 1995

10 Palmatosides C and F Nil Gangan et al., 1994

11 Tinocordifolioside, Tinocordifolin

Nil Maurya and Handa,

1998

12

Tinosporafuranol, Tinosporafurandiol,

Tinosporaclerodanol, Tinosporaclerodanoid

Nil Ahmad et al., 2010

B. Alkaloids

1 Magnoflorine, Tembetarine, Berberine,

Nil Srinivasan et al., 2008

2 Choline, Palmatine, Jatrorrhizine

immunomodulation Sharma et al., 2012


3 1,2-Substituted pyrrolidine hypoglycaemic Mahajan et al., 1985

4 Choline Nil Bisset and Nwaiwu, 1983

5 N-formylannonain Immunomodulation Sharma et al., 2012

C. Lignan

1 3(a,4-dihydroxy-3-methoxybenzyl)-4-(4-hydroxy-3-methoxy benzyl)

Nil (Hanuman et al.,

1986b)

D. Glycosides

1 Tinoside, Tinocordiside, and tinocordifolioside

Increased phagocytic activity

(Sharma et al., 2012)

E. Steroids

1 Giloinsterol Nil (Kidwai et al., 1949)

2 ß-Sitosterol Nil (Khaleque et al., 1970)

3 20α- Hydroxy ecdysone Nil (Pathak et al., 1995)

4

δ-sirosterol, 2β,3β,14α,20β,22α-25hexahydroxyl-

5β-cholest-7-en-6-one

Nil (Ahmad et al., 2010)

F. Others

1 Giloin, Giloinin Nil Kidwai et al., 1949

3 Tinosporan acetate, Tinosporic acid, Tinosporal acetate

Nil Hanuman et al., 1986a

4 Tinosporidine, Heptacosanol, Cordifolone

Nil Khaleque et al., 1970

5 Tinosponone Nil Maurya et al., 1995

6 Tinosporic acid, tinosporal, tinosporon,

Nil Pradhan et al., 1997

7 Arabinogalactan immunomodulation Chintalwar et al., 1999

8 Policosanol immunomodulation (Thippeswamy et al.,

2008)

9 (1,4)-α-D-glucan immunostimulation (Koppada et al., 2009)

Chapter 3

Materials and Methods


4.3.1 Materials

LC–MS grade solvent, acetonitrile was purchased from Fisher Scientific

(Fair Lawn, New Jersey, USA). HPLC grade solvents were purchased form

Merck Specialities, India. Samples and solvents were filtered using the 0.45-µm

nylon filter (Millipore, USA). Ultra-pure water was prepared by using an ultra-

pure water purification system, PURELAB Option-Q, ELGA LabWaters, India.

The HPLC analysis was carried out in a SHIMADZU Prominence HPLC

LC20AP system (Shimadzu, Tokyo, Japan) with a UV-VIS detector SPD20A set

at 210 nm and the LC solution software program (Shimadzu, Japan). Enable

C18G column with 250 x 4.6 mm internal diameter, 5 µm particle size and 120 A˚

pore size from Shimadzu-Spinco Biotech, India was used for HPLC analysis. The

structure identification studies were conducted with the Waters Xevo G2 QTof

mass spectrometer (Waters, Milford, MA, USA) with an ACQUITY UPLC™

BEH C18 column (50 mm x 2.1 mm x 1.7 µm; Waters, Milford, MA, USA)

4.3.2 HPLC Analysis of T. cordifolia extracts

The Bioactive Fractions (F11 and F9) were analysed for the separation

of phytoconstituents in the solvent and polar/non-polar nature of the compounds

by HPLC analysis. The instrument used was the SHIMADZU Prominence

HPLC LC20AP system (Shimadzu, Tokyo, Japan) with a UV-VIS detector

SPD20A set at 210 nm. The LC solution software program (Shimadzu, Japan)

was used for instrument control and data analysis. The column and autosampler

temperatures were kept at 40°C and room temperature, respectively. The

reverse-phase chromatography was performed with an analytical Enable

C18G column (250 mm by 4.6 mm internal diameter, 5 µm particle size and 120

A˚ pore size). Mobile phase was filtered, degassed by passage through a 0.45-

µm nylon filter (Millipore, Bedford, MA) under vacuum, and sonicated for 30

min. The optimized method used a binary-gradient mobile phase with water

Materials and Methods 197

containing 0.1% TFA as mobile phase A and methanol as mobile phase B. The

following gradient program was used starting with 100% A and going to 0-5

min with 100-25% A, 5-15 min with 25% A, and 15-25 min with 25-100% A.

The flow rate was 0.9 ml/min, and the injection volume was 5 µl.

4.3.3 Structure Identification with UPLC system and Chromatographic conditions

The fractions with highest and least activity at active concentrations

were analysed for its chemical constituents with the Waters Xevo G2 Q-TOF

mass spectrometer which is a quadrupole orthogonal acceleration time-of-flight

(TOF) tandem mass spectrometer. The mass spectrometer consists of a Waters

Acquity H class Ultra Perfomance Liquid Chromatography (UPLC) system,

equipped with a quarternary pump solvent management system, an online

degasser and an autosampler. Chromatographic analysis was performed on a All

separations were carried out on an ACQUITY UPLC™ BEH C18 column (50

mm x 2.1 mm x 1.7 µm; Waters, Milford, MA, USA) at 30˚C. The mixture of

(A) 0.1% formic acid in water and (B) acetonitrile was chosen as the mobile

phase using a gradient program:

0 – 6 min : 10-95% B

6 - 6.5 min : 95% B

6.5 –7.5 min : 95-10% B.

The back pressure was set at 1500 psi. The nebulizer gas, nitrogen was

set at 900 L/h and the temperature at 350 ˚C under both negative and positive

ion electrospray ionisation (ESI) modes. The cone gas was nitrogen and was set

at a flow rate of 50 L/h, and the source temperature was set at 135˚C. The

capillary voltage was set at 3.5 kV and the cone voltage at 35 V. The ToF data

were collected between m/z 50 and m/z 1000 and the MS/MS experiments were

performed using a range of collision energies for fragment ion information. The


flow rate was 0.2 mL/min and the injection volume was 10 ul. All analyses were

acquired using an independent reference spray via the Lock Spray interference

to ensure accuracy and reproducibility. The use of the Lock Spray allows the

accurate determination of the molecular masses of the precursor ion and the

product ions and consists of leucine-enkephalin ([M+H]+m/z 556.2771) as a

reference compound (Sztáray et al, 2011 and Zhou et al, 2014)

The data was collected with the use of MSE centroid technology was

used in which two separate scan functions were programmed for the MS

acquisition method. The first scan function was set at low collision energy (6

eV) which provided parent ions, and the second scan function was set a high

collision energy (ramped from 20 to 30 eV) which provided fragment ions. The

sample analysis was performed in triplicate to test the repeatability, and the

results are acceptable. The accurate mass and composition for the precursor ions

and for the fragment ions were calculated using the Mass Lynx V4.1 software

incorporated in the instrument.

4.3.3.1 Optimisation of chromatographic and MS conditions

The chromatographic conditions were optimised for the better separation

of the components in each fraction. The MS spectra were studied in both

positive and negative electrospray ionization modes. The positive mode is being

shown as it had better sensitivity of ion response than the negative mode,

making it easier to confirm and identify the molecular ions in each peak. The

MS and MS/MS data of active dilutions of F11, F9 were characterized and

identified based on their retention behaviour and MS/MS data.

Chapter 4

Results


4.4.1 HPLC Analysis of the T. cordifolia extracts

The HPLC analysis of the T. cordifolia bioactive fractions were

conducted using a binary gradient of water containing 0.1% TFA and

Methanol in a ratio of 25 : 75%. The chromatograms were analysed for the

presence of phytoconstituents. The bioactive fraction 11 shows 8 major

peaks and fraction 9 shows 4 major peaks.

1. Bioactive Fraction of T. cordifolia (F11)

Figure 4.1: HPLC Chromatogram and Peak Table of Fraction 11

Results 201

2. Bioactive Fraction of T. cordifolia (F9)

4.4.2 Separation and Identification of chemical constituents by UPLC / Q-TOF-MSE

The mass spectrometry data obtained for the T. cordifolia methanol

extract, water extract and bioactive fractions were processed using the

Waters Mass Lynx V4.1 software. The UPLC-MS data were detected and

noise-reduced in both the UPLC and MS domains such that only true

analytical peaks were further processed by the software. The Lock Spray

dual electrospray ion source optimizes the co-introduction of analyte and

lock mass compound directly into the ion source, providing authenticated

exact mass in MS and MS/MS modes to within 5 ppm RMS mass. The

increased specificity associated with exact mass measurement provides

improved efficacy in compound or fragment identification. Using Mass

Lynx, the calculated masses, mass accuracy (ppm) and FIT Conf % values

(the confidence of the i-FIT value which is the likelihood that the isotopic


pattern of the elemental composition matches a cluster of peaks in the

spectrum) were generated and studied. The mass error for molecular ions of

all identified compounds was within ±6 ppm The MS/MS data along with the

retention time of compounds are summarized in Table 4.2.

The study of the MS/MS data has led to 12 structures being identified

in F11, and F9 of which 7 compounds were found in both the fractions. The

structures identified were not reported earlier in T. cordifolia but were

reported in other plant sources with actions that justify medicinal properties

and reported activities in Tinospora. TC1, TC3-TC5, TC7-TC9 were found

in both the fractions while TC2 was found only in F11 and TC6 only in F9.

The details of the mass spectrum are given in Table 4.2 and the name and

structure in Table 4.3. The total ion chromatogram is given as Fig 4.3 and

Fig 4.4 while the MS/MS spectrum and structure identification of each

compound is given as Fig 4.5.A to 4.5.I.

Results 203

Table 4.2: MS/MS Data of Compounds Identified in bioactive fractions of T. cordifolia extracts

The table represents the retention time of each parent ion identified and the molecular weight determined for each mass. The mass error is indicative of the accuracy of the determined mass.

Name of Compound Plant Extract

tR

(min)

Elemental Composition

[M+H] +

Measured Mass

Theoretical Mass

Mass Error (ppm)

TC1 F11, F9 3.01 C20H19NO4 338.1377 338.1386 -2.66

TC2 F11 4.13 C14H20O5 269.1390 269.1383 2.16

TC3 F11, F9 4.19 C20H43NO3 346.3316 346.3315 0.28

TC4 F11, F9 4.65 C27H38O10 523.2524 523.2537 -2.48

TC5 F11, F9 4.66 C24H51NO4 418.3908 418.3890 4.3

TC6 F9 4.68 C29H38O8 515.2627 515.2639 -2.32

TC7 F11, F9 4.7 C24H36O9 469.2436 469.2432 0.85

TC8 F11, F9 6.2 C18H30O7 359.2038 359.2064 -7.23

TC9 F11, F9 6.2 C31H52O8 551.3572 551.3578 1.08


Fig 4.3: Total Ion Chromatogram of F11

Results 205

Fig 4.4: Total Ion Chromatogram of F9


Figure 4.5.A.1: MS/MS Spectrum at Mass 338

Results 207

Figure 4.5.A.2: MS/MS Spectrum of TC 1


Figure 4.5.B.1: MS/MS Spectrum at Mass 269

Results 209

Figure 4.5.B.2: MS/MS Spectrum of TC 2


Figure 4.5.C.1: MS/MS Spectrum at Mass 346

Results 211

Figure 4.5.C.2: MS/MS Spectrum of TC 3

Results 213

Figure 4.5.D.1: MS/MS Spectrum at Mass 523


Figure 4.5.D.2 MS/MS Spectrum of TC 4A

Results 215

Figure 4.5.D.3: MS/MS Spectrum of TC 4B


Figure 4.5.E.1: MS/MS Spectrum at Mass 418

Results 217

Figure 4.5.E.2: MS/MS Spectrum of TC 5


Figure 4.5.F.1: MS/MS Spectrum at Mass 515

Results 219

Figure 4.5.F.2: MS/MS Spectrum of TC 6


Figure 4.5.G.1: MS/MS Spectrum at Mass 469

Results 221

Figure 4.5.G.2: MS/MS Spectrum of TC 7

Results 223

Figure 4.5.H.1: MS/MS Spectrum at Mass 359


Figure 4.5.H.2: MS/MS Spectrum of TC 8A

Results 225

Figure 4.5.H.3: MS/MS Spectrum of TC 8B

Results 227

Figure 4.5.I.1: MS/MS Spectrum at Mass 551


Figure 4.5.I.2: MS/MS Spectrum of TC 9A

Results 229

Figure 4.5.I.3: MS/MS Spectrum of TC 9B


Table 4.3: Structure and Names of Proposed Compounds in Bioactive Fractions of T. cordifolia

Sl. No.

Name of Compound Name of Proposed Structure Proposed Structure

1 TC1 DihydroBerberine

2 TC2 1-(2,3,5,6,8,9-hexahydro1,4,7,10 -benzotetraoxacyclododecin-12-yl) ethanol

3 TC3 N-(2-Hydroxyhexadecyl) diethanolamine

Results 231

4 TC4

TC4A 3β,11α-Di-O-Acetylouabagenin

TC4B

Ester Of Glaucarubolone

[(1β,11β,12α,15β)-1,11,12-Trihydroxy-2,16-dioxo-11,20-epoxypicras-3-en-15-yl 2-ethyl-2-(hydroxymethyl) butanoate]

5 TC5 3,3'-(Octadecylimino) bispropane-1,2-diol


6 TC6 3β-Acetoxydeoxyangolensic Acid, Methyl ester

7 TC7 Cardivin B

8 TC8 TC8A (3a'S,4'R,5a'R,9a'R,9b'S)-4'-(Methoxymethoxy)-8',8'-dimethyltetrahydro-3a'Hspiro[cyclohexane-1,2'-[1,3]dioxolo[4,5-h][1,3]benzodioxin]-5a'(6'H)-ol

Results 233

TC8B (3a'R,4'R,5'R,7'R,7a'S)-7'-(Methoxymethoxy)-2'',2''-dimethyltetrahydro-'Hdispiro[cyclohexane-1,2' [1,3]benzodioxole-5',4''-[1,3]dioxolan]-4'-ol

9 TC9

TC9A

(2R, 3S,4aR,5S,8aS)-3-[(6-Deoxy-α-L-mannopyranosyl)oxyl]-5-[(3E)-5-hydroxy-3-methyl-3-penten-1-yl]-1,1,4a,6-tetramethyl-1,2,3,4,4a,5,8,8a-octahydro-2-naphthalenyl (2Z)-2-methyl-2-butenoate (sesquiterpene)

TC9B Blancasterol (secosteroid)

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