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Phytochemical studies and Biological activity of Goniothalamus macrophyllus
Nurfarahen binti Jamil (21981)
Bachelor of Science with Honours (Resource Chemistry)
2012
FacultyofResourceScienceandTechnology
Phytochemical studies and Biological activity of Goniothalamus macrophyllus
Nurfarahen Binti Jamil (21981)
The project is submitted in partial fulfillment requirement for the degree of Bachelor of
Science with Honors (Resource Chemistry)
Bachelor of Science with Honors (Resource Chemistry)
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
2012
ii
DECLARATION
No portion of the work referred to in this dissertation has been submitted in support of an
application for another degree of qualification of this or any other university or institution of
higher learning.
______________________
Nurfarahen Binti Jamil
Program of Resource Chemistry
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
iii
ACKNOWLEDGEMENT
First of all, I would like to take this opportunity to express my gratitude to my supervisor,
Prof. Dr. Fasihuddin Ahmad for his guidance and support in helping me to carry out this final
year project.
I would also like to thank Mr. Benedict, Mdm Norhayati Ms. Leida and Mr. Wahab for their
kind assistance especially during the operation of equipments and machines in the laboratory.
Last but not least, my appreciation also goes up to my family and friends for their endless
support throughout this project.
iv
TABLE OF CONTENTS
Declaration…………………………………………………………….. ii
Acknowledgement……………………………………………………... iii
Table of contents………………………………………………………. iv
List of Abbreviation and Symbols….…………………………………. vi
List of Tables…………………………………………………………… vii
List of Figures….………………………………………………………. viii
Abstract……………………………………………………………….. ix
Abstrak………………………………………………………………… x
1.0 Introduction………………………………………………………… 1
1.1 Problem Statement…………………………………………. 2
1.2 Objectives…………………………………………………... 2
2.0 Literature Review…………………………………………………... 3
2.1 Annonaceae………………………………………………… 3
2.2 Goniothalamus……………………………………………… 5
2.2.1 Uses of Goniothalamus sp. in traditional medicine. 7
2.2.2 Phytochemical study and biologically studies……. 8
2.2.2.1 Styryl-lactone…………………………… 8
2.2.2.2 Acetogenins……………………………... 12
2.2.2.3 Alkaloids……………………………….. 13
2.2.2.4 Flavonoid………………………………. 15
3.0 Materials and Method………………………………………………. 16
3.1 Plant Material………………………………………………. 16
3.2 General Procedures………………………………………… 16
3.3 Extraction………………………………………………….. 17
3.4 Isolation and Purification…………………………………... 17
3.4.1 Column Chromatography (CC)………….......... 17
3.4.2 Thin Layer Chromatography (TLC)…………… 18
3.4.3 Preparative Thin Layer Chromatography (PTLC) 18
v
3.5 Purity Detection………….………………………………… 18
3.5.1 Melting Point………………….………………. 18
3.6 Structure Elucidation………………………………………. 19
3.6.1 Fourier Transform Infrared Spectrometer…....... 19
3.6.2 Gas Chromatography-Mass Spectroscopy…….. 19
3.6.3 Nuclear Magnetic Resonance Spectroscopy….. 19
3.7 Biological Activities………………………………………. 20
3.7.1 Brine Shrimp Toxicity Test……………………… 20
4.0 Results and Discussion…………………………………………….. 21
4.1 Extraction of Goniothalamus macrophyllus……………….. 21
4.2 Isolation and Purification of crude extract………………… 21
4.2.1 Solvent partition of crude extract………………… 21
4.2.2 Column Chromatography of G. macrophyllus…… 22
4.2.2.1 Dichloromethane crude extract………… 22
4.2.2.2 Methanol crude extract………………… 26
4.3 Structure Elucidation………………………………………. 29
4.3.1 IDCM fraction…………………………………… 29
4.3.1.1 FTIR Result…………………………….. 29
4.3.1.2 GC-MS Result………………………….. 31
4.3.1.3 Melting Point…………………………… 32
4.3.1.4 NMR Result……………………………. 33
4.3.2 CMeOH fraction…………………………………. 37
4.3.2.1 FTIR Result……………………………. 37
4.3.2.2 GC-MS Result…………………………. 38
4.3.2.3 Melting Point……...…………………… 41
4.3.2.4 NMR Result……………………………. 41
4.4 Biological assay……………………………………………. 46
4.4.1 Brine Shrimp Toxicity Test………………………. 46
5.0 Conclusion…………………………………………………………. 49
6.0 References…………………………………………………………. 50
vi
LIST OF ABBREVIATION AND SYMBOLS
g = gram
kg = kilogram
mg = milligram
% = percentage
˚C = degree celcius
IR = infrared
UV = Ultraviolet- Visible
NMR = Nuclear Magnetic Resonance
CC = column chromatography
TLC = Thin-Layer Chromatography
vii
LIST OF TABLE
Table Page
Table 2.1 Medicinal Uses of some Annonaceae’s genus species 4
Table 2.2 Distribution of Goniothalamus sp. in Malaysia 5
Table 4.1 Weight of crude extract for leaves obtained by solvent partition 21
Table 4.2 Solvent systems used for column chromatography of DCM crude
extract obtained from the leaves of Goniothalamus macrophyllus 23
Table 4.3 Solvent system used to develop TLC plate for fractions from column
chromatography of DCM crude extract and the Rf value 24
Table 4.4 Combined fractions, colour and weight obtained from DCM crude
extract fractions for the leaves of Goniothalamus macrophyllus 25
Table 4.5 Solvent system used to develop TLC plate for fractions from column
chromatography of MeOH crude extract and the Rf value 27
Table 4.6 Combined fractions, colour and weight obtained from MeOH crude
extract fractions for the leaves of Goniothalamus macrophyllus 28
Table 4.7 Predicted functional group present in Compound 1 isolated from the
leaves of Goniothalamus macrophyllus 30
Table 4.8 Possibility type of H for Compound 1 35
Table 4.9 Possibility type of C for Compound 1 35
Table 4.10 NMR comparison spectral data of Compound 2 44
Table 4.11 Percentage of average death of Artemia salina and the LC50 value 46
viii
LIST OF FIGURES
Figure Page
Figure 4.1 FTIR spectrums for pure compound of white needle IDCM isolated
from the leaves of Goniothalamus macrophyllus 29
Figure 4.2 Gas chromatogram of white needle crystal of IDCM from leaves of
Goniothalamus macrophyllus (Compound 1). 31
Figure 4.3 Mass fragmentation of Compound 1 32
Figure 4.4 1H NMR for Compound 1 from leaves of Goniothalamus macrophyllus 33
Figure 4.5 13
C NMR for Compound 1 from leaves of Goniothalamus macrophyllus 34
Figure 4.6 FTIR spectrum for compound 2 isolated from leaves of Goniothalamus
macrophyllus. 37
Figure 4.7 Gas chromatogram of white solid from CMeOH from leaves of
Goniothalamus macrophyllus (Compound 2) 38
Figure 4.8 Mass fragmentation of Compound 2 39
Figure 4.9 Mass fragmentation pattern for peak at Rf 17.515 min 40
Figure 4.10 Mass fragmentation pattern for peak at Rf 19.135 min 40
Figure 4.11 1H NMR for Compound 2 from leaves of Goniothalamus macrophyllus 43
Figure 4.12 13
C NMR for Compound 2 from leaves of Goniothalamus macrophyllus 44
Figure 4.13 Graph of percentage death of Artemia salina vs Log concentration 48
ix
Phytochemical studies and Biological activity of Goniothalamus macrophyllus
Nurfarahen binti Jamil
Resource Chemistry Programe
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
Phytochemical and biological studies have been carried out on the extract obtained from the
leaves of Goniothalamus macrophyllus. Extraction using methanol gave dark green extract of
33.27 g (7.25%). Solvent partition was carried out using solvents with increasing polarity in
the order of hexane, dichloromethane and ethyl acetate. Isolation and purification were
performed on dichloromethane and methanol partition using column chromatography. Two
semipure compound namely Compound 1 and Compound 2 corresponding to molecular mass
of 128 g/mol and 200 g/mol, respectively were isolated. Compound 1 was isolated as white
crystal needle with melting point in the range of 115.0-117.0 C and Rf value of 0.52 in
dichloromethane:ethyl acetate (1:1). Infrared spectrum revealed strong absorption band at
frequency 1689 cm-1
that was assigned to C=O. Based on the NMR spectrum, the compound
have 11 protons and 10 carbons. Due to impurities, the information was not enough to
elucidate the structure of Compound 1. Compound 2 was isolated as white solid with melting
point in the range 84.5-86.0 C and Rf value of 0.82 in dichloromethane. Based on the 1H and
13C NMR spectrum, it indicates there were 12 protons and 13 carbons. Based on the spectral
data on published data, Compound 2 has been identified as goniothalamin. The brine shrimp
toxicity test showed that none of the extracts were toxic to Artemia salina.
Keywords : Goniothalamus macrophyllus, chromatography, spectroscopy,
goniothalamin, brine shrimp toxicity test
x
ABSTRAK
Kajian fitokimia dan biologi telah dijalankan ke atas ekstrak daun Goniothalamus
macrophyllus. Proses pengekstrakan telah dilakukan menggunakan metanol dan memberikan
sebanyak 33.27 g (7.25%) ekstrak berwarna hijau gelap. Pemfraksian telah dilakukan
menggunakan pelarut dengan pertambahan kepolaran iaitu heksana, diklorometana dan etil
asetat. Pemisahan dan penulenan telah dilakukan ke atas partisi diklorometana dan metanol
menggunakan kromatografi turus. Dua sebatian yang dilabelkan sebagai sebatian 1 dan
sebatian 2 dengan jisim molekul 128 g/mol dan 200 g/mol masing-masingnya telah berjaya
dipisahkan. Sebatian 1 dengan jisim molekul 128 g/mol diperolehi sebagai kristal jarum putih
dengan takat lebur 115.0-117.0 C dan memberikan nilai Rf 0.52 dalam pelarut
diklorometana:etil asetat (1:1). Spektrum inframerah menunjukkan penyerapan kuat pada
frekuensi 1689 cm-1
yang menunjukkan kehadiran ikatan C=O. Berdasarkan maklumat
spektroskopi NMR, Sebatian 1 mempunyai 11 hidrogen dan 10 karbon. Pembinaan struktur
sebatian 1 tidak dapat dilakukan kerana sebatian yang kurang tulen. Sebatian 2 dengan jisim
molekul 200 g/mol telah diperolehi sebagai hablur putih dengan takat lebur 84.5-86.0 C dan
memberikan nilai Rf 0.82 dalam pelarut diklorometana. Berdasarkan maklumat 1H dan
13C
NMR spektrum, sebatian 2 mempunyai kehadiran12 proton dan 13 serta berdasarkan data
spektroskopi dan perbandingan dengan data yang telah diterbitkan, sebatian 2 telah
dikenalpastikan sebagai goniothalamin. Ujian ketoksikan ke atas anak udang menunjukkan
kesemua ekstrak tidak toksik terhadap larva Artemia salina.
Kata kunci: Goniothalamus macrophyllus, kromatografi, spektroskopi, goniothalamin, ujian
ketoksikan anak udang.
1
1.0 INTRODUCTION
1.1 General
Herbal plants are the sources of natural remedy for traditional medicine long time
before modern science occupied our today’s livelihood. Goniothalamus is one of the genus in
the Annonaceae family and it is also known as the custard apple family, a floral family consist
of 160 genera and over 2000 species found in Africa, South America, Southeast Asia and
Australia (Wiart, 2007). Many part of the genus had been used to treat various illnesses such
as the bulbs, stems, roots, and leaves (Wiart, 2007). Therefore, it had been widely studied by
chemist for its chemical constituent due to their various uses in traditional medicine. Previous
studies have shown that styrylpyrone found within the Annonaceae family is one of the
bioactive styryl-lactone, which possess medicinal properties against various diseases and
cancer (Jewers et al., 1972; Nasir et al., 2004).
In Malaysia, decoctions of the roots of Goniothalamus macrophyllus are used to stop
early pregnancy and as protective medicine after parturition, while the leaves is beneficial for
steaming the body when fever (Wiart, 2000).
Wiart (2007) reported that out of 160 species of Goniothalamus, around 22 species
have been phytochemically investigated and the two distinct classes of secondary metabolites
that had been isolated are acetogenins and styryl-lactones. Therefore, there are still a lot of the
species that are scientifically uninvestigated and lack of its chemical constituents information.
2
1.2 Problem statement
Based on available informations, there are many benefits from Goniothalamus spp.,
which mostly contribute to the traditional medicinal uses and previous studies showed that
acetogenins, styryl-lactone and alkaloid from Goniothalamus spp. are cytotoxic against cancer
cell lines. Since only a limited number of scientific researches had been performed on the
phytochemical and biological activities on Goniothalamus spp., especially in Sarawak,
Malaysia, this study is important to obtain comprehensive information on the secondary
metabolites and biological activity of Goniothalamus sp.
1.3 Objectives
The general objectives of this study are to extract, isolate, purify and elucidate the
chemical structures and to perform biological test on the leaves of Goniothalamus
macrophyllus. The specific objectives are:
- To extract leaves of Goniothalamus macrophyllus by using different solvent
system.
- To isolate and purify the chemical constituent using various chromatography
procedures.
- To elucidate the structure of purified compound by using various spectroscopy
information such as infrared spectrometry, mass spectrometry and nuclear
magnetic resonance.
- To perform biological activity testing of the crude extracts, partitions, semi-pure
compounds and pure compounds isolated against brine shrimp, Artemia salina.
3
2.0 LITERATURE REVIEW
2.1 Annonaceae
Annonaceae are mostly found in the tropical and subtropical region and it is a large
family of trees and shrubs. Other than that, it is also known as the Custard Apple family.
Mostly they are important sources of edible fruits and perfume material. The Annonaceae
flowers have three sepals and two whorls of three petals. The flowers have bisexual system,
with numerous free stamens and carpels. The stamens have large apical connectiveness that is
variable in shape and the apocarpus fruits are taxonomically very important, with variation in
size, shape, indumenta, peduncle and stripe length (Saunders, 2003).
Annonaceae plants are also used widely as traditional medicine as antimicrobial (Khan
et al., 2002; Rahman et al., 2005); insecticidal (Khan et al., 2002; Rahman et al., 2005);
antiparasitic (Sahpaz et al., 1994) and also anticancer properties (Alali et al., 1999). Previous
studies shows that Annonaceous acetogenins have antitumor, anti-parasiticidal, pesticidal,
antimicrobial and immunosuppressive activities (Alali et al., 1999). Furthermore, acetogenins
inhibits the ubiquinone-linked NADH oxidase that is constituently present in the cell
membrane of cancer cells, but only transiently expressed in normal cells (Morre et al., 1995).
Other than that, there are many genus under the Annonaceae family and several are
known to have medicinal uses other than Goniothalamus spp. Table 2.1 shows the medicinal
uses of some members in the Annonaceae family.
4
Table 2.1: Medicinal Uses of some members in Annonaceae family.
Species Medicinal Uses
Annona muricata - Leaves used as insecticide, externally used for
rheumatism and applied for scabies and skin
diseases.
- Green barks and leaves used to wash ulcer and
wound.
Cananga orodata - Barks used for scabies.
- Essential oil of the plant used as one of the
ingredients for cephalgia, ophtalmia and gout.
- Flower used to cure malaria.
- Paste of the flower used to treat asthma by applying
it to the chest.
Cyathostemma argenteum - Used to treat cancer
- Barks used as antispasmodic
Desmos chinensis - Roots of the plant used for treating diarrhea,
dysentery, vertigo and post partum medicine.
Fissistigma sp.
- Root and flower used in treating stomach-ache.
Polyalthia cauliflora - Paste of the leave used to cure skin diseases.
Uvaria grandifolia - Treating stomach-ache and as post partum
medicine.
Adapted from Wiart (2000). *continued from table 2.1
5
2.2 Goniothalamus
Goniothalamus is one of the largest genera in the Annonaceae family (Saunders,
2003). There are about 44 species of Goniothalamus found in Malaysia (Din, 2000). Table 2.2
shows the distribution of Goniothalamus species found in Malaysia.
Table 2.2: Distribution of Goniothalamus spp. in Malaysia.
Species Borneo (Sabah and Sarawak) Peninsular Malaysia
G. andersonii Endemic in Borneo Not found
G. borneenis Endemic in Borneo Not found
G. bracteosus Endemic in Sabah Not found
G. calcareous Endemic in Sarawak Not found
G. calycinus Not found Easily found
G. clemensii Endemic in Sabah Not found
G. crockerensis Endemic in Sabah Not found
G. curtisii Not found Endemic
G. cylindrostigma Endemic in the south of Sarawak Not found
G. dolichocarpus Endemic in Sarawak Not found
G. fasciculatus Endemic in Borneo Not found
G. fulvus Not found Easily found
G. giganteus Not found Easily found
G. gigantifolius Endemic in Sabah Not found
G. holttumii Not found Bukit Fraser
6
Species Borneo (Sabah and Sarawak) Peninsular Malaysia
G. kinabaluensis Endemic in Sabah Not found
G. longistipites Endemic in Sarawak Not found
G. macrophyllus Easily found Easily found
G. malayanus Easily found Easily found
G. marcanii Not found Endemic
G. montanus Easily found Easily found
G. nitidus Endemic in Sabah Not found
G. parallelivenius Endemic in Sarawak Not found
G. puncticulifolius Endemic in the East of Sabah Not found
G. ridleyi Not found Easily found
G. roseus Endemic in Gunung Kinabalu Not found
G. rostellatus Endemic in Sabah Not found
G. rotundisepalus Not found Endemic
G. rufus Easily found Easily found
G. scortechinii Not found Easily found
G. sinclairianus Endemic in Sarawak Not found
G. stenopetalus Endemic in Gunung Kinabalu Not found
G. stenophyllus Endemic in Sarawak Not found
G. subevenius Not found Endemic
G. tapisoides Endemic in Sarawak Not found
G. tavoyensis Not found Endemic
7
Species Borneo (Sabah and Sarawak) Peninsular Malaysia
G. tenuifolius Not found Easily found
G. tortilipetalus Not found Easily found
G. umbrosus Not found Endemic
G. undulates Not found Endemic
G. uvaroides Easily found Easily found
G. velutinus Endemic in Borneo Not found
G. woodii Endemic in Borneo Not found
G. wrayi Not found Easily found
Adapted from Din (2000). *continued from table 2.2
The botanical characteristics of Goniothalamus species are simple, and it is strongly
aromatic bark, having few simple, alternate and exstipulate leaves. Furthermore, the
secondary nerves are oblique, straight and parallel with scalariform reticulations (Wiart,
2006). The flowers are axillary and characteristically woody, fusiform and often dark green
(Saunders, 2003).
2.2.1 Uses of Goniothalamus spp. in traditional medicine
Goniothalamus spp. is widely used in traditional medicine by the local folks. Local
woman in the hilly regions of India consumed dried powdered leaves of G. sesquipedalis
during labor pain and for external uses and the leaves were used as mosquito repellents (Goh
et al., 1995). The seeds extract of G. amuyon has been used for the treatment of edema and
rheumatism (Kan, 1979).
8
Decoction of G. macrophyllus was used to nurture the blood by invigorating the body
by the ethnic group of Sakai (Thonghom, 1993). In Malaysia, various types of plant for
example, G. macrophyllus, G. malayanus and G. scortechinii are often used as post-natal
treatment and for abortions (Burkill, 1966). The leaves of G. macrophyllus was used to treat
cold and fever Wiart (2000) and the roots of G. malayanus was used for the treatment of
rheumatism and fever, while the barks has been used to treat measles and as insect repellents
(Mat Salleh and Ahmad, 1989). In Borneo, Goniothalamus spp. had been used ever since,
especially in treating diarrhea, fever, skin diseases, antidotes and most commonly for
postparturation aids and as abortifacient medicine (Ahmad, 2004).
2.2.2 Phytochemical studies of Goniothalamus spp.
So far, about 22 out of 160 species of the Goniothalamus spp. had been
scientifically investigated (Wiart, 2007). Some of the Goniothalamus spp. that had been
studied phytochemically resulted in the isolation of various interesting secondary metabolites
especially styryl-lactones, alkaloids (Zhang et al., 1999; Hisham et al., 2000; Ahmad, 2004),
and Annonaceous acetogenins (Fang et al., 1992).
2.2.2.1 Styryl-lactone
Styryl-lactone is a secondary metabolite that consist at least one six-membered ring of
benzene, mainly isolated from the Goniothalamus genus. Sam et al., (1987) reported the
isolation of goniothalamin (1) and goniothalamin oxide (2) from the stem bark of G.
macrophyllus. (5S,6R,7S,8S)-Goniotriol (3) and (6R,7R,8R)-goniodiol-8-monoacetate (4)
9
were isolated from the leaves of G. amuyon (Lan et al., 2003). Three other compound of
styryl-lactone characterized as goniobutenolides A (5), B (6) and goniofupyrone (7) have been
isolated from the bark of G. gigantues (Fang et al., 2001). Complex styryl-lactone such as
goniolactones A-D (8-11) were isolated from the roots of G. cheliensis (Wang et al., 2002),
while cardiopetalolactone (12), cardiobutanolide (13), altholactone (14), and goniopypyrone
(15) were isolated from of G. cardiopetalus (Hisham et al., 2000). Four derivatives of
goniothalamin identified as goniotriol (3), altholactone (14), goniopypyrone (15) and
goniofufurone (16) were isolated from the fruit peel of G. scortechinii (Abdullah et al., 2009).
O O
O
O O
(1) (2)
O
OH
O
OH
HO
O
OH
O
OAc
(3) (4)
11
O
O
O
O
HO
HO
OH
O O
HO
O
OOH
HO
HO
(10) (11)
O
O
OH
O
O
HO OH
HO
HO
(12) (13)
O
O
O
OH
O
O
O
OH
HO
(14) (15)
12
HO
O
O
O
OH
(16)
2.2.2.2 Acetogenins
Annonaceous acetogenins are promising new antitumor and pesticidal agents (Alali et
al., 1999). This class of acetogenins usually possesses a terahydrofuran (THF) ring with one
or two hydroxyl and various terminal lactone rings (Kojima and Tanaka, 2009). Other than
that, it is also known as natural polyketides. Many acetogenins had been isolated from
Goniothalamus species, such as goniothalamusin (17) from G. gardneri (Seidel et al., 1999),
giganin (18) from G. giganteus (Fang et al., 1993), donnaienin A (19) and donnaienin B (20)
from G. donnaiensis (Jiang et al., 1997). Compound (18) was believed to be the first bioactive
annonaceous acetogenin that lacks tetrahydrofuran or epoxide rings along the aliphatic chain
(Fang et al., 1993).
O
O
HOH2C
(17)
(CH2)13
OH
OH
(CH2)5
OH
O
O
OH
(18)
13
OO
OH
OH
OOH
OH
(19)
O
OH
O
OH
O
OH
OH
OH
(20)
2.2.2.3 Alkaloids
Alkaloids are a group of chemical compound that contain nitrogen in the molecules.
For example, azaanthraquinones such as, marcanine A-E (21-25) and dielsiquinone (26) from
G. macrophyllus (Soonthornchareonnon et al., 1999) and griffithazanone A (27) from G.
griffthii (Zhang, 1999) have been isolated. Various aristolactams such as taliscanine (28),
aristolactam AII (29), cepharanone B (30) and velutinum (31) and griffithinam (32) from G.
griffthii have been isolated (Jun, 1999; Zhang, 1999). Griffithdione (33) from G.griffthii (Jun
et al., 1999) and amino-napthoquinones (34) are some of the alkaloids that had been reported
in Goniothalamus genus (Phetkul, 2009).