faculty of resource science and technology - ir.unimas.my studies and biological... ·...

Phytochemical studies and Biological activity of Goniothalamus macrophyllus

Nurfarahen binti Jamil (21981)

Bachelor of Science with Honours (Resource Chemistry)

2012

FacultyofResourceScienceandTechnology


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



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


Nurfarahen binti Jamil

Resource Chemistry Programe



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


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


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)

10

OH

OHO

O

O

O

OH

OH

(5) (6)

O

O

OH

OHO H

H

(7)

O O

O

O

OO

OH

O

O

O

O

HO

OH

HO

(8) (9)

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).

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