PHYTOCHEMICALAND BIOWGICAL STUDIES ON GONIOTHALAMUS MALAYANUS
Hamdan Bin Asfiar
Bachelor of Science with Honours QIC 861 (Resource Chemistry) 8198 2005 2005 Pusat Khidmat Makiumat Akade UNIVERS1TI MALAYSIA SARAWA 94 ()() Kola Samarahan
P.KHIDMA TMAKLUMATAKADEMIK UIIMAS 1111111111111111111111111 1000127066 PHYTOCHEMICAL AND BIOLOGICAL STUDIES ON GONIOTHALAMUS MALAYANUS
HAMDAN BIN ASFIAR
This project is submitted in partial fulfillment of the requirements for the degree of Bachelor of Science with Honours (Resource Chemistry)
Faculty of Resource Science and Technology UNIVERSITI MALAYSIA SARAWAK 2005 DECLARATION
No portion of the work referred to in this dissertation has been submitted in support or nn application for another degree of qualification of this or any other university or institution of
higher learning.
Hamdan bin Asfia r Program of Resource Chemistry Faculty of Resource Science and Technology Univcrsiti Malaysia Sarawak
II ,.....
ACKNOWLEDGEMENTS
Firstly, to the God , for His Humanity and Power to His creatures to let me finished this tinal year
project. Then , I would like to express my highest gratitude to my respective supervisor, Assoc.
Prof. Dr. Fasihuddin Ahmad tor all invaluable guidance and suppOli in the fulfillment of thi s
tinal year project and to all lectures for their precious information. This special
acknO\",ledg~mcnt also goes to everyone who always giving me inspirations and hopes l:specially
for the laboratory assistants for the help while working in the laboratory and to my fellow frit:nd s
who always giving me information related to my project. Not forget, to my loving family who
always praying for my success upon their prayer and support.
III TABLE OF CONTENTS
DECLARATION II
ACKNOWLEDGEMENTS III
TABLE OF CONTENTS IV
ABSTRACT VI
ABSTRAK VI
CHAPTER 1: INTRODUCTION
CHAPTER 2: LITERATURE REVIEW 3
CHAPTER 3: MATERIALS AND METHODS 8
3.1. Plant materials 8
3.2. General 8
3.3. Extraction 9
3.4. Isolation and purification 9
3.5. Structural determination II
3.6. Bioassay II
3.6.1. Brine shrimp toxicity test II
3.6.2. Antifungal toxicity test 12
CHAPTER 4: RESULTS AND DISCUSSION 13
4.1. Extraction, isolation and purification of G. 111l11uy anlls sample 13
4.2. Structural determination of isolated compounds 16
4.3. Brine shrimp toxicity test 17
IV 404. Antifungal toxicity test 20
CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS 24
REFERENCES 25
APPENDICES
v Phytochemical and biological studies on Goniothalamus malayanus
Hamdan bin Asfiar
Resource Chemistry Faculty of Resource Science and Technology Universiti Malaysia Sarawak
ABSTRACT
Phytochemical and biological studies have been carried out on the stem bark of COlliol/wlUlIIIlS IIll1lc~\ ·( lI1l1s. In the study, extraction has been carried out using three different rypes of <;olvents; hexane, et hyl acetate and ethanol, followed by thlctionation and puri tication. Ethyl acetate crude ('xtract was subjected to Tl,C analysis and yielded 4 spots with Rr values of' 0.75. 0.81. 0.87 and 0.91 in dichlowmethane-ethyl acetate-acetone (4 :2: I) solvent system. For column chromatography, 19 combined Ira tions were obtained. Fraction GCO I was subJected to PTLC analysis ami resulted 3 compounds namely GCOI (i), GCOI (ii) and GCOI (iii) ',.\ith Rr values 01'0.77, 0.90 and 0.93. Compound GCOI (iii) was the most pure compound than GCOI (ii) and GCOI (iii) with the highest peak at retention time or '2_.799 min, Brine shrimp toxicity test performed on crude extracts and lI'actions revealed that crude extracts and ti'actions have low toxicity on Arlemia salina except for fraction GCO I which gave LC -II value of 100 pg/mL.. All the crude extracts also showed moderate toxicity against fungus PI'CI/OP0I'IIS spp. and .\"chi=ophyllunI comrnllne.
Keywo rds: (JOl1iolhu/omlls ll1u/oYUI1US, phytochemical studies, column chromatography, thin layer chromatography, biological activity.
ABSTRAK
Kldial/ ,lilokill1i(/ d(/II hi%gi Iclah dija/ankon ke alas kulil hal({ng GrJlliolha/al11l1.1 I/w/am/ll/s. PmsL's /II!IIgl'kslrakulI ((:/ali Ji/akukan d(!llg011 lI1enggltnukUI1 3 j(!l1is edaml yang ha/uil/all iailll heksw/(J, eli/ lIsdal dun dallo/ diikuli dellgall proses pemfi'oksia/1 dall penll/enal1. Anu/isis KLN I(!/uh dilukllkul1 ke (flus rksll'lIk klislIr eli/ uselal dun --I lilik dmgull lIilaj R( {). 75. O.SI. 0.87 dan 0.91 £la/am sis/em p(!/al'lll Jik/oromelal1£1-c/i/ uselal-useIO// (4:2: I) telah dipero/ehi. Km/7/ulogr(!fi IlIrlIS Ie/ah Jnl!l71herikall ga/?lIngal1 /9 paksi. Frob-i CCr)f lelah dillji dengan sis/em KLNP meng/wsi/kun 3 kOI11/)()J1f! 1I dUll di/alle/aka" sehugui (J( 'f) I (i). C( '() / Oi) dOli ceo I (iii) dellgol/ jiJk(or pl:'lIu/llmUII 0. 77. a.l)() dUll U 93 lIl£lsillg-lJ1asingn,l'u. Sl!hulia/1 CCO I (i) ({datah sehalian YOllri paling fltll!n herhanJing (J( '0 I (ii) dUll (iC() / (iii) dl!ngan pUl1cok lerlinggi pm/a ni/oi jJellahonon 22.7W. Ujian keloksikall diia/ullk({1/ ke ulav eks/rak k(/sa/' dall .!i-aksi lIIell1l11jllkkan ke(oksikun .l'Ul1g renda/i ler/wdap Arleflliu .w/il1({ keel/u/i ./i'aksi (j( 'O J yal1g melllherikan LC, I\,I/a k1l116: (jolliol/1l1/0111I1S I//alavunlIS, kajial'l filakimia. kromalogl' !II III/,u.I'. kroll1ologro/i /api.IUlI l7iJlis. ;"i/i hi% gi. VI CHAPTER 1 INTRODUCTION GoniOfhlJ/alllus is the second largest genus of Annonaceae and comprises I 15 species of , hrubs and trees growing in Asia cspecially South-East Asia and many of them arc used in the t{ II,. medicine in sevcral countries (Surivct and Yatele. 1999). More than 150 taxa have becn desc ribcd in Gonioflw/lJn1us; about 120 species and 10 subspecies are currently recognized (Mat Sallch. 1993). Members orthe genus are widely used in traditionalmcdicinalpractices in Asia. Many novel and clinically bioactive compounds have been isolated from Gonioflia/lIl11us species (Mat Salleh. 1993). They are widely employed in traditional medicine. alone or as part of herbal mixtures. as post-partum protective remedies for women after delivery, abortifacients and insects' repellcnts (Seidel el a/., 2000). In Malaysia, a decoction of the roots is used for treating colds and likewise a steam bath IS Llsed for treating fevers. For example, Goniotha/an1l1s n1a/ayanlls is used as ab0l1iracient. treatment atter delivery, to cure headache, fever, diarrhea, measles and rheumatism (Mat Salleh and Ahmad, 1989 ; Latiff. 1991). A decoction of Goniofha/an1us l11ac;rophyllus roots is taken orally to stOp early prcgnancy and as protective drugs after childbirth (Wiart. 2000). (ioniofha/amlls n1a/ayalllls and Goniolha/amus scorlechinii are believed to be effective as agents of abortion or contraception (Mat Salleh, 1993). Goniotha/an1l1s curfisii is one of the most popular concoctions for the treatment of stomachaches (Mat Salleh, 1993). Plant parts from some species of this genus have been used traditionally as insecticides. The smoke from the barks can be used to repel mosquitoes, snakes and other wild animals. ror e .Imple. the bark of (joniotha/ol11l1s macrophyllus is used to repel mosquitoes (Secoy and Sm ith. J983 ; Morton, 1987). The fragrance associated with the stems of Gvnio/halamus, c pecially GOl7io/halamlls vell//inus is said to keep away bad spirits and dangerous wild animals 'uch as snakes, elephants or tigers (Mat Salleh, 1993). Most of the Gonio/halamlls are concentrated in Borneo with high percentage of endemics. Borneo has a remarkable representation of the richness and diversity of the genus (Mat Salleh. 19(3). A total of 30 species, including new species, of the genus are recogn,ized from Borneo. Twenty four species are local endemics and 10 species from Borneo are known from very few localities. This shows that most of the Gonio/halumus species are locally evolved in Borneo. Some of the Gvnio/hulamus species are Goniv/haLamus m alayan us, Goniv/haLaml/s Kigunlells. GOl1io/halamLis uvaroides. Gonio/haiamlls do/ichvcarpus, Gvnio/huiwl1us jt,,\'(:icl1luntus. Gonio/halamus tomcn/OSlIS, Goniv/haial1111S lapis. Goniothalall1l1s macroph.vllus. (ionio/halal11l1s s/el/ope/aILls, Goniothalamus c1emensii and Gvniv/haiamus montanus. The objectives of this project are to isolate, purify and characterize biological active compounds frol11 GOl1io/halamus malayanus and to perform toxicities studies on brine shrimp, Ar/emill salina and fungus, Pycnopol'1ls spp. and SchizophvLLum CO/11/1l/.l/1 e. 2 ,.... r CHAPTER 2 LITERATURE REVIEW (joniofhuIUf11I1S maluyantls Hook f. & Thoms is a small tree of about 5-10 m tall with smooth bark gro\Vs mostly in the swampy areas (Mat Salleh, 1993). The distribution of GOl1io/halamlls sp cies is endemic throughout Malaysia. The di stribution of Gonio/ha/amus spp. ill Malaysia is sho\\n in Table I. Table 1: Distribution of (Jol1iolhalamlls spp. in Malaysia (Mat Salleh. 1993). Species Locations - U curtisii Kelantan, Perak, Selangor (j. IIl/1hmSlIS Peninsular (j. giJl,un/el1s Peninsular, Sarawak (j. Ina/ayanus Peninsular except Perl is & Malacca, Sarawak, Sabah Ci. rid/e.vi Peninsular, Sarawak, Sabah (i. Inacrophylllls Peninsu'lar except Perl is, Sarawak, Sabah (i. IIvamides Kedah, Perak, Pahang, Sarawak, Sabah G. tapis Penang, Perak, Johor, Sarawak U. undersonii Sarawak 0 . horneensis Sarawak 0. sindairianlls Sarawak (i. ve/1I1inus Sarawak, Sabah ( i. / asciculu/lls Sarawak 3 vemiolha/amus spp. are widely used in traditional medicine in Malaysia. For example. (ioniolhala177l1s gigal1leus is used for the treatment after giving birth (Wiart. 2001). The uses of some other 'pecies of Goniolha/all1us in traditional medicine are given in Table 2. Table 2: Medicinal uses of some (}oniot/wlamus species Species Medicinal uses G. lI1a/ayal1 /I.~ Agents of ab0l1ion or contraception. lIsed to cure measles. rheumatoid, as treatment of fever, typhoid and post partum medicine G. seorlechinii Agents of abortion or contraception and post partulll medicine Ci. curlisii Treatment of stomachaches and d i.arrhea G. W11l(\ /OI1 Treatment of rheumatism, scabies and other dermatitis (i. IIl'amides Treatment of stomachache, diarrhea and post parturition G. giga171e/I\' Abortifacient (i. lapis As tonic. to reduce cough, ab0l1ifacient and treatment for stomachache The gen us (}oniolhalamus (Annonaceae) is well known as an interesting source of various bioactive compounds sllch as styryl-Iactones (Sam el al., 1987 ; Fang el a/.. 19903 : Fang III al .. 1993 ). acctogenins (Fang el al .. 1992; Gu el a/ .. 1994), alkaloids (Taiapatra ef a/. , 1988 ; Din el al.. 1990 ; Omar el al.. 1992), and llavanoids. About 20 species of Goniolha/al17l1s have been investigated and the pr~dolllinallt isolates ve been styryl-Iactones (Bermejo e/ a/.. 1998). acetogenins (Zafra ~ Polo e/ ul.. 1(98). and 4 isoquinoline-dcrived alka'ioids (Omar e{ af., 1992) with significant cytotoxic, insecticidal and 4 antimicrobial activities. Styryllactones and acetogenins possess complex stereochemistry and appear in different stereoisomeric forms with varying levels of cytotoxicity towards several human tumor cell lines (Hisham el af., 2000) . . e eral turano-pyrone semisynthetic derivatives found to possess cytotoxicities and inhibitory 011 L-1210 leukemia line (Bermejo e{ of.. (999).The styryl-pyrone derivatives found abundanti) in the genus Goniolhafalllus species have also been investigated for cytotoxic and antitumor properties (Ali el 01.,1997; Cao el al., 1998; lIawariah and Stanslas, 1998; Bermejo el til.. 19(9). Plant styryl-Iactone derivatives isolated from Goniolhalalllus spp. are potential compound for cancer chemotherapy (Inayat-Hussain el £If., 2002). Styryl-Iactones are an interesting gr up of bioactive compounds with significant cytotoxicities against several human tumor cell li nes (Blazquez eI 01.. 1999). For example, altholactone (Inayat-Hussain el £II., 2002) isolated from Gonioll/{/lwlIlIS mtlft~ranlls inh ibited the mechanism ofapoptosis on human HL-60 promyclocytic leukemia cells. Goniothalam in, a plant styryl-Iactone isolated from Goniolhalamus mafayanus is a potential cytotoxic c mpound especially by inducing apoptosis in a variety of tumor cell lines (Ali e/ al.. 1997: lIa\\ariah and Stanslas, 1998; Inayat-Hussain el (/1.. 2000). 3-acctylaltholactone isolated from Gonio/ha/wlIlIs arvensis and two other known compound previously isolated from several Gonio{h£lfamlls spp. and 5-acetoxyisogoniothalamin oxide showed cytotoxicity based on inhibition of mammalian mitochondrial respiratory chain (Pcris el al.. 2000). Thus, they might be exploited for biomedical research, antitumor therapy and lchemical pest control like other respiratory chain inhibitors (Peris el al., 2000). 5 Cdgunin isolated from the bark of Goniothalamus gigal1lells also showed strong cytotoxici l. (Fang el al., 200 I a). Gonioheptol ides A and B isolated from (Jo17iolhalal1111S gigcmlell.'i showed marginal cytotoxicities to celtain human solid tumor cells in culture (Fang el ClI.. 200 I b). Goniobutenolides A and Band goniofupyrone ('rom Coniolhalamlls giganlells shO\\cd cyt toxicity to human tumor cells in culture (Fang el al., 200 I c). Gi gantransenins A. Band C isolated from Goniolhalamus giganleus showed selective inhibitory ctl~c ts on the human breast tumor cell line (MCF-7) (Zeng el al., 1998a). Several acctogenins showed interesting biological activities. Bioactive compounds such as cis gigantrioncni n and 4-acetyl gigantetrocin A isolated from C;oniolhalallllls giganlells showed significant hioactivities on various human solid tumor cell lines (Zeng el al., 1998b). Goniothalesdiol isolated from Goniolhalamlls hurneensis showed cytotoxic activities against P3 88 mouse leukemia cells and insecticidal activities (Cao el al., 1998). Goniothalenol. pyranicin. pyragonicin and goniotrionin are cytotoxic compounds isolated from GOl1ioiho/lIf1lllS spp. showed cytotoxicity on various cell lines (Alali el al., 1998). The chemical structures of some compou nd · isolated from Goniolhalamus spp. are shown in Figure I. 6 HO OH OH HO OH Cardiobutanolide Goniothalesdiol o o OH (+ )-garvensintriol (+ )-etharvendiol NH HO OH OMe Goniothalactam Gonioheptolides A OH H OH 0 ~O -:::::: HO 0 0 ~ 0 H Goniobutenolides A Gon iofupyrone igure t: Chemical structures of some biological active compounds isolated from Gonio/hulumus spp. 7 CHAPTER 3 MATERIALS AND METHODS 3.1. Plant Material Goniothulumm mulayul1l1s was collected from the forests around UNIMAS. Voucher specimen was prepared for the purpose of ,identification. The plants were separated into leaves, stem, barks and roots. Pl ants parts were air-dried before ground to fine powder. Stem barks was used for funher studies. 3.2. General Milling of the amples was performed by using General Electric Grinder (Model 5KH39 QN5525). Gas Chromatography/Flame Ionization Detector (GC/FID) was lIsed to determine the purity of sam ples. Rotavapor evaporator (BUCHI type R-200) was used to concentrate the crude extract. Spots on chromatogram were visual ized using U Itra-Violet Spectrometer (U V P CC-I 0). Silica gel 60 (M t: rck, 230-400 mesh) was used for column chromatography (CC). Thin layer chromatography (TLC) and preparative thin layer chromatography (PTLC) wcre pcrformcJ on silica gel plate (Kieselgel 60F~54 , 0.25 mm and 1.00 mm, Merck). Toxicity test was carried out using Artf!lI1iu salina. Antifungal test was performed on different crude extracts and fractions by using two types of fungus, pycl1oporus spp. and Schizophylllll11 commune. 8 1.30 kg of (ioniolhalamlls malayanus dried sample was pulverized and percolated with at room temperature tor three days and filtered . This method was repeated tor three times the resulting solution was combined. The solvents \-vas removed and evaporated to dryness reduced pressure by using rotavapor. The residue \vas next extracted using ethyl acetate. tmction was carried out for three days at room temperature and repeated for three times and •.t'iltcred The solution was combined and concentrated using rotavapor. This was 10 II owed by ethanol extraction for three days at room temperature. Ex traction repeated lor three times and the solution was then combined and dried using rotavapor. Th e weight and the percentage of all the extracts were determined. Hexane. ethyl acetate and ethanol crude extracts were used for bioassay . Ethyl acetate crude extract was selected for fUl1h er ion and purification purposes. 3.4. Isolation and purification and purilication were carried out using thin layer chromatography (TLC) and column ~ ..h lrnn~"'t'''..r'''''''hy (CC). Silica gel plate F254 (0.25 mm thickness) was used for TLC. Sample was . :>tt€:d onto TLC plate using capillary tube. The plates were de veloped llsing suitable sol vent dilTerent polarity. This procedure was used in order to tind solvent that gave the hest and table separation of components on the TLC plate. Solvent system that consist mixture of loromethane-ethy l acetate-acetone (4:2:1) was found to gave the best separation of ...... n.n..·nh: on the TLC plate. The retention factor. Rr for each spot was determined atter th e was visualized under UV light. 9 The reten tion factor, Rrfor each spot was determined after the plate was vis Uialized under V light. For colu mn chromatography (CC), column with 60.0 em length and diameter or 3.2 cm J8Ckea with silica ge l (230-400 mesh) was used. The column was first rinsed with acetone and Jlowed by hexane (100%). Control valve at the bottom of the column was closed \vhen the luting solvent reached the cotton level. Column was filled with the slurry of silica gel that has been made in hexane. Silica gel slurry was prepared by adding 220 g of silica gel into 350 mL hexane. Glass rod was used to knock off the silica gel slowly into the column to make sure the Ica gel compacted and no air bubble present in the column. The silica gel column was rinsed hexane. About 10.0 g of ethyl acetate crude extract was added into the column ancr being dissolved in the et hyl acetate. Solvent system with increasing polarity was us~d for the elution 'DrCK:eS;S. Eluent of 20 mL each was collected. Each fraction was tested using TLC and I:ractions similar Rrval ue were combined and dried. The weight for each fraction was recorded. Preparati I: thin layer chromatography (PTLC) was carried out using silica gel plate 6O h5.j (1.00 mm thickness) with 20 x 20 em size. Fraction GCO 1 was selected for further alysis. Abo~. t 100 mg of fraction GCOI was dissolved in 5mL of ethyl acetate and applied on plate. Dried PTLC plate was developed using mixture of dichloromethane-ethyl : 1Ci~tatle-al(:et()ne (4:2: I). PTLC plate was removed when the solvent reached the appropriate s. Three bands were observed when visualized under UV light. All the bands \vcre and dissolved in dichloromethane-ethyl acetate-acetone (4:2: I), filtered lIsing gravity IrltrDtllon and tinall y dri ed with rotavapor. The weight and Rr values of the components wcre 10 Structural determination chromatography-flame ionization detector (GC-FID) was used to determine the purity of the pies. About 1.0 mg of GCO I (i), GCO I (ii) and GCO I (iii) were diluted with 50 )J.L hexane then sonicated to obtain the mixture. 1.0 ~lL of each diluted samples in hexane was injected the gas chromatography. The initial temperature for GC-FID was programmed at 50T and reased to 350 C then maintained for about 50 minutes. 1. Brine shrimp toxicity test by McLaughlin (1991) was carried out to determine th e icity of crude extracts and fractions against Arlemia sa/ina larvae. About 20.0 g of Arlemia eggs (Biomarine) was added into an aquarium containing 9 liter of seawater (salinity ppt. temperature 27.83°C and pH 7.77) for hatching process. Air was provided tinuously into the aquarium and left for 24hrs. Hexane. ethyl acetate and ethanol crude extracts and all combined fractions were used in toxicity test. Ea h extract (2 mg) was dissolved in 2 ml of methanol. From these solutions. ilL. 50 ~ll and 5 III samples were transf~rred to vials in triplicate. Solvent was removed ng rotavapor and 5 mL sea\vater was added to each vial, resulting in final concentrations of 100 IlglmL. 10 Ilglm L and I Ilg/mL. Exactlly 2 mL sample was transferred into each NUNC Atler that. 10 Arlcmia sa/ina larvae were added into each NUNC l11ultidi sh. was carried out for the first three hours. After 24 hours contact, amount of th e II ivors \vas counled and LC so value was determined. Controls were performed by similar way using seawater. " ,'U'M' Antifungal have b en used to test for antifungal activity of the crude extracts. The fungus _ ''''',''"1IFl''''/fo: spp, and Schizophyl/uin commune were used for the antifungal activity. The fungi inoculated on MEA (Malt Extract Agar) containing different concentrations ranging from 5.10.25.50 and 100 ppm of the crude extracts. The growths of the fungus were determined measuring colon y diameter. Diameter on the agar plates 'were measured everyday until the fully co ered the whole surface of agar in the plate dishes. The growth rates for each - 'I'\nrpnlratiolls of crude extract were calculated based on the following equation. rate = ( do - dn-I ) + ( dn-I - dn-2 ) + ( dn-2 - dn-3 ) + ...... ( d3 - d2 ) + ( d2 - d I ) 11-1 12 CHAPTER 4 RESULTS AND DISCUSSION . Extraction, i olation and purification of Gon;othalamlls malayanus sample extraction of tern bark of Gonio/halamlls malayal1l1s were carried out using solvents with ranges of polarity. In the first step of extraction, hexane was llsed followed by etinyl and tinall wi th the most polar solvents, ethanol. The purpose of using hexane \vas to less polar compounds such as lipids and fatty acids which might interfere the extraction . Ethyl acetate ",,'as used to isolate the intermediate polar compounds wh j,ch may present. Hn~ln"l \\as the most polar solvent and could remove all the polar compounds hased on its to obtain the desired compounds. The hexane, dhyl acetate crude extracts were black in while the ethanol crude extract was red. A II the crude extracts were concentrated under tdulced pressure b rotavapor until dryness and the weight of all extracts were determined. The and percentage yield of the extracts are shown in the Table 3. 3: The weight and percentage yield of extracts from Gonio/halamus malayanus Weight (g) Percentage yield (%) 8.693 0.67 21.331 1.64 30.367 2.34 13 acetate crude extract was used for further isolation in order to isolate intermediate polarity s. The ethyl acetate crude extract was diluted with ethyl acetate and was subjected to thin layer chromatography (TLC) (0.25 mm thickness. 20 x 20 cm). The suitable solvent gave best cparation for ethyl acetate crude extract was dichloromethane-ethyl acetate- with the ra tio of 4:2: I. There were 4 spots observed after the plate was developed. A II spots are shown in Appendix I. The plate was vi sualized under UV light and the RI- values determined. The Rrvalues for each spots are given in Table 4. 4: Rrvalues fo r compounds observed in ethyl acetate extract of Goniotha!wnus ll1u/ayul1l1s in the solvent system of dichloromethane-ethyl acetate-acetone (4:2: I). Rr 0.75 0.81 0.87 0.91 crude extract was next separated by column chromatography with increasing solvent. The olvent used in the column chromatography began with the less polar until increasing polarity of solvent system. Each 20 mL of eluent were collected and each fradion tested with TLC and fractions with similar Rr values were combined. All together 19 fractions were obtained. The weights of combined fractions obtained from column are gi ven in Table 5. 14 Pusat Khidmat Maklumat Akademll UNfVERSITI MALAYSIA SARAWAK 94100 Kota. Samarahan 5: The weight of combined fractions obtained from ethyl acetate crude extract of uoniolhuiul11l1 . mula,vunlls. Fraction Weight (mg) 13-15 213 .0 16-20 419.6 21-25 567.5 26-30 920.0 31-32 162 .3 33 282.0 34-35 374.0 36-39 718.1 40 177.8 41-43 261 .6 44-46 356.6 47-50 382.0 51-53 342.2 54-56 430.8 57-60 821.0 61-62 385.0 63-64 256.0 65-70 331.0 71-75 452.0 OCM: EtOAc: GB=EtOAc: GC=EtOAc: EtOH; GD=EtOH 15 selected for further purification by preparativc thin layer chromatography (PTLC). Ivent system. dichloromethane-ethyl acetate-acetone (4 :2: I) was used for PTLC. Three re observed. scrapped. dissolved, ti Itrated and dried separately. The weight for the IMinf"n t~ namely GCOI (i). GCOI .(ii) and GCOI (iii) wcre determined and TLC was carried determine the Rrvalues (Appendix 2-4). Table 6 below provides the Rrvalues and weight 3 components isolated by PTLC. he weight and R, values of compounds isolated from fraction GCO I Rr t (mg) 0.77 101 0.90 97 0.93 135 ~, !\l'r...·fllIl'fIIl determination of isolated compounds MPlM1e!Bt GCOI (i), GCO I (ii) and GCOI (iii) were subjected to GC/FID to determine the these compounds. Ba ed on the peak shown in the chromatogram, the purity of GCO I 1 (ii) and GCOI (iii) was determined. The GC chromatogram tor GCO I (i). GeO I (ii) (iii) were shown in Appendix 5-7. GC chromatogram of GCO I (i) shmved one sharp ~IJliI!dlest peak at retention time of 22.799 min and some smal'l peaks present in the GCOI (ii) gave one highest peak at retention time of 25.596 min which was 01 (iii) chromatogram . Thus, it can be concluded that GCO 1 (ii) and GCO I (iii) arc mpound. For the GCOI (i) chromatogram. compound presents was shown to be pure 16 n the highest peak at retention time of 22 .799 min. The structure determination of GCO I be determined due to lacks of spectroscopy information available for GCO I (i). BriDe shrimp toxicity test rude extracts and fractions were used in the bioassay (Appendix 8). Table 7 summarizes the death of Arlemia salina at different concentrntion of crude extracts and fractions while 2 shows the av rage death of Arlemia salina as a function of concentrations. Based on the test on fracti on GCO I showed 50 % death of Arle/llia salina larvae at concentration of pglmL which gave LC50 at 100 j..lg/m L. The LC 50 va ~ ues for the crude extracts and other were higher than 100 j..lg/mL. Overall, crude extracts and fractions except for GCO I low toxicity I vel on Arlemia salina. The overall range of death of Arlemia salina was 10-30%. Based 011 the results, it can be concluded that Goniolhalamus malc~ranlls stcm _ l1teltroI1en'n and gigantrionenin are example of acctogenins which have been isolated i'rom bark of v(J.niolhalamlis giganlells and showed toxicity against brine shrimp (Fang eI al.. I-azaanthraquinone and 3-aminonaphthoquinone which have been isolat~d from the stem f voniolhalamlls marcullii also showed brine shrimp toxicity (Soonthornchareonnon el al. , nnonacin is a type of acetogenins which have been found to have cytotoxicity potential isolated from various vOl1iolhalamlls species such as Conio/halal11us vell/linlls . .....'Jtnult-'ml~/.\' malayanlls and vonio/holaml/s do/ichocarjJlIs (Goh, 1998). 17