Extracts obtained from three Malaysian Labiatae plants, including Ocimum sanctum, Mentha

arvensis and Orthoshiophon staminea, were investigated for their biological activity. The volatile fraction

of each plant was isolated and the major components were characterized by gas chromatography (GC),

gas chromatography-mass spectrometry (GC-MS), and nuclear magnetic resonance (NMR). The antibacterial

and antifungal activity, insecticidal activity and inhibition of the germination of seeds of the

volatile fraction and residue were studied

Sukari, Mohd Aspollah

Takahashi, Shozo

Universiti Putra Malaysia Institutional Repository

Pertanika 11(2), 249-253 (1988)Biological Activity of Some Malaysian Plant ExtractsMOHD ASPOLLAH SUKARI and SHOZO TAKAHASHI lChemistry DepartmentFaculty ofScience and Environmental StudiesUniversiti Pertanian Malaysia43400 UPM Serdang, Selangor, Malaysia.Key words: Biological activity; plant extracts, antifungal activity; insecticidal activity; Ocimum sanctum;Mentha arvensis; bioassay.ABSTRAKEkstrak yang diperoleh daripada tiga tumbuh-tumbuhan Labiate Malaysia, termasuk Ocimumsanctum, Mentha arvensis dan Ortoshipon staminea, telah dikaji keaktifan biologinya. Pecahan mudahmeruap daripada setiap tumbuhan telah dipisahkan dan komponen utamanya telah dicirikan dengan kro-matografi gas (KG), kromatografi gas-spektrum jisim (KG-Sf) dan resonans magnet nukleus (RMN).Kesan daripada setiap pecahan mudah meruap dan sisa ekstrak terhadap keaktifan antibakterio. dan anti-fungi, keaktifan keracunan serangga dan penghalangan percambahan biji benih dikaji.ABSTRACTExtracts obtained from three Malaysian Labiatae plants, including Ocimum sanctum, Menthaarvensis and Orthoshiophon staminea, were investigated for their biological activity. The volatile fractionof each plant was isolated and the major components were characterized by gas chromatography (GC),gas chromatography-mass spectrometry (GC-MS), and nuclear magnetic resonance (NMR). The anti-bacterial and antifungal activity, insecticidal activity and inhibition of the germination of seeds of thevolatile fraction and residue were studied.INTRODUCTIONMalaysia has about 10,000 species of seedplants and 1000 species of seedless plants (Latif,1985). Of these at least 15% have been claimed tohave some medicinal uses (Latif et at., 1984).The last decade has witnessed a resurgence ofinterest and research in plant products, especiallyin those which show some biological activity.Active phytochemical screening programmes havebeen carried out by Carrick et al. (1968); Chanand Teo (1969, 1972); Chan et al. (1977). Morerecently, survey on alkaloid, saponin and triter-penes/steroid contents of plants collected fromvarious parts of West and East Malaysia have beenconducted (Rahmani et al., 1985; Lajis et aL,1985). Several studies on the pharmacologicalscreening of biologically active ingredients inplants have also been reported (Nakanishi et al.,1965; Yeoh, 1983). This paper discusses the resultof antibacterial and antifungal activity test,insecticidal activity test and inhibition of thegermination of seed test of three MalaysianLabiatae plant extracts.MATERIALS AND METHODSExtractionThe air-dried leaves of each plant were immersedin methanol for several days, filtered and extractedwith ethyl acetate. The volatile fraction was steam-distilled to separate the essential oil, while theresidue was extracted with ethyl acetate. The oilof each species was purified by High Performance1 Pesticide Research Institute, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan.MOHO ASPOLLAH SUKARI AND SHoZO TAKAHASHINuclear Magnetic Resonance (NMR) spectrawere recorded on a lEOL FX-90Q for solutionsin CDC13 with TMS as internal standard.Infrared (IR) analysis 'was performed as thinfllms on a Beckman IR Acculab 7 spectrophoto-meter.Gas Chromatography (GC)Gas chromatography was conducted Usinga Hewlett Packard chromatograph on a capillarycolumn (OV - 101, 0.3 mm Ld., 25 m long)equipped with a flame ionization detector (F 10).The column temperature was programmed from800 to 2400 C at 50 /min. Helium was used -as thecarrier gas at a flow rate -of 50 ml/min.Gas chromatograph-mass spectrometry (GC-MS)Mass spectra were measured using a massspectrameter HITACHI M-90 combined with agas chromatograph.High Performance Liquid Chromatography (HPLC)The oils of O. sanctum and M. arvensis werepurified by HPLC on silicic acid column (Nucleosil8 mm x 30 cm) eluting with 6% ether in hexane(5 ml/min), and the major components wereseparated.RESULTS AND DISCUSSIONGC-MS analysis of essential oil ofO. sanctum showed the presence of methyleugenol(2) as the main constituent (86%~he~­distillate), eugenol (1) (0.5%) and {3-caryo-phyllene (3) (3%) by comparison of their retentiontime (capillary GC on OV-IOI) with the authen-tic samples. The presence of methyleugenol (2)in O. sanctum and its attractiveness to 9 speciesof Dacus has already been reported (Tan, 1982).The steam distillate of O. sanctum containingmethyleugenol is a good source of Dacus attrac-tant.Similar investigations on the soil of M.arvensis have resulted in the characterization ofpiperitenone oxide (4) as the major component(ca 87% of the volatile) on the basis of its1H NMR and IR data, which were in agreementwith the literature (Yukawa and Ito, 1973).Among many cultivars of M. arvensis, there aremany different varieties that produce menthol,menthone, piperitenone oxide and piperitoneoxide as the major terpene compound (Ikeda etPERTANIKA VOL. 11 NO.2, 1988250Liquid Chromatography (HPLC) (in case ofO. santum and M. arvensis) and analyzed byCG-MS to characterize the major components.The extract (oil and residue) of each plant wasthen subjected to bioassay.BioassayAntifungal and Antibacterial Test. Differentconcentrations of oil and residue of each extractwere tested against Colletotrichum lagenarium(Passerini) Ellis et Halsted, Pyricularia oryzaeCavara, Cochliobolus miyabeanus (Ito et Kuri-bayashi) Drechsler ex Dastur, Trichophytonmertagrophytes (Robin) Blanchard and Bacillussubtilis (Ehrenberg) Cohn by using the standardPetri dish method. All the fungi and a bacteriumused in the tests are from a stock culture of thePesticide Research Institute, Kyoto University.A pulp disc (8 mm diameter) impregnated withsample (at specific concentrations) was put intofungal or bacterial culture in agar medium in aPetri dish and left for 24 hours at 240 C (for fungi)and 370 C (for bacteria). Benzylcornium chloridewas used as the control, and the size of the clearzone around the disc was compared as shown inTable 1.Inhibition of the Seed Germination Test.Rice seeds (Oryza sativa, variety 'tanginbouzu'),which lack the gibberellin synthesis pathway wereused for seed growth and root elongation test.Ten rice seeds were placed on filter paper soakedwith plant extract (solution containing 0.5 mg and5 mg of sample) inside a bottle (35 mm Ld. and100 mm high). Water (1 ml) was added to thebottle, covered and kept in the dark at 300 C for3 days. Seeds grown with gibberellin solution wereused as control. The germination of each seed wasdetermined by observing the appearance of bothshoot and root.Insecticidal Activity Test. The insecticidalactivity of each plant extract was tested againstGerman cockroach (Blattella germanica L.) (firstinstar nymph) and red bean weevil (Callosobrun-chus chinensis L.) (adult). The extract (10 mg) inethyl acetate was sprayed onto the bottom ofcylindrical flasks (area of 96.8 cm2) and eva-porated off the solvent. The test insects (about 20)were kept in the treated flasks and monitored for72 hrs. to observe for mortality of the insects.The control test was carried out without sampletreatment.BIOLOGICAL ACTIVITY OF SOME MALAYSIAN PLANT EXTRACTSal., 1971). Malaysian M. arvensis belongs to avariety which produce piperitenone oxide as themajor terpene but the origin of the variety was notidentified. From the volatile fraction of O. sta-minea, methyleugenol (2) (3%) eugenol (1) (8.3%)and ()- caryophllene (3) (2.2%) were confIrmedas the essential oil constituents. Other constituentsof the oil were left unidentified.III©r~~ELWO..(1)H¥ .~PlPERlIDOC OXltE (4)study could most probably be due to the presenceof piperitenone oxide too. A parallel experimenthas demonstrated that piperitenone oxide sepa-rated from oil of M. arvensis inhibited the germi-nation of Phaseohls aureus seed (Yusof, 1986).As for the insecticidal activity, the contacttoxicity test indicated that application of 10 mgeach of the essential oil from O. sanctum andM. arvensis showed 100% mortality in B. germani-ca and C. chinensis within 24 hrs, (about 20insects were used in each test). On the otherhand, the oil from O. staminea and the residuesof the three plant extracts investigated did notindicate any toxicity effect on the test insects.ACKNOWLEDGEMENTThe work carried out in this report is partof an activity of Japan Society for the Promotionof Science-Vice-Chancellors' Council (JSPS-VCC)programme. The authors gratefully acknowledgeJSPS for a travel grant. They are also gratefulto Mr. M. Hajika, Dr. T. Takabayashi and Dr. M.Tsuda for their assistance and discussion.REFERENCESThe results from antibacterial and antifungalassay shown in Table 1 indicate that O. sanctumand M. arvensis extracts exhibit some degree ofIinhibitory activity to T. mertagraphytes andB. subtilis growth at the concentrations above104 ppm. On the other hand, no effect wasobserved on three other organisms used in thetest. Ocimum gratissimum which contains high /"amount of eugenol (1) (81.24% of essential oil)has been shown to be fungitoxic to betelvinepathogenic fungi (Alternaria alternata, Colleto-trichum capsici and Sclerotium rolisil) at theconcentration of 100 ppm (Tripathi et al., 1985).This has been attributed to the presence of euge-nol as the main component.The results in Table 2 show that essentialoil from M. arvensis strongly inhibited the germi-nation of rice whereas oil from O. santum showeda slight activity at the higher concentration. Inhi-bitory activity of piperitenone oxide and piperi-tone oxide toward lettuce seed germination hasbeen reported (Shimizu, 1986). The inhibitoryactivity of M. arvensis essential oil in the presentCARRICK, J., K.C. CHAN and H.T. CHUNG. (1968):A New Phytochemical Survey of Malaya. I. ChemicalScreening. OJem. Pharma. Bull., 16 2436-2441.CHAN, K.C., K.F. MAK and L.E. TEO. (1977): A NewPhytochemical Survey of Malaya. IV. ChemicalScreening. Chem.l'PlanM. Bull., 25 U26:"'1829.CHAN, K.C. and L.E. TEO. (1969): A New Phytoche-mical Survey of Malaya. II. Chemical Screening.Chem. Pluzrma. Bull. 17.1284-1286; 1972, A NewPhytochemical Survey of Malaya. Ill. ChemicalScreening. Chem. Phanna Bull. 20: 1528-1529.IKEDA, N., S. SHIMIZU, D. KARASAWA, T. ORIGASAand S. ONO. (1971): Mentha arvensis L. Va!. pi-perascens MAL. which grows wild in the North-eastern Part of Japan. The Scientific Reports ofthe Faculty of Agriculture Okayama University,No. 37,pp 1-8.LAHS, N. and L. DIN. (1985): The PhytochemicalSurvey Workshop, Universiti Pertanian Malaysia,Serdang. Report On The Phytochemical SurveyConducted at The Krau Game Reserve, 'Lancang,Temerloh, Pahang.LATIF, A. (1985): The l'Plytochemical Survey Workshop,Unlversiti Pertanian Malaysia, Serdang. Some Malay-sian Plants of Medicinal Value.LATIFF, A., G. ISMAIL" M. OMAR, 1M. SAID andA. lCADIR. (1984): A multi-variate approach to .thePERTANIKA VOL. 11 NO.2, 1988 251NV>NTABLE l.Antifungal and antibacterial assay of plant extract at various concentrations.Concentration (ppm)Fungus & Bacterium Ocimum sanctum Men rha arvensis Orrhoshiphon srammeaOil Residue Oil Residue Oil Residue5xl02 103 104 lOs 5xl02 103 104 5xl02 103 104 lOS 5xl02 103 104 lOS 5xl02 103 104 5xI02 103 104Trichophyton mertagrophytes- -. ±- - • - - . . - - • • - - • - - .Bacillus subtilis - - ± ++ - - ± - - ± + - - + ++ - - - - - -Conuol O.sanctum M. arvensis O. stamineaAcOEt GA-3 Oil Residue Oil Residue Oil Residue0.5 mg 5 mg 0.5 mg 5 mg 0.5 mg 5 mg 0.5 mg 5 mg 0.5 mg 5 mg 0.5 mg 5 mgS R S R S R S R S R S R S R S R S R S R S R S R S R S R2.5 (3+) 15.5 (3+) 0.4 (+) 0 (-) 2.3 (3+) 3.2 (3+) 0 (-) 0 (-) 3.5 (3+) 2.5 (2+) 3.6 (+) 2.7 (±l) 4.0 (3+) 3.1 (2+)2.3 (3+) 16.1 (3+) 1.7 (+) 0 (-) 2.6 (3+) 2.4 (3+) 0 (-) 0 (-) 2.1 (3+) 3.2 (2+) 3.5 (+) 3.5 (± I) 3.6 (3+) 3.1 (2+)2.2 (3+) 16.0 (3+) 0.6 (+) 0 (-) 2.4 (3+) 2.8 (3+) 0 (-) 0 (-) 2.4 (3+) 2.9 (+) 3.5 (+) 3.2 (± I) 2.9 (3+) 2.9 (2+)2.6 (3+) 15.0 (3+) 0.7 (+) 0 (-) 2.9 (3+) 2.4 (3+) 0 (-) 0 (-) 2.7 (3+) 3.0 (2+) 3.1 (+) 3.2 (+) 2.8 (3+) 2.9 (2+)2.7 • (3+) 16.0 (3+) 0.8 (+) 0 (-) 2.7 (3+) 2.8 (3+) 0 (-) 0 (-) 3.1 (3+) 0.5 (+) 3.0 (+) 3.1 (±l) 2.7 (3+) 2.5 (2+)2.7 (3+) 0.9 (+) 0 (-) 3.0 (3+) 2.1 (3+) 0 (-) 0 (-) 2.3 (3+) 0.9 (-) 2.0 (+) 2.9 (+) 2.4 (3+) 2.0 (-)2.8 (3+) 0.6 (+) 0 (-) 2.6 (3+) 2.3 (3+) 0 (-) 0 (-) 2.1 (3+) 0 (-) 1.8 (+) 2.6 (+) 0 (-) 1.0 (+)3.0 (3+) 0.8 (+) 0 (-) 2.8 (3+) 2.6 (3+) 0 (-) 0 (-) 2.6 (3+) 0 (-) 1.2 (+) 2.5 (~ 0 (-) 0 (-)2.7 (3+) 0 (-) 0 (-) 2.9 (3+) 2.7 (3+) 0 (-) 0 (-) 1.0 (3+) 0 (-) 0 (+) 2.5 (±) 0 (-) 0 (-)3.2 (3+) 0 (-) 0 (-) 2.4 (3+) 2.5 (3+) 0 (-) 0 (-) o (-) 0 (-) 0 H 0 (-) 0 (-) 0 (-)TABLE 2.Seedling growth (S, cm) and root elongation(R) of Oryza sativa grown on plant extract."0m;Q-l»:>':»<or-z91'-'-a0000•clear zone was not observed. ±Not tested.Clear zone less than 10 mm (width). + Clear zone ca. 10 mm (width). ++ Clear zone ca. 15 mm (width).s:o:r:o»Vl"0or-r-»:r:Vlc;.::»e!»zoVl:r:oNo~~:r:~:;(3+) Clear elongation observed.(-) No elongation observed.BIOLOGICAL ACTIVITY OF SOME MALAYSIA PLA T EXTRACTSstudy of medicinal plants in Malaysia. J. SingaporeNatL Acad. Sci., 13.NAKANISHI, K., S·1. SASAKI, AJ. KIANG, 1. GOH,H. KAKISAWA, M. OHASHI, M. GATO, J·M.WATANABE, H. YOKOTANI, C. MATSUMURAand M. TOGASHI. (1965): Phytochemical Surveyof Malaysian Plants. Preliminary Chemical andPharmacological Screening. Chem Pharm Bull.•13: 882-890.RAHMANI, M., R. KIEW, N. LAJlS, R. OTHMANand R.F. TOIA. (1985): A Contribution to thePhytochemical Survey of Peninsular Malaysia.Pertanika. 8(3): 347-357.SHIMUZU, S. (1986): Chemical Constituents of Mentharotundifolia and the Mints Related to rotundifolia.Memoirs oflida Women's College. 8: 13-28.TAN, K.H. (1982): CEC/IOBO Symposium, Athens.Response of Dacus to Ocimum sanctum Extractsand Different Synthetic Attractants in Penang,Malaysia,513.TRIPATHI, R.D., R. BANERJI, M.L. SHARMA, Y.R.BALASUBRAHMANYAM and S.K. NIGAM.(1985): Toxicity of Essential Oil from a New Strainof Ocimum gratissimum against Betelvine PathogenicFungi. Agric. Bioi. Chem. 49: 2277 -2282.YEOH, P.N., M. MARZIAH (Ed) (1983): Proceedings ofthe Seminar on Scientific Approach to TraditionalMedicine, Universiti Pertanian Malaysia, Serdang,Selangor.YUKAWA, Y and S. ITO, (Ed.), (1973): Spectral Atlasof Terpenes and The Related Compounds, HirokawaPublishing Inc., Tokyo, pp 186-187.YUSOF, o. (1986): Phytochemical and biological Acti·vity Surveys of Malaysian Plant Extracts, Under·graduate Research Project Report, Chemistry De-partment, Universiti Pertanian Malaysia.(Received 3 September. 1987)PERTANIKA VOL. II NO.2, 1988 253

Biological Activity of Some Malaysian Plant Extracts

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Biological Activity of Some Malaysian Plant Extracts

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