Phytochemical and bioactivity studies of the leaves of Melastoma malabathricum L. (Melastomataceae) have been investigated. The n-hexane extract yielded α-amyrin, patriscabatrine and auranamide, ethyl acetate extract gave quercetin and quercitrin, and methanol extract gave quercitrin and kaempferol-3-O-(2”,6”-di-O-p-trans-coumaroyl)glucoside. The crude extracts and isolated compounds were screened for their antioxidant and cytotoxic activities. The antioxidant assay was carried out by FTC and DPPH radical scavenging method. Kaempferol-3-O-(2”,6”-di-Op- trans-coumaroyl)-glucoside, quercetin and quercitrin showed strong activities with inhibition more than 90% in the FTC method. Quercetin was found to be the most active as radical scavenger in DPPH method with IC50 of 0.69 µM. α-Amyrin and kaempferol-3-O-(2”,6”-di-O-p-transcoumaroyl) glucoside demonstrated the strongest activities in the anti-inflammatory assay of TPA mouse ear oedema with IC50 of 0.11 and 0.34 mM/ear, respectively

Ahmad, Farediah

Darnis, Deny Susanti

Mat Ali, Rasadah

Mohd. Sirat, Hasnah

English

The International Islamic University Malaysia Repository

Jurnal Ilmiah Farmasi Vol.5 No.1 Tahun 2008 1BIOACTIVE CONSTITUENTS FROM THE LEAVES OF MELASTOMAMALABATHRICUM L.Deny Susanti1*, Hasnah M. Sirat2, Farediah Ahmad3, Rasadah Mat Ali31Department of Biomedical Science, Faculty of Science, International Islamic University Malaysia,25200 Kuantan, Malaysia.2Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, 81310 Skudai, Johor,Malaysia3Medicinal Plant Division, Forest Research Institute Malaysia, 52109 Kepong,Kuala Lumpur, Malaysia*e-mail: deny@iiu.edu.myABSTRACTPhytochemical and bioactivity studies of the leaves of Melastoma malabathricum L.(Melastomataceae) have been investigated. The n-hexane extract yielded-amyrin, patriscabatrineand auranamide, ethyl acetate extract gave quercetin and quercitrin, and methanol extract gavequercitrin and kaempferol-3-O-(2”,6”-di-O-p-trans-coumaroyl)glucoside. The crude extracts andisolated compounds were screened for their antioxidant and cytotoxic activities. The antioxidantassay was carried out by FTC and DPPH radical scavenging method. Kaempferol-3-O-(2”,6”-di-O-p-trans-coumaroyl)-glucoside, quercetin and quercitrin showed strong activities with inhibition morethan 90% in the FTC method. Quercetin was found to be the most active as radical scavenger inDPPH method with IC50 of 0.69 M. -Amyrin and kaempferol-3-O-(2”,6”-di-O-p-trans-coumaroyl)glucoside demonstrated the strongest activities in the anti-inflammatory assay of TPAmouse ear oedema with IC50 of 0.11 and 0.34 mM/ear, respectively.Keywords: Antiinflammatory, Antioxidant, Flavonoids, Melastomataceae, Melastomamalabathricum, TerpenoidINTRODUCTIONM. malabathricum L. (senduduk) is a very common herb or shrub found throughout thetropic in the moist part mostly from India, Thailand and Malaysia, where it grow as small trees 12-13ft. high, occasionally even up to 20 ft. (Burkill, 1966). This species has at least three varieties, i.e.large, medium and small size flower with dark purple-magenta petals, light pink-magenta petals andthe rare variety white petals as reported by Corner, 1951.The chemistry of Melastomataceae is poorly known. Previous studies of this family havecharacterized hydrolysable tannin. Several hydrolysable tannins have been isolated from the dryleaves of M. malabathricum with light pink-magenta petals. The main tannin was oligomers namednobotanin B, dimmers named malabathrins B, malabathrins C and malabathrins D, monomersnamed 1,4,6-tri-O-galloyl--D-glucoside, 1,2,4,6-tetra-O-galloyl--D-glucoside, strictinin,casuarictin, pedunculagin, nobotanin D, pterocarinin and oligomers nobotanin G, nobotanin H,nobotanin J (Yoshida et al., 1992), -sitosterol, amyrin, uvaol, sitosterol-3-O--D-glucopiranoside, quercetin, quercitrin and rutin (Nuresti et al., 2003). To our knowledge this is theonly reported chemical composition of Melastoma with light pink-magenta petals reported so far.Jurnal Ilmiah Farmasi Vol.5 No.1 Tahun 2008 2METHODSPlant Material. Melastoma malabathricum was collected from Johor, Malaysia in 2002 andidentified by Dr. Rusdi Tamin of Universitas Andalas, Padang, Indonesia. A voucher specimen(YANTI-2061) was deposited the Herbarium of Universitas Andalas (ANDA) Padang, Indonesia.General methods. Mps. (uncorr.) were determined using Leica Gallen III apparatus. UVwas recorded on Shimadzu UV-160 spectrophotometer in methanol. IR spectra were recorded onPerkin Elmer 1650 FTIR spectrophotometer. NMR spectra were recorded on JEOL JNH A500Spectrometer measured at 500 MHz and 125 MHz, respectively. Vacuum liquid chromatography(VLC) and column chromatography (CC) were carried out using silica gel 230-400 mesh, Merck9385 and 70-230 mesh, Merck 7734. Sephadex LH-20 was purchased from Pharmacia (Uppsala,Sweden). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was from Fluka (Switzerland), vitamin E andvitamin C were purchase from Merck (Darmstadt, Germany), 12-O-tetradecanoylphorbol-13-acetate(TPA) was from Sigma Aldrich (St. Louis, MO, USA).Extraction and Isolation. The air-dried leaves of M. malabathricum (2.09 kg) of white petalswas successively extracted with n-hexane, EtOAc and MeOH. The chromatographic separations byvacuum liquid chromatography (VLC) and column chromatography (CC) on silica gel of the n-hexane and ethyl acetate extracts have afforded 1 - 5. Purification of the MeOH extract by VLC andCC has resulted in the isolation of 5 and 6. The structures of 1 - 6 were elucidates as -amyrin,patriscabratine, auranamide, quercetin, quercitrin and kaempferol-3-O-(2”,6”-di-O-p-trans-coumaroyl)--glucoside by interpretation of the spectral data (IR, FABMS, 1D and 2D NMR) as wellas by comparison of the data with literature values (Mahato and Kundu, 1994; Gu, et al., 2002;Banerji and Ray, 1981; Ishiguro et al., 1991; Slowing et al., 1994; Skaltsa et al., 1994). -Amyrin,quercitrin and quercetin have been isolated previously from M. malabathricum with light pink-magenta petals (Nuresti et al., 2003). Kaempferol-3-O-(2”,6”-di-O-p-trans-coumaroyl)--glucosidehas been encountered previously in leaf hairs of Quercus ilex (Fagaceae) (Skaltsa et al., 1994).Antioxidant Activity, FTC (Ferric Thiocyanate) Method. The detection of lipid peroxide andpreparation of solutions were carried out according to method described by Kikuzaki and Nakatani(1993). A mixture of 4.0 mg of sample in 4.0 mL of 99.5% ethanol, 4.1 mL of 2.5% linoleic acid in99.5% ethanol, 8.0 mL of 0.02 M phosphate buffer (pH 7.0) and 3.9 mL of water contained in ascrew-cap vial (38 x 75 mm) was placed in an oven at 40C in the dark. The final concentration ofsample was 0.02% w/v. To 0.1 mL of the mixture, 9.7 mL of 75% (v/v) ethanol and 0.1 mL of 30%ammonium thiocyanate was added. Three minutes after the addition of 0.1 mL of 2.0 10-2 Mferrous chloride in 3.5% hydrochloric acid to the reaction mixture, the absorbance was measured at500 nm at every 24 hours interval until 1 day after the absorbance of the control reached itsmaximum value.DPPH Method. The method established by Tagashira and Ohtake (1998) was used withslight modification. Stock solution of sample was prepared at 1 mg/mL in methanol. A test samplesolution (200 L) was added to 3.8 mL of 50 M DPPH methanolic solution (to give a finalconcentration of 500, 250, 125, 62.5, 31.3 and 7.8 g/mL). After vortexing, the mixture wasincubated for 30 minutes at room temperature. The changes of DPPH in colour (from deep violet toJurnal Ilmiah Farmasi Vol.5 No.1 Tahun 2008 3light yellow) were measured at 517 nm. The difference in absorbance between a test sample and acontrol (methanol) was expressed as % inhibition and taken as activity. The activity was shown asIC50 value (50% inhibitory concentration).The percentage inhibition was calculated by the following formula:Percentage inhibition =Anti-inflammatory Activity. TPA (12-O-tetradecanoylphorbol-13-acetate )-induced MouseEar Oedema method. This method was carried out according to the method described by NikMusa’adah et al. (2000). The mice were weighed, marked and arrange to six mice in one group.The crude extracts and pure compounds (20 L) was applied topically to the inner surface of theright ear of the mice 40 minutes before TPA (25 mg/mL in Me2CO) was applied. The left ear whichacted as control was applied with the same volume of Me2CO. The resulting of oedema wasmeasured after six hours TPA application. The mice were killed by cervical dislocation and a 7 mmdiameter section of both ears was punched out and weighed.The swelling induced by TPA was assessed in terms of the increase in the weight of theright ear punch biopsy over that of the left ear. The inhibitory effect (IE %) of each samples werethan calculated as the ratio of weight increase of the ear sections, according to the followingformula:Inhibitory effect (IE%) =Where:L = weight of left ear which was treated with TPA onlyR = weight of right ear which was treated with TPA and thetest sampleC = weight of normal ear (untreated)C is the calculated weight. It has been found that treating a normal earwith 0.5 g TPA resulted in a 2.41 times increase in the weight of theear.Sample which has the percent of inhibition more than 50% was considered to have the anti-inflammatory activity.RESULTS AND DISCUSSIONPhytochemicals Investigation. The dried and powdered leaves of M. malabathricum (2.09kg) was extracted by soxhlet extraction for 18 hours with n-hexane, EtOAc and MeOH,successively. The n-hexane and EtOAc extract were subjected to several chromatographictechniques (VLC and CC) to yield triterpenoid 1, two alkaloids 2 –3 and two flavonoids 4 - 5, whilethe MeOH extract after purification afforded flavonoids 5 and 6.%100... )(  DPPHsampleDPPHDPPHAbsAbsAbs100)41.2(LLRL100CLRLOHOOH OOROHOHBy correlating with melting points and spectral data (UV, IR, 1H, 13C NMR and MS) ofliterature values, compounds 1 - 6 were identified as -amyrin (1) (Mahato, 1994) , patriscabratine(2) (Gu, et al., 2002), auranamide (3) (Banerji and Ray, 1981), quercetin (4) (Ishiguro, et al., 1991),quercitrin (5) (Slowing, et al., 1994) and kaempferol-3-O-(2”,6”-di-O-p-trans-coumaroyl)glucoside(6) (Skaltsa, et al., 1994), respectively.This is the first report on isolation of -amyrin (1), patriscabratine (2), auranamide (3),quercetin (4), quercitrin (5) and kaempferol-3-O-(2”,6”-di-O-p-trans-coumaroyl)--glucoside (6) fromthe leaves of M. malabathricum with white petals. Kaempferol-3-O-(2”,6”-di-O-p-trans-coumaroyl)--glucoside (6) in the leaves of M. malabathricum of white petals was found for the first time inMelastoma species. The presence of-amyrin (1), quercetin (4), quercitrin (5) and kaempferol-3-O-(2”,6”-di-O-p-trans-coumaroyl)--glucoside (6) in the leaves of M. malabathricum with white petalsis the chemotaxonomic significant of this plants.Antioxidant Activity. FTC (Ferric Thiocyanate) Method. FTC method measures the amountof peroxide produced during the initial stages of oxidation which are the primary products ofoxidation. In this method, the reaction mixture of linoleic acid, ethanol, phosphate buffer andantioxidant (sample and standard) is incubated at 40C and the peroxide value is determined bymeasuring the absorbance at 500 nm after reaction between FeCl3 and thiocyanate.(4) R = H(5) R = RhaHO (1)C NCHCH2O HCOO CH2CHNCH2H COR(2) R = CH3(3) R = PheOOHOOHOHOOOO OHOHCCOOOHOH(6)Jurnal Ilmiah Farmasi Vol.5 No.1 Tahun 2008 5Figure 1 shows the results of the antioxidant activity of the flavonoids, isolated from theleaves of M. malabathricum. These flavonoids showed stronger antioxidative activities than vitaminE. The percentage inhibition of these flavonoids was between 92.2-96.1% (table 1).Incubation Time (Day)Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9Absorbanceat500nm0.00.51.01.52.02.5QuercitrinQuercetinKaempferol-3-O -(2",6"-di-O -p-trans-coumaroyl glucoseControlVitamin EFigure 1. Antioxidant activity of flavonoids from the leaves of M. Malabathricum asmeasured by the FTC method (final sample concentration, 0.02% w/v)DPPH Method. The DPPH method is a rapid and cheap method to measure antioxidantcapacity. The radical scavenging activity was determined from the reduction in the opticalabsorbance at 517 nm due to scavenging of stable free radical of DPPH. Positive DPPH testsuggests that the extracts and compounds are free radical scavengers.Table 1.The absorbance values and percentage of linoleic acid peroxidation of the flavonoids asmeasured by FTC antioxidant assaySample Absorbance* Percent InhibitionQuercetin 0.061 96.1Quercitrin 0.092 94.1Kaempferol-3-O-(2”,6”-di-O-p-trans-coumaroyl)--glucoside0.122 92.2Vitamin E 0.339 78.3Control 1.560 0*Absorbance reading on the 9th day (one day after control reached maximum)Jurnal Ilmiah Farmasi Vol.5 No.1 Tahun 2008 6Table 2 shows the activity of isolated compounds from two species that have free radicalscavenging properties. Quercetin (4) with IC50 0.69 M was found as the most active free radicalscavenger compared to the positive control, vitamin E and vitamin C and the other flavonoids. Thepercent inhibition of quercetin (4) at concentration 7.8g/mL was 57.60%Table 2. The IC50 of the isolated compounds from M. malabathricum by UV SpectrophotometrymethodSampleIC50(meanSD)Percent inhibition 7.8g/ml(meanSD)Quercetin 0.69M1.4 57.60.5Quercitrin 74.1M0.4 15.41.8Kaempferol-3-O-(2”,6”-di-O-p-trans-coumaroyl)--glucoside308.1M1.7 7.51.2Vitamin E 17.1 mM2.5 7.40.4Vitamin C 8.3M1.2 30.80.7Data represent meanSD of three independent experiments performed in triplicateAnti-inflammatory Activity with TPA (12-O-tetradecanoylphorbol-13-acetate )-inducedMouse Ear Oedema method. The topical application of TPA onto a mice ear induced a long lastingoedema formation. The majority of TPA actions appear to involve or be dependent on arachidonicacid release and metabolism. At the biochemical level, PGI2, PGE2 and PGF2 are elevated withinminutes to hours after TPA application. Phospholipase inhibitors, cyclooxigenase inhibitors andlipoxygenase inhibitors as well as corticosteroids are effective at oedema suppression after topicalapplication of high dose of TPA, confirming a role of arachidonic acid release and metabolism(Jeenapongsa, et al., 2003).Oedema was induced by topical application of 0.5 mg per ear of TPA. The crude extracts(2 mg per ear); the isolated compounds (0.5 mg per ear) and the standard drug indomethacin (0.5mg per ear) were applied topically, simultaneous with TPA. TPA-induced oedema formation wasclearly observed at six hour after the topical application. The inhibitory effect of crude extracts,isolated compounds and positive control are given in table 3. Data are expressed as IC50 values.Jurnal Ilmiah Farmasi Vol.5 No.1 Tahun 2008 7Table 3. IC50 and inhibitory effect of the isolated compounds on mice ear oedemaSample IC50 Percent inhibition-amyrin 0.34 mM/ear1.1 79.2110.6Kaempferol-3-O-(2”,6”-di-O-p-trans-coumaroyl)--glucoside0.11 mM/ear0.4 99.838.1Indomethacin 2.10 M/ear0.5 76.006.0In this test, kaempferol-3-O-(2”,6”-di-O-p-trans-coumaroyl)glucoside (6) was found to beactive. It gave higher percentage of oedema inhibition (99.83% SD 8.1) at a dose of 0.5 mg/mLcompared to the reference drug, indomethacin, 76.00% (SD 5.0) at the same dose. The IC50value of kaempferol-3-O-(2”,6”-di-O-p-trans-coumaroyl)glucoside (6) was 0.11 mM/ear (SD 0.4)and indomethacin as a positive control was 2.10 M/ear (SD0.5).CONCLUSIONSPhytochemical study, which employed various chromatographic techniques, resulted in theisolation of a series of flavonoids, triterpenoids and alkaloids. These compounds were successfullyisolated and characterized using modern spectroscopic techniques. The compounds are -amyrin(1), patriscabratine (2), auranamide (3), quercetin (4), quercitrin (5) and kaempferol-3-O-(2”,6”-di-O-p-trans-coumaroyl)--glucoside (6).Traditionally, the Melastomataceae family used for the treatment of many ailment, such asdiarrhea, puerperal infection, dysentery, leucorrhoea, wound healing, post partum treatment andhaemorrhoids. Interistingly the in vitro and in vivo assay, the isolated compounds from this plantexhibited many biological activities such as anti lipid peroxidation, radical scavenger and anti-inflammatory.ACKNOWLEDGEMENTThe authors wish to thank IRPA via project no 74051 for financial support.REFERENCESBurkill, I. H. (1966). A dictionary of the economic products of the Malay Peninsula. Kuala Lumpur,Singapore (pp. 1462-1464). Government of Malaysia and Singapore by the Ministry ofAgriculture and Co-OperativesBanerji, A. and Ray, R. (1981). Auranamide, a New Phenylalanine Derivative Isolated from Piperaurantiacum Wall. Indian J. Chem. 20B: 597-598Corner, E.J.H. (1951). Wayside trees of Malaya. Malayan Nature Society: Kuala Lumpur. Vol 1. (pp445-453)Jurnal Ilmiah Farmasi Vol.5 No.1 Tahun 2008 8Gu, Z. B., Yang, J. G., Liu, W. Y., Li, T. Z., Qiu, Y. and Zhang, W. D. (2002). A New Alkaloid fromPatrinia scabra. Chin. Chem. Lett. 13(10): 957-958Ishiguro, K., Nagata, S., Fukumoto, H., Yamaki, M., Takagi, S. and Isoi, K. (1991). Phytochemistry.30(9): 3152-3154Jeenapongsa, R., Yoovathaworn, K., Sriwatanakul, K. M., Pongprayoon, U. and Sriwatanakul, K.(2003). Anti-inflammatory Activity of (E)-1-(3,4-dimethoxyphenyl)butadiene from Zingibercassumunar Roxb. J. Ethnopharmacol. 87(2-3): 143-148Kikuzaki, H. and Nakatani N. (1993). Antioxidant Effect of Some Ginger Constituents. J. Food Sci.58(6): 1407-1410Mahato, S. B. and Kundu A. P. (1994). 13C NMR Spectra of Pentacyclic Triterpenoids- ACompilation and some Salient Feature. Phytochemistry. 37(6): 1517-1575Nik Musa’adah, M.and Rasadah, M.A. (2000). Inhibitor Effect of Solanum torvum on TPA-InducedMouse Ear Oedema. J. Trop. Forest Prod. 6: 222-224Nuresti, S., Baek, S. H. and Asari, A. (2003). Chemical Components of Melastoma Malabathricum.ACGC Chemical Research Communications. 16, 28-33Skaltsa, H., Verykokidou, E., Harvala, C., Karabourniotis, G. & Manetas, Y. (1994). UV-B ProtectivePotential and Flavonoid Content of Leaf Hairs of Quercus ilex. Phytochemistry. 37(4), 987-990Slowing, K., Sollhuber, M., Carretero, E. and Villar, A. (1994). Flavonoid Glycoside from Eugeniajambos. Phytochemistry. 37(1): 255-258Tagashira, M. and Ohtake, Y. (1998) A New Antioxidative 1,3-Benzodioxole from Melissa officinalis.Planta Med. 64(6): 555-558Yoshida, T., Nakata, F., Hosotani, K., Nitta, A., Okuda, T. (1992). Dimeric Hydrolysable Tannin fromMelastoma malabathricum. Phytochemistry, 31, 2829-2833

Bioactive constituents from the leaves of Melastoma Malabathricum L

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Bioactive constituents from the leaves of Melastoma Malabathricum L

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