Indonesian J. Pharm. Vol. 25 No. 2 : 68 – 75 ISSN-p : 2338-9427 DOI: 10.14499/indonesianjpharm25iss2pp75 Research Article BIOACTIVE COMPOUNDS IN BENGKOANG (Pachyrhizus erosus) AS ANTIOXIDANT AND TYROSINASE INHIBITING AGENTS Endang Lukitaningsih1*, Ulrike Holzgrabe2 1Dept. of Pharmaceutical ABSTRACT Chemistry, Faculty of In Indonesia, the roots of bengkoang (Phacyrhizus erosus) Pharmacy, Gadjah Mada have been used as the excipient for sun screening and skin University, Sekip Utara, whitening paste. Since the active compounds exhibiting skin Bulaksumur, whitening or sun screening effect have not previously been Yogyakarta 55281, Indonesia studied, the aim of this study was to identify compounds with 2 Dept of Pharmacy and antioxidant and tyrosinase inhibitor activities. Soxhlet extraction Food Chemistry, Würzburg was used as the method of isolation with petroleum ether as the University, Am Hubland, solvent and it was followed by fractionation using ethyl acetate 97074, Würzburg, Germany to obtain three isoflavonoids (i.e. daidzein (2); daidzein-7-O-ß- glucopyranose (3); 5-hydroxy-daidzein-7-O-ß-glucopyranose Submitted: 11-11-2013 (4)), and a new pterocarpan (i. e. 8,9-furanyl-pterocarpan-3-ol Revised: 10-01-2014 Accepted: 15-03-2014 (1)) which antioxidant activities (SC50% values) of 2.11; 11.86; 0.69 and 7.86 respectively. All compounds showed tyrosinase *Corresponding author inhibiting activities with IC50 values of 4.38; 5.35; 7.49 and Endang Lukitaningsih 22.20 mM, respectively for compound 4, 2, 1 and 3. These compounds can be used as antioxidant and skin whitening Email : materials. [email protected] Key words: Pachyrhizus erosus, antioxidant, tyrosinase inhibitor, flavonoids INTRODUCTION Melanin is classified into two main Bengkoang is a species of Pachyrizus and groups: the black and brown eumelanins which grows naturally in many tropical and are insoluble in water and the yellow and subtropical countries such as America and Asia. reddish-brown phaeomelanin which is alkali It is usually consumed directly or sometimes soluble. Both melanins were derived from with salt, lemon juice and powdered chilli. In tyrosine by the same initial step, namely Indonesia, bengkoang roots have been oxidation process at the phenolic system traditionally used as a cosmetics material for (Bleehen et al., 1995; Parvez et al., 2007; centuries empirically. They have been used as Kobayashi et al., 1994); is starting from the skin whitening materials. Yet, the active conversion of the L-tyrosine to L-3,4- compounds in bengkoang roots having skin dihydroxyphenylalanine (L-DOPA) and whitening activity have not previously been followed by the subsequent oxidation of L- investigated (Lukitaningsih, 2009). DOPA to produce an ortho-quinone (dopa- Skin whitening compounds are in a close quinone) by tyrosinase. Dopaquinone is further relationship with melanin, the major pigment transformed via several reactions to yield for colour of skin, hair and eye (Briganti et al., brown to black melanin which is responsible 2003). The production of melanin depends on for the colour of mammal’s skin (Okombi et al., UV light or sun exposure. It is a natural 2006; Lee 2002; Ohguchi et al., 2003; Wang and protective mechanism of the skin against too Hebert, 2006). Another two melanogenic much UV light penetrating the human skin. enzymes, tyrosine-related protein1 (TRP1) and Too much UV irradiation causes sunburn, tyrosine-related protein2 (TRP2), also named disrupts the synthesis of precursors necessary dopachrome tautomerase (DCT) (Solano et al., for DNA synthesis and increases the amount of 1994), are involved in the melanin biosynthesis free radicals. Melanin captures free radicals and (Kobayashi et al., 1994; Parvez et al., 2007). participates in other oxidation-reduction pro- Another strategy for maintaining skin cesses in the human body (Bleehen et al., 1995). whiteness is to avoid ultraviolet exposure. UV 68 Volume 25 Issue 2 (2014) Endang Lukitaningsih radiation can also induce the formation of meter (Rheinstetten, Germany), Shimadzu various radicals (Matsuura et al., 2006), primarily GC/MS-QP 20105 gas chromatography reactive oxygen species (ROS) in the skin such (Kyoto, Japan), Agilent 1100 series HPLC as singlet oxygen and superoxide anion, apparatus (California, USA) equipped by promoting biological damage in exposed tissues column Zorbax SB-C18 (25cm, i.d. 0.46 cm, via iron-catalyzed oxidative reactions. These 5µm, Agilent, California, USA), UV absorbance radicals play important roles in the activation of detector and ESI-MS detectors, Agilent 1100 tyrosinase in human skin and therefore enhance series preparative-HPLC (California, USA) melanin biosynthesis via induction of the equipped by column Zorbax SB-C18 (7μm, proliferation of the melanocytes. The radicals 21.2X150 mm, Agilent, California, USA). also cause the damage of DNA. Furthermore, ROS scavengers or inhibitors, such as Plant material and extraction antioxidant, may reduce hyperpigmentation and Bengkoang was collected from Purwo- can also be used as whitening materials (Wang rejo, Central Java, Indonesia on the dry season et al., 2006). Therefore, it is necessary to from July until September on 2007. The roots combine sun screen compounds and anti- (45kg) were peeled and washed with water, oxidant compounds in cosmetic products to subsequently dried at 60°C and milled into fine obtain an optimal whitening effect. powder. The fine powder (4.75kg) was extracted by Soxhlet using 6L petroleum ether. MATERIALS AND METHODS The residue was extracted using methanol to Chemicals and solvent achieve the semi polar and polar compounds. The chemicals used in the detection and The extracts were filtered and concentrated in isolation methods were mushroom tyrosinase vacuum evaporator. The concentrated 4187IU/mg, L-DOPA (dihydroxy phenyl methanol extract was added with water and alanine), kojic acid (Fluka, Seelze, Germany), then partitioned with ethyl acetate. The ethyl dimethylsulfoxide extra pure (Acros® organic, acetate phase was further concentrated. Geel, Belgium), DPPH (1,1-diphenyl-1- picrylhydrazine), catechin, Dulbeco’s phosphate Compound isolation of ethyl acetate buffered saline, (purchased from Sigma extract Aldrich, Steinheim Germany), ascorbic acid The ethyl acetate extract (31.1gram) was subjected to silica gel column chromatography (Sigma Aldrich, Steinheim, Germany), Sephadex LH20 (Aldrich, Steinheim, Germany), Silica gel and eluted using the gradient mixture of 60 (particle sizes 0.063-0.200mm, Merck, petroleum ether-ethyl acetate and ethyl acetate- Darmstadt, Germany), TLC Aluminium sheets, methanol producing 35 fractions with 100mL silica gel 60 F254 (layer thickness 0.2mm, of eluents. Merck, Darmstadt, Germany). Fractions 6-11 which had an Rf value of Solvents for separation were petroleum 0.17 (positive with DPPH) were collected and evaporated. The fractions may contain ether, ethyl acetate (Fisher Scientific, antioxidant compounds because the spot was Leichestershire, UK), methanol (Merck, Darmstadt, Germany), chloroform, dichloro- able to reduce DPPH. The concentrated methane and n-butanol (Fluka, Seelze, fraction was then purified using column Germany). sephadex LH-20 chromatography and methanol as an eluent, producing 50 fractions. Equipments Fractions 9-15 were further subjected into Melting point SMP3 Stuart® apparatus preparative HPLC and yield 670mg yellow (Staffordshire, UK), Cary 50 Bio UV-Visible crystal (1) that is identified as 8,9-Furanyl- spectrophotometer (Varian, California, USA), pterocarpan-3-ol. Fractions 37-40 from JASCO FT/IR-6100 Spectrometer (Gross- column chromatography with an Rf value of Umstadt, Germany), Thermo Mixer Comfort 0.35 on TLC were collected and purified using 5355 V.2.12 Eppendorf (Hamburg, Germany), sephadex column chromatography giving 19 ALPHA II-12 Freeze dryer (Osterode, fractions. Fractions 7-11 of this Germany), Bruker Avance 400 NMR spectro- chromatography had the Rf value of 0.85 Volume 25 Issue 2 (2014) 69 Bioactive Compounds in (Pachyrhizus erosus) (positive with DPPH) then purified using 1H NMR (500 MHz, MeOH-d4): 6.74 (s, 1H, preparative-HPLC giving 33 mg of compound H8), 6.76 (d, 2H, H3’, H5’), 6.85 (d, 1H, H6), 7.26 2. Based on the GC/MS and NMR (d, 2H, H2’, H6’), 7.95 (d, 1H, H5), 8.02 (s, 1H, spectroscopic data, compound 2 was identified H2); 13C NMR (125 MHz, MeOH-d4): 153.04 as Daidzein. (C2), 124.32 (C3), 176.57 (C4), 128.53 (C5), Fractions 17-24 of the first column 116.47 (C6), 164.62 (C7), 103.24 (C8), 158.72 chromatography with an Rf value of 0.52 on (C9), 118.23 (C10), 125.98 (C1‘), 131,24 (C2‘), TLC were collected and evaporated. The 116.24 (C3‘), 159.82 (C4‘), 116.24 (C5‘), 131.42 concentrated fraction was purified by (C6‘). preparative HPLC giving two compounds. The COSY data shows the correlations of compound with a retention time of 11.4min H6-H5; H2’-3’ and H6’-H5’. HMBC data shows (compound 3) was assigned to Daidzein-7- the correlations between H2 and C1’ (3JC-H), H2 O-ß-glucopyranose, while the other and C4 (3JC-H), H2 and C9 (3JC-H), H5 and C7 (3JC- compound having a retention time of 12.6min H), H2’ and C1’ (2JC-H), and H6’ and C1’ (2JC-H). was assigned to 5-hydroxy-daidzein-7-O-ß- glucopyranose (compound 4). Compound 3 (Daidzein-7-O-ß-glucopyranose) Yellow powder; MW 416; UV (MeOH) Identification of isolated compounds λmax 260 nm; IR (cm-1): 3324, 2921, 1617, 1516, New compound 1 (8,9-Furanyl-pterocarpan- 1445, 1374, 1249, 1070, 1044, 887, 836, 785; 3-ol) ESI-MS m/z (% intensity): 417 ([M+H]+,