Tyrosinase inhibitory lignans from the methanol extract of the roots of Vitex negundo Linn. and their structure-activity relationship.
Phytochemical investigation of the methanol extract of Vitex negundo afforded eight lignans; negundin A 1, negundin B 2, 6-hydroxy-4-(4-hydroxy-3-methoxy)-3-hydroxymethyl-7-methoxy-3,4-dihydro-2-naphthaledehyde 3, vitrofolal E 4, (+)-lyoniresinol 5, (+)-lyoniresinol-3[alpha]-O-[beta]-D-glucoside 6, (+)-(-)-pinoresinol 7, and (+)-diasyringaresinol 8. The structures of these compounds were elucidated unambiguously by spectroscopic methods including 1D and 2D NMR analysis and also by comparing experimental data with literature data. The tyrosinase inhibitory potency of these compounds has been evaluated and attempts to justify their structure-activity relationships have been made in the present work. The compound 5 was found to be the most potent (I[C.sub.50] = 3.21 [micro]M) while other compounds demonstrated moderate to potent inhibitions. It was found that the substitution of functional group(s) at C-2 and C-3 positions and the presence of the -C[H.sub.2]OH group plays a vital role in the potency of the compounds. The compound 5 can act as a potential lead molecule to develop new drugs for the treatment of hyperpigmentation associated with the high production of melanocytes.
[c] 2005 Elsevier GmbH. All rights reserved.
Keywords: Vitex negundo; Lignans; Tyrosinase inhibition; Hyperpigmentation; Melanocytes; Structure-activity relationship
Tyrosinase (EC 184.108.40.206) is a multifunctional copper-containing enzyme widely distributed in plants and animals. It catalyses the oxidation of monophenols, O-diphenols and O-quinones. Tyrosinase is known to be a key enzyme for melanin biosynthesis in plants and animals. Tyrosinase inhibitors therefore can be clinically useful for the treatment of some dermatological disorders associated with melanin hyperpigmentation. They also find uses in cosmetics for whitening and depigmentation after sunburn. In addition, tyrosinase is known to be involved in the molting process of insects and adhesion of marine organisms (Shiino et al., 2001). Melanin is a heteropolymer of indole compounds that is produced inside melanosomes by the action of the tyrosinase enzyme on the tyrosinase precursor material in melanocytes. It has recently been shown that other factors such as metal ions and the TRP-1 and TRP-2 enzymes also contribute to the production of melanin. However, tyrosinase plays a critical regulatory role in melanin biosynthesis. Therefore, many tyrosinase inhibitors that suppress melanogenesis have been actively studied with the aim of developing preparations for the treatment of hyperpigmentation (Masamoto et al., 2003).
Vitex negundo Linn. (syn: V. inesia Lam.) which is a deciduous shrub belongs to family Verbenaceae, chiefly occurring in Pakistan, India and Ceylon (Watt, 1972, Nasir and Ali, 1974). Literature survey of V. negundo revealed the presence of volatile oil (Singh et al., 1999), triterpenes (Chawla et al., 1992b), diterpenes (Chawla et al., 1991), sesquiterpenes (Vishnoi et al., 1983), flavonoids (Achari et al., 1984), flavone glycosides (Misra and Subramanian, 1980), iridoid glycosides (Dutta et al., 1983), (Sehgal et al., 1982), (Sehgal et al., 1983), stilbene derivative (Banerji et al., 1988) and lignan (Chawla et al., 1992a). Though almost all plant parts are used, the extract from leaves and roots is the most important in the field of medicine and is sold as drugs (Chandramu et al., 2003). The leaf extract is used in Ayurvedic and Unani system of medicine (Kapur et al., 1994). The decoction of leaves is considered as tonic, vermifuge and is given along with long pepper in catarrhal fever (Chandramu et al., 2003). Water extract of mature fresh leaves exhibits anti-inflammatory, analgesic and antihistamine properties (Dharmasiri et al., 2003). The methanol extract of roots possesses potent snake venom neutralizing capacity (Alam and Gomes, 2003). The acetone extract of V. negundo was found to possess insecticidal, ovicidal, feeding deterrence, growth inhibition and morphogenetic effects against various life stages of a noxious lepidoteron insect-pest, Spilarctia obliqua Walker (Prajapati et al., 2003). A poultice of this plant is used by local cosmetic practitioners as cosmetic for the treatments of hyper pigmentation such as melasma and ephilides. However, no scientific reason has so far been described. The methanolic extract of this plant showed significant inhibitory activity against tyrosinase. On further fractionation, the major activity was located in chloroform soluble fraction. We, therefore, have attempted to isolate tyrosinase inhibitors from the chloroform soluble fraction of this plant. The [.sup.1]H-NMR of the crude chloroform fraction showed the presence of phenolic lignans. Since the phenolic compounds are mainly responsible for tyrosinase inhibitory activity, therefore our main focus was on isolation, characterization and tyrosinase inhibitory activity of individual phenolic lignans. The present paper describes the isolation, characterization, their tyrosinase inhibitory potentials and the structure-activity relationships (SAR) of eight lignans isolated from the roots of V. negundo.
Materials and methods
The roots of V. negundo Linn. were collected in November 2001 from Bannu district and identified by Prof. Abdur Rehman (Plant Taxonomist), Department of Botany, Govt. Post Graduate College Bannu, Pakistan. A voucher specimen (No. 318b) has been deposited at the herbarium of the Botany Department of Post Graduate College, Bannu, Pakistan.
General experimental procedures
The [.sup.1]H- and [.sup.13]C-NMR, HMQC and HMBC spectra were recorded on Bruker spectrometers operating at 400 MHz for [.sup.1]H and 100 MHz for [.sup.13]C-NMR respectively. MS and HR-MS were obtained on a JMS-HX-110 with a data system and on JMS-DA 500 mass spectrometers. The UV spectra were recorded on a Hitachi UV-3200 spectrophotometer ([[lambda].sub.max] in nm). Flash silica (230-400 mesh) was used in flash column chromatography. TLC plates and pre-coated silica gel G-25-UV[.sub.254] plates were used to check the purity of the compounds. Visualization of the TLC plates was carried out under UV at 254 and 366 nm and by spraying with ceric sulphate reagent (with heating). The IR spectra were recorded on a 460 Shimadzu spectrometer.
Extraction and isolation
The shade dried roots (40 kg) of V. negundo were extracted three times, 7 days each, with methanol. The combined methanolic extract was evaporated in vaccuo. The resulting residue (1.5 kg) was suspended in water and extracted successively with n-hexane, chloroform, ethyl acetate and n-butanol.
The chloroform soluble fraction was subjected to column chromatography over column silica eluting with hexane-ethyl acetate and then ethyl acetate-methanol in increasing order of polarity. As a result fractions 1-30 were obtained. The fractions showing similar profiles on TLC were combined to afford fractions A-D. Repeated column chromatography of fraction B using hexane-ethyl acetate (3:1) resulted is the isolation of compounds 1, 3, 4, 7. Repeated Column chromatography of fraction C using hexane-ethyl acetate (7:3) solvent system resulted in the isolation of compounds 2, 5, and 8. The n-butanol soluble fraction was subjected to column chromatography over silica gel, eluting with CH[Cl.sub.3]-MeOH in increasing order of polarity. The fraction which eluted with CH[Cl.sub.3]-MeOH (87:13) was subjected to preparative TLC plates using CH[Cl.sub.3]-MeOH (85:15) to afford compound 6 (Fig. 1).
Compound 1: Amorphous white solid (18 mg), mp 125[degrees]C. UV [[lambda].sub.max] (MeOH) nm (log[epsilon]): 256.2 (4.64), 288.6 (4.07), 313.6 (4.10). IR (KBr) [cm.sup.-1]: 3529-3266, 2925, 1679, 1621, 1512, 1461, 1266, 1157, 1024. [.sup.1]H and [.sup.13]C NMR (Azhar-ul-Haq et al., 2004). HREIMS: m/z 352.0911, calcd. for [C.sub.20][H.sub.16][O.sub.6], 352.0942.
Compound 2: Amorphous white solid (15 mg), [[alpha]][.sub.D.sup.26] -56[degrees] (c = 0.11, MeOH). UV [[lambda].sub.max] (MeOH) nm (log [epsilon]): 283 (4.34), 222 (4.70). IR (KBr) [cm.sup.-1]: 3340, 2924, 1599, 1512, 1452, 1225, 1126, 1024. [.sup.1]H and [.sup.13]C NMR (Azhar-ul-Haq et al., 2004). HREIMS: m/z 358.1421 calcd. for [C.sub.20][H.sub.22][O.sub.6] 358.1416.
[FIGURE 1 OMITTED]
Compound 3: Yellowish powder (30 mg), mp 126-127[degrees]C. [[alpha]][.sub.D.sup.26]--176.0[degrees] (c = 0.005, MeOH). UV [[lambda].sub.max] (MeOH) nm (log [epsilon]): 255 (4.31), 359 (4.24). IR (KBr) [cm.sup.-1]: 3390, 2840, 1650, 1620, 1565, 1515. [.sup.1]H and [.sup.13]C NMR (Chawla et al., 1992a). HREIMS: m/z 356.1270 calcd. for [C.sub.20][H.sub.20][O.sub.6], 356.1259.
Compound 4: Yellowish amorphous solid (10 mg). IR (KBr) [cm.sup.-1]: 3600-3000, 1684, 1508, 1270, 1211. UV [[lambda].sub.max] (dioxane) nm (log [epsilon]): 320 (4.1), 267 (4.5). [.sup.1]H and [.sup.13]C NMR (Kawazoe et al., 2001). HREIMS: m/z 324.0981 calcd. for [C.sub.19][H.sub.16][O.sub.5], 324.0998.
Compound 5: White powder (20 mg) mp 117-118[degrees]C. [[alpha]][.sub.D.sup.23] + 68[degrees] (MeOH; c = 0.1). UV [[lambda].sub.max] (MeOH) nm (log [epsilon]): 219 (2.32), 278 (0.40). IR (KBr) [cm.sup.-1]: 3380, 1598, 1490, 1463 1295, 1195. [.sup.1]H and [.sup.13]C NMR (Zhang et al. 1999). HREIMS: m/z 420.1773 calcd. for [C.sub.22][H.sub.28][O.sub.8], 420.1784.
Compound 6: Amorphous powder (80 mg). [[alpha]][.sub.D.sup.22] + 22.4[degrees] (MeOH; c = 1.01). IR (KBr) [cm.sup.-1]: 3600-3100, 1570,1515,1460. [.sup.1]H and [.sup.13]C NMR (Miya-mura et al. 1983; (Achnbach et al., 1992). HREIMS: m/z 582.2308 calcd. for [C.sub.28][H.sub.38][O.sub.13], 582.2313.
Compound 7: Colourless needles (11 mg), mp 168-170[degrees]C. [[alpha]][.sub.D.sup.26] + 0[degrees] (MeOH; c = 0.50). IR (KBr) [cm.sup.-1]: 3600-3200, 1606, 1510, 1460. [.sup.1]H and [.sup.13]C NMR (Casabuono and Pomilio, 1994), HREIMS: m/z 358.1410 calcd. for [C.sub.20][H.sub.22][O.sub.6], 358.1416.
Compound 8: Amorphous powder (16mg). mp 170-172[degrees]C, [[alpha]][.sub.D.sup.25] + 110[degrees] (c = 0.1, CH[Cl.sub.3]), UV [[lambda].sub.max] (MeOH) nm (log [epsilon]):212 (4.11), 240 (4.16), 278 (4.09). IR (KBr) [cm.sup.-1]: 3400, 1600, 1500[cm.sup.-1]. [.sup.1]H and [.sup.13]C NMR (Chang et al. 1998), HREIMS: m/z 418.1634 calcd. for [C.sub.22][H.sub.26][O.sub.8], 418.1627.
Tyrosinase inhibition assay
Tyrosinase inhibition assays were performed in 96-well microplate format using SpectraMax 340 micro-plate reader (Molecular Devices, CA, USA) according to the previously developed method (Hearing, 1987). The compounds were initially screened for the O-diphenolase inhibitory activity of tyrosinase using L-DOPA as substrate. All the active inhibitors from the preliminary screening were subjected to I[C.sub.50] studies. Compounds were dissolved in methanol to a concentration of 2.5%. Thirty units of mushroom tyrosinase (28 nM) were preincubated with the compounds in 50 nM Na-phosphate buffer (pH 6.8) for 10 min at 25[degrees]C. Then the L-DOPA (0.5 mM) was added to the reaction mixture and the enzyme reaction was monitored by measuring the change in absorbance at 475 nm (at 37[degrees]C) due to the formation of the DOPAchrome for 10 min. The percent inhibition of the enzyme was calculated as follows, by using MS Excel[R][TM] 2000 (Microsoft Corp., USA) based program developed for this purpose:
Percent inhibition (%) = [B - S/B] x 100.
The B and S are the absorbances for the blank and samples, respectively. After screening of the compounds, median inhibitory concentration (I[C.sub.50]) was also calculated. All the studies have been carried out at least in triplicates and the results represent the mean[+ or -]S.E.M. (standard error of the mean). Kojic acid and L-mimosine were used as standard inhibitors for the tyrosinase. All the chemicals and reagents were purchased from Sigma Chem. Co., MO, USA.
Results and discussion
Tyrosinase inhibition studies on the structurally similar lignans isolated from the roots of V. negundo Linn. have been carried out and their structure-activity relationships (SAR) also studied.
Compound 1 (negundin A) which contains lactone functionality at C-2 position exhibited moderately potent (I[C.sub.50] = 10.06 [micro]M) inhibition against the enzyme tyrosinase when compared with the standard tyrosinase inhibitors Kojic acid (I[C.sub.50] = 16.67 [micro]M) and L-mimosine (I[C.sub.50] = 3.68 [micro]M). Compound 2 (negundin B) which contains -C[H.sub.2]OH group at C-2 position and a double bond between C-1 and C-2, exhibited potent (I[C.sub.50] = 6.72 [micro]M) inhibition against tyrosinase when compared with Kojic acid (I[C.sub.50] = 16.67 [micro]M). When C[H.sub.2]OH group was replaced with CHO functionality at C-2 position the resulting compound 3 exhibited lesser degree of inhibition (I[C.sub.50] = 7.81 [micro]M) than that of 2. Interestingly when the C[H.sub.2]OH group at C-3 position was removed and a double bond introduced between C-3 and C-4 the potency of the compound 4 was further decreased (I[C.sub.50] value 9.76 [micro]M).
The compound 5 where both C-2 and C-3 positions contain C[H.sub.2]OH group in opposite orientations, exhibited highly potency (I[C.sub.50] = 3.21 [micro]M) inhibition. Very interestingly, glycosidation of C[H.sub.2]OH group at C-3[alpha] of compound 6 did not show any inhibition against tyrosinase. This may be due to the presence of the bulky glucose moiety which probably hinders the penetration of the molecule into the active site of the enzyme. It can be concluded that the presence or absence of functional group(s) at C-2 and C-3 positions is crucial for the inhibitory potency of the compounds.
Compound 7 exhibited moderate inhibition (I[C.sub.50] = 15.13 [micro]M). However when 5'-H and 3"-H were replaced with -OC[H.sub.3] group then the resulting compound 8 exhibited potent inhibition (I[C.sub.50] = 5.61 [micro]M) against tyrosinase when compared with the standard tyrosinase inhibitors.
All these results are summarized in Table 1 and Fig. 2.
From this study, it can be concluded that the lignans (especially compounds 5) isolated from the roots of V. negundo Linn., can be effective inhibitors of tyrosinase enzyme and exhibit potential to be used for the treatment of hyperpigmentation associated with the high production of melanocytes.
[FIGURE 2 OMITTED]
Keeping in view the fact that the plant poultice is locally applied by cosmetic practitioners, it appears that the active lignans may better be used in the form of ointment.
The authors acknowledge Higher Education Commission Islamabad, for financial support through Merit Scholarship Scheme for Ph.D. Studies in Science & Technology.
Achari, B., Chowdhury, U.S., Dutta, P.K., Pakrashi, S.C., 1984. Two Isomeric flavonones from Vitex negundo. Phytochemistry 23, 703-704.
Achnbach, H., Lowel, M., Waibel, R., Gupta, M., Solis, P., 1992. New ligan glucosides from Stemmadenia minima. Planta Med. 58, 270-272.
Alam, M.I., Gomes, A., 2003. Snake venom neutralization by Indian medicinal plants (Vitex negundo and Emblica officinalis) root extracts. J. Ethnopharmacol. 86, 75-80.
Azhar-ul-Haq, Malik, A., Anis, I., Khan, S.B., Ahmed, E., Ahmed, Z., Nawaz, S.A., Choudhary, M.I., 2004. Enzyme inhibiting lignans from Vitex negundo. Chem. Pharm. Bull. 52, 1269-1272.
Banerji, J., Das, B., Chakrabarty, R., 1988. Isolation of 4, 4'-dimethoxy-trans-stilbene & flavonoids from leaves & twigs of Vitex negundo Linn. Indian J. Chem. 27B, 597-599.
Casabuono, A.C., Pomilio, A.B., 1994. Lignan and a stilbene from Festuca argentina. Phytochemistry 35, 479-483.
Chandramu, C., Manohar, R.D., Krupadanam, D.G.L., Dashavantha, R.V., 2003. Isolation characterization and biological activity of betulinic acid and ursolic acid from Vitex negundo L. Phytother. Res. 17, 129-134.
Chang, F.R., Chao, Y.C., Teng, C.M., Wu, Y.C., 1998. Chemical constituents from Cassytha filiformis. J. Nat. Prod. 61, 863-866.
Chawla, A.S., Sharma, A.K., Handa, S.S., 1991. Chemical investigation and antiinflammatory activity of Vitex negundo seeds: Part I. Indian J. Chem. 30B, 773-776.
Chawla, A.S., Sharma, A.K., Handa, S.S., Dhar, K.L., 1992a. A lignan from Vitex negundo seeds. Phytochemistry 31, 4378-4379.
Chawla, A.S., Sharma, A.K., Handa, S.S., 1992b. Chemical investigation and anti-inflammatory activity of Vitex negundo seeds. J. Nat. Prod. 55, 163-167.
Dharmasiri, M.G., Jayakody, J.R.A.C., Galhena, G., Liyanage, S.S.P., Ratnasooriya, W.D., 2003. Anti-inflammatory and analgesic activities of mature fresh leaves of Vitex negundo. J. Ethnopharmacol. 87, 199-206.
Dutta, P.K., Chowdhury, U.S., Chakravarty, A.K., Achari, B., Pakrashi, S.C., 1983. Studies on Indian medicinal plants--Part LXXV. Nishindaside, A noval irridoid glycoside from Vitex negundo. Tetrahedron 39, 3067-3072.
Hearing, V.J.J., 1987. Mammalian monophenol monooxygenase (tyrosinase): purification, properties, and reactions catalyzed. Methods Enzymol. 142, 154-165.
Kapur, V., Pillai, K.K., Hussain, S.Z., Balani, D.K., 1994. Hepatoprotective activity of 'JIGRINE' (an Unani polypharmaceutical herbal formulation) on liver damage and lipid peroxidation caused by alcohol-carbon tetrachloride and paracetamol in rats. Indian J. Pharm. Sci. 56, 160.
Kawazoe, K., Yutani, A., Tamemoto, K., Yuasa, S., Shibata, H., Higuti, T., Takaishi, Y., 2001. Phenyl naphthalene compounds from the subterranean part of Vitex rotundifolia and their antibacterial activity against methicillin-resistant Staphylococcus aureus. J. Nat. Prod. 64, 588-591.
Masamoto, Y., Ando, H., Murata, Y., Shimoishi, Y., Tada, M., Takahata, K., 2003. Mushroom tyrosinase inhibitory activity of esculetin isolated from seeds of Euphorbia lathyris L. Biosci. Biotechnol. Biochem. 67, 631-634.
Misra, G.S., Subramanian, P.M., 1980. Three new flavone glycosides from Vitex negundo. Planta Med. 38, 155-160.
Miyamura, M., Nohara, T., Tomimatsu, T., Nishioka, I., 1983. Seven aromatic compounds from bark of Cinnamo-mum cassia. Phytochemistry 22, 215-218.
Nasir, E., Ali, S.I., 1974. Flora of West Pakistan. Department of Botany, University of Karachi, Karachi No.77, p. 1.
Prajapati, V., Tripathi, A.K., Khanuja, S.P.S., Kumar, S., 2003. Anti-insect screening of medicinal plants from Kukrail Forest, Luknow, India. Pharm. Biol. 41, 166-170.
Sehgal, C.K., Taneja, S.C., Dhar, K.L., Atal, C.K., 1982. 2'-p-Hydroxybenzoyl mussaenosidic acid, a new irridoid glucoside from Vitex negundo. Phytochemistry 21, 363-366.
Sehgal, C.K., Taneja, S.C., Dhar, K.L., Atal, C.K., 1983. 6'-p-Hydroxybenzoylmuss-aenosidic acid-an irridoid glucoside from Vitex negundo. Phytochemistry 22, 1036-1038.
Shiino, M., Watanabe, Y., Umezawa, K., 2001. Synthesis of N-substituted N-nitrosohydroxylamines as inhibitors of mashroom tyrosinase. Bioorg. Med. Chem. 9, 1233-1240.
Singh, V., Dayal, R., Bartley, J.P., 1999. Volatile constituents of Vitex negundo leaves. Planta Med. 65, 580-582.
Vishnoi, S.P., Shoeb, A., Kapil, R.S., Popli, S.P., 1983. A furanoeremophilane from Vitex negundo. Phytochemistry 22, 597-598.
Watt, G., 1972. A Dictionary of the Economic Products of India. Cosmo Publications, Delhi-6 India. 4, pp. 248-249.
Zhang, Z., Guo, D., Changling, L., Zheng, J., Koike, K., Jia, Z., Nikaido, T., 1999. Gaultherins A and B, two lignans from Gautheria yunnanensis. Phytochemistry 51, 469-472.
Azhar-ul-Haq (a), A. Malik (a,*), M.T.H. Khan (b), Anwar-ul-Haq (c), S.B. Khan (a), A. Ahmad (a), M.I. Choudhary (a)
(a) International Center for Chemical Sciences, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan
(b) Department of Biochemistry and Molecular Biology, Centre for Biotechnology, University of Ferrara, Via L. Borsari, 46, I-44100 Ferrara, Italy
(c) Technische Universitat Chemnitz, Institute fur Chemie, D-09107 Chemnitz, Germany
Received 28 July 2004; accepted 29 September 2004
E-mail address: firstname.lastname@example.org (A. Malik).
Table 1. Tyrosinase inhibitory activities of the lignans from V. negundo, as compared with the standard inhibitors Compounds I[C.sub.50][+ or -]S.E.M. (a) (in [micro]M) 1 10.06[+ or -]1.064 2 6.72[+ or -]0.652 3 7.81[+ or -]1.0971 4 9.76[+ or -]1.1024 5 3.21[+ or -]0.1654 6 NA 7 15.13[+ or -]1.9521 8 5.61[+ or -]0.3551 Kojic acid (b) 16.67[+ or -]0.519 L-mimosine (b) 3.68[+ or -]0.02234 (a) S.E.M. is the standard error of the mean. (b) Are the standard inhibitors of the enzyme tyrosinase.