Tyrosinase Enzyme Inhibitory Activity of selected Indian Herbs

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701Buffer: Potassim dihydrogen orthophosphate,Himedia, India, store at RT; Potassium hydroxide(KOH) Lenoid chemicals Pvt. Ltd. India store at RT;Positive control: Kojic acid, store at 2-8 o c. Otherchemicals were of the highest grade commerciallyavailable.2.2. MaterialsIndian herbal medicines were purchased from localmedicinal markets and identity was confirmed byNatural Remedies Pvt. Ltd, Bangalore shown inTable No-1.2.3. Preparation of extractsDried traditional Indian herbal medicines werepulverized in a grinder and extracted by refluxingwith methanol, of 1:3 ratio and then filtered. Theprocedure was repeated two times. The filtrates werecombined and were concentrated under reducedpressure, vaccum-dried, and stored in a closedcontainer until use. Same procedure followed forwater extraxtion.2.4. PrincipleL-DopatyrosinaseDopachromeSpectrophotometric quantification at 475 nm.2.5. Preparation of working solutionsPhosphate buffer potassium (50mM, pH 6.5 at25o c):680.45mg of Potassium dihydrogenorthophosphate is dissolved in 100ml of deionizedwater by adjusting the pH to 6.5 with 1M KOH.1M KOH : 5.611gm of potassium hydroxide isdissolved in 100ml of de-ionised water. Enzyme :Stock1(25000U/250ul)Workingsolution(5600units/ml): 50ul of stock1 is made up to1ml with 50mM Potassium phosphate buffer, pH6.5.Substrate (4.25mM) : 4.19mg L-DOPA isdissolved in 5ml of de-ionised water. Positivecontrol(1mg/ml): 5mg of Kojic acid is dissolved in5ml of 50mM Potassium phosphate buffer,pH6.5.2.6. MethodThe assay is carried out as per the procedure of withslight modifications 8 . In brief, add 112.5ul 50uMpotassium phosphate buffer pH 6.5 / test sample /positive controle of various concentrations, 85ul ofdeionised water and 2.5ul of enzyme (5600 units/ml),mix and pre-incubation, add 50ul substrate (4.25mM)mix and incubate at 37 o c for 8 minutes. FollowingPre-incubation, add 50ul substrate (4.25mM) mix andincubate at 37 o c for 10 minutes. The absorbance ismeasured at 475nm.A control reaction is carried outwithout the test sample.% inhibition =Absorbance (control) – Absorbance (test) x 100Absorbance (control)Final assay concentration in 250ul reaction volume,the final concentration are 23.04mM of potassiumphosphate buffer pH 6.5, 0.85mM of L-Dopa and 14units of mushroom tyrosinase. All reaction is carriedout in 96 microwell plate, Tarsons.Assay quality control parameters: Control OD shouldbe greter than 1.1. Blank value should be less than0.05. IC 50 positive control should be in between 20 to45 ug/ml.3. RESULTS AND DISCUSSIONIn previous papers, inhibition of tyrosinase by avariety of compounds has been studied, with theresult that several inhibitors are now used as cosmeticadditives or as medicinal products forhyperpigmentation 9-10 . Recently, natural substancessuch as green-plant products have been in increaseddemand in the global market for new agents fordepigmenting, cosmeceutical, and skin-lighteningpurposes 11 . Traditional Indian herbal medicines havebeen used in clinical practice for centuries; they areoften used to maintain good health or used to treatvarious diseases. In the present study, these materialswere selected based on compiled ethnobotanical datathat revealed the agents are usually used clinically asskin applications (Table-1). Therefore, we evaluatedtheir effects on tyrosinase enzyme as its percentageinhibition activities. The selected traditional Indianherbal medicines were extracted with methanol, withextract yields ranging from 2.1 to 33.5%.In the present study, we used tyrosinase enzyme as anin vitro model because of the need to measure %inhibition. An assay for inhibition was employedbefore further in vitro testing in skin melanocyteswas done to test melanin content. We used l-DOPAas the substrate to detect any tyrosinase inhibitoryeffect. Among the tested extracts, 7 extracts,Glycyrrhiza glabra (rhizome), Azadiracta indica(bark), Aesculus indica (fruits), Camelliasinensis(leaves), Nelumbo nucifera( seed), Acasiacatechu(bark), Mangifera indica(leaves), showedrelative % inhibition, with tyrosinase above 30% withVol. 3 (3) Jul – Sep2012 www.ijrpbsonline.com 978

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701Table 2: Data showing tysonase inhibitory activity of methanolic andaqueous extracts of selected plant parts at a concentration of 1000µg/mlS.No.Scientific name Part used MI *(Methanol ext.)MI *(Aqueous ext.)1. Acacia catechu (Mimosaceae) B 44.40 12.782. Aesculus indica (Hippocastanaceae) Fr. 38.35 WI3. Aloe vera (Liliaceae) Lf. 08.30 -4. Azadiracta indica ( Meliaceae) Lf.B10.1043.5907.50WI5. Bacopa monniri (Scrophulariaceae ) WP 09.50 04.606. Berberis aristata (Berberidaceae ) S 20.80 WI7. Bixa orellana (Bixaceae) Sd 08.20 WI8. Camelia sinensis (Theaceae) Lf. 32.76 WI9. Centella asiatica (Apiaceae) WP 11.28 -10. Cocos nucifera (Arecaceae) K 23.82 WI11. Curcuma longa (Zingiberaceae) Rhiz. NA 14.3412. Curcuma zeodoria (Zingiberaceae) Rhiz. 04.60 WI13. Cyprus rotandus (Cyperus rotandus) Rhiz. 11.82 WI14. Glycyrrhiza glabra (Fabaceae) Rhiz. 76.53 -15. Hedychium spicatum (Zingiberaceae) Rhiz. 17.87 07.0016. Hemidesmus indicus (Asclepiadaceae) R 14.80 03.2017. Lawsonia inermis (Lythraceae) Lf. 14.62 -18. Mangifera indica (Anacardiaceae) Lf. 32.85 24.4319. Michelia champaka (Magnoliaceae) Flw. 07.00 -20. Nelumbo nucifera (Nymphaeaceae) Flw.Sd.11.9133.5703.40WI21. Nyctanthus arbortistis (Oleaceae) Lf. 16.33 01.8022. Ocimum santum (Lamiaceae) Lf. 07.30 04.3023. Prunus amugdalus (Rosaceae) Sd. 10.92 WI24. Punica granathum (Punicaceae) Fr.P14.1634.11WIWI25. Rosa alba (Roseaceae) Flw. 19.67 WI26. Rubia cordifolia (Rubiaceae) RS14.8009.20-05.3027. Symlocos racemosa (Symplocaceae) B 06.60 03.6028. Tinospora cordifolia (Menispermaceae) S 14.35 WI29. Trigonella foenum graceum (Fabaceae) SdLf.13.3516.1509.9004.6030. Vetivera zizconoides (Poaceae) R 11.28 05.7031. Vitis vinifera (Vitaceae) Fr. 08.60 02.6032. Withania somnifera (Solanaceae) R 14.89 -Abb :- B (Bark), Fr. (Fruits), Lf.( Leaf), R (Root), Rhiz. (Rhizome), S (Stem), Sd (Seed), WP (Whole plant), K (Kernel), Flw (Flower), P (Peel).MI:-Mean % inhibition, NA:-Not active, Note: The value of >50% inhibition is indicated in bold.*Determinations were done in duplicate.Table 3: Data showing IC 50 of reference standard (Kojic acid)for all methanolic and aqueous extractsS. No. Concentration in Kojic acid µg/ml % Inhibition1. 5 µg/ml 14.552. 10 µg/ml 23.203. 25 µg/ml 42.854. 50 µg/ml 66.965. 100 µg/ml 79.52IC 50 = 28.60 (23.78 – 34.71)Table 4: Data showing IC 50 of the methanolic extract of Glycyrrhiza glabraS. No. Concentrations of Glycyrrhiza glabra methanolic extract % Inhibition1. 100 µg/ml 23.542. 500 µg/ml 63.583. 1000 µg/ml 82.05IC 50 = 286.02 (222.91 - 356.74 )Vol. 3 (3) Jul – Sep2012 www.ijrpbsonline.com 980

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-370180Glycyrrhiza glabra% Inh 76.53%706050% Inhibition.4030201001 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37Methanolic Extracts of 32 plantFig. 1: Data showing % Inhibition of tyrosinase enzyme by 37 methanolic extracts ofselected Indian medicinal plants100 µg/ml500 µg/ml1000 µg/mlIC 50 = 286.02 µg/ml.Fig 2: IC 50 value for Glycyrrhiza glabra4. CONCLUSIONIn the present study, 34 selected Indian herbalmedicines were investigated for potentialeffectiveness as skin-whitening agents and inmaintaining skin health. Extract of Glycyrrhizaglabra (rhizome) was shown to be potent tyrosinaseinhibitors in human skin. In addition to extracts ofAzadiracta indica (bark), Aesculus indica (fruits),Camellia sinensis(leaves), Nelumbo nucifera( seed),Acasia catechu(bark), Mangifera indica(leaves), (seegraph of Fig. 1 & 2) which are currently in use ascosmetic , results of this study indicate that exract ofGlycyrrhiza glabra (rhizome) likely to be useful forcosmetic applications and products. Their bioactivityVol. 3 (3) Jul – Sep2012 www.ijrpbsonline.com 981

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701guided isolated components may prove to haveconsiderable value as cosmetics additives in thefuture.5. REFERENCES1. Hearing VJ. Biogenesis of pigment granulesa sensitive way to regulate melanocytefunction. Journal of Dermatological Science.2005;37:3-14.2. Briganti S, Camera E and Picardo M.Chemical & instrumental approaches to treathyperpigmentation. Pigment cell research.2003;16:101-110.3. Sturm RA, Tesdeal RD and Box NF. Humanpigmentation genes identification, structureand consequences of polymorphic variation.Gene. 2001;277:49-62.4. Perluige M, DeMarco F, Foppoli C, CocciaR, Blarzino C, Marcante ML, Cini C.Tyrosinase protectes human melanocytesfrom ROS- generating compounds.Biochemical & Biophysical researchcommunications. 2003;305:250-256.5. Seiberg M, Paine C, Sharlow E, Andrede-Gordon P, Costanzo M, Eisinger M andShairo SS. Inhibition of melanosometransfer results in skin lightning. Journal ofInvestigative Dermatology. 2000;115:162-167.6. Shiino M, Watanabe Y and Umezawa K.Synthesis of N-substituted N-nitrosohydroxylmines as inhibitors ofmushroom tyrosinase. Bioorg Med Chem.2001;9:1233-1240.7. Kiken DA and Cohen DE. Contactdermatitis to botanical extracts. AmericanJournal of Contact Dermatitis. 2002;13:148-152.8. Lida K, Hase K, Shimomura K, Sudo S,Kadota S and Nambati T. potent inhibitionof tyrosinase activity and melaninbiosynthesis from Rheum officinale. Plantamed. 1995;65:425-428.9. Rescigno A, Sollai F, Pisu B, Rinaldi A andSanjust E. Tyrosinase inhibition: general andapplied aspects. Journal of EnzymeInhibition and Medicinal Chemistry.2002;17:207–218.10. An BJ, Kwak JH, Park JM, Lee JY, Park TS,Lee JT, Son JH, Jo C and Byun MW.Inhibition of enzyme activities and theantiwrinkle effect of polyphenol isolatedfrom the persimmon leaf (Diospyros kakifolium) on human skin. DermatologicSurgery. 2005;31:848–854.11. Aburjai T and Natsheh FM. Plants used incosmetics. Phytotherapy Research.2003;17:987–1000.12. Sharma PV. Charaka Samhita, vol-1.Chaukhambha orientalia, Varanasi; 1994;p.26.13. Varier VPS. Indian medicinal plants, acompendium of 500 species.Vol.1-5. OrientLongman Ltd., Madras; 1993.14. Bunny S. The Illustrated Book of Herbs.Octopus Books Ltd., London. 1984; p.54.15. Murthy KRS. Bhavaprakasa of bhavamisra.Vol I. Krishnadas Academy. Varanasi; 1998.16. Sharma PC, Yelne MB, Dennis TJ. Databaseon medicinal plants used in Ayurveda. Vol.1-5. Central Council for research inAyurveda & Siddha, New Delhi; 2000.17. Sharma PV. Dravyaguna vijnana. Vol 2.Chaukhambha bharathi academy, Varanasi;1993; p.537.Vol. 3 (3) Jul – Sep2012 www.ijrpbsonline.com 982