Screening of Peruvian Medicinal Plants for Tyrosinase Inhibitory Properties: Identification of Tyrosinase Inhibitors in Hypericum laricifolium Juss.

Tyrosinase inhibitors are of far-ranging importance in cosmetics, medicinal products, and food industries. Peru is a diverse country with a wide variety of plants that may contain excellent anti-tyrosinase inhibitors. In the present study, the tyrosinase inhibitory properties of 50 medicinal plant extracts from Peru were investigated using tyrosinase assay. Among plant extracts, those that showed an inhibition rate >50% were Hypericum laricifolium Juss., Taraxacum officinale F.H.Wigg., and Muehlenbeckia vulcanica Meisn., with H. laricifolium Juss. showing the greatest anti-tyrosinase activity. Although H. laricifolium Juss. has been widely used as a medicinal plant by Peruvians, little is known regarding its bioactive components and effects on tyrosinase activity. For this reason, we attempted to discover tyrosinase inhibitors in H. laricifolium Juss. for the first time. The bioactive components were separated by Sephadex LH-20 chromatography and eluted with 100% methanol. Eight compounds were discovered and characterized by high-performance liquid chromatography coupled with diode array detection (HPLC-DAD): protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanilic acid, caffeic acid, kaempferol 3-O-glucuronide, quercetin, and kaempferol. In addition, the concentration of these compounds required for 50% inhibition (IC50) of tyrosinase activity were evaluated. Quercetin exhibited the strongest tyrosinase inhibition (IC50 14.29 ± 0.3 μM). Therefore, the Peruvian plant H. laricifolium Juss. could be a novel source for anti-tyrosinase activity.


Introduction
Tyrosinase (E.C.1.14.18.1), also known as polyphenol oxidase, is a multifunctional glycosylated copper-containing enzyme which is widespread in many organisms including animals, plants, and microorganisms [1]. Tyrosinase is mainly involved in the initial steps of the pathway which catalyzes the orthohydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) (monophenolase activity) and the oxidation of L-DOPA to dopaquinone (diphenolase activity), which is then converted to the end-product melanin [2,3]. Melanin is the main component responsible for the darkening of the skin and hair, and plays an important role against ultraviolet (UV) ray damage. However, the accumulation of an excessive level of melanin can cause skin damage, such as age spots or malignant melanoma. It has also been associated with Parkinson's disease [4]. Additionally, the browning of fruits and vegetables is related to the oxidation of phenolic compounds catalyzed by tyrosinase, which results in a loss of market value for the food [2][3][4][5]. Therefore, tyrosinase inhibitors are important in cosmetics (hyperpigmentation), medicinal products, and food industries. To date, despite the existence of a large number of tyrosinase inhibitors, only a few are marketed as safe [3,6]. Thus, the search for new tyrosinase inhibitors is important for the treatment of hyperpigmentation, development of skin-whitening agents, and use as preservatives in the food industry.
Peru is a developing country characterized by a rich biodiversity, where medicinal plants still represent the main therapeutic tool in traditional medicine, especially in many ethnic groups. It is estimated that 17,144 flowering plant species exist in Peru, of which 5354 (31.3%) are endemic, while the rest are native or introduced [7,8]. Some of these may be good candidates for the development of new anti-tyrosinase agents and/or standardized phytomedicines, and may be effective as future therapies for various diseases. However, to date, no report has been published regarding the tyrosinase inhibitory activity of Peruvian plants.
For that reason, as part of our ongoing efforts to find new tyrosinase agents, we measured the anti-tyrosinase activity of 50 Peruvian plants traditionally used as medicinal infusions by Peruvian people, based on the ethnobotanical information provided by literature sources [9][10][11][12][13][14][15][16][17]. Among the extracts that were screened, we selected the most active plants against tyrosinase based on spectrophotometric analyses of their bioactive compounds. Subsequently, we found that Hypericum laricifolium Juss. (A10) extract showed significant tyrosinase inhibition. H. laricifolium Juss., is a species of Hypericum (Clusiaceae) which can be found in tropical regions of the world at high altitudes, particularly in South America and Africa. In South America, H. laricifolium Juss., is distributed from western Venezuela, along the Andes of Colombia and Ecuador through central Peru and into Bolivia. In Peru it is called "Hierba de la fortuna" and used for good health, fortune, or luck in love, while in Ecuador it is called "Romerillo" or "Hierba de San juan" and used as a diuretic and for inducing menstruation [18,19].
In one study, from the aerial parts of H. laricifolium Juss., twelve xanthones were isolated and identified [20]. Biological investigations also revealed the presence of phenolic acids, flavonoids, triterponoids [21], and dimeric and monomeric acylphloroglucinol derivatives [22][23][24]. Additionally, studies in Venezuela reported the chemical composition of H. laricifolium Juss. essential oils [25]. Scientists in Peru also found that the leaves of H. laricifolium Juss. had an anti-depressive effect in rats [22]. To the best of our knowledge, this plant has not previously been studied relative to its effect on tyrosinase. In this study, for the first time, we investigated inhibitory effects and characterization of compounds with an effect on tyrosinase extracted from H. laricifolium Juss.

Ethnopharmacological Data
Plants represent a rich source of bioactive chemicals, many of which are largely free from harmful adverse effects [26,27], but their individual activity is not sufficiently potent to be of practical use. Recently, safe and effective tyrosinase inhibitors have become important for their potential applications in improving the quality of food, preventing pigmentation disorders, and preventing other melanin-related health problems in human beings, in addition to cosmetic applications [28]. We selected 50 species to be studied which were bought in popular markets in Lima, Peru. These plants were chosen due to the fact that there have been no previous studies on their effect on tyrosinase activity. Table 1 provides the scientific name, the common name, the family name, as well as the traditional use of each plant. Many of the plants had two or more names which could be found in Spanish or in the native language Quechua. All the plants chosen are used by Peruvians, and have different traditional uses, such as for treatment of liver diseases, use as an anti-inflammatory, or for curing cancer, diabetes, and other diseases. These 50 species belong to 29 botanical different families. Asteraceae was the family with the largest number medicinal species (18%), followed by the Fabaceae, Lamiaceae, and Euphorbiacea families (10%, 8%, 8%).

Screening of Tyrosinase Inhibitory Activities of Methanol Extracts
Crude extracts of 50 plants were prepared using 70% methanol and their yields were from 1.1% to 57.1% as listed in Table 2. For the extraction of the 50 plants, an assortment of plant parts were used in our study, including leaves, flowers, aerial parts, seeds and roots, according to the traditional use by Peruvians. The effect on tyrosinase inhibition of the 50 crude methanol extracts were investigated at a concentration of 500 µg/mL and the results are reported in Table 2. Arbutin was used as the positive control. Of the 50 extracts assayed, 36 extracts demonstrated an effect on tyrosinase activity, among which three showed an inhibition rate >50%, 5 showed inhibition rates between 40% and 50%, and the rest showed in inhibition rate <40%. The best inhibitory activities were observed in extracts of leaves of H. laricifolium Juss. which showed 74% ± 2.1% inhibition, followed by Taraxacum officinale F.H. Wigg (P49, inhibition percent is 60.8% ± 4.1%), Muehlenbeckia vulcanica Meisn (P82, inhibition percent is 57.1% ± 3.0%), Salvia hispanica L. (P4, inhibition percent is 44.3% ± 3.4%), Senna sp. (A7, inhibition percent is 44.1% ± 2.3%), Anacardium occidentale L. (A41, inhibition percent is 42.5% ± 7.2%), Matricaria recutita L. (A12, inhibition percent is 40.49% ± 2.1%) and Ch. pilosa Goldm (P17, inhibition percent is 40.35% ± 2.0%).
In Korea, one study reported on the antioxidant capacity and tyrosinase inhibition activity of water extracts of various parts of T. Officinale. The leaf extract (1 mg/mL) showed the highest tyrosinase inhibition (34.2%), while the roots and whole plant showed tyrosinase inhibition of more than 20% [32]. M. volcanica contains anthraquinone-O-glycosides, and according to some investigations, some anthraquinones have an anti-tyrosinase effect [33,34]. S. hispanica L. contains compounds such as quercetin, kaemperol and caffeic acids, and these are known to have anti-tyrosinase activity [35]. A. occidentale L. was found to exhibit anti-tyrosinase activity [36], while M. recutita L. showed an ability to reduce ultraviolet B (UVB)-induced pigmentation in vivo [37]. There are no reports on anti-tyrosinase activity in the other samples.
As Figure 1 shows, the extracts which showed an inhibition rate >50%, were selected and their IC 50 (concentration of each extract required for 50% inhibition of the enzyme activity) was determined with H. laricifolium Juss., T. officinale F.H. Wigg, and M. vulcanica Meisn, with an IC 50 of 120.9 µg/mL, 280.1 µg/mL, and 290.4 µg/mL, respectively. Because of its low IC 50 and due to its high availability, H. laricifolium Juss. was selected as a potential source of a new tyrosinase inhibitor. For the extraction of the 50 plants, an assortment of plant parts were used in our study, including leaves, flowers, aerial parts, seeds and roots, according to the traditional use by Peruvians. The effect on tyrosinase inhibition of the 50 crude methanol extracts were investigated at a concentration of 500 μg/mL and the results are reported in Table 2. Arbutin was used as the positive control. Of the 50 extracts assayed, 36 extracts demonstrated an effect on tyrosinase activity, among which three showed an inhibition rate >50%, 5 showed inhibition rates between 40% and 50%, and the rest showed in inhibition rate <40%. The best inhibitory activities were observed in extracts of leaves of H. laricifolium Juss. which showed 74% ± 2.1% inhibition, followed by Taraxacum officinale F.H. Wigg (P49, inhibition percent is 60.8% ± 4.1%), Muehlenbeckia vulcanica Meisn (P82, inhibition percent is 57.1% ± 3.0%), Salvia hispanica L. (P4, inhibition percent is 44.3% ± 3.4%), Senna sp. (A7, inhibition percent is 44.1% ± 2.3%), Anacardium occidentale L. (A41, inhibition percent is 42.5% ± 7.2%), Matricaria recutita L. (A12, inhibition percent is 40.49% ± 2.1%) and Ch. pilosa Goldm (P17, inhibition percent is 40.35% ± 2.0%).
In Korea, one study reported on the antioxidant capacity and tyrosinase inhibition activity of water extracts of various parts of T. Officinale. The leaf extract (1 mg/mL) showed the highest tyrosinase inhibition (34.2%), while the roots and whole plant showed tyrosinase inhibition of more than 20% [32]. M. volcanica contains anthraquinone-O-glycosides, and according to some investigations, some anthraquinones have an anti-tyrosinase effect [33,34]. S. hispanica L. contains compounds such as quercetin, kaemperol and caffeic acids, and these are known to have anti-tyrosinase activity [35]. A. occidentale L. was found to exhibit anti-tyrosinase activity [36], while M. recutita L. showed an ability to reduce ultraviolet B (UVB)-induced pigmentation in vivo [37]. There are no reports on anti-tyrosinase activity in the other samples.
As Figure 1 shows, the extracts which showed an inhibition rate >50%, were selected and their IC50 (concentration of each extract required for 50% inhibition of the enzyme activity) was determined with H. laricifolium Juss., T. officinale F.H. Wigg, and M. vulcanica Meisn, with an IC50 of 120.9 μg/mL, 280.1 μg/mL, and 290.4 μg/mL, respectively. Because of its low IC50 and due to its high availability, H. laricifolium Juss. was selected as a potential source of a new tyrosinase inhibitor.
In other studies, the ethanol extracts of leaves of H. laricifolium Juss. showed a weak minimum inhibitory concentration (MIC) of 250 µg/mL against Candida albicans, but the bioactive compounds were not identified [38]. In the literature, no information has been found regarding tyrosinase activity in H. laricifolium Juss., but there are reports on one species of Hypericum of which St. John's Wort (Hypericum perforatum L.) is the most well-known. It is a medicinal herb with antidepressant activity and is possibly associated with anti-cholinesterase, anti-tyrosinase, and antioxidant properties [20,39].
Its known that genus Hypericum has a variety of molecules with different biological activities, among them the content of hypericin and various phenolics is considerable, exhibiting wide pharmacological activities such as anti-inflammatory, anti-cholinesterase, antimicrobial, antiviral and antioxidant properties [40]. The species Hypericum humifusum and Hypericum perfoliatum have great content of hypericin and phloglucinols. Hypericin is used against viruses and retroviruses such as the human immunodeficiency virus (HIV) 35, and influenza A, while phloroglucinols (hyperforin and adhyperforin) show potential antibacterial, antidiabetic, and cytotoxic activities [40,41].
According to our results mentioned above, we decided to compare the differences in anti-tyrosinase activities between 70% and 50% methanol extracts, and a methylene chloride extract of H. laricifolium Juss. at concentrations of 100, 500, and 1000 µg/mL, and arbutin. As shown in Table 3, we found that the methylene chloride extract did not show any activity and the 50% methanol extract showed low inhibition at 1000 µg/mL (22.3%). The 70% methanol extract demonstrated a good inhibition of tyrosinase activity; the inhibition rate was >50% at 500, and 1000 µg/mL (IC 50 is 122.1 µg/mL). The IC 50 value for arbutin was 42.0 µg/mL. According these results, a 70% methanol extract should be studied to determine the constituents responsible for tyrosinase inhibition.  Figure 2 shows the HPLC chromatograms of crude H. laricifolium Juss. extracts. All the sample components were separated in less than 40 minutes, with retention factors depending mainly on structural hydrophobicity, as previously demonstrated by a number of studies employing the HPLC for highly efficient separations of complex samples [42]. After the isolation, we identified and isolated eight compounds as candidates for tyrosinase inhibition, eluting in different retention times. From compound (1) to compound (5), peaks appeared between 12 and 19 min, and from compound (6) to compound (8), peaks appeared between 20 and 30 min. Figure 3 shows the chemical structures of these isolated compounds. From the 70% methanol extract of the plant, protocatechuic acid was obtained and identified as compound (1), p-hydroxybenzoic acid as compound (2), chlorogenic acid as compound (3), vanillic acid as compound (4), caffeic acid as compound (5), kaempferol 3-O-glucuronide as compound (6), quercetin as compound (7) and kaempferol compound as (8). These compounds were positively identified both on the basis of information provided by the literature, by direct comparisons with authentic materials available commercially, and by comparison with data from similar species in the same family [21,43,44].

Effect of Bioactive Compounds on Tyrosinase Inhibition
The tyrosinase inhibitory activities of the major bioactive components of H. laricifolium Juss. were confirmed using a tyrosinase assay. As shown in Table 4, all compounds except quercetin (compound 7), did not show any anti-tyrosinase activity. Quercetin showed strong tyrosinase inhibition with an IC50 of 14.29 ± 0.3 μM, which is seven times lower than that of arbutin (IC50 = 110.4 ± 1.9 μM) but a little higher than that of kojic acid (IC50 8.0 ± 0.5 μM), which is also a positive control produced by many species of fungi such as Aspergillus and Penicillium sp. [27]. Although some other compounds showed some tyrosinase inhibitory activities at 500 μg/mL or 1000 μg/mL (data not shown), there were no other strong inhibitors.

Effect of Bioactive Compounds on Tyrosinase Inhibition
The tyrosinase inhibitory activities of the major bioactive components of H. laricifolium Juss. were confirmed using a tyrosinase assay. As shown in Table 4, all compounds except quercetin (compound 7), did not show any anti-tyrosinase activity. Quercetin showed strong tyrosinase inhibition with an IC50 of 14.29 ± 0.3 μM, which is seven times lower than that of arbutin (IC50 = 110.4 ± 1.9 μM) but a little higher than that of kojic acid (IC50 8.0 ± 0.5 μM), which is also a positive control produced by many species of fungi such as Aspergillus and Penicillium sp. [27]. Although some other compounds showed some tyrosinase inhibitory activities at 500 μg/mL or 1000 μg/mL (data not shown), there were no other strong inhibitors.

Effect of Bioactive Compounds on Tyrosinase Inhibition
The tyrosinase inhibitory activities of the major bioactive components of H. laricifolium Juss. were confirmed using a tyrosinase assay. As shown in Table 4, all compounds except quercetin (compound 7), did not show any anti-tyrosinase activity. Quercetin showed strong tyrosinase inhibition with an IC 50 of 14.29 ± 0.3 µM, which is seven times lower than that of arbutin (IC 50 = 110.4 ± 1.9 µM) but a little higher than that of kojic acid (IC 50 8.0 ± 0.5 µM), which is also a positive control produced by many species of fungi such as Aspergillus and Penicillium sp. [27]. Although some other compounds showed some tyrosinase inhibitory activities at 500 µg/mL or 1000 µg/mL (data not shown), there were no other strong inhibitors. The inhibition percent of original methanol extract of H. laricifolium Juss. (data not shown) and quercetin at 100 µg/mL of concentration were compared, the methanol extract show around 50% inhibition, while quercetin show high inhibition over 95%. Therefore, it is revealed that quercetin has a significant inhibition on tyrosinase activity compared to the other compounds and is the main determinant of the observed activity for H. laricifolium Juss. The x-axis and y-axis is fuzzy.
In previous studies on flavonoids, compounds such as quercetin and kaempferol were shown to interfere with the activity of tyrosinase through chelation of copper in its active site [28]. It has been demonstrated that quercetin and kaempferol can suppress melanogenesis, directly inhibiting tyrosinase activity or reducing tyrosinase expression [45]. In our study, quercetin proved to be a good tyrosinase inhibitor, while kaempferol was a weaker inhibitor than arbutin. Figure 4 shows % inhibition of quercetin using L-tyrosine as a substrate, we decided to study quercetin's ability to inhibit the activity of tyrosinase at different times and in different concentrations (2 min, 5 min, 10 min, and 3 µg/mL, 6.25 µg/mL, and 12.5 µg/mL). At a concentration of 12.5 µg/mL, quercetin inhibited >50% tyrosinase at 2 min, 5 min, and 10 min, with the greatest inhibition being 88.1%.
The inhibition percent of original methanol extract of H. laricifolium Juss. (data not shown) and quercetin at 100 μg/mL of concentration were compared, the methanol extract show around 50% inhibition, while quercetin show high inhibition over 95%. Therefore, it is revealed that quercetin has a significant inhibition on tyrosinase activity compared to the other compounds and is the main determinant of the observed activity for H. laricifolium Juss. The x-axis and y-axis is fuzzy.
In previous studies on flavonoids, compounds such as quercetin and kaempferol were shown to interfere with the activity of tyrosinase through chelation of copper in its active site [28]. It has been demonstrated that quercetin and kaempferol can suppress melanogenesis, directly inhibiting tyrosinase activity or reducing tyrosinase expression [45]. In our study, quercetin proved to be a good tyrosinase inhibitor, while kaempferol was a weaker inhibitor than arbutin.  Figure 4 shows % inhibition of quercetin using L-tyrosine as a substrate, we decided to study quercetin's ability to inhibit the activity of tyrosinase at different times and in different concentrations (2 min, 5 min, 10 min, and 3 μg/mL, 6.25 μg/mL, and 12.5 μg/mL). At a concentration of 12.5 μg/mL, quercetin inhibited >50% tyrosinase at 2 min, 5 min, and 10 min, with the greatest inhibition being 88.1%. As quercetin is the only active compound in H. laricifolium Juss. responsible for tyrosinase inhibition, the 70% methanol extract, with the highest concentration of quercetin, showed the strongest activity against tyrosinase, more than the 50% methanol extract and methylene chloride extract. This study is a starting point for further investigations on tyrosinase inhibitors with respect As quercetin is the only active compound in H. laricifolium Juss. responsible for tyrosinase inhibition, the 70% methanol extract, with the highest concentration of quercetin, showed the strongest activity against tyrosinase, more than the 50% methanol extract and methylene chloride extract. This study is a starting point for further investigations on tyrosinase inhibitors with respect to Peruvian plant extracts. Simultaneously, we suggest that more Peruvian plants should be explored in order to identify more useful chemical compounds.

Ethnobotanical Search
The plants were selected according to the ethnobotanical bibliography provided by different literature sources [9][10][11][12][13][14][15][16][17][29][30][31], particularly those with a lack of scientific information about their activity. Scientific names, common names which can be found in Spanish or Quechua (native language in Peru), and traditional uses for Peruvian population are summarized in Table 1.

Chemicals
Mushroom tyrosinase (120 kDa), L-tyrosine, propylene glycol, dimethyl sulfoxide, arbutin, and kojic acid were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Potassium phosphate dibasic (K 2 HPO 4 ), and potassium phosphate monobasic (KH 2 PO 4 ), were obtained from Junsei Chemical Co. (Tokyo, Japan). Deionized distilled water used for all solutions, dilutions, and HPLC analysis was obtained from a Milli-Q system (Millipore, Bedford, MA, USA) with a resistance of over 18.2 MΩ cm. The Sephadex LH-20 column was purchased from GE Healthcare (Uppasala, Sweden). All organic solvents were HPLC grade and were obtained from J.T. Baker (Phillipsburg, NJ, USA). All other chemicals and solvents, unless otherwise specified, were guaranteed reagent grade and purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

Plant Materials
The 50 dried plant samples were purchased from different local markets in Lima, Peru from May to October 2015. The vouchers of all samples were deposited at the Center for Efficacy Assessment and Development of Functional Foods and Drugs, Hallym University (Chuncheon, South Korea). The samples were identified according to macroscopic characters using available literature in English and Spanish and verified by Paul H. Gonzales Arce from the Museo de Historia Natural, Universidad Nacional Mayor de San Marcos, Lima, Peru.

Preparation of Extracts and Isolation of Plant Samples
The dried Peruvian plants (20-50 g) were pulverized at room temperature and then were extracted by maceration at room temperature with 70% methanol for 72 h. The supernatants were filtered through filter paper (Hyundai Micro, Seoul, Korea) and evaporated under vacuum at 37 • C by means of a rotary evaporator (Eyela, Tokyo Rikakikai CO, Tokyo, Japan). The resultant extract was stored at −4 • C until use. Fifty extracts were prepared, and the extractions were performed in duplicate. A yield for each extract was obtained after the solvent was removed and expressed as the calculated weight of air-dried crushed plant material with respect to the starting material.

HPLC Analysis
HPLC analysis was carried out on a Dionex system (Dionex, Sunnyvale, CA, USA) consisting of a P850 pump, an ASI-100 automated sample injector, a Synergi Hydro-RP 80A column (150 mm × 4.6 mm, 4 µm; Phenomenex, Torrance, CA, USA) maintained at 30 • C, and an UVD170S detector. Briefly, the mobile phase was composed of 0.1% trifluoroacetic acid (A-line) and methanol (B-line). The gradient elution system was modified as follows: 0-35 min, starting with 5% B-line, programmed to reach 40% B-line at 15 min using a linear gradient, followed by 100% B-line from 15-35 min. The flow rate was 0.7 mL/min and 10 µL was the sample injection volume. The detector monitored the eluent at wavelength 254 nm.

NMR Analysis
Two compounds were separated from the 70% MeOH extract of H. laricifolium Juss. by Sephadex LH-20 column chromatography. Kaempferol 3-O-glucuronide (compound 6) and quercetin (compound 7) were identified by comparing 1 H-NMR and 13 C-NMR spectra with previously reported data. 1 H-NMR spectra of these two isolated, pure compounds were recorded with a Bruker AV600 instrument (Mundelein, IL. USA), using DMSO-d 6 as a solvent. The detailed structural information was listed as following:  13 C-NMR data for compound 6 are identical to those reported previously [46]. Compound 6 was identified as kaempferol 3-O-glucuronide.  13 C-NMAR data for compound 7 are identical to those reported previously [47,48]. Compound 7 was identified as quercetin.

Tyrosinase Assay
Tyrosinase activity was determined by spectrophotometry, with minor modifications [26]. First, 80 µL of 0.1 M potassium phosphate buffer (pH 6.6), 20 µL of sample dissolved in dimethyl sulfoxide at the concentrations needed (final concentrations 100-500 µg/mL), and 50 µL of L-tyrosine buffer (1.5 mM) solutions were mixed. Then, 50 µL of mushroom tyrosinase solution (200 unit/mL in phosphate buffer) was added to each mixture, which was then incubated at 25 • C for 15 min. The absorbance of the mixture was observed at 450 nm using a 96-well reader and an EL-800 Universal microplate reader (Bio-Tek Instrument Inc. Winooski, VT, USA). Arbutin and kojic acid were used as positive controls. All the samples were first tested at 500 µg/mL and those showing 50% inhibition or higher in three different repetitions were further evaluated for the concentration necessary for 50% inhibition (IC 50 ). The percent inhibition of tyrosinase was calculated as follows: where A is the absorbance of the reaction mixture of the enzyme but without test samples, B is the absorbance of the buffer solution without test samples and enzyme, C is the absorbance of the test samples and enzyme, and D is the absorbance of the test sample but without enzyme.

Statistical Analysis
The results were expressed as mean ± standard deviation (SD), and were repeated three to five times. The inhibitory concentration (IC 50 ) was calculated by log-Probit analysis.