Introduction

Tyrosinase (EC 1.14.18.1; polyphenol oxidase, PPO) is a multifunctional copper-containing enzyme (mono- and diphenolase activities) that is involved in the synthesis of melanin. It catalyzes the hydroxylation of tyrosine to o-diphenol and the oxidation of o-diphenol to o-quinone. The formation of melanin has been reportedly important not only in insect defensive and developmental processes (Kramer and Hopkins 1987; Solano 2014; Sugumaran and Barek 2016), but also in mammalian cells (Xu et al. 1997; Ali and Naaz 2018).

In wounded plants, tyrosinase-induced pigmentation is responsible for enzymatic browning, which leads to undesired color quality and loss of nutritional value of plant-derived foods and beverages (Sánchez-Ferrer et al. 1995; Mayer and Harel 1998; Kim and Uyama 2005; Chang 2009). Many naturally occurring tyrosinase inhibitors have been studied for the prevention of enzymatic browning in the food industry, as well as for the reduction of hyper-pigmentation in the cosmetic (Maeda and Fukuda 1991; Samaneh Zolghadri et al. 2019); and medical industries (Mosher et al. 1983; Parvez et al. 2006, 2007). The modes of inhibitory effect for naturally occurring tyrosinase inhibitors were observed as competitive (Kubo and Kinst-Hori 1999; Jiménez et al. 2001; Samaneh Zolghadri et al. 2019), noncompetitive (Kubo and Kinst-Hori 1998; Masum et al. 2019), or mixed (Chen et al. 1991a, b; Samaneh Zolghadri et al. 2019), mediated through any of the following mechanisms: (1) inhibition of PPO (Kahn and Andrawis 1986; Masum et al. 2019); (2) reduction of o-quinones to diphenols (Golan-Goldhirsh et al. 1984; Masum et al. 2019) or Cu2+ to Cu+; (3) interaction with the formation of o-quinone products (Ferrer et al. 1989; Chang 2009; Samaneh Zolghadri et al. 2019) or (4) decrease in the uptake of oxygen for the reaction (Embs and Markakis 1965; Kim and Uyama 2005).

The degree of inhibitory effect is variable because of differences in the sources of tyrosinase and phenolics substrates studied (Chen et al. 1991a, b; Fan et al. 2019). Several tyrosinase inhibitors, including arbutin, ascorbic acid, 5-hydroxymethyl-2-furfural (5-HMF) and kojic acid, had been studied and used for the purpose of skin whiting (Maeda and Fukuda 1991; Mishima et al. 1988; Sharma et al. 2004; Smit et al. 2009; Samaneh Zolghadri et al. 2019). However, there is always a need for new skin-whiting agents. During our continual search for new tyrosinase inhibitors. In our previous report, we found that among 243 crude medicinal plant extracts, Galla Rhois has potent inhibitory activity against mushroom tyrosinase (Seo et al. 2003; Parvez et al. 2007). Thus tyrosinase principal was assayed and was identified the presence of inhibitory bioactive compound by HPLC assays.

Galla Rhois is a commonly used medicine in East Asia for the treatment of several diseases, Galla Rhois is considered to have medicinal properties, to treat diarrhea and bleeding (Zhu 1998) and showed promising hepatoprotective activity (Ren-Bo et al. 2005). Water extract of Galla Rhois with steaming process enhances apoptotic cell death in human colon cancer cells (Yim et al. 2016), Galla Rhois water extract inhibits lung metastasis by inducing AMPK-mediated apoptosis and suppressing metastatic properties of colorectal cancer cells (Mun et al. 2019), gallotannin-enriched extract Isolated from Galla Rhois may be a functional candidate with laxative effects for the Treatment of Loperamide-Induced Constipation of SD Rats (Kim et al. 2016).

However, little work has been carried out on the effects of Galla Rhois derived materials against mushroom tyrosinase activity, despite its excellent pharmacological action (Zhu 1998; Ren-Bo et al. 2005). Tyrosinase inhibitors properties of Galla Rhois may be a good source for leading compounds as a alternatives for the non-natural tyrosinase inhibitors currently used and these effects have not been reported previously in the literature.

The objective of this study was to isolate bioactive compounds from natural source and characterize the nature of tyrosinase inhibitors from Galla Rhois and the HPLC analysis study of Galla Rhois to identify the presence of ascorbic acid, 5-HMF and arbutin (was not identified earlier) and is the first HPLC analysis report. Galla Rhois exert varying degrees of inhibition on tyrosinase-dependent and these result suggesting that Galla Rhois may serve as candidates for depigmenting agents and further study is needed their application on the melanocytes.

Materials and methods

Chemicals

Ascorbic acid, kojic acid, arbutin, 5-hydroxymethyl-2-furfural (5-HMF), mushroom tyrosinase, and l-DOPA were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Arbutin, l-DOPA and Mushroom tyrosinase, were purchased from Sigma Chemical Co. (USA). The analytical solvents, methanol, water, and acetic acid glacial for the quantitative analysis by HPLC were purchased from J. T. Baker (Mallinckrodt Baker Inc., Phillipsburg, NJ, USA) as HPLC reagents. And the solvents—n-hexanes, ethyl acetate (EtOAc), n-butanol (n-BuOH), methylene chloride (MC) methanol (MeOH) etc.—for solvent partition were used the technical grade of Ducksan Pharmaceutical Industry Co. Ltd. (Ansan, Korea).

Plant materials

The sprayed dried extracts of herbal medicines used in this study were purchased from Sun Ten Pharmaceutical Company (Taipei, Taiwan).

Bioassay

Mushroom tyrosinase (EC 1.14.18.1) used for the bioassay was differs somewhat from other sources, this fungal source was used for the experiment because it is readily available. Because the mode of inhibition depends on the structures of both the substrate and inhibitor, l-DOPA was used as the substrate in these experiments, unless otherwise specified. The activity of mushroom tyrosinase, and their effect on the enzyme was determined by spectrophotometry (dopachrome formation at 490 nm). Tyrosinase activity was determined by spectrophotometry with small modifications. 40 μl of 5 mM l-DOPA solution, 80 μl of 67 mM phosphate buffer (pH 6.8) and 40 μl of the test sample solution with or without 40 μl of oriental herbal extract, were mixed. Then, 40 μl of mushroom tyrosinase (100 U/mL) was added. The increase in absorption at 490 nm due to the formation of DOPA chrome (ε490 = 3600 M−1 cm−1) was monitored as a function of time, using a spectrophotometer (molecular devices, precision micro plate reader, USA). The initial rate was used as a measure for the tyrosinase activity. The same procedure was applied with the extract.

The percentage of tyrosinase inhibition activity was calculated as follows:

$$ \% {\text{ inhibition}} = \left[ {\left( {{\text{A}} - {\text{B}}} \right)/{\text{A}}} \right] \times 100 $$

where A = absorbance at 490 nm without test sample (40 μl l-DOPA Solution, 80 μl of 67 mM phosphate buffer (pH 6.8) and after incubation 40 μl of mushroom tyrosinase (100 U/mL) and B = absorbance at 490 nm with test sample (40 μl of herbal extract).

Kinetic study

Inhibition kinetics studied using the Lineweaver–Burk (double-reciprocal) plot method (Piao et al. 2004), The experiments were carried out with the same protocol used in the tyrosinase assay, except for the concentration of l-DOPA (substrate). The reaction velocities (V) were measured with (mM) and without an inhibitor on the linear part of the kinetics (initial rates), where the amount of substrate is not the limiting parameter. The results were analyzed according to the Lineweaver and Burk plot method that allows the determination of the Michaelis constant (Km) and maximum velocity (Vm). The inhibition constant (Ki) according to Michaelis constant equation.

Extraction and fractionation of Galla Rhois

Dried and chopped Galla Rhois (1000 g) was extracted three times with 50% aquous MeOH (5L) by the ultra-sonication (IEC Centra GP8R, Thermo Electron Corporation, Milford, MA, USA) at room temperature for 1 h.

The methanol was removed by the evaporation under reduced pressure, and the resulting soluble material was filtered off with Whatman No. 2, in vacuo. The filtrate was applied onto an Diaion HP-20 (Merck 70-230 mesh, 500 g, 70 cm × 5.5 i.d) column, and then fractionated stepwise with distilled H2O, MeOH–H2O (20:80 v/v), MeOH–H2O (50:50, v/v, 28.9 g), and MeOH. Then solution was partitioned and sequentially extracted twice with (10 L) ethyl ether (12.80%), (10 L) methyl chloride (MC) 0.07%, (2 L) ethyl acetate (E.A) 28.65%, water (24.73%) and then saturated, with (2 L) Butane (n-BuOH) 2.02%, successively. Finally, the each partitioned layer was evaporated at 40 °C, and freeze-dried for 48 h (Labconco/ICE-O-M, Labconco Co., Kansas, Missouri, USA). Finally, each fraction samples was bioassays.

The yield (g) and recovery rate of the each partition were as ethyl ether (85.73 g), MC (0.463 g), ethyl acetate (191.93 g), n-BuOH (13.54 g), and water layer (165.69 g) respectively. The partition procedure result was shown in Scheme 1.

Scheme 1
scheme 1

The solvent fractionation procedure of Galla Rhois

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The ethyl acetate fraction (191.93 g) was chromatographed on a silica gel column (Merck 70-230 mesh, 500 g, 70 cm × 5.5 i.d.) and successively eluted with a stepwise gradient of ethyl acetate/methanol (0, 5, 10, 15, 20 and 25%). The active 5% fraction was chromatographed on a silica gel column and eluted with a stepwise gradient of chloromform/methanol (0, 10, 20, 30, 40 and 50%). The active 30% fraction was collected and analysed by TLC (hexane/ethyl acetate, 3:1). Fractions with identical Rf values were combined. This was chromatographed on a Sephadex LH column (Pharmacia 25-100 mesh, 200 cm × 3.5 i.d) and eluted with methanol/chloroform 4:1. For further separation of the biologically active substance (s), a waters Delta Prep 4000 HPLC was used. The column C18 (Gemini, 150 mm × 4.6 mm (I.D), Phenomenex, USA) (Waters) using a stepwise gradient of methanol/water (30, 40, 50, 60, 70, 80, 90 and 100%) at a flow rate of 1 mL/min. Finally, one potent active principle F2 was isolated in the 30% methanol/water fraction, and the other major potent active principle was isolated in the 60% methanol water fraction (F1 and F3).

HPLC analytical methods

The standards, Kojic acid, ascorbic acid, arbutin and 5-hydroxymethyl-2-furfural (5-HMF) etc., were quantitatively analyzed of Galla Rhois using HPLC as follows.

Firstly, kojic acid, ascorbic acid, arbutin, and 5-hydroxymethyl-2-furfural (5-HMF) as each standard materials were weighted 1.0 mg, respectively and were dissolved according to the analysis condition of standard materials. The dissolved standard solution was diluted as 0.01, 0.05, and 0.1 mg/mL respectively, and then each standard HPLC chromatograms were obtained. The relationship between the concentration and the peak-area was measured by the minimum square method (R2 value). The HPLC apparatus was a Waters Breeze System (717 + Auto sampler, TCM, 2996 Photodiode Array Detector, 1525 Binary HPLC Pump, Waters Co., Milford, USA), and the associated Waters Empower System (Ver. 5.00, Waters Co., Milford, USA) was used for data acquisition and integration.

Samples preparation

In order to quantify the amount of ascorbic acid, arbutin, 5-HMF, and kojic acid from Galla Rhois, we prepared samples as follows:

That is, the spray dried crude Sun Ten product of about 100.0 mg of Galla Rhios and its solvent partition, and its samples were accurately weighed, put in a test tube, then dissolved in 10 mL of water, then filtered using a 0.45 μm syringe filter (PVDF, Waters, USA).

And then the prepared samples were quantitatively analyzed by HPLC.

The HPLC analysis condition as follows:

Standards, Galla Rhois, and its fermented sample were analyzed by reverse-phased HPLC on a C18 column (Gemini, 150 mm × 4.6 mm (I.D), Phenomenex, USA) with a graduated condition 2.5% (for 5 min), 12% (for 10 min), successively, of MeOH/H2O containing with 2% acetic acid (v/v) at 1 mL/min, with a detection at PDA UV 205–600 nm, resolution 1.2 nm, and with a injection in portions of 25 μL and the column temperature was 25 °C.

The solvent partition of Galla Rhois Sun Ten products

In order to obtain better active fraction from Galla Rhois than Crude spray dried Sun Ten products, we carried out the solvent partition by the solvent polarity as follows:

Firstly, we weighted about 1 kg of Galla Rhois Sun Ten products (as 670 g of Galla Rhois ex.), and then we solvent-extracted with 50% MeOH aqueous solution 5 L by the ultra-sonication (IEC Centra GP8R, Thermo Electron Corporation, Milford, MA, USA) at room temperature for 1 h, twice. The extracted solution was filtered with Watman No. 2, in vacuo, and then filtered solution partitioned with ethyl ether 10 L, twice. Secondly, 50% MeOH layer was partitioned with MC 10 L, twice, again, and then evaporated at 40 °C and dissolved with water 2 L Thirdly, the dissolved water layer was partitioned with the water-saturated EtOAc 2 L, twice, and it was done with the water-saturated n-BuOH 2L, twice, successively.

Finally, the each partitioned layer was evaporated at 40 °C, and freeze-dried for 48 h (Labconco/ICE-O-M, Labconco Co., Kansas, Missouri, USA) The yield (g) and recovery rate of the each partition were ethyl ether (85.73 g, 12.80%), MC (0.463 g, 0.07%), EtOAc (191.93 g, 28.65%), n-BuOH (13.54 g, 2.02%), and water layer (165.69 g, 24.73%) respectively. The partition procedure result was shown in Scheme 1.

The quantitative analysis formula of standard materials

To quantify of standards, arbutin, ascorbic acid, 5-HMF, and kojic acid contained in Galla Rhois, we calculated it by the following formula:

$$ \begin{aligned} & {\text{Amount}}\,\left( {\text{mg}} \right)\,{\text{of}}\,{\text{standards }} \\ & \quad = \{{\text{ quantitative}}\,{\text{amount}}\,\left( {\text{mg}} \right)\,{\text{of}}\,{\text{standard}}\,{\text{materials }} \\ & \quad \quad \times {\text{ the}}\,{\text{purity}}\,{\text{of}}\,{\text{standards}} \times {\text{AT/AS}} \\ & \quad \quad \times 1/{\text{the}}\,{\text{dilution}}\,{\text{of}}\,{\text{samples }} \\ & \quad \quad \times {\text{ the}}\,{\text{amount}}\,\left( {\text{mg}} \right)\,{\text{of}}\,{\text{samples }}\}/n\quad \left( {n = 3} \right) \\ \end{aligned} $$

(cf., AT: the peak-area of Galla Rhois containing standards, AS: the peak-area of standards) by Steel et al. (1997).

Instrumental analysis

UV–visible absorption spectra in MeOH were recorded on a spectrophotometer (Shinco, S2030, Korea) in MeOH. The IR spectra were taken on an FS 120 HR/FRA infrared spectrophotometer (Bruker Germany) as KBr discs, and the absorbance frequency was expressed in cm−1. H-NMR (500 MHz) and 13C-NMR (125 MHz) were taken on a Bruker AMX-500 spectrometer in CD3 OD containing tetramethylsilane (TMS) as an internal standard. The fast atom bombardment mass (FAB-MS) was measured with a JEOL JMS-AX-505 WA spectrometer (Japan Electron and Optics, Tokyo, Japan), using glycerol as a mounting matrix.

Results and discussion

Tyrosinase inhibition activity of Galla Rhois

In our routine screening, using mushroom tyrosinase, we observed that Galla Rhois showed significant inhibition of l-DOPA oxidation (Parvez et al.2007; Seo et al. 2003; Kim and Uyama 2005). Tyrosinase activity represented by OD (490 nm) versus various substrate concentration of Galla Rhois or kojic acid, ascorbic acid and arbutin as a positive control which exhibited the tyrosinase inhibitor effects with IC50 values for Galla Rhois, kojic acid, ascorbic acid and arbutin were 0.163, 0.019, 0.316 and 1.520 mg/mL, respectively and (Table 1), when l-DOPA was used as substrate, these result are in corresponding with Jeong and Shim (2004), Chang (2009) and Loizzo (2012) of tyrosinase inhibitor of Zanthoxylum piperitum. Anti-tyrosinase activity of Galla Rhois is higher about two and nine-fold of anti-tyrosinase activity of ascorbic acid and arbutin, while lesser than kojic acid, enzyme used (0.04 mL of 100 U/mL). This result indicates that Galla Rhois, arbutin, kojic acid and 5-HMF exhibited a competitive, competitive, mixed and noncompetitive respectively, inhibition for l-DOPA oxidation by mushroom tyrosinase (Table 1).

Table 1 Inhibition effect of Galla Rhois or Kojic acid, arbutin, ascorbic acid and 5-HMF on the activity of mushroom tyrosinase
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In a comparison of their IC50 values and mushroom tyrosinase inhibition activity, Kojic acid exhibited superior activity compared to that of arbutin, ascorbic acid and Galla Rhois (Table 1 and Fig. 1). Arbutin and kojic acid were consistent with previous reports wherein the kojic acid is apparently higher inhibitory activity (Piao et al. 2004; Chang et al. 2005; Sharma et al. 2004; Wang et al. 2018.

Fig. 1
figure 1

Effect of Galla Rhois (GR) compared with kojic acid or arbutin, on the mushroom tyrosinase for the catalysis of l-DOPA (enzyme concentration 100 U/mL). While R2 values of Galla Rhois and kojic acid or arbutin is 0.9318, 0.9739 and 0.8374 respectively

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The inhibition kinetics of Galla Rhois was analyzed by lineweaver-Buk Plot exhibited a noncompetitive inhibition for l-DOPA oxidation by mushroom tyrosinase (Fig. 2). The initial reaction velocities of Galla Rhois was measured, with (0.003 and 0.042 mM) and without the inhibitor. The Michaelis constant (Km) and maximum reaction velocity (Vmax) of mushroom tyrosinase activity were determined from the slop and intercept of the straight lines. The Vmax (O. D. 490 nm/min) was 0.0770, and the Km was 1.055 mM while in the presence of Galla Rhois at 0.003 and 0.042 mM, respectively. This result indicates that Galla Rhois exhibited a noncompetitive inhibition for the oxidation of l-DOPA catalyzed by mushroom tyrosinase. It is belived that tyrosinase contains a binuclear copper active site Sánchez-Ferrer et al. (1995). The Galla Rhois is a reversible noncompetitive may bind preferentially to the copper center with the hydroxy groups.

Fig. 2
figure 2

Lineweaver–Burk plots showing the reciprocal of the velocity (1/V) of mushroom tyrosinase reaction versus the reciprocal of the substrate concentration (1/S) with l-DOPA as a substrate. Concentration of inhibitor Galla Rhois (GR) 0.125 mg/mL (in reaction mixture) is showing the noncompetitive behavior

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The anti-tyrosinase activity was measured every 5 min for a total of 35 min with three concentrations, 0.5, 0.125 and 0.163 mg/mL of Galla Rhois and a pre-incubation of tyrosinase for 10 min (Fig. 3). The inhibition activity result showed a high dose –dependent correlation with kojic acid and the IC50 value was calculated as 0.163 and 0.019 mg/mL (Fig. 1). The correlation between the activity and reaction time was very was similar to that in Fig. 3, but the present percent inhibitions were some what reduced (data not shown). This observation suggested that Galla Rhois interacts with site other than the active site of tyrosinase.

Fig. 3
figure 3

Tyrosinase activity represented by OD 490 versus time. The tyrosinase was preincubated with Galla Rhois for 10 min control corresponds to tyrosinase activity without Galla Rhois

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The optimum time for mushroom tyrosinase enzyme reaction (post incubation) in the presence of Galla Rhois or Kojic acid and arbutin was between 0 and 10 min (data not shown) and tyrosinase inhibition activity was at enzyme concentration units was 100 U/mL (Fig. 4).

Fig. 4
figure 4

Optimum time for enzyme reaction (post incubation) of Galla Rhois (G.R) using 0.25 mg/mL at various enzyme unit concentration. This data represent the result of three readings

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HPLC analysis of Galla Rhois constituents

Figure 5a and Table 2 shows the HPLC chromatogram of analysis of Galla Rhois (1 g) contains L-ascorbic acid, arbutin and 5-HMH 0.67, 0.707 and 0.050 mg/mL respectively. While Fig. 5b is the representation of standard for ascorbic acid, arbutin and 5-HMF in the retention time 15 min for all samples.

Fig. 5
figure 5

HPLC chromatogram. a Mixture of Galla Rhois and standards, b chromatograms of ascorbic acid, arbutin and 5-HMF (standards)

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Table 2 HPLC quantitative analysis of Galla Rhois derivative components (mg/dried ex. 1 g)
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Sharma et al. (2004) studied the inhibition of mushroom tyrosinase by methanolic extract of Dictyophora indusiata and the bioactive component was characterized and identified as 5-(hydroxymethyl)-2-furfural (HMF). 5 HMF is naturally occurring compound (Sharma et al. 2004) and is used caramel, which is widely used coloring agent in food and pharmaceutical syrups (Wilcox et al. 1985) and it not pose a serious health risk (Wilcox et al. 1985) 5-HMF is suggested by various researchers in cosmetics and food (Kubo et al. 1998; Kubo and Kinst-Hori 1999; Sharma et al. 2004).

In the present study, we demonstrated Galla Rhois to be a new inhibitor of mushroom tyrosinase. We believe that this natural compound can be used as an excellent food-browning preventing agent and skin-whitening agent instead of arbutin due to the following reasons. First, it showed potent anti-tyrosinase activity (IC50 = 0.163), which is nine-fold higher than that of arbutin (IC50 = 1.520). Second, the Galla Rhois demonstrated to contain antioxidant which has role as antityrosinase inhibitor (Hewala et al. 1993; Di Petrillo et al. 2016; Parvez et al. 2007) and is used in the traditional healthy food tempeh (Janzowski et al. 2005), indicating that the compound is safe for human usage. Arbutin and kojic acid are tyrosinase inhibitors (Maeda and Fukuda 1991; Mishima et al. 1988; Clark and Siyamani 2016; Pillaiyar et al. 2017). In particular, arbutin is widely used as whitening agent in cosmetics and was reported to inhibit the enzyme activity competitively (Maeda and Fukuda 1991, Tomita et al. 1990; Céline and Laurence 2016; Pillaiyar et al. 2017).

Safety is of prime concern for tyrosinase inhibitors, especially when they are used in large quantities on a regular basis as food and cosmetic products. HPLC ananlysis indicated that Galla Rhois is constitutents of arbutin, ascorbic acid and 5-HMF (Fig. 5a and Table 2). Arbutin is, hydroquinone-O-beta-d-glucopyranoside (1) isolated from the fresh fruit of California buckeye, Aesculus californica Tomita et al. (1990) is reported by various researchers to inhibit the oxidation of l-DOPA catalyzed by mushroom tyrosinase and effective in the topical treatment of various cutaneous hyperpigmentations characterized by hyperactive melanocyte function (Chakraborty et al. 1998; Hori et al. 2004; Tomita et al. 1990; Clark and Siyamani 2016).

Recent study indicated that arbutin inhibit melanin synthesis by inhibition of tyrosinase activity this appears to be because of the inhibition of melanosomal tyrosinase activity, rather than the suppression of the synthesis and expression of this enzyme (Yang et al. 1999; Clark and Siyamani 2016) and the structure formula is illustrate (Fig. 6). Ascorbic acid as Ascorbyl form has been tested extensively and reported to inhibit the production of the melanin (Janzowski et al. 2005; Carsberg et al. 1994), chemical structure is demonstrated (Fig. 6) and HMF is a naturally occurring compound and has been identified in honey, apple juice, citrus juices and tomato products and is used as a decoloring agents in food and pharmaceutical syrups (Hewala et al. 1993).

Fig. 6
figure 6

a Lineweaver–Burk plots of mushroom tyrosinase of various concentration of Galla Rhois. 1 to 0.5 mg/mL conc. Of inhibitor (GR) is showing the maximum non-competitive behaviour, while conc. Lesser than 0.25 to 0.6 having less non-competitive behaviour. b Effects of various fractions (derivative from Galla Rhois) on tyrosinase reactions using l-DOPA as a substrate while EA ethyl acetate, EE ethyl ether, MC methylene chloride, MB n-BuOH layer, GR Galla Rhois

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Extraction and fractionation of Galla Rhois components for tyrosinase inhibition activity

In fractionation, guided by tyrosinase inhibitory activity, chloroform and ethyl acetate fractions from methanol extracts showed, respectively, moderate and strong inhibitory activity against mushroom tyrosinase (Scheme 1). Little or no activity was present in the hexane, butane, and water fractions.

Bioassays with pure ethyl acetate (EA) fraction of Galla Rhois showed a dose-dependent inhibitory effect l-DOPA oxidation by mushroom tyrosinase, and the IC50 of Galla Rhois and ethyl acetate fraction values were established as 0.035 and 0.163 mg/mL respectively (Table 2 and Fig. 7a). These results indicate that ethyl acetate extracted fraction is approximately four times more effective than Galla Rhois as an antityrosinase agent (Fig. 7b). Furthermore, the inhibition kinetics of ethyl acetate fraction and Galla Rhois were analyzed by a Lineweaver–Burk plot as shown in Fig. 8b, c. The three lines, obtained from the uninhibited enzyme and from the two different concentrations of Galla Rhois and ethyl acetate (EA) fraction intersected on the horizontal axis. This result indicates that Galla Rhois and exhibited a noncompetitive inhibition for l-DOPA oxidation by mushroom tyrosinase. Nevertheless, Galla Rhois and its derivative component by ethyl acetate may be useful in controlling/mediating antityrosinase activity.

Fig. 7
figure 7

a Effects of Galla Rhois (GR) or Ethyl acetate (EA) fraction on the activity of mushroom tyrosinase for the catalysis of l-DOPA. Both Galla Rhois and Ethyl acetate (E.A) fractions are exhibiting the non-competitive behavior. While E.A: ethyl acetate fraction and GR: is Galla Rhios or none is without inhibitors. b Lineweaver–Burk plots showing the reciprocal of the velocity (1/V) of mushroom tyrosinase reaction versus the reciprocal of the substrate concentration (1/S) with l-DOPA as a substrate and in the presence or in the absence of inhibitor. c Kenitic of Galla Rhois showing hyperbola of [s] and velocity, v behaviour with or without substrate (none). While R2 values for Galla Rhois and Ethyl acetate fraction were 0.9601 and 1.00 respectively

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Fig. 8
figure 8

Effects of various concentration of (second fraction of Galla Rhois), F1 fraction one, F2 fraction 2 and F3 is fraction third on the activity of mushroom tyrosinase for the catalysis of l-DOPA

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Finding potent tyrosinase inhibitory activity with Galla Rhois and with its derivative components led us to evaluate other well-known tyrosinase inhibitors such as Kojic acid, arbutin and ascorbic acid (Pillaiyar et al. 2017; Samaneh Zolghadri et al. 2019). There was a significant difference in the inhibitory effect on the tyrosinase activity among the treatments. In a comparison of all Galla Rhois derivative components of their IC50 values, inhibition activity, component derived by ethyl acetate (EA) exhibited superior activity compared to that of Galla Rhois and arbutin (Table 2 and Fig. 8).

Furthermore, in the inhibition kinetics of various fractions, Methylene chloride (M.C), n-Butanol (n-BuOH), exhibited competitive inhibition for l-DOPA oxidation by mushroom tyrosinase, whereas ethyl ether (E.E), ethyl acetate (E.A) and Galla Rhois (G.R) use a control displayed non-competitive inhibition (Table 3). The IC50 values were approximately 0.227, 0.0356, 0.3309, 0.33306, 0.3308 and 0.1625 while the IC50 values of arbutin was 1.520 (Table 3 and Fig. 9). It should be noted that further studies are required to understand the effect of Galla Rhois substitution and the mode of inhibition on tyrosinase inhibitory activity.

Table 3 IC50 and mode of inhibition of Galla Rhois derived or fractionation components
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Fig. 9
figure 9

a HPLC chromatogram of Galla Rhois (second fraction) EtOAc F1. b HPLC chromatogram of Galla Rhois EtOAc F2 and c is HPLC chromatogram of Galla Rhois EtOAc F3 at PDA UV 280 nm (10 mg/mL)

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These results suggest that not only methanol, but also a mixture of organic solvents might be effective for extracting the tyrosinase-inhibiting materials from Galla Rhois. A mixture of an organic solvent and water has been reported effective for extracting the tyrosinase-inhibiting materials from Chinese traditional medicines (Parvez et al. 2006, 2007; Kim et al. 2016; Pillaiyar et al. 2017). Poor extraction efficiency of water, M.C: Methylene chloride, MB: n-BuOH and E.E: ethyl ether, indicating less than 50% inhibition activity while water extract showed about 50% inhibitory activity, indicating that Galla Rhois might contain water-soluble active compounds as well. Some oriental traditional medicines have been used as cosmetics for beauty treatment, and tyrosinase-inhibitory activity was reported (Céline and Laurence 2016; Hae et al. 2019). The 50% tyrosinase inhibitory concentration of the Galla Rhois methanol extract was found to be 0.1625 mg/mL this figure is comparable to those of Chinese medicinal plant materials (Hsieh et al. 2015). As a positive control, the 50% inhibitory concentration of standard arbutin was detected as 1.520 mg/mL.

To evaluate the inhibitory activity of ethyl acetate partition was further extracted with three kinds different solvents for comparison (Scheme 1), the similar fraction of Rf values was combined and obtained total three fractions from EtOAc layer; F1—n-hexane: EtOAc = 10:0 − 9:1 (v/v), F2—n-hexane: EtOAc = 8:2 − 0:10 (v/v), and F3—methanol 100% (Scheme 2). The yield and recovery rate of each fraction from EtOAc layer (191.93 g) was F1 (0.12 g, 0.062%), F2 (5.25 g, 2.73%), and F3 (152.31 g, 79.36%), respectively and each fraction was bioassay, highest inhibitory activity was found in fraction, F2 (Fig. 10), the fractions were analyzed Rf values of each fractions by TLC (Silicagel, Kieselgel 60 F254, 20 × 20 cm, chloroform/methanol = 9:1, v/v). The origin of the above 80% tyrosinase-inhibitory activity found in the Galla Rhois while other three fraction samples with ethyl acetate fraction were F1, F2 and F3 was 58.11,94.64 and 87.61% indicating that ethyl acetate extract of F2 have moderate ability for extracting the tyrosinase inhibiting material from these samples (Fig. 10a–c).

Scheme 2
scheme 2

Second/sub fraction of Ethyl acetate solvent extraction of Galla Rhois fractionation procedure

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Fig. 10
figure 10

Galla Rhois Sub-fraction: the HPLC chromatogram. a Galla Rhois EtOAc F2, b sub-fraction (F2-1) and c sub-fraction, F2-2, (10 mg/mL) at PDA 290.2 nm

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Safety is of prime concern for tyrosinase inhibitors, especially when they are used in large quantities on a regular basis as food and cosmetic products. HPLC ananlysis indicated that Galla Rhois is constituents of arbutin, ascorbic acid and 5-HMF (Fig. 5b and Table 3). Arbutin is, hydroquinone-O-beta-d-glucopyranoside (Di Petrillo et al. 2016) isolated from the fresh fruit of California buckeye, Aesculus californica (Clark and Siyamani 2016; Parvez et al. 2006) is reported by various researchers to inhibit the oxidation of l-DOPA catalyzed by mushroom tyrosinase and effective in the topical treatment of various cutaneous hyperpigmentations characterized by hyperactive melanocyte function (Samaneh Zolghadri et al. 2019).

From the result of standard calibration curve, R2 values and progressive linear equation of each standard were arbutin + kojic acid (0.993, Y = 14,596,850.0X − 229,186.0), nicofinamide (0.993, Y = 684,117.4X − 6168.3), L (+)-ascorbic acid (0.992, Y = 608,840.3X − 16,405.5), which shown the high linear relationship with passing through zero point. The amounts (mg) of each standard for quantitative analysis of Galla Rhois were calculated as shown in Table 3.

Recent study indicated that arbutin inhibit melanin synthesis by inhibition of tyrosinase activity this appears to be because of the inhibition of melanosomal tyrosinase activity, rather than the suppression of the synthesis and expression of this enzyme (Pillaiyar et al. 2017; Chang 2009). Ascorbic acid as Ascorbyl form has been tested extensively and reported to inhibit the production of the melanin (Telang 2013; Pullar et al. 2017; Ravetti et al. 2019) and HMF is a naturally occurring compound and has been identified in honey, apple juice, citrus juices and tomato products and is used as a decoloring agents in food and pharmaceutical syrups Nico (Smit et al. 2009; Chang 2009).

On the basis of our limited data and some earlier findings, the inhibitory action of Galla Rhois against tyrosinase activity may be an indication of at least one of the biological actions of Galla Rhois and the extract of the Galla Rhois consistently showed the highest tyrosinase-inhibitory activity, and for its active constituents three major phenolic compounds were isolated, and one of them exhibiting tyrosinase inhibitory activity stronger than L-ascorbic acid (IC50 = 90 μg/mL) was identified as Methyl gallate (IC50 = 40 μg/mL) by UV, IR, C-NMR, and FAB-MS spectroscopy (Fig. 11a–c). The compound isolated from Galla Rhois exhibited non-competitive inhibition against oxidation of l-3-4-dihydroxyphenylalanine (l-DOPA) by mushroom tyrosinase. As a naturally occurring tyrosinase inhibitor, Methyl gallate may be useful as a new agents to inhibit the oxidation of l-DOPA by mushroom tyrosinase.

Fig. 11
figure 11

a Methyl gallate Molecular weight = 184…15UV,IR H/C-(NMR and FAB-MS spectroscopy), b Mushroom tyrosinase inhibition activity of methyl gallate, c 3,4,5-trihydroxybenzoic acid methyl ester (methyl gallate): Amorphous solid; 1H-NMR (400 MHz, CD3OD) δ: 3.81 (3H, s, OMe), 7.04 (2H, s, H-2/H-6). 13C-NMR (100 MHz, CD3OD) δ: 52.4 (OMe), 110.2 (C-2/C-6), 121.6 (C-1), 139.9 (C-4), 146.7 (C-3/C-5), 169.2 (C=O)

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