Tyrosinase

abdosia serra has been used in traditional Chinese medicine for centuries. In order to illustrate the pharmaceutical activity of R. serra as hypoglycaemic and skin-whitening agents, rosmarinic acid (confirmed as the major compound in R. serra), methyl rosmarinate and pedalitin isolated from R. serra were evaluated for their inhibitory effects and mechanisms on tyrosinase and α-glucosidase. The inhibitory effects on both tyrosinase and α-glucosidase were in decreasing order, pedalitin>methyl rosmarinate>rosmarinic acid. The IC50 values for the tyrosinase and α-glucosidase activity inhibited by pedalitin were 0.28 and 0.29mM, respectively. Both rosmarinic acid and methyl rosmarinate were considered as noncompetitive inhibitors of tyrosinase, while pedalitin was suggested to be a mixed-type inhibitor of tyrosinase. In the assay of α-glucosidase inhibition, rosmarinic acid was found to be a competitive inhibitor, whereas both methyl rosmarinate and pedalitin were considered as mixed-type inhibitors[1].

Assay of inhibitory effects of purified compounds on mushroom tyrosinase [1]
Inhibitory effects of phenolics on mushroom tyrosinase were determined by the method of Yang, Zhao, and Jiang (2008) with some modifications. l-DOPA was used as the substrate for the diphenolase activity assay. The phenolics isolated from R. serra were accurately weighed, and dissolved by DMSO to give the final concentration of 2 mM. Samples tested were subsequently diluted with distiled water to 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, and 0.4 mM, respectively. Sample (20 μl) was added in a well of 96-well microtitre plate. Fifty micrlitres of mushroom tyrosinase (200 U/ml in 50 mM NaH2PO4–Na2HPO4 buffer (pH 6.8)) was added to the same well, and the mixture was preincubated 2 min at 30 °C. Finally, 180 μl of 0.5 mM l-DOPA in 50 mM NaH2PO4–Na2HPO4 buffer (pH 6.8) was added at zero time. The reaction was carried out at a constant temperature of 30 °C. The increase in absorbance from 0 to 5 min was measured at 475 nm. All the above measurements were performed on a Varioskan Flash spectral scan multimode plate reader. Averages of three replicates are presented. The enzymatic activity assayed without phenolics from R. serra was defined as 100% relative activity. Relative enzymatic activity (%) = (slope of reaction kinetics equation obtained by reaction with inhibitor)/(slope of reaction kinetics equation obtained by reaction without inhibitor) × 100.

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Assay of inhibitory effects of purified compounds on yeast α-glucosidase [1]
Inhibitory effects of phenolics on yeast α-glucosidase were determined by the method of Kerem, Bilkis, Flaishman, and Sivan (2006) with some modifications. P-nitrophenyl-α-d-glucopyranoside was used as the substrate for enzymatic activity assay. The phenolics isolated from R. serra were accurately weighed, and dissolved by DMSO to give the final concentration of 2 mM. Samples tested were subsequently diluted with distiled water to 0.01, 0.05, 0.1, 0.15, 0.2, and 0.4 mM, respectively. Sample (20 μl) was added in a well of 96-well microtitre plate. Thirty μl of yeast α-glucosidase (0.7 U/ml in 100 mM NaH2PO4–Na2HPO4 buffer (pH 6.8)) was added to the same well, and the mixture was preincubated 5 min at 37 °C. Finally, 200 μl of 0.8 mM p-nitrophenyl-α-d-glucopyranoside in 100 mM NaH2PO4–Na2HPO4 buffer (pH 6.8) was added at zero time. The reaction was carried out at a constant temperature of 37 °C. The increase in absorbance from 0 to 5 min was measured at 400 nm. All the above measurements were performed on a Varioskan Flash spectral scan multimode plate reader. Averages of three replicates are presented. The enzymatic activity assayed without phenolics from R. serra was defined as 100% relative activity. Relative enzymatic activity (%) = (slope of reaction kinetics equation obtained by reaction with inhibitor)/(slope of reaction kinetics equation obtained by reaction without inhibitor) × 100.

[1]. Comparative evaluation of rosmarinic acid, methyl rosmarinate and pedalitin isolated from Rabdosia serra (MAXIM.) HARA as inhibitors of tyrosinase and α-glucosidase. Food Chem. 2011 Dec 1;129(3):884-9.

Methyl rosmarinate is a hydroxycinnamic acid.
Methyl rosmarinate has been reported in Salvia miltiorrhiza, Perilla frutescens, and other organisms with data available.

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The competitive inhibition type of rosmarinic acid indicated that inhibition arised from binding in the enzyme active site. Thus, the combination between enzyme and substrate might be inhibited by rosmarinic acid. Both Methyl rosmarinate and pedalitin were determined as mixed-type inhibitors. Although Methyl rosmarinate and rosmarinic acid have a similar structure except for the OCH3 fragment binding to C-1, the inhibitory activity and type of methyl rosmarinate was different with those of rosmarinic acid.
Wang et al. (2010) investigated the inhibitory effects of a series of flavonoids on α-glucosidase. They found that the free 3-position hydroxyl group was a key functional group for inhibiting α-glucosidase. In addition, the inhibitory activity increased considerably with the increase of the number of hydroxyl groups on the B-ring. The fact that pedalitin possessing the 3′,4′-dihydroxyl groups substituent exhibited excellent inhibitory effects on α-glucosidase was fully consistent with their conclusion.
In conclusion, the inhibitory effects and mechanisms of rosmarinic acid, Methyl rosmarinate, and pedalitin on tyrosinase and α-glucosidase were evaluated. The inhibitory effects on mushroom tyrosinase and α-glucosidase were in a decreasing order, pedalitin > Methyl rosmarinate > rosmarinic acid. Their inhibitory types on tyrosinase and α-glucosidase were obtained by their Lineweaver–Burk plots, which clearly illustrated their inhibitory mechanisms on tyrosinase and α-glucosidase. Additionally, R. serra as hypoglycaemic and whitening agent may relate to enzymatic inhibition. Results from this study will provide a better understanding of R. serra as hypoglycaemic and skin-whitening agents and to expand its potential application as tyrosinase and α-glucosidase inhibitors. On the basis of results obtained from this study, further work on developing new technologies (transforming rosmarinic acid into its methyl ester) and enriching pedalitin are in progress. [1]

C19H18O8

374.3414

374.1

99353-00-1

20283-92-5 (Rosmarinic acid); 537-15-5 (Rosmarinic acid racemate)

6479915

White to off-white solid powder

1.5±0.1 g/cm3

655.4±55.0 °C at 760 mmHg

236.5±25.0 °C

0.0±2.0 mmHg at 25°C

1.668

Salvia miltiorrhiza, Perilla frutescens

2.12

4

8

8

27

534

1

COC(=O)[C@@H](CC1=CC(=C(C=C1)O)O)OC(=O)/C=C/C2=CC(=C(C=C2)O)O

XHALVRQBZGZHFE-BBOMDTFKSA-N

InChI=1S/C19H18O8/c1-26-19(25)17(10-12-3-6-14(21)16(23)9-12)27-18(24)7-4-11-2-5-13(20)15(22)8-11/h2-9,17,20-23H,10H2,1H3/b7-4+/t17-/m1/s1

methyl (2R)-3-(3,4-dihydroxyphenyl)-2-[(E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxypropanoate

Methyl rosmarinate; 99353-00-1; methyl (2R)-3-(3,4-dihydroxyphenyl)-2-[(E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxypropanoate; CHEMBL241405; Benzenepropanoic acid,a-[[(2E)-3-(3,4-dihydroxyphenyl)-1-oxo-2-propenyl]oxy]-3,4-dihydroxy-,methyl ester, (aR)-; (R)-3-(3,4-Dihydroxyphenyl)-1-methoxy-1-oxopropan-2-yl (E)-3-(3,4-dihydroxyphenyl)acrylate; Methylrosmarinic acid; (+)-Methyl rosmarinate;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~267.14 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.68 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.68 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)