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| Year : 2011 | Volume
: 5
| Issue : 2 | Page : 103-106 |
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| Tyrosinase kinetic in the presence of three inhibitory medicinal plants |
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Mehdi Ansari1, Farzaneh Etelaei2, Fariba Sharififar3
1 Department of Pharmaceutics, Kerman University of Medical Sciences, Pharmaceutical Research Center, School of Pharmacy, Kerman, Iran
2 Department of Pharmaceutics, Kerman University of Medical Sciences, Herbal and Traditional Medicines Research Center, School of Pharmacy, Kerman, Iran
3 Department of Pharmacognosy, Kerman University of Medical Sciences, Herbal and Traditional Medicines Research Center, School of Pharmacy, Kerman, Iran
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| Date of Submission | 06-Mar-2011 |
| Date of Acceptance | 18-May-2011 |
| Date of Web Publication | 21-Sep-2011 |
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 Abstract | | |
Quercus infectoria, Terminalia chebula and Linum usita-tissimum. Different concentration of the extracts was examined against l-tyrosine oxidation in comparison to kojic acid and their IC 50 were calculated by probit analysis. The tyrosinase inhibition kinetics, analyzed by Lineweaver-Burk plots, and the parameters of K m and V max for each extract were calculated using the Linweaver--Burk equation. The results show that the methanolic extracts of Q. infectoria (MEQI) and T. chebula (METC) potentially inhibit mushroom tyrosinase with IC 50 values of 3.34 and 3.87 μg/ml respectively in comparison to kojic acid (IC50 value = 1.56 μg/ml). The MEQI and METC exhibited the most V max (81.96 and 78.74 μg/ml/min respectively). This activity is comparable with one of kojic acid (Vmax = 103 μg/ml/min ). The MEQI and METC also have shown the lowest value of K i (0.20 and 0.44 respectively) in comparison to kojic acid (K i = 0.18). The MEQI and METC would be developed further as a natural source of tyrosinase inhibitors.
Keywords: Linum usita-tissimum , Quercus infectoria, tyrosinase kinetic, Terminalia chebula
How to cite this article:
Ansari M, Etelaei F, Sharififar F. Tyrosinase kinetic in the presence of three inhibitory medicinal plants. Int J Green Pharm 2011;5:103-6 |
| Introduction | |  |
The formation of melanin is due to the tyrosinase activity which catalyzes the hydroxylation of l-tyrosine to 3,4-dihydroxyphenyl)-l-alanine (DOPA) and induces pigments of melanin. [1] Tyrosinase inhibitors are becoming increasingly important due to their antipigmenting effects. [2] We previously reported the tyrosinase inhibitory effects of Quercus infectoria Olive and Terminalia chebula Retz. [3] Linoleic acid (C18:2) is a major component of biological cell membrane which can lighten the hyper pigmented skin induced by UV. [4],[5] So the present work aims to study the tyosinase kinetic in the presence of aqueous and methanolic extracts of Quercus infectoria, Terminalia chebula, and Linum usita- tissimum.
| Materials and Methods | | |
Chemicals
l-tyrosine, tyrosinase, and kojic acid were purchased from Sigma-Aldrich, USA. Methanol and all other chemicals used were of analytical grades.
Plant Materials
The dried galls of Q. infectoria, fruits of T. chebula and seeds of L. usita-tissimum were purchased from the local market and euthanatized by Dr. Sharififar in Department of Pharmacognosy, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran.
Preparation of Plant Extract
A total of 200 g of each plant was extracted with methanol and water separately using percolation method for 72 hours at room temperature. The resultant extracts were lyophilized using freeze dryer. The extracts were stored at -20°C with suit adsorbent until testing.
Enzymatic Assay of Tyrosinase
The inhibitory activity of the samples against hydroxylation activity of the mushroom tyrosinase was determined in accordance with the methods of Chang et al. [6] A total of 880 μl of 2 mM substrate (l-tyrosine) dissolved in a 50 mM phosphate buffer (pH=6.8) was mixed with 100 μl of the tested extracts (different concentrations in methanol) at 25°C for 2 minutes. Then, 20 μl of tyrosinase (1000 U/ml in a phosphate buffer) was added to initiate the reaction. The assay mixture was incubated at 25°C for 10 min. The increase in absorbance at 475 nm, due to the formation of dopachrome was measured with a spectrophotometer. The percentage of inhibition of tyrosinase activity was calculated as follows: % Inhibition=(A−B/A) Χ100, where A is the absorbance at 475 nm with methanol instead of the extract and B is the absorbance at 475 nm with the extract. The concentration of a compound at which 50% of the enzyme activity was inhibited (the IC 50 value) was obtained by probit analysis program.
Tyrosianse Kinetic in a Time-dependent Manner
The activity of tyrosinase has been evaluated on the basis of conversion of tyrosine to dopa chrome. The enzyme activity of tyrosinase was determined in a time-dependent manner as described by Baek with some modifications. [7] A mixture of 0.1 ml of different concentrations of each extract (25, 50, and 100 μg/ml concentrations) and 0.1 ml of tyrosinase (33 units in a phosphate buffer, pH= 6.8) was incubated at 31°C for 10 min. A total of 2.8 ml of tyrosine (0.5 mM in a phosphate buffer, pH=6.8) was warmed to 31°C and added to the mixture. Kojic acid and the solvent were used as positive and negative controls. The activity of tyrosinase was measured continuously for 30 minutes using a UV spectrophotometer (Shimadzu, Japan). For determining the kinetic parameters of enzyme inhibition in fixed substrate concentration in a time-dependent manner, the absorbance was measured at wavelength 492 nm (1-minute intervals) at 31°C in the presence of each extract. The individual parameters for each curve data were calculated. [8],[9] The percentage of enzyme inhibition was determined according to the following equation: where A is the absorbance of negative control in the time of t n , B the absorbance of the extract in the time of t n . The test was done in triplicate and data were reported as mean±SD.
The kinetic parameters of tyrosinase were determinedin the presence of the tested extract using thelineweaver-burk equation. Affinity constant (K i) was calculated in the following way:
where [S] is the concentration of substrate.
Statistical Analysis
Results are expressed as mean±S.E.M. Differences between the control and treated groups were tested for significance using a one-way analysis of variance (ANOVA).
| Results | | |
Inhibitory Activity Against Tyrosinase in Hydroxylation of l-tyrosine
The inhibitory effect against tyrosinase activity in hydroxylation of l-tyrosine for both aqueous and methanolic extracts of tested plants was determined at different concentrations. The IC 50 value for each extract and the results of extraction have been given in [Table 1]. These results indicated that the methanolic extracts of Q. infectoria (MEQI) and T. chebula (METC) have shown the least IC 50 values (3.34 and 3.87 μg/ml respectively) in comparison to that of kojic acid (IC 50 = 1.56 μg/ml). These two extracts inhibited the tyrosianse activity in a concentration-dependent manner. Among the tested extracts the most percentage of inhibition was shown by MEQI (85.9%) in concentrations of 100μg/ml [Figure 1]. The least of inhibition was exhibited by the methanolic extract of L. usita-tissimum (MELU) and aqueous extract of L. usita-tissimum (AELU). The methanolic extracts of the all of three plants exhibited more tyrosinase inhibition activity than the aqueous ones. | Table 1: The results of extraction and IC50 values for tyrosinase inhibition of different tested extracts
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 | Figure 1: Time- course oxidation of l-tyrosine by tyrosinase in the presence of different concentratins of kojic acid. After adding tyrosinase, incubated at 25°C for 10 min. Increase in absorbance at 475 nm, due to dopachrome formation was measured
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The Results of the Kinetic Study of Tyrosinase
The tyrosinase kinetic was studied in the presence of tested extracts for 30 minutes. [Figure 1],[Figure 2],[Figure 3] show the results of a time- response inhibition for kojic acid, MEQI, and METC at the concentrations of 25, 50, 100 μg/ml respectively. As shown in the [Figure 1] and [Figure 2], kojic acid and MEQE potentially can inhibit tyrosinase activity at three used concentrations which increased with time. The kinetic parameters were analyzed under our experimental conditions and K m and V max of each extract were calculated using the Linweaver--Burk equation by plotting the reverse of velocity versus reverse concentration. More V max indicated more inhibition potency. The results have been given in [Table 2]. These results indicated that among the tested extracts, the MEQI and METC exhibited the most inhibition of tyrosinase (V max = 81.96 and 78.74 μg/ml/min respectively). This activity is comparable with that of kojic acid (V max = 103 μg/ml/min). The lowest value of K i also belongs to MEQI and METC (0.20 and 0.44 respectively) in comparison to kojic acid (K i = 0.18) [Table 2] which indicates the low affinity of binding while the AELU showed the highest capacity of binding (K i = 1243.29) | Table 2: The results of kinetic parameters for tyrosinase inhibition in the presence of different tested extracts
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 | Figure 2: Time- course oxidation of l-tyrosine by tyrosinase in the presence of different concentratins of MEQI. After adding tyrosinase, incubated at 25°C for 10 min. Increase in absorbance at 475 nm, due to dopachrome formation was measured
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 | Figure 3: Course of oxidation of L-tyrosine by tyrosinase in the presence of different concentratins of methanol extract of Terminalia chebula (METC)
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| Discussion | | |
Kojic acid is a secondary metabolite of some species of Aspergillus and Penicillium which has been used in cosmetic preparations as a hypopigmentation agent [10] and competitively inhibits the tyrosinase. In the previous study using mushroom tyrosinase, MEQI and METC significantly inhibited the oxidation of both l-tyrosine and l-DOPA. [3] In the present work, the kinetic behaviors of the oxidation of l-tyrosine catalyzed by mushroom tyrosinase were studied at different concentrations of aqueous and methanolic extracts of three medicinal plants. The results indicated that among the tested extracts, two extracts of MEQI and METC can potentially inhibit tyrosinase activity. The IC 50 of the extracts, as a parameter of potency, indicated that the MEQI and METC with least IC 50 exhibited the most tyrosinase inhibition. As shown in [Table 1], the IC 50 of these two extracts are two times more than those of kojic acid. The aqueous and methanolic extracts of L. usita-tissimum exhibited the highest IC 50 value. The results of the kinetic study of tyrosinase in the presence of kojic acid and different extracts have been given in [Table 2]. Under these conditions, K m and V max of the l-tyrosine oxidation by the tyrosinase in the presence of MEQI were found to be 5.4 μg/ml and 81.96 μg/ml/min respectively. The V max of MEQI is comparable with the one of kojic acid (V max = 103 μg/ml/min). The highest value of V max obtained in the recent work is due to MEQI followed by METC (V max= 78/74 μg/ml/min), whereas AELU exhibited the least V maxvalue (22.62 μg/ml/min). The K i of each extract was determined from the kinetic study of tyrosinase. The MEQI has shown the lowest value of K i among the tested extracts which indicates the most its effective capacity for binding to the tyrosinase whereas MELU and AELU showed the least binding capacity followed by METC and AEQI in decreasing order.
The plot of the percentage of inhibition versus the time of incubation for each extract shows that when the concentration increases, the more inhibition induces. The most inhibition was considered within the time of 15-25 minutes after incubation for all the extracts whereas in the last 5 minutes of incubation, the least of inhibition occurs. As IC 50 and K i of these two plants indicate, these plants show less effective tyrosinase inhibition than kojic acid, which might be due to the presence of a complex of phytochemicals with different biological functions. Flavonoids and phenolic acids were found to be the main constituents of galls of Q. infectoria and fruits of T. chebula. Major phenolic acids are gallic acid and ellagic acid.[11],[12] Ellagic acid shows high affinity to two copper ions of tyrosinase. [13] Furthermore the flavonoid derivatives chelate the copper ions of tyrosinase. [14] Tyrosinase activity depends on the function of copper ions, so the agents with affinity to binding to these ions such as kojic acid or gallic acid can inhibit the activity of tyrosinase. [15] In the present work, two extracts of MEQI and METC show similar activity to kojic acid but with lesser potency. The high yield of extraction for MEQI and METC (34 and 29% respectively) indicates that each of these plants would be a valuable source of tyrosinase inhibitors. Our previous study showed the high antioxidant effects of MEQI and METC [3] which somehow may be related to their potential tyrosinase inhibition effects. The results of this work also suggest that these extracts are worthy for further studies and would be developed further as a natural source of tyrosinase inhibitors.
| Acknowledgments | | |
The present article is a part of a research project which has been supported by the Kerman University of Medical Sciences, Vice Cancellor for Research. The authors are grateful for financial support of this work.
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Correspondence Address:
Fariba Sharififar
Department of Pharmacognosy, Herbal and Traditional Medicines Research Center, School of Pharmacy, Kerman
Iran
DOI: 10.4103/0973-8258.85165
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2] |
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