Antioxidant and Anti-Tyrosinase Activity of Some Medicinal Plants

Arun Duraisamy, Nithya Narayanaswamy and K.P. Balakrishnan*

ITC R&D Centre, ITC Limited, Peenya Industrial Area, Bangalore 560 058 (Karnataka)

ABSTRACT:

Exposure to UV radiation becomes inevitable these days due to the depletion of ozone layer. Skin being the largest and outermost organ of the body, gets much exposed as well as affected due to these radiation. The absorption of high energy UV radiation by the cells results in the liberation of free radicals. The formed free radicals damage the major macromolecules like proteins, lipids and DNA through its chain reaction mechanism. Free radicals’ being the signal transducers of the cells promotes or enhances the expression of the enzyme tyrosinase. Tyrosinase, the key enzyme in melanogenesis promotes the biosynthesis of melanin. The melanin formed is an endogenous antioxidant produced in the cell to protect itself from the free radical toxicity. Due to over exposure to UV radiation or the improper trigger in the biogenesis of melanin results in the hyperpigmentation disorders. Hence finding the inhibitor for tyrosinase becomes a key target for the cosmetic industries. Thus the present study opts for searching such novel tyrosinase inhibitor from herbal origin, which can attenuate the free radical toxicity by being a potent antioxidant.

KEYWORDS: UV radiation, tyrosinase, hyperpigmentation, melanogenesis and antioxidant.

1. INTRODUCTION:

Aging is a physiological change in an organism that leads to senescence i.e., decline of biological functions and the organism’s ability to adapt to metabolic stress with time (Encyclopedia Britannica, 2004). Beyond this intrinsic aging process, sun exposed areas such as face, neck, dorsum of hands and forearms encounter additional damaging effects due to long term UV exposure. Many of the functions of skin that decline with age show an accelerated decline in photoaged skin (Yaar et al., 2003).

The well-known situation is the production of reactive oxygen species (ROS) by the skin exposed to ionizing or ultraviolet radiation (UVR) under aerobic conditions (Black, 1987). The UVR absorbed in the skin excites the ground-state O₂, resulting in the production of a number of ROS. The production of ROS like singlet oxygen (Cunningham et al., 1985), hydroxyl radical and H₂0₂ in the cells ultimately induces DNA damage and lipid peroxidation (Halliwell and Gutteridge, 1984). Membrane lipids are the major site of attack for the free radicals due to the unsaturated fatty acid composition. Thus free radical mediated damage to lipids results in the altered fluidity of cell, which reflects in the texture of skin (Akiu et al.,1991).

The exposure to UVR increases vascular permeability leading to leakage, disorganization and desquamation of the epidermis and leucocyte infiltration.

There is an alteration in lysosome structure and associated enzymes, cell proliferation and increased melanin production associated with increase in melanocyte size and number (Gilchrest et al., 1996). This increase in melanocyte number ultimately increases the melanin turnover in the skin.

The overexposure to UVR triggers the biosynthesis of an endogenous, nonenzymic antioxidant called melanin. Tyrosinase being the key regulatory enzyme in the melanogenesis, it oxidizes the conversion of L-tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and DOPA to DOPA quinone, which further polymerises to form the brown pigments namely eumelanin and pheomelanin(Kahn 1995).

The protective role of melanin pigment should not be under estimated. The difference between the black and white skin is in the size, number and aggregation pattern of melanosomes within melanocytes and keratinocytes (Rijken et al.,2004). Compared with the black skin, the dermal DNA photo damage, infiltrating neutrophils, expression of MMP and IL-10 seems to be more in white skin (Szabo, 1959).Thus the distribution of melanin is thought to provide protection from sunburn, photoaging and carcinogenesis by absorbing and scattering the detrimental UV rays (Kaidbey,1979).

On the other hand, overexposure to UV radiation can lead to a pathological increase in melanin production. Such hyperpigmentation leads to the clinical conditions like melasoma, solar lentigines and ephelides(Maeda and Fukuda, 1991). Skin being an important component of body image and it can be a source of significant emotional distress due to hyperpigmentation (Baharav et al., 1996).

This led to the development of products with skin-lightening tyrosinase inhibitors by the cosmetics and pharmaceutical companies (Yoshida et al., 2000). Most of the current commercial products for skin whitening contain kojic acid, arbutin, catechins, hydroquinone (HQ) and azelaic acid as one of the essential ingredient (Maeda and Fukuda, 1996). The adverse effects of these synthetic compounds are irreversible (Kumano et al., 1998). Hence paved the way for the search of plant based safer skin lightening actives with potent antioxidant property (Badria and Gayyar , 2001).

2. MATERIALS AND METHODS:

2.1 PLANT EXTRACT PREPARATION:

Plants studied: Tribulus terrestris (TT)-seeds, Berberis aristata(BA)-Root, Inula racemosa (IR) -Root, Symplocos racemosa (SR)-Bark, Cyperus scariosus (CS)-Root.

Plant Collection: The plant parts were either collected from the field or from the commercial source. The collected plant materials were shade dried and powdered with the help of a mortar and pestle or a mixer. The powder is sieved to remove the coarse materials. The fine powder is used for the analysis

Extraction: The solvents used for extraction are water (AQ), ethanol (ET) and petroleum ether (PE). The extracts were prepared at 10 % concentration in the respective solvents. The herbals with respective solvents are placed in a boiling water bath at 60 ° C for 1 hour. The hot solutions of the plant material are centrifuged at 6500 rpm for 10 mins. The supernatant was collected, filtered and used for the assays.

2.2. Skin whitening assay:

2.2.1. Antityrosinase assay: (Lee et al., 2003)

Tyrosinase (Phenoloxidase activity) which catalyses the transformation of L-tyrosine into L-DOPA by hydroxylation and further into O-dopaquinone by oxidation. Then, through a series of non-enzymatic reactions, O-dopaquinone is rapidly transformed into melanin, which is measured at 492 nm in a spectrophotometer. Each plant extract was assayed for tyrosinase inhibition by measuring its effect on tyrosinase activity using a 96-well reader. The reaction was carried out in a 50 mM potassium phosphate buffer (pH 6.8) containing 20 mM L-tyrosine and 312.5 U/mL mushroom tyrosinase at 30°C. The reaction mixture was pre-incubated for 10 min before adding the enzyme. The reaction mixture without the enzyme serves as blank. The reaction mixture with the corresponding solvents (without plant material) serves as control. The change of the absorbance at 492 nm was measured. The percent inhibition of tyrosinase was calculated as follows:

OD of Control- OD of Test

% Inhibition of tyrosinase = X100

OD of Control

2.3. Antioxidant assays:

The antioxidant activity of plant material was evaluated by employing the following methods.

2.3.1. DPPH radical scavenging assay (Mensor et al., 2001)

DPPH (2, 2-diphenyl-1-picryl hydrazyl) is a commercially available, commonly used, stable free radical, which is purple in colour. Antioxidant molecules when incubated, reacts with DPPH and converts it into di-phenyl hydrazine, which is yellow in colour. The degree of discoloration of purple to yellow was measured at 520 nm, which is a measure of scavenging potential of plant extracts. 5 µl of plant extract was added to 195 µl of DPPH solution (0.1mM DPPH in methanol) in a microtitre plate. The reaction mixture was incubated at 25⁰ C for 10 minutes, after that the absorbance was measured at 520 nm. The DPPH with corresponding solvents (without plant material) serves as control. The methanol with respective plant extracts serves as blank. The DPPH radical scavenging activity of the plant extract was calculated as the percentage inhibition.

OD of Control- OD of Test

% Inhibition of DPPH radical = X100

OD of Control

2.3.2. Determination of total phenolics (Mallick and Singh, 1980) Phenols react with phosphomolybdic acid in Folin-ciocalteau reagent in alkaline medium and produce a blue colored complex (molybdenum blue) that can be estimated colorimetrically at 650 nm. Pipetted out different aliquots (0.1 to 0.5 ml) of plant extracts into the test tubes. Made up the volume in each tube to 3.0 ml with water. Added 0.5 ml of Folin-Ciocalteau reagent. After 3 minutes, added 2.0 ml of 20% sodium carbonate solution to each tube. Mixed thoroughly, placed the tubes in a boiling water bath for exactly 1 minute, cooled and measured the absorbance at 650nm against reagent blank.

2.4. Statistical analysis:

Samples were analyzed in triplicate and the results were given as Mean ± S.D.

3. RESULTS AND DISCUSSION:

The skin whitening potential and the antioxidant activity of the aqueous, ethanol and petroleum ether extract of the selected five herbs were depicted in following division.

3.1. AQUEOUS EXTRACTS:

3.1.1. Anti-tyrosinase activity:

The tyrosinase inhibiting ability of the aqueous extract of the herbals are depicted in the Fig.1, it can be inferred that the aqueous extract of the Cyperus scariosus (exerts 23.4% tyrosinase inhibition) is a potent skin lightening extract among the selected medicinal plants. The percentage inhibition of tyrosinase by the aqueous extract of other herbs falls in the range of 6-10 %. When compared to other herbals CS-AQ shows better tyrosinase inhibition.

Fig 1: Percentage inhibition of tyrosinase by the aqueous extract of few herbs

3.1.2. DPPH radical scavenging activity:

The antioxidant potential of the aqueous extract of the herbs were examined using the DPPH radical scavenging assay. The assay results are depicted as percentage inhibition in the following Fig.2. Among the screened aqueous extracts, the Cyperus scariosus shows to be the potent antioxidant by exerting 89.85 % inhibition in scavenging DPPH radical. The aqueous extract of Inula racemosa shows 44.35% inhibition whereas the DPPH radical scavenging by Symplocos racemosa and Tribulus terrestris is 32.73%.

3.1.3. Total phenol content:

The antioxidant ability of a plant extract is due to the presence of the secondary metabolites like alkaloids, flavonoids and terpenes etc. The total phenol content of the plant material was quantified using Folins Ciocalteau method. It is expressed in terms of catechol equivalents. The unit used to express the quantified total phenols is mg per gram of plant material. Among the aqueous extract of the herbs screened, the Cyperus scariosus contains the maximum phenol content in it (10.96 mg equivalent of catechol / g of plant material), whereas other extracts shown to have 4-7mg/g of plant material. The results are depicted in Fig 3.

Fig 2 DPPH radical scavenging activity of aqueous extract of few herbs

Fig 3 Total phenolic content of the aqueous extract of few herbs

From the above sections, it is clear that the aqueous extract of the Cyperus scariosus was found to be superior in the aspect of skin whitening and antioxidant properties. The aqueous extract of Cyperus scariosus was found to be rich in phenolic content too. This suggests that the skin whitening and antioxidant potential of the aqueous extract of Cyperus scariosus may be due to the high content of water soluble phenols in it.

3.2. ETHANOLIC EXTRACTS:

3.2.1. Anti-tyrosinase activity

The skin lightening potency of the ethanolic extract of the selected medicinal plants were shown in the following Fig 4. The tyrosinase inhibition by the ethanolic extract of herbs namely Tribulus terrestris, Symplocos racemosa and Cyperus scariosus falls in the range of 39%.The ethanolic extract of the Berberis aristata(23.74%) and Inula racemosa (30.58%) has exerted a good tyrosinase inhibition potential when compared to the aqueous extract of the same. This data suggest that the compounds responsible for the tyrosinase inhibition are readily soluble in the solvent like ethanol whereas the compounds in Cyperus scariosus are readily soluble in both ethanol and water.

Fig 4 Percentage inhibition of tyrosinase by the ethanolic extracts of few herbs

3.2.2. DPPH radical scavenging activity:

The DPPH radical scavenging potential of the ethanolic extract of the selected medicinal plants is depicted in the Fig 5. The percentage inhibition pattern by the ethanolic extract of the selected herbs in the tyrosinase inhibition assay and the DPPH radical scaveniging assay are not one and the same. The radical quenching by the ethanolic extract of Cyperus scariosus seems to be superior (92.76%) when compared to the other ethanolic extracts (percentage inhibition values ranged from 22-64 %).

3.2.3. Total phenol content:

The total phenol content estimated in the ethanolic extract of the selected medicinal plants are depicted in the following Fig 6. The phenolic content in the ethanolic extract of Cyperus scariosus is shown to be 14.43 mg of catechol equivalents/g of plant material. The other herbal materials show the phenolic content in the range of 1-7 mg of catechol equivalents /g of plant material. The phenolic content of the ethanolic extract of Cyperus scariosus is 14 mg whereas the aqueous extract is 10 mg of catechol equivalents/g of plant material. This suggests that the compounds in Cyperus scariosus, contributing for the antioxidant potential and the tyrosinase inhibition are readily soluble in ethanol compared to aqueous.

From the above results it can be inferred that the antioxidant potential and tyrosinase inhibition potential of the ethanolic extract of Cyperus scariosus is due to the high phenolic content in it, whereas the activity of Tribulus terrestris and Symplocos racemosa, might be due to the presence of nonphenolic compounds present in it. Eventhough the tyrosinase inhibition potential of the Cyperus scariosus, Tribulus terrestris and Symplocos racemosa are in the same range of 39%, the ethanolic extract of the Cyperus scariosus was found to be better option because of its antioxidant efficacy and rich phenolic content when compared to the other two.

Fig 5 DPPH radical scavenging activity of ethanolic extract of few herbals

Fig 6 Total phenolic content of the ethanolic extract of few herbs

3.3.PETROLEUM ETHER EXTRACTS:

3.3.1. Anti-tyrosinase activity:

The results of anti-tyrosinase activity of petroleum ether extracts of the selected medicinal herbs were depicted in the following Fig 7. The percentage inhibition of tyrosinase by petroleum ether extract of Inula racemosa was 10.27%, which is superior among the petroleum ether extract screened. The petroleum ether extract of Cyperus scariosus and Tribulus terrestris has shown no tyrosinase inhition. From this data, it can be inferred that the compounds which are contributing for the tyrosinase inhibition by Cyperus scariosus extracts are highly insoluble in the solvent petroleum ether.

Fig 7 Percentage inhibition of tyrosinase by the petroleum ether extract of few herbs

Fig 8 DPPH radical scavenging activity of petrolum ether extract of few herbs

3.3.2. DPPH radical scavenging activity

The DPPH radical scavenging ability of the petroleum ether extract of the selected medicinal plants is depicted in the following Fig 8. The percentage inhibition of DPPH radical scavenging by petroleum ether extract of Cyperus scariosus shows to be 18.54%, which is superior among the petroleum ether extracts screened, whereas this is found to be the less percentage when compared to the aqueous and ethanolic extract of the same plant. Hence, the compounds responsible for the antioxidant property are highly soluble in ethanol and water rather than petroleum ether

3.3.3. Total phenol content:

The total phenolic content of the petroleum ether extract of the selected medicinal plants are very low (negligible) when compared to the aqueous and ethanolic extracts. Hence there is no graphical representation made.

4. CONCLUSION:

From this preliminary screening of herbs for skin whitening and antioxidant property, the following recommendations were made.

1. The ethanolic extract of the herbs namely Cyperous scariosus, Tribulus terrestris and Symplocos racemosa can be shortlisted because of its good skin whitening potential in the same percentage level.

2. By considering the antioxidant efficacy of the shortlisted herbs, despite of the solvents used for extraction the Cyperous scariosus seems to be rich in phenol content and radical scavenging ability.

3. Hence the ethanolic extract of the Cyperous scariosus can be used for treating the hyperpigmentation disorders once the dermal safety is ensured.

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Received on 17.12.2010

Accepted on 27.12.2010

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Research Journal of Pharmacognosy and Phytochemistry. 3(2): March- April 2011, 86-90