Photochemoprotective Activity of Alcoholic Extract of Camellia sinensis

MATERIALS AND METHODS

The dried leaves of the plant Camellia sinensis were purchased from local authentic herbal distributor and were authentified with the help of herbarium of the Pharmacognosy department of University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur. Ethanol, trichloroacetic acid, potassium ferricyanide, L-ascorbic acid and ferric chloride all were of analytical grade. Double distilled water was used for all experiments. Microcentrifuge (RM-12 C DX, Remi) and UV spectrophotometer (UV-Visible spectrophotometer 1700-Pharmaspec, Shimadzu, Japan) were used for the present study.

Preparation of extract (WHO, 2004; Rajpal, 2004): Extraction of the green tea leaves was done by hot extraction process using Soxhlet apparatus. The 350 g of weighed green tea leaves were taken, made to coarse powder and packed in the Soxhlet apparatus. The defatting was done by petroleum ether at 60-80°C. Then extraction was performed with ethyl alcohol (90%) at temperature 50-60°C. The extract was concentrated by recovering the solvent and then dried the extract and weighed to get actual percent yield of the extract. It was calculated from the obtained mass and initial powdered drug.

Antioxidant activity determination: The antioxidant activity of the extract was assessed by reducing power estimation method in which the antioxidant activity of extract was compared with that of standard (ascorbic acid).

The relative reducing activity in terms of antioxidant activity of green tea extract was determined by using 5 mg of extract as well as its combination with equal amount of ascorbic acid. The extract (5 mg) and ascorbic acid (5 mg) were dissolved separately in 1.0 mL of deionized water with phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and 1% potassium ferricyanide (2.5 mL). The mixture was incubated at 50°C for 20 min. Aliquotes of trichloroacetic acid (2.5 mL, 10% w/v) were added to the mixtures and centrifuged at 3000 rpm for 10 min. The upper layer of solution (2.5 mL) was mixed with distilled water (2.5 mL) and a freshly prepared ferric chloride solution (0.5 mL, 0.1%). The complete mixture was diluted upto 10 mL with distilled water and further took 1 mL and diluted upto 10 mL with distilled water. The absorbance was measured at 700 nm by UV-visible spectrophotometer (1700-Pharmaspec, Shimadzu, Japan) by taking as blank the mixture of all the reagents in the same quantity as added while preparing samples. Increased absorbance of the reaction mixture indicated increased antioxidant activity via reducing power with reference to equal amount of standard ascorbic acid (Rajeshwar et al., 2005; Ashawat et al., 2007b). Similar procedure was repeated to know combination antioxidant power of extract with ascorbic acid as shown in Table 1.

Sun Protection Factor (SPF) determination: The efficacy of a sunscreen is usually expressed by the Sun Protection Factor (SPF) which is defined as the UV energy required to produce a Minimal Erythema Dose (MED) on protected skin, divided by the UV energy required to produce a MED on unprotected skin:

The Minimal Erythemal Dose (MED) is defined as the lowest time interval or dosage of UV light irradiation sufficient to produce a minimal, perceptible erythema on unprotected skin (Aburjai and Natsheh, 2003; Bendova et al., 2007). The higher the SPF, the more effective is the product in preventing sunburn. In vitro screening methods may represent a fast and reasonable tool reducing the number of in vivo experiments and risks related to UV exposure of human subjects, when the technical test parameters are adjusted and optimized (Wood and Murphy, 2000).

The in vitro SPFs were determined according to the method described by Mansur et al. (1986), Santos et al. (1999), Ashawat et al. (2006) and Kaur and Saraf (2010).

Table 1:	Antioxidant activity of ethanolic extracts
Values are Mean±SD for n = 3, ***p<0.001

Table 2:	SPF determination of green tea extract
Σ EE (λ)xI (λ)xA = 1.810±0.005, SPF = Σ EE(λ)xI (λ)xAx10 (Correction Factor), SPF = 1.8101x10 = 18.10±0.05

For sample preparation 100 mg of green tea extract was weighed and made up the volume upto 10 mL with ethanol which gave 10,000 micro g mL^-1 of extract. Then 1 mL was taken out of it and made up the volume upto 10 mL which gave 1000 μg mL^-1. Further took 2 mL of the above dilution and made up the volume upto 10 mL which produced 200 μg mL^-1 of the extract. Then absorbance values of each aliquot prepared were determined from 290-320 nm, at 5 nm intervals, taking ethanol as blank for extract aliquots and distilled water for cream aliquots, using Shimadzu UV-Visible spectrophotometer (Shimadzu 1700, Japan) values are shown in Table 2.

The observed absorbance values at 5 nm intervals (290-320 nm) were calculated by using formula:

where, CF is correction Factor (10), EE (λ) is erythmogenic effect of radiation with wavelength λ and and Abs (λ) is spectrophotometric absorbance values at wavelength λ. The values of EE (λ)xI (λ) are constants. The obtained absorbance values Abs (λ) were multiplied with the respective EE (λ)xI (λ) values as given by Sayre et al. (1979). Then their summation was taken and multiplied with the correction factor (10) as depicted in Table 2.

Statistical analysis: Statistical analysis was carried out using STAT software14, the measurements were taken thrice and the values were expressed in Mean±Standard deviation. All parameters were statistically analyzed at 95% confidence level. One-way ANOVA analysis, with Tukey correction was used to determine significant differences for the comparison. Differences were considered statistically significant if p<0.001.

RESULTS AND DISCUSSION

On taking initially 350 g of green tea leaves coarse powder, after complete extraction 63.875 of concentrated green tea alcoholic extract was obtained. Thus practical yield obtained was 18.25% w/w which was appreciably good amount. The antioxidant activity of the extract was assessed by reducing power estimation method in which the antioxidant activity of extract was compared with that of standard (ascorbic acid). As observed in the Table 1 the absorbance value for ascorbic acid was considered 100% antioxidant activity and when we compared absorbance of green tea extract as compared to ascorbic acid it produced 51.12±1.8%. Similarly when equal amount of ascorbic acis and green tea extract was taken the antioxidant activity came to be 158.44±0.20,which clearly shows the additive synergistic effect with ascorbic acid. As work done by Ashawat et al. (2007a) on A. catechu and P. granatum and Adesegun et al. (2008) on Sapium ellipticum. It was observed that antioxidant activity was produced due to the presence of phenolic compounds. Similarly it could be concluded that as C. sinensis containes polyphenolic flavonoids named as epicatechins which can scavenge ROS (reactive oxygen species, such as lipid free radicals, superoxide radicals, hydroxyl radicals, hydrogen peroxide and singlet oxygen). Afaq and Mukhtar (2006) have reported in their publications that topical application or oral feeding of a polyphenolic fraction prepared from green tea prevents photocarcinogenesis. The assessment of antioxidant activity proves the photoprotective nature of green tea extract.

Antioxidant properties of medicinal plants have been shown to be due to high content of phenolic compounds (Vinson et al., 2001; Meyers et al., 2003). Polyphenols have a high redox potential which allows them to act as reducing agents, hydrogen donors and singlet oxygen quenchers (Kahkonen et al., 1999). The antioxidant effects of the extract may be due to its phenolic content. In the reducing power assay, the antioxidants in the extract would cause the conversion of Fe³+/ferricyanide complex to the ferrous form. The reducing power increased with increasing concentration of the extract and the standards. This shows that the reducing ability of green tea extract was evident. This suggests that the extract is an electron donor and could neutralize free radical (Zhu et al., 2001).

Second parameter taken was in vitro SPF determination. The SPF is a quantitative measurement of the effectiveness of a sunscreen formulation. To be effective in preventing sunburn and other skin damage, a sunscreen product should have a wide range of absorbance between 290 and 400 nm. The in vitro SPF is useful for screening test during product development, as a supplement of the in vivo SPF measure. In this research alcoholic green tea extract was evaluated by UV spectrophotometry applying Mansur mathematical equation. SPF values of samples obtained using the UV spectrophotometric method are shown in Table 2. We can clearly see that high absorbance values were obtained at 290-320 nm wavelength range. The value of SPF obtained was 18.10±0.05 which is very appreciating when we compare with that of other herbal extracts as reported by various authors. For e.g., SPF of Boerhavia diffusa was between 3.539±0.0213 to 7.1747±0.0038 (Ashawat et al., 2006) and of fresh Aloe vera gel was 0.995±0.221 (Ashawat et al., 2008). In contrast, Khazaeli and Mehrabani (2008) determined in vitro SPF values of extracts of sixteen medicinal plants and reported that extracts of leaves of Dracocephalum moldavica L. and flowering tops of Viola tricolor L. had high SPFs i.e., 24.79 and 25.69, respectively and concluded that these plants had high amounts of phenolic compounds and flavonoids which could be the cause for their high SPF.

It has been reported that topical application of green tea extract prior to UV B radiation result in significant protection against UVB induced cutaneous edema and erythema. Hence green tea extract could form an important constituent of photoprotective formulations. By adding various constituents producing synergistic effects with herbal extract and its photoprotective activity high range herbal photoprotective formulations could be designed.

CONCLUSION

By the antioxidant activity determination it was found that Camellia sinensis extract is an effective antioxidant and it also produced synergistic effect with ascorbic acid. Thus it could be used in herbal topical photoprotective formulations. Thus natural antioxidants alone or in combination with synthetic chemical antioxidants may produce multiple effects on skin. The proposed UV spectrophotometric method is simple, rapid, employs low cost reagents and can be used in the in vitro determination of SPF values in many cosmetic formulations. The proposed methodology may be useful as a rapid quality control method. It can be used during the production process in the analysis of the final product and can give important information before proceeding to the in vivo tests. Similarly the high value of SPF obtained also supports to the photoprotective action of green tea extract. Thereby, we can say that natural photoprotectives could be successfully used as they will produce effect and reduce the side effects produced by the long term effects of chemical antioxidants.

ACKNOWLEDGMENTS

One of the author wishes to thank DST FIST Scheme, New Delhi for financial support and Director, University Institute Of Pharmacy, Pt. Ravishankar Shukla University, Raipur for providing other facilities.