MATERIALS AND METHODS

Chemicals
The chemicals i.e., dichromate acetic acid, ethylenediaminetetraacetic acid (EDTA), sodiumcyanide, nitroblue tetrazolium, riboflavin, thiobarbituric acid, di-nitrophenylhydrazine, 2-4 di-nitrophenylhydrazine, thiourea, bovine serum albumin, folins ciocalteus (AR grades) were purchased from, Fluka and Merck India Ltd.

Formulation Studied
An optimized herbal aqueous base cream was prepared accordingly to method (Ashawat et al., 2006a). The optimized formula consisting of natural base like sesame oil 2%, almond oil 3%, honey 2%, jajoba oil, tea tree oil 0.5% each with blend of herbal extracts; C.asiatica 0.64 %, G.glabra 0.64%, P. granatum 0.17%, A. catechu 0.5%, C. zeylanicum 0.35%, P. corlifolia 0.64%, C. tora 0.64%, C. longa 0.17% and fresh A. vera gel 0.65%.

Animal Housing
Albino rats (Wistar strain) were used for the present study (body weight 175 to 200 g). The animals were kept in well-ventilated area of the period of experiment till thirty days. The animals were housed in polypropylene cages at controlled temperature (25±2°C), relative humidity (60±5%) and light. Rats were fed with standard laboratory diet and drinking water was given through drinking bottles through out the experiment. Care was taken to avoid coprophagy (by using metabolic cages). The animals were maintained as per CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) regulations and the experiment was cleared by IAEC (Institutional Animal Ethics Committee).

Animal Grouping
To evaluate the protective effects of the herbal cream on the oxidative stress markers, the three groups of six animals each were prepared i.e., E1, E2 and E3. The study pattern was designed according to following manner.

Methods of Biological Estimation
UV Treatment to Animals
In present study two broad ultraviolet range lamps (200-400 nm) were used to induce photo stress on the skin area of rats. The intensity of light (irradiance) was regularly checked before each test, by using a suitable broadband solarimeter (UV meter). The intensity of exposed radiation was calibrated by using solarimeter. The irradiance of 3.6±0.4 mW cm^-2 was exposed to achieve the 5 J cm^-2 intensity of exposed light radiation. Irradiance of source was determined by using the Surya mapi (SPW.QA CEL India).The doses of 2.5 mg cm^-2 cream formulations were applied to E3 group animals before irradiation to UV light.

The time of light exposure is calculated in the following way accordingly to OECD Test Guidelines:

Tissue Preparation for Biochemical and Histological Studies
After thirty days study, rats were sacrificed by decapitation and their skins were surgically dissected out under aseptic conditions. The excess fascia and blood clots from the skin were removed by washing with chilled physiological saline buffer (pH 7.2).

Skin tissues were weighed and minced on glass plate over ice bags to make colloid and then allowed to homogenate. The homogenate were centrifuged at 5000 rpm for 10 min and supernatant fractions were collected and stored in deepfreeze for study of different biochemical parameters.

For histological studies, skin tissues were fixed in freshly prepared 10% chilled neutral formalin for 12 h at 4°C. After fixation, skin tissue was rinsed in distilled water for several times and dehydrated in graded alcohol series, cleared in zylene and embedded in paraffin wax to make tissue blocks. All the tissue blocks were labeled accordingly to groups. The 8 μm thick series sections were prepared by using rotary microtome. Sections were stained with hematoxylin and eosin stains for demonstration of ultra structural epidermal and dermal changes (Meenakshi et al., 2005).

UV protecting effects of cream formulation was studied by estimation of biochemical parameters i.e., Catalase (Sinha, 1972), Superoxidedismutase (SOD) (Winterbourn et al., 1975), Malondialdehyde (MDA) (Buege and Aust, 1978), Ascorbic acid (Natelson, 1971) and Total Protein (Adeghate, 1999) in each set of experiment.

Statistical Analysis
The statistical analysis was carried out by using SPSS soft ware (version 14.0) and results were expressed as mean±SEM. The significance of difference between mean values for the various treatments was tested using the unpaired student t-test p<0.05 was considered as statistically significant (Mahajan, 1989).

RESULTS AND DISCUSSION

The antioxidant enzymes, such as SOD, catalase and glutathione peroxidase constitute a mutually supportive team of defense against Reactive Oxygen Species (ROS). The high reactivity of ROS can trigger a host of disorders in biological systems. Endogenous antioxidant enzymes are responsible for preventing or neutralizing the free radical induced damages of tissues.

In the present investigation the elevated level of end products of lipid peroxidation in skin of rats treated with UV radiations were observed. The increase in MDA level in skin tissues suggests enhanced lipid peroxidation leading to tissue damage and failure of antioxidant defense mechanisms to prevent formation of excessive free radicals (Table 1). Pretreatment with cream (2 mg cm^-2day topically) significantly reversed these changes. Veerendra et al. (2002) showed a significant decrease the levels of malondialdehyde (MDA) after the treatement of C. asiatica aqueous extracts. Hence it is possible that extracts exert their effects due to the antioxidant activity.

Table 1:	Effect of CAA4 on Malondialdehyde (MDA) ascorbic acid and total protein in UV treated rats
All values are represented as Mean±SEM (n = 6), p-value: +<0.001; ++<0.01 When compared with control untreated animals, *<0.001; **<0.01 When compared with UV treated group

Table 2:	Effect of CAA4 on Catalse (CAT) and superoxidedismutase (SOD) in UV treated rats
All values are represented as Mean±SEM (n = 6), p-value: ++<0.01; +++<0.05 When compared with control untreated animals, ***<0.05 when compared with UV treated group

Present study also showed that the catalase level was significantly (p<0.01) decreased in UV induced oxidative stress, but the level of catalase was significantly increased (p<0.05) after the treatment with cream as compared to E2 group rat model (Table 2).

The level of superoxidedismutase (SOD) was significantly low in UV induced oxidative stress rats when compared to control untreated animals. The level of SOD in E3 group was found to be significantly high as compared to E2 group (Table 1).

The reduction in total protein level appears to correlate with the reduction in collagen matrix or degeneration of tissues bundles. Total protein level was found significantly reduced (p<0.01) as compared to control group as shown in Table 1, irradiation of UV radiations noticeably inhibited total protein level. It has been reported that UVB irradiation showed an inhibitory effect of UV on decreased amino acid uptake (Hirotomo and Shinji, 2003).

The enzyme catalase, is also responsible to catalyze the reduction of H₂O₂ to H₂O and O₂ (Cotgreave et al., 1988). In oxidative stress the level of catalase are reduced. Oxidative stress is an outcome of imbalance between ROS production and antioxidant defenses, which is turn, evokes a series of events to regulating the cellular functions (Bandopadhyay and Banerjee, 1999). The Gr-E2 showed the declined levels of catalase as compared to Gr-E1, while found significantly (p<0.01) increased level of catalase were observed in gr-E3. The level of Ascorbic acid (Vitamin-C) was significantly (p<0.01) reduced after the UV exposure when compared with control untreated animals. While the level of ascorbic acid were found significantly high in group Gr-E3, therefore it was clearly indicated that the extracts present in cream scavenges the free radicals by showing the increase of ascorbic acid level as compared to UV induced oxidative stress in rats (Table 2).

The histological changes were observed after the ultra structural studies. From the histological studies it was noted that the features of UV irradiated skin (Gr E2) were similar to that of a chronic allergy contact dermatistis, showed moderate to severe spongosis (Hirose et al., 2006).

It was evidenced from the literature that some cases with vesiculation and a superficial perivascular lymphohistocytic infiltrate with exocytosis. Ultra structural changes in collagen fibers bear a remarkable similarity to those seen in photodamaged skin. The collagen was damaged to an extent (Fig. 1) are shown in UV exposed group samples.

The elastosis was observed severe, but was not only confined to the upper dermis, as it is with the UVB, but more dense were found deeply in to the dermis, which are the sign of UVA. The cellular structure of Gr-E2 (Fig.1) showed that the thickness of epidermis, this might be due to the eruption of photo allergic dermatitis and hyperkeratosis and photoaging of the follicular mouths (Jones and Veale, 2004).

Fig. 1:	Microphotograph of rat skin after UV exposure

Fig. 2:	Microphotograph of rat skin after formulation treatment

The thining of dermis area was occur in the Fig.Gr-E2 might be due to plunging of the follicular mouths. Such conditions also occurred due to topical application of allergens and chronic exposure to UV radiations (Lisa et al., 1997). The Gr-E2 photograph showed the papillary dermis, there was an increased number of fibroblasts and vertically oriented collagen bundles. This discontinuous and fibroblastic collagen bundle structure might be due to UV intensity and chronicity of UV dose. The changes in epidermal region were found in-group Gr-E2 as compared to group E1 due to the UV irradiation. The cellular structure of UV exposed area showed liquefaction degeneration of basal cell layer of the epidermis region (Fig. 1). The normal skin showed normal arrangement of connective tissues, while the UV exposed and Gr-E3 treated animal skin showed regenerative changes in the connective tissues, oedema and fibrinoid changes were marked immediately below the epidermis. Figure 1 of Gr-E2 showed drastic changes in the collagen degeneration as compared to Gr-E1 and Gr-E3, this might be due to prevention of premature elastotic degeneration of collagen, which clearly indicated the chronic UV exposure symptoms (Lewis et al., 2004). The Fig. 2 of Gr-E3 histological evidences showed remarkable protection level of collagen bundles and elastic fibers. Many polyphenolic components like epigallocatechin, stilbenes, coumarins, cinnamic acids, coca, tea etc. were tested for the prevention of melanoma skin cancer (Linden et al., 2003; Kim et al., 2001; Krutmann, 2001).

Heinrich et al. (2006) shows the photoprotective efficiency of flavanols and found that high flavanols groups showed the significant decrease in minimal erythemal dose (Campos et al., 2006). Bioflavonoids are helpful to maintain the skin biological activity in terms of photoprotection, because the association of bioflavonoids acted by different mechanism such as antioxidant and absorbs ultraviolet rays, which suggest photoprotective effects of botanical extracts.

It is evident from the results of the present investigation that the herbal extracts in cream formulation possess photo protective and anti aging properties.

CONCLUSION

Study suggests that extracts containing cream formulation may also have a role in preventing the aspect of photo aging attributable to enzymic and nonenzymic mediated dermal damages. Such formulations should also address the merits of cosmetic in relation to sun protecting formulation.

ACKNOWLEDGMENTS

The authors wish to thank AICTE(RPS) and UGC (major research project) for providing instrumental facility. The authors are also thankful to Director, Head Cosmetics Laboratory. Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur (CG) for providing experimental facilities.