Photo Protective Role of Wild Edible Plants on Skin of Mice from Harmful Effects of Ultraviolet Type-B Irradiation

Aim : The aim of this study was to determine the protective antioxidant capacity of edible wild plants in the decreasing effect of ultraviolet B (UVB) on the skin of albino mice. Study Design: Thirty skin biopsies were taken from the mice to detect and compare the hyperplastic effects between the groups. Place and Duration of Study: Animal House of College of Veterinary Medicine, Sulaimani University, Histopathology Lab of Consultative Hospital/Sulaimani Governorate, Anatomy and Histopathology Department, College of Veterinary Medicine, Sulaimani University. Original Research Article Hassan et al.; BJAST, 15(4): 1-10, 2016; Article no.BJAST.23989 2 Methodology: Thirty mice (Mus musculus species, BALB/c strain) underwent this study and were divided into six groups from A-F, according to the UVB exposure, species of plant, and routes of administration. Mice from all groups (Exposure and plant administration except control group) were subjected to UVB irradiation four days/week (20 minutes/day, four weeks), and treated with edible wild plants (Gundelia tournefortii and Malva sylvestris) by different routes. Results: According to the Student’s t-test (Paired) and Pearson’s correlation coefficients we found that the effect of Gundelia tournefortii and Malva sylvestris were highly significant in reducing the hyperplastic effect of UVB irradiation. Conclusion: This study gives an overview of traditional uses of Gundelia tournefortii and Malva sylvestris against the adverse effects of UVB radiation.


INTRODUCTION
Reactive oxygen species can be generated by daily exposure to ultraviolet light and may cause some sub chronic and chronic disorders [1]. It has been known for decades that reactive oxygen species (ROS) produced in the body following UV irradiation are key mediators of oxidative damage. Cell damage from UV also occurs through many mechanisms that include: peroxidation of membrane lipids via generation of lipid peroxides, rapid depletion of several endogenous enzymes and antioxidants such as glutathione reductase and catalase [2]. Exposure to UV has also been shown to induce proinflammatory cytokines, such as IL-1α, and matrix metalloproteinase (MMPs) in skin cells such as keratinocytes and fibroblasts [3].
The human and animal body have multiple mechanisms and antioxidant systems that protect the cellular molecules against damage induced by free radical [4]. In fact, the balance between free radicals production and antioxidants is thought to be strongly related to lifespan [4]. However, these systems don't exercise sufficient protection against oxidative stress. Most of the exogenous antioxidants are constantly required to maintain an adequate level of antioxidant so that the ROS in the human and animal body are balanced [5]. Antioxidants are usually classified into two groups, specifically, synthetics and natural, in addition, numerous synthetic antioxidants have been developed. The phenolic compounds such as vitamin E, flavonoids, tocopherols, carotenoids as well as ascorbic acids are typical antioxidants that have been used [6,7]. Synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxyl toluene (BHT) have been used since the beginning of this century. The diet rich in polyphenolic compounds and flavonoids is associated with longer life expectancy because according to the most research discovery, antioxidant compounds have properties such as anticancer, antiviral, anti-inflammatory activities [8].
Nowadays, there has been an increasing interest in consuming wild food plants [9,10]. In the face of the agricultural society's primary dependence in collecting plants, the tradition of eating wild plants has not completely disappeared and their nutritional role and health benefits are being reported in many surveys world-wide [11][12][13].
Malva sylvestris belongs to the mallow genus in the family Malvaceae mortician. It is commonly known as mellow in Europe, gulkhaira or vilayatti kangani in Pakistan and India [14,15], recently Malva is well-known using plants because many researchers are focusing on its therapeutic properties, such as, antioxidant, antiinflammatory, anticancer, and, attenuation of skin disorder symptoms [16,17].
The aim of this study was to determine the antioxidant protective role of Gundelia tournefortii and Malva sylvestris in decreasing the damaging effect of UVB on the skin of albino mice.

Animal Model
Adult albino Mus musculus species, BALB/c strain (15 males and 15 females) was used in this experiment, which was fed with standard pellet diet (Pico Lab) and provided with water and ad libitum. Animals were housed in the animal house of College of Veterinary Medicine, Sulaimani University under a controlled room temperature of about 25ºC and photo-periodicity of 12 hours light/dark system. Animals were assigned into six groups: Group A (Control group, n=5) which were not exposed to UVB and not administrated with plants, Group B (n=5) exposed to UVB irradiation only, Group C (n=5) which were exposed to UVB light and administrated with Malva sylvestris orally before UVB irradiation, Group D (n=5) which were irradiated with UVB light and painted with Malva sylvestris before UVB irradiation, group E (n=5) which were exposed to UVB light and administrated orally with Gundelia tournefortii before UVB irradiation, and Group F (n=5) which were irradiated with UVB light and painted with Gundelia tournefortii before UVB irradiation. All Groups except control one were exposed to UVB for about 20 minutes, four days/week for one month consecutively.

Collection of Plants
Malva sylvestris and Gundelia tournefortii were collected from the local market of Sulaimani/Kurdistan Regional Governorate (KRG).

Malva sylvestris
Malva sylvestris, which was distributed throughout most villages of Sulaimani/KRG, which was named by toleke (Fig. 1a). Fresh Malva sylvestris (whole plant) was collected from the market and washing many times by plenty water, The 3 kg fresh palnt has been boiled in a container that contains 1 L of tab water for about 10 minutes, then the fluid or juice of plants were filtered and their stored in refrigerator in 8ºC till the time of its using, each mouse from group C were administrated orally with 1ml of Malva sylvestris before UVB irradiation and also each mouse from group D were painted with Malva sylvestris by cotton for about 5 minutes before UVB irradiation throughout the experimental study.

Gundelia tournefortii
Gundelia tournefortii L., Which was named by Kangar ( Fig. 1b), were scattered throughout most villages of Sulaimani/ KRG. Fresh (whole plant) were collected and washed, the 3 kg of this plant mixed with 1 L of water and boiled for about 15 minutes, then it was filtered and their juice stored in 8ºC degree of temperature till the time of its using, each mouse from group E were administrated orally with 1 ml (3 mg/1 mL) of Gundelia tournefortii, before UVB exposing and mice from group F were painted with Gundelia tournefortii by cotton for about 5 minutes before UVB irradiation throughout the experimental study.

UVB Lamp
The Lamp that was used in this experiment was about 312 nm wavelength, 15 Watts, VILBER-LOURMAT-FRANCE, with a calculated power 80 mj/ Sec. with exception of control group mice from all groups were exposed to UVB light for 20 minutes 4 days/week (4 weeks) consecutively, and this was done after shaving the mouse's back skin (2×5 cm).

Sampling Method
At the end of the experiments, the animals were euthanized using (Xylazine-Ketamine: 0.1 mL/10 gm of body weight) as recommended dose intraperitoneally and cervical dislocation. Tissue samples were taken from skin tissue. The tissues specimens were fixed in 10% neutral buffered formalin for at least 24 hours and then routinely processed that were performed in the Histopathology Lab of Consultative Hospital/Sulaimani Governorate. The samples were paraffin embedded and a section of 5µm thickness to detect and compare any abnormal lesions formed by UVB and in administrating plants' groups in skin tissue.

Morphometric Study
The epidermal thickness was measured Haematoxylin and Eosin-stained sections at a magnification of 100 using the image analyzer (Scope image software 9.0 "H3D" computer system-England, digital binocular compound microscope), in the Anatomy and Histopathology Department, College of Veterinary Medicine, Sulaimani University. Five different fields of the epidermis were randomly chosen section. Epidermal thickness was defined distance between the basement membrane and the apical surface of the uppermost layer (from stratum basale to the top layer stratum corne

Statistical Analysis
For each case, the epidermal thickness was measured in five high power fields by, then mean was obtained and at the end the measurements of each epidermal skin thickness (Mean ±S. E. M) of different groups were compared with corresponding epidermal skin thickness of the control group and between each Student's t-test (Paired) and Pearson's correlation coefficients were used to estimate the skin thickness of the back region and analyzed, a P = 0.05 was considered to be significant. . Five different fields of the were randomly chosen from each was defined as the distance between the basement membrane and the apical surface of the uppermost layer (from stratum basale to the top layer stratum corneum). thickness was measured in five high power fields by, then mean was obtained and at the end the measurements of each epidermal skin thickness (Mean ±S. E. M) of different groups were compared with a epidermal skin thickness of the control group and between each group. The test (Paired) and Pearson's correlation coefficients were used to estimate the skin thickness of the back region and in all considered to be

RESULTS AND DISCUSSION
by palpation back skin for estimation of thickness in all groups and visual inspection of any lesions that developed in the backs' skin as shown in Fig. 2. In group A, skin showed delicate and thin appearance (Fig.  2a). While in the group B different lesions were observed due to UVB exposure that ranged from erythema, edema, ulcer of the exposed area and epidermal thickness as shown in (Fig. 2b), in comparing to group B the skin thickness of the groups; C, D, E and F developed a variable epidermal thickness that detected during palpation as in (Fig. 2c-f). Our result result that proved oxidative damage of contributes to a variety of skin illnesses, including inflammation, degenerative aging, hyperplasia and cancer [27]

Light Microscopic
The results of this study have demonstrated different histopathological changes shown in Fig. 3. Examination of sections stained with H&E from the control animals (Group revealed the normal histological structure of the epidermis and dermis layers of the thin skin. The epidermis was composed of stratified squamous epithelium, formed mainly by keratinocytes that appeared to be arranged in 2border between the epidermis and clearly demarcated. The underlying papillary layer of the dermis had abundant capillaries and connective tissue cells, whereas the inner reticular layer of the dermis was composed of a denser connective tissue rich in fiber as (Fig. 3a). In group B, there was a highly epidermal proliferation or hyperplastic epidermis in a responded to UVB exposure as in (Fig. 3b), however, in group C and D (Fig. 3c,d), there was ; Article no.BJAST.23989 species visual inspection of any lesions that developed in the backs' skin as shown in Fig. 2. In group A, skin showed delicate and thin appearance (Fig. While in the group B different lesions were observed due to UVB exposure that ranged from , ulcer of the exposed area and epidermal thickness as shown in (Fig. 2b), in comparing to group B the skin thickness of the groups; C, D, E and F developed a variable epidermal thickness that detected during Our result was by the oxidative damage of UVB that contributes to a variety of skin illnesses, including inflammation, degenerative aging, hyperplasia The results of this study have demonstrated different histopathological changes that are 3. Examination of sections stained with H&E from the control animals (Group A) histological structure of the epidermis and dermis layers of the thin skin. The of stratified squamous epithelium, formed mainly by keratinocytes that 3 layers. The the epidermis and dermis was clearly demarcated. The underlying papillary layer of the dermis had abundant capillaries and connective tissue cells, whereas the inner reticular layer of the dermis was composed of a fiber as shown in In group B, there was a highly epidermal proliferation or hyperplastic epidermis in a responded to UVB exposure as in (Fig. 3b), however, in group C and D (Fig. 3c,d), there was a mild-moderate epidermal hyperplasia due to administration of Malva sylvestris orally and painted routes, in the group E and F developed normal-mild epidermal hyperplasia, due to treatment by Gundelia tournefortii orally and painted routes (Fig. 3e, f).
The observed lesion that found in the dermis was shown diffusely infiltration of the inflammatory cells including neutrophils, macrophages, and lymphocytes in the dermal layer of group B.
While in the group C and D, there was mildmoderate infiltration of inflammatory cells when comparing to group E and F that show normalmild infiltration of inflammatory cells as shown in (Fig. 4a-f).
This study reported an extremely significant association between UVB irradiation and increasing epidermal thickness as we detected epidermal thickness in exposure group 12.93 times greater than the control epidermal thickness, this finding agrees with previous studies [28,29], which demonstrated that UVB radiation on the skin induces a variety of changes in the epidermis, including keratinocyte proliferation that leads to epidermal thickness.

. Diffuse infiltration of inflammatory cells in the exposed group; c,d. Mildly-moderate infiltration of inflammatory cells in orally administered and painted with Malva sylvestris group; e,f. Normal-mildly infiltration of inflammatory cells in orally administered and painted with Gundelia tournefortii group (X400, H&E stains)
Another useful role of Gundelia tournefortii and Malva sylvestris administration, despite the reduction of the epidermal thickness effect of UVB in this present study is their role in decreasing the inflammatory cell infiltration, this finding is in agreement with the previous study, which reported the role of both edible plants as an anti-inflammatory agent, and specially the Gundelia tournefortii species that have shown an extremely significant effect in decreased inflammatory cells [30][31][32]. The present study was first to display the role of both edible plants on reducing inflammatory cells in mice by chronic UVB irradiation.

Morphometric Analysis
As assessed by the image analyzer. The epidermal measurements for the cases and control groups were presented in Table 1. The control group was measured as 7.065±0.129. There was a highly significant increase in the mean thickness of the epidermis in group B (t=-17.052, P=0.000) and their mean was (91.416±4.825), when compared with the control group and its' full epidermal thickness was 12.93 greater than the control group, the mean thickness of the epidermis in group C was ( In this current study, we provided protection against the UVB-induced epidermal thickness by oral and painted administration of Malva sylvestris and Gundelia tournefortii species. Due to the biological activity of these plants that may be attributed to antioxidants, such as polyphenols, vitamin C, vitamin E, carotene, and other important phytochemicals, which protect skin against UVB irradiation [33]. The present finding showed that protective effect of Gundelia tournefortii species was highly significant in reducing the hyperplastic effect of UVB than the Malva sylvestris The result of this finding agrees with the publications of [34], who reported that free radical scavenging activity values of Gundelia tournefortii that includes Total phenol, flavonoid, alkaloid and ascorbic acid exhibited stronger value than those found in Malva sylvestris species. The similar result [35][36][37], which reported that the total phenolic content and antioxidant activity in this plant are attributed to Gallic Acid and Quercetin with typical flavonoids that include; Kaempferol and Quercetin. These antioxidant components inhibit glutathione-S-transferase activity and suppress the generation of reactive oxygen species.
In this study, we proved the effect of Gundelia tournefortii Species orally was more effective than painted one and showed highly significant by reducing the epidermal thickness to 3.82 times, therefore using this edible plant has a powerful value in protecting skin from harmful effect of UVB. Our study in agreement with studies [38][39][40][41], who proved the oral administration of Gundelia tournefortii is useful in treating several disorders including; liver diseases, diabetes, chest pain, heart stroke, gastric pain, vitiligo, diarrhea, and bronchitis. It is also reported to have hypoglycemic, antiinflammatory, anti-parasite and antiseptic. To my knowledge, no study has been reported the antioxidant effect of this plant in reducing the role of UVB exposure.
Our results it found using of Malva sylvestris was effective in reducing UVB-inducing epidermal thickness development, it revealed that the oral administration showed highly significant association that was 4.09 time, when compared to UVB group and this demonstration proved that this plant is an excellent source of antioxidant against oxidative stress of UVB, and this result is in agreement with the publication of [42], who showed that Kaempferol-3-Orutinoside and quercetin-3-O-rutinoside were the main flavonoid component in this edible plant, this finding is inconsistent with the previous studies, who reported that oral administration of Malva sylvestris has lowered highly oxidizing reactive oxygen species, due to presence of flavonoids, phenolic component that usually involves several antioxidant potential activities [43,44]. The results of this study were the first of its kind to be reported.
Epidermal thickness according to Pearson correlation coefficient test as shown in Table 2. In group B epidermal thickness was increased in comparison to the group A, and shown a negative, high correlation between them (r=-0.936, p=0.000), also, a negative, strong correlation was found between the group C, D  (2-tailed) and group B (r=0. -0.937, r=-0.952=P=0. 000), that showed its protective effect on UVB radiation. In group E and F also a negative, strong correlation was found (r=-0.858, r= -0.957, P=0.000) when compared to Group B that was more efficient than group C and D, there was a strong, positive correlation that also found between individual groups

CONCLUSION
The result obtained in the present study indicates that these edible wild plants it considered as a protective valuable traditional using plants against the full range effects of UVB radiation.