The effect of Medicago sativa extract and light on skin hypopigmentation disorders in C57/BL6 mice

Vitiligo is a common depigmentation skin disease that affects the quality of life in many patients.

accompanied by loss of pigments such as Vitiligo which is a skin disorder characterized by a lack of melanocytes, and leads to pigment dilution in the affected areas of the skin. The affected area is a nonscaly, amelanotic, chalky-white with distinct margins. 2 Vitiligo is not a curable disease, different treatment choices with different mechanisms for enhancing the pigmentation process or inhibiting the depigmentation process include topical or systemic corticosteroids or immunomodulators, phototherapy, and surgery. 2 It has also been reported that psoralens or linear furanocoumarin are photosensitizing agents that improve skin sensibility by enhancing melanin production. In conjugation with phototherapy, the former has been reported therapeutically effective in treating vitiligo. 3 In addition, several studies reported that coumarins have effective photosensitivity which may positively influence the treatment of vitiligo when combined with ultraviolet radiation exposure. 4 Herbal medicine, in addition to other treatment options, was used for the treatment of vitiligo, especially using herbs that contain furanocoumarins such as Khellin, Green Tea Polyphenols, St. John's wort naphthodianthrones, and others of high Coumarin composition. 5 These remedies seem to be related to their anti-inflammatory, immunomodulatory, and antioxidant characteristics as well as photosensitizing properties. 6 Moreover, several agents have been found to act as tyrosinase inducers to increase pigmentation. For example, Daphne gnidium extracts are essential oils that contain fatty acids, coumarins, flavonoids, terpenoids, and alkanes. Moricandia arvensis leaves contain saponins, tannins, flavonoids, cardenolides, and alkaloids. 7 Vernonia anthelmintica contains steroids, flavonoids, carbohydrates, fatty acids, sesquiterpene lactones, and terpenes. 8 Medicago sativa L. is a large genus of the Leguminosae tribe, with around 83 distinct species. M. sativa has long been used in China, Turkey, and America as a popular herbal remedy for a range of ailments. 9 M. sativa contain a variety of phytochemicals including flavonoids, alkaloids, phytoestrogens, digestive enzymes, triterpene, saponins, and coumarins. Several research attempts concerning coumarin use for skin hypopigmentation disorders and vitiligo treatment have been reported Bora & Sharma. 10 Bergapten has also been successful in the treatment of vitiligo. 11 Thus, this study will focus on

| Plant material collection and extraction
Medicago Sativa plant samples were collected from commercial farms at Al-Azraq, south of Jordan. Samples were sent to the Royal Society for the Conservation of Nature (Amman, Jordan) for identification and authentication. The plant was dried under the sun for 3 days then grinded using a blender and stocked in a dark, dry place at room temperature. A 50 g of dry plant material was soaked in methanol for 24 h. The prepared extract was filtered and evaporated until completely dried. The extract was stored in a dark place at room temperature until used.

| Determination of total phenols content
Total phenol was determined using Folin-Ciocalteu method. 12 Briefly, 1 ml of serial dilutions of the plant extract was added into 4 ml of 7.5% Na 2 CO 3 and 5 ml Folin-Ciocalteu reagent. These mixtures were covered and kept at room temperature for 1 h, and absorbance was recorded for each extract concentration using UV-V spectrophotometer at 765 nm. All concentrations were expressed as mg equivalents to gallic acid per gram of plant extract (mg/g) based on Equation 1:

| Determination of total flavonoids content
Total flavonoid content was determined by the colorimetric method described by Cosmulescu et al. 13 Briefly, 1 ml of serial dilution from each sample was added into 0.5 ml AlCl 3 , 0.5 ml NaNO 2 , 2 ml NaOH, and 4 ml distilled water. After 15 min, the absorbance was recorded using a UV-V spectrophotometer at 510 nm (Shimadzu, Japan). All concentrations were expressed as mg equivalents to quercetin per gram of plant extract (mg/g) based on Equation 2:

| Determination of antioxidant
The principle of the antioxidant assay was based on the method described by Pramod et al. 14

| Rheological testing
The viscosity and rheological behavior of the prepared creams were determined using a cone and plate viscometer (Anton Paar, Rheometer Germany GmbH, Model MCR 101). All measurements were carried out at a temperature of 25 ± 1°C, using spindle CP 35.
The formulations were loaded on the plate at an amount of 0.5 g and allowed to reach the stable temperature for 5 min. To assure accuracy, the rheometer was calibrated and programmed via a computercontrolled RheoCompass software (Anton Paar).

| Temperature sweep test
The heating range is set as 14-37°C, the heating rate is 5°C/min, the strain is fixed at 1%, and the oscillation frequency is 1.0 rad/s.
All measurements were carried out in triplicate, and average values were reported. After acclimatization, mice were randomized into six groups (n = 8)

| Animal model and experimental design
as follows: negative control treated with neither formulas nor exposed to UVA light, control receiving vehicles only, placebo receiving vehicles and exposed to artificial UVA light (Philaquin 4%, Philadelphia Pharmaceuticals, Amman, Jordan), a group receiving Bergaptin (0.002%) as a positive control to depigmentation models, and two groups receiving different concentration of the study plant extract (10% and 20%). On the first day of the study, animals' hair was shaved off the dorsal region using an electronic trimmer. Thereafter, animals were treated with either vehicle or 4% hydroquinone (HQ) for 15 consecutive days for induction of skin depigmentation "vitiligo model." The treatment continued even after confirmation of depigmentation in the animal models on Day 9; thus, they were treated for another 6 days. On Day 9 from the initiation of the study, signs of vitiligo were noted; therefore, animals were prepared for hair shaving and were treated with corresponding study treatments. Thereafter, animals

| Skin and hair depigmentation model
The depigmentation of skin was evaluated using a scoring method ( for the flavonoid content, a yield of 21.97 ± 0.08 mg/g was calculated in the dry extract as quercetin equivalence.

| Antioxidant activity
Medicago sativa methanol extract exhibited an antioxidant inhibition activity of 71% when compared to Trolox.

| Rheological test and flow curve
All tested formulations demonstrated a typical shear-thinning behavior of decreased viscosity with increased shear rate, as shown in Figure 1.  Figure 2).

| In vivo pigmentation activity of M. sativa
At the termination of the dosing period, animals were examined for overall skin color as well as hair color and growth. Skin color was assessed after shaving the inspected area, and results were scored and interpreted as presented in Figure 3.  (Figures 3 and 4).
Observing the hair growth among groups (Figure 3), some mice of the control group presented a slower growth rate in comparison F I G U R E 3 Effect of treatments on skin color., hair color, and growth. Scored observations of animals are interpreted in percent animals based on parameters of (A) skin color; (B) hair color, and (C) hair growth. Negative control. Treated with neither formulas nor exposed to UVA light. Control: treated with vehicles cream and exposed to UVA light. Placebo: treated with Hydroquinone cream and exposed to artificial UVA light. Bergapten: treated with Bergapten (0.002%) cream and exposed to artificial UVA light. 10% plant extract: treated with cream containing plant extract 10% and exposed to artificial UVA light. 20% plant extract: treated with cream containing plant extract 20% and exposed to artificial UVA light.  Figure 6 shows the skin histopathology of mice exposed to UVA, the layers of skin with magnification of 10×, specifically; Figure 6A the stratum corneum (SC) followed by the stratum spinosum (SS). Figure 6B shows the papillary dermis (PD) is thinner than the reticular dermis (RD) and both contain hair follicles (HF) and eccrine sweat glands (SG). Below, are layers of the adipose white dermis (WD) preceding the panniculus carnosus (PCM). Figure 6C,D shows the melanocytes are normally restricted to hair follicles, and after exposure to UVA light, the epithelial matrix around the hair follicles increases and causes darker hair to grow.

| Skin and hair morphology
For the Bergapten-treated group (Figure 7), the hair shaft was observed as black and white, while the root was white. For the 10% plant extract cream-treated group, the hair shaft and roots were observed black. The effect of herbal cream treatments against hydroquinone-induced vitiligo can be observed in both Figure 7A,B, in which the 10% plant extract cream-treated group showed a protective effect against the growth of white hair. The same result was seen in the 20% plant extract cream-treated group.

| Skin irritation/corrosive potential
Following the OECD guideline and recommendation of conducting skin irritation/corrosive potential for dermal exposure of animals to study formulation, the M. sativa containing formulas were concluded safely on skin, described in Tables 3 and 4.

| DISCUSS ION
Plants that contain coumarins and their derivatives have been used for re-pigmentation of the skin in Egypt, India, and other countries, including Ammi majus L., Psoralen corylifolia L., and Facus carica L. 4 In this study, methanol was used for the extraction of M. sativa, due to its ability to extract phenolic acids, coumarins, and flavonoid compounds, which are the most valuable active constituent in M. sativa. 15 The presented data showed high phenol and flavonoids content, as well as antioxidant activity in the methanolic extract of M. sativa.

F I G U R E 4
Effect of treatments on hair color and recovery to black color in extract-treated animals. Presentation of percent animals showing a change in hair color and gradual recovery to normal (black). Animals showed (A) mixture of black and white hair; (B) some showed more black hair and some had recovered into (C) black hair. Bergapten: treated with Bergapten (0.002%) cream and exposed to artificial UVA light. 10% plant extract: treated with cream containing plant extract 10% and exposed to artificial UVA light. 20% plant extract: treated with cream containing plant extract 20% and exposed to artificial UVA light.
Similarly, previous research found that methanol was the best solvent to extract phenolic compounds from M. sativa flower, which has also been reported to pose anti-inflammatory and antioxidant activities. 16 In the current study, HPLC-MS/MS analysis revealed the presence of 18 different phytochemical compounds detected in the methanol extract, of these seven compounds were not previously detected in the study plant species. In that regard, succinic acid, 3′,4'-Dimeth oxy-7-hydroxyflavanone, and isoferulic acid were detected and are known for their antioxidant effects. In addition, benzoic acid and 4-Hydroxybenzoic acid were also observed which are usually used in pharmaceutics and cosmetic products due to their antimicrobial activity. 17,18 Nevertheless, salicylic acid was among the constituents, and well-documented for its capacity in exfoliation, therefore, is often used in photodamage, melasma, freckles, and lentigines treatment. 19 In addition, vanillin was also detected and has been reported to enhance psoriatic skin inflammation was detected 20 and 2,5 dihydroxybenzoic acid, which is widely used in cosmetics to treat pigmentary disorders. 21 As well, rutin which has the ability to increase skin elasticity and decrease wrinkles, 22 and saponarin, 7-Glu Chrysoeriol, ISO-Orientin were also detected and are reported to have anti-inflammatory activity and are used for inflammatory skin diseases. 23 ISO-Orientin may be accounted to the inhibition of UVB-light induced skin injury in mice, and genistin might be the mediator in treating skin keloid scars. 24 Of the most important is the detection of P-Coumaric acid in the studied plant extract, which poses an antioxidant effect, reducing UVinduced erythema formation and subsequent pigmentation in human F I G U R E 5 Pictures of mice from all the tested groups at days 0, 8, 16 and 29 of the experiment periods. Negative control: treated with neither formulas nor exposed to UVA light. Control: treated with vehicles cream and exposed to UVA light. Placebo: treated with Hydroquinone cream and exposed to artificial UVA light. Bergapten: treated with Bergapten (0.002%) cream and exposed to artificial UVA light. 10% plant extract: treated with cream containing plant extract 10% and exposed to artificial UVA light. 20% plant extract: treated with cream containing plant extract 20% and exposed to artificial UVA light.
skin, 25,26 and is considered a concentration-dependent tyrosinase inducer. 27 Tyrosinase activators, which have stimulating effect on melanin formation, are beneficial in the treatment of depigmentation diseases. 28 Plants that have compounds that act as tyrosinase inducers such as Daphne gnidium, Moricandia arvensis, and Vernonia anthelmintica were previously used to increase skin pigmentation. 29 The including protecting human skin from ultraviolet (UV) radiation. 28 Melanogenesis is a complex pathway that involves melanin synthesis, transport, and release. Melanin synthesis is stimulated by variable factors, including α-melanocyte-stimulating hormone and cyclic AMP enhancers. Loss of hair shaft melanin is associated with decreased tyrosinase activity in melanocyte bulbs. 28 Therefore, the black color of the skin which was observed in bergapten-treated mice can be explained by the ability of Bergapten to increase skin pigmentation by stimulating melanin synthesis. 30 Restoration of black skin color was also observed in the 20% plant extract cream treated mice, which may be due to the ability of M. sativa extract to increase the pigmentation by inducing the tyrosine enzyme activity. In addition, 31 reported that UVA treatment would enhances epidermal melanocyte activity and reduces melanocyte degeneration. In melanocytes, the photo conjugation of psoralens and DNA promotes the differentiation and proliferation of melanocytes and enhances the synthesis of tyrosinase by regulating the activity of cAMP, thus increasing the formation of melanosomes. 30 In addition, UVA exposure results in increased melanin content in the deeper layer of the epidermis. 32 The latter could be another proposed mechanism, suggesting a photosynthesizing effect for the plant extract similar to psoralens.
The adopted model is a classical in vivo model for mimicking vitiligo induced in C57BL/6 mice through the topical application of hydroquinone daily for several consecutive days, which might in some studies reach up to 60 days. The use of hydroquinone-induced hypopigmentation is a practical, efficient, and simple method to mimic the phenotypic characteristics of vitiligo, yet is considered limited to the phenotypic changes of vitiligo but not the actual etiology. However, more sophisticated and expensive models like the utilization of spontaneous animal models are considered the ideal approach to studying vitiligo. 33 Nevertheless, several chemical compounds were studied using chemically induced models before translating them into clinical settings. 34 In the current study, hair color of mice observed at the end of experiments indicated that plant extract had a concentrationdependent activity of restoring black hair color in hydroquinone depigmentation model, in comparison with bergapten-treated animals.
Hydroquinone has been reported capable of reducing the number of basal melanocytes, melanin-containing follicles, and melanincontaining epidermal cells. The melanocytes that cause hair pigmentation are located in the bulb of the hair follicles, where they transfer melanin to the cortical keratinocytes of the hair shaft and the melanin-containing hair follicles determines the color of the hair. 33 Nevertheless, melanin-containing hair follicles were less due to the activity of hydroquinone on tyrosine.

| CON CLUS ION
Phytochemical analysis results confirm that M. sativa extracts are considered a good source of many important natural compounds including phenol, flavonoids, P-coumaric acid, and antioxidants.
Methanol extract showed high total phenols, flavonoids, and antioxidant content. P-coumaric acid which is known to act as a tyrosinase inducer and may have a good effect on depigmentation and hypopigmentation disorder was detected in the study plant extract. Based on the findings of the in vivo study, cream with M. sativa extract had the capacity of restoring pigmentation of both the skin and hair without causing any skin irritation. Therefore, the current knowledge suggests that M. sativa might be a potential treatment for the treatment of vitiligo and other skin hypopigmentation or pigmentation disorder, thus, to be implemented in pharmaceutical formulations.

AUTH O R CO NTR I B UTI O N S
All authors contributed to the study conception and design. All authors contributed to the material preparation and data collection.
Azal Hussein Najm Ubaydee, Reem Issa, Bayan Y. Ghanim, and Faisal Al-Akayleh contributed to the data analysis. The first draft of the manuscript was written by Azal Hussein Najm Ubaydee, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

ACK N OWLED G EM ENTS
The

CO N FLI C T O F I NTE R E S T
The authors report no conflicts of interest in this work.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C A L A PPROVA L
The protocol of the animal testing on mice was reviewed and ap-