Archives Des Sciences Vol 66, No. 5;May 2013 686 ISSN 1661-464X Biosafty of Ziziphus Lotus Extract as Hair and Body Wash: Animal Study Bassem Y. Sheikh1,2 and Wael M. Elsaed1,3,4 1MABL Chair for Scientific Miracles of Prophetic Medicine. 2Depatement of Surgery and 3 Department of Anatomy, Faculty of Medicine, Taibah University, Kingdom of Saudi Arab. 4 Department of Anatomy, Faculty of Medicine, Mansoura University, Egypt. *Corresponding Author Prof. Bassem Yousef Sheikh, MD, FRCS, FICS Professor of Vascular Endovascular Neurosurgery Director, Al-Moalim MA Bin Ladin (MABL) chair for Scientific Miracles of Prophetic Medicine, Faculty of Medicine, Taibah University P.O. Box: 30001, Al-Madinah Al-Nabawiah, Saudi Arabia E-mail: askdrsheikh@gmail.com Archives Des Sciences Vol 66, No. 5;May 2013 687 ISSN 1661-464X Background: Zizphus lotus (Sidr) tree is commonly used in traditional medicine for treatment of multiple diseases particularly of skin. Its extract is rich in beneficial chemicals that help wound healing and treatment of boils and fruncles. The emollient properties of the extract are thought to be valuable as a hair and body wash in prolonged use. Materials and methods: Water extract from the green leaves of the plant was prepared in two concentrations (50% and 100%). The extract was added to the commercial base of shampoo. The produced wash was applied to the skin of rabbits. 12 rabbits were used. They were divided into 3 groups. Group I served as the control. Group II subjected to 50% of the wash and group III to 100%. The wash was applied for 30 consecutive days then skin samples were collected after animal scarification and examined histologically. Results: Safety of 50% concentration of sidr wash was proofed. It induced no signs of allergy or inflammation. No change in epidermal thickness or inflammatory cell infiltration was noted in comparison to the controls. However 100% concentration induced epidermal thickness and inflammatory cell infiltration of the dermis. Small areas of focal hemorrhage occurred in dermis. Conclusion: Ziziphus extract is a promising material to formulate a new hair and body wash (Shampoo). Our results showed that 50% conc. hair and body wash is save for utilization. Key wards: Ziziphus lotus extract, Skin, Hair follicles, Prophetic medicine, Complementary, Alternative, Scientific chair. Archives Des Sciences Vol 66, No. 5;May 2013 688 ISSN 1661-464X Introduction Ziziphus lotus is a tropical evergreen tree with shiny green leaves. It can reach a height of 2-5 metres (6.6–16 ft). It is abundantly present in the Mediterranean region, throughout Libya to Morocco, Algeria and southern European countries like Spain, Sicily, Greece and Cyprus (Pottier Alapetite, 1981).The edible fruit is a globose dark yellow drupe 1–1.5 cm diameter called a nabk. Its common Arabic name is sidr (Dafni et al. ,2005). The plant extract has been studied (Ikram et al.,1976 and Aynehchi and Mahoodian, 1973) and its chemical composition is well-known (Younes et al., 1996 and Mahran et al., 1996). The main constituents of the essential oil are alpha-terpineol (16.4%) and linalool (11.5%);of the hydrocarbons are n-pentacosane forms (81%); of methyl esters include methyl palmitate, methyl stearate and methyl myristate; of aglycones are beta-Sitosterol, oleanolic acid and maslinic acid; of alkaloids include: zizyphine-F, jubanine-A and amphibine-H. The plant leaves also contains saponin glycosides (Mahran et al., 1996; Abdel-Galil and El-Jissry., 1991). Extracts from different parts of the tree is commonly used in traditional and ancestral medicine for treatment of several pathologies including digestive disorders, weakness, liver complaints, obesity, urinary troubles, diabetes, skin infections, fever, diarrhoea and insomnia (Lahlou et al., 2002). Also it has sedative, antidiabetic and hypoglycaemic effects (Tschesche and Kaubmann, 1975, Anand et al., 1989 and Zakaria et. al., 1999). In Saudi Arabia it is used for the treatment of ulcers, wounds, eye diseases and bronchitis (Adzu et. al., 2001). The leaves of this plant have been used for washing the hair and body and for healing skin diseases such as atopic dermatitis (Amin, 1991 and Nafisy, 1989). The medicinal properties of this plant depend on the part of the plant concerned (root, leaf stalk and pulp or fruit) and the extract used (ethanolic, butanolic etc.). Anti-inflammatory, analgesic and anti-ulcerogenic activities of Zizyphus lotus have been demonstrated in rodents (Borgi et al. 2007 and Borgi et al. 2008). The 10% methanol extract of the stem bark of the plant showed antidiarrheal effects (Adzu et. al., 2003). Different extracts and fractions of the leaves, fruits and seeds of Ziziphus showed antiviral, antifungal and antibacterial activities (Shaht et. al., 2001). The aqueous extract of the plant is reported to Archives Des Sciences Vol 66, No. 5;May 2013 689 ISSN 1661-464X possess antinociceptive activity (Adzu et. al., 2001). It also has strong mollusuicidal activity (Lahlou et. al., 2002). The plant extract containing cyclopeptides and peptide alkaloids form was found to induce sedative activity (Hwang et. al., 2001). Fruit has been used for its emollient properties, and a mixture of dried leaves and fruits is applied topically in the treatment of boils (Le Floc’h, 1983 and Benammar et al. 2010). It was also proofed to have antibacterial and antifungal properties (Pandey and Devi, 1990). Benammar et al. 2010 reported a high concentration of vitamin A and C. They also proofed a notifiable, antioxidant and immunosuppressive activities of the extract. Anti-inflammatory, analgesic and anti-spasmodic activities of this plant were demonstrated in rodents (Borgi et al., 2008 and Borgi and Chouchane, 2009). The Quran mentioned the tree twice (LIII: 13–18; LVI: 28– 32) and it is highly respected by the Muslims throughout the world (Farooqi, 1997). The use of the leaves extract on skin is limited to wound healing, boils and furuncles treatment. No studies on prolonged use of the extract on healthy skin are available. The variable uses and benefits of extract uses made us think that the use of the leaves extract as hair and body wash will have good results on skin. The aim of this study is to investigate the effect of ziziphus lotus extract on healthy skin and hair of rabbits as preliminary step to use it as a regular human shampoo specified for scalp hair common problems as hair dandruff, greasy hair or scalp infections as boils or frunclosis. Material and methods Plant material: The leaves of Ziziphus lotus were collected from the trees in Almadinah Alnbawiah locality. Only green apparent healthy leaves were selected for the study. Preparation of the leaves extract: The authenticated leaves were shade dried at room temperature until total dehydration. Dried aerial parts of plants were blended into fine powder and stored in the dark at a dry place. The powdered Archives Des Sciences Vol 66, No. 5;May 2013 690 ISSN 1661-464X aerial part of plants were extracted in a 1/10 (w/v) of water for 12 h under a continuous reflux set-up in a Soxhlet extractor (hot) or at room temperature (cold) for 24 h (Bouaziz et al. 2009). The extracts were concentrated by rotary evaporation (Büchi Rotavapor and Büchi Laboratories, Switzerland) at 40°C under vacuum to dryness and the yield of extraction was determined. All the dried extracts were preserved in the refrigerator until further use. Plant extracts were dissolved in water (20/80, v/v) at 50 mg/ml before use (Kokate at al., 2003). Two concentrations of the extract were prepared. Concentration 1: 50% and 2: 100%. They were added to a commercial base of shampoo to form two models of the Sidr hair and body wash. Animals: Twelve (12) rabbits were used in this experiment. These were divided into three groups I, II, III. Each group included 4 rabbits. A skin area of 4 cm- 5cm of back of all animals was marked by a permanent ink pen. Group I served as control and had application of 3 ml. of water daily with a soft brush for 30 days. Rabbits of group II were applied with 3 ml. of Sidr hair and body wash (Conc. 1) on the marked area of skin for 30 days daily with a soft brush. In group III same procedure was used with 3 ml. of the hair and body wash (Conc. 2). After wash application, the test area was covered with a non-occlusive dressing (a gauze patch) and then a semi-occlusive bandage for 24 hours and then washed thoroughly with tape water before next application. The process in then repeated daily for 30 days. At the end of the exposure period, any residual test substance was removed by distilled water, and carefully dried (fig. 1 a and b). Animals were observed for signs of inflammation and toxicity once daily. At the end of the experiment, animals were sacrificed. The tissue samples from skin of the application area were fixed in neutral buffered formalin solution and latter processed for section cutting at five micron sections. These were stained with Heamatoxylene and Eosin (Hx & E) and Periodic Acid Schiff (PAS). Histological examination of the skin of the tested area was compared to the control. Archives Des Sciences Vol 66, No. 5;May 2013 691 ISSN 1661-464X Fig. 1 a: Ziziphus hair and body wash applied to skin on the back of the animal after skin shaving at 4 cm- 5cm area of skin. b: After application of the test substance, the test area were covered with a non-occlusive dressing (a gauze patch) and then a semi-occlusive bandage for 24 hours. Results: At the of the examination period, animals of group II (50% Ziziphus extract conc.) showed no signs of allergy or discomfort as compared to the controls. Naked eye examination of the tested area of skin did not show any signs of inflammation (redness or oedema). Histological examination of the skin sections of group I (the control group) showed the normal histological features of skin. It was seen to be formed of epidermis with its stratified keratinized squamous epithelium. Below lays the dermis which is rich in collagenous and elastic fibers. It contains blood vessels and hair follicles (Fig2 a &b). Fig. 2 a: Normal skin stained with Hx & E. the epidermis is thin. Dermis is rich in blood vessels and hair follicles. b: Normal skin stained with PAS. The epidermis is thin and rich in bluish stained keratine scales. a b a b Archives Des Sciences Vol 66, No. 5;May 2013 692 ISSN 1661-464X The abundant hair follicles in rabbit skin appeared in cross sections formed of external root sheath, internal root sheath and cortex. The hair itself is seen formed of medulla, cortex and cuticle of the hair (Fig. 3 a &b). Fig. 3 a: Hair follicle of normal rabbit skin stained with Hx & E. it is formed of external and internal root sheathes and cortex. b: PAS stained sections showed longitudinally lied hairs formed of Keratin filaments. Histological examination of the skin sections of group II showed almost the same histological features of the control group. Epidermis is of the same thickness. No congestion of the dermal blood vessels was observed. None of the inflammatory cells was seen invading the dermal tissue (Fig. 4 a &b). Fig. 4 a: Group II skin section stained with Hx& E showing almost normal skin. b: PAS stained section showing normal dermis with normal hair follicle. b a b a Archives Des Sciences Vol 66, No. 5;May 2013 693 ISSN 1661-464X In contrast, group III (50% Ziziphus extract conc.) showed signs of inflammation of the skin in the form of epidermal thickness, congestion of the dermal blood vessels with extravasations of the red blood cells. Inflammatory cells (plasma cells and esinophils) are seen invading the tissues around the blood vessels (Fig. 5 a, b & c). Higher magnifications showed thickness of the external root sheath of the hair follicle. Hair shafts showed no changes compared to the controls (Fig6 a & b). Fig. 5 a: Hx& E skin section of group III showing thickened epidermis and congestion of the dermal blood vessels. b: PAS stained section at higher magnification showing small areas of hemorrhage in the dermis. C: PAS stained section shows inflammatory cells invading the nearby area close to the blood vessels. a b c c Archives Des Sciences Vol 66, No. 5;May 2013 694 ISSN 1661-464X Fig.6 a: Hx & E stained section of group III shows hair follicles with thickened external root sheath compared to the control group. b: PAS higher magnification no changes in the hair shaft compared to the other groups. Discussion: Corneal layer thickness is one of the measures to assess the efficiency of body washes and shampoos (Groux and Bensignor, 2000). Our experiment showed that the 50% conc. of sidr skin and body wash did not change the thickness of the epidermis as a whole. However higher concentration (100%) increased the thickness to a notifiable extent (Fig. 6 a & b). Although the clinical improvement is the main criterion to evaluate the efficacy of shampoos (Curtis, 1998), the Ziziphus extract at 50 % proved to be safe on the histological level. Oils in Ziziphus (alpha-terpineol and linalool) are helpful to soften and rehydrate the skin, and reduce transcutaneous water loss. It also helps softening and moisturizing. Its major effect is probably by incorporation of essential fatty acids (especially linoleic acid) into stratum corneum ceramides (Campbell and Kirkwood, 1993). The hydrocarbon n-pentacosane in Ziziphus extract is believed to be helpful in prevention of skin diseases as boils and fruncles. Similar studies on the same hydrocarbon extracted from Capparis decidua edgew (Verma et al., 2011) proofed this effect on skin. b b a Archives Des Sciences Vol 66, No. 5;May 2013 695 ISSN 1661-464X Methyl esters are known to cause skin irritation in high concentration. However its presence in Ziziphus hair and body wash is believed to be non toxic as its level does not reach the toxic level (Hewitt et al., 2000). Aglycones play a preventive role in age-related skin changes accelerated by the menopause without the harmful oestrogenic side effects on reproductive tissues (Cassidy, 2003). It showed consistent efficacy in managing conditions of oestrogen deprivation, and its cosmetic creams were actually used to improve skin dryness and wrinkles (Rona et al., 2004). As Ziziphus contains saponin glycoside, it has been shown to decrease inflammation and granular tissue formation in rats (Patel et al. 2011). Our results add to the fact that the plant in its raw form is already used in many parts of the world for the care of the skin (Abbiw, 1990). The chemical composition and phytochemicals present in the plant caused the cultural spread and wide uses particularly in soothing hair and skin (adsu et al., 2001). Recommendation: From our results, Ziziphus extract is a promising material to formulate a new hair and body wash (Shampoo). Our results showed that 50% conc. hair and body wash is save for utilization. The proposed shampoo must be left on skin and hair for several minutes, to allow the active ingredients to be properly absorbed and reach adequate levels in the deep cellular layers. This length of time should be not less than 5 minutes. The skin should then be rinsed thoroughly, for at least 5 minutes, to prevent irritation and to enable the skin to become adequately hydrated. The proposed shampoo may be applied several times a week. Acknowledgment: The authors would like to express the gratitude for the MABL chairn for their financial support (Grant No. SMPM1434/A0106) and to Mr. Hany Salah and Mr. Mohamed Abdulsamad for their clerkal work. Archives Des Sciences Vol 66, No. 5;May 2013 696 ISSN 1661-464X References: 1. Abbiw D.K. (1990) Useful plants of Ghana – West African use of wild and cultivated plants. Intermediate Technology Publications and the Royal Botanic Gardens Kew. 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