Burn Wound Healing Effect and Hair Growth Promoting Activity of Lawsonia inermis L. and Honey in Oryctolagus cuniculus Rabbits

Corresponding Author: Zouhir Djerrou Department of Sciences of Nature and Life, Faculty of Sciences, University of August 20th 1955, Skikda, Algeria and Laboratory of Pharmacology and Toxicology, University of Mentouri Constantine, Algeria Email: z.djerrou@univ-skikda.dz zouhir21265@yahoo.fr Abstract: This study aimed to assess Lawsonia inermis L. and honey mixture effects on burn wounds and hair growth in rabbits. Nine male Oryctolagus cuniculus local rabbits were allocated randomly in 3 groups. After antisepsis and local anesthesia, four circular burns were created on the animal’s backs. Immediately after burning, the wounds were covered with honey in three rabbits (HON group), another group was treated first with honey and then L. inermis powder was added on honey (LI_HON group). Three untreated animals were used as control (CRL group). The treatment of rabbits was applied once daily (6/7 days) until 24th day post burns. The animals were observed for their general state, aspects of wounds and the healing times were also recorded. At 35th day post burns, the hair growth was investigated in the different groups. LI_HON group has recorded the best results compared to other groups; a short significant necrosis duration (LI_HON Vs CRL, p<0.001; LI_Hon Vs HON, p<0.05), precocious start of 1st crust detachment (LI_HON Vs CRL, p˂0.05; LI_Hon Vs HON, p<0.001), a quick complete detachment of the first crust (LI_HON Vs CRL, p>0.05; LI_Hon Vs HON, p<0.05) and a significant reduction of healing time (LI_HON Vs CRL, p<0.05; LI_Hon Vs HON, p<0.01). LI-HON group has shown the best promoting activity of hair growth in term of recovered surface percentage (LI_HON Vs CRL, p = 9,13038E −7 ; LI_Hon Vs HON, P = 1,70745E −7 ) and hair length (LI_HON Vs CRL, p<0.01; LI_Hon Vs HON, p≤0.001). The study concludes that the topic use of L. inermis powder and honey simultaneously accelerates burn wound healing process in rabbit’s model. Henna has also shown a remarkable hair growth promoting activity in term of hair recovered area and hair length.


Introduction
Wound healing is a natural and dynamic process; it occurs in three fundamental steps including: inflammation, proliferative and remodeling phases (Wulff and Wilgus, 2013). Inflammation includes coagulation and inflammatory cell recruitment. The fibrin clot formed contains fibrin molecules, fibronectin, vitronectin and thrombospondins; it hosts the active arriving cells and different cytokines and growth factors (Reviewed by Broughton et al., 2006). Macrophages are present throughout all stages of the healing process, producing a variety of substances that regulate healing including growth factors, prostaglandins and complement factors (Nathan, 1987). Numerous growth factors, cytokines and chemokines implicated in the execution and regulation of the wound healing stages were investigated. (Barrientos et al., 2008). The physiological processes of angiogenesis, granulation tissue formation, epithelialization, collagen synthesis and wound contraction characterized the proliferative phase (Broughton et al., 2006). The wound healing cascade finishes by remodeling phase which occurs in three weeks to several years according to wound's complexity (Clark, 1988). The apoptosis of cells in this step prevent the appearance of hypertrophic scar or keloïd; in normal conditions a mature wound was formed and characterized as avascular and acellular (Broughton et al., 2006).
According to Khorasani et al. (2008), thermal burn injury represents a major cause of death and disability and may cause a high costs in health care. The conventional drugs used in the management of burn injuries were reported to exert unwanted side effects (Fan et al., 2015).
Many of today's modern drugs have their origin in traditional plant medicine (Blanks et al., 1998). The use of plants for the healing purposes cited around 3000 B. C, in China, Egypt and subcontinent (Morse, 1934).
Henna is a tall shrub or small flowering tree. The scientific name of this plant is Lawsonia inermis L., which belongs to Lythraceae family. It is a shrub that grows in North Africa and Middle East (Gibbons et al., 2005). Henna is native to a number of tropical regions in Asia, Northern Africa and Australia. It is naturalized and cultivated in the tropics of America, Egypt, India and parts of the Middle East (Muhammad and Muhammad, 2005). The different parts of henna have been reported to exert antioxidant, hypoglycemic, antidiabetic, hepatoprotective, antibacterial, antifungal, anticancer and wound healing properties (Majtan et al., 2013;Hadagali and Chua, 2014;Rajashri and Sachin, 2014;El Bergadi et al., 2015;Devasvaran and Yong, 2016;Mohamed et al., 2016).
The aim of the present study is to evaluate the effect of a combination of honey bee and Lawsonia inermis on burn wound healing and to investigate the possible hair growth promoting activity in rabbit's model.

Materials and Methods
Drugs 200 g of commercial Lawsonia inermis L. powder was purchased from local market. It is an imported product from Pakistan under the name "Special mehndi", fabrication date: October, 2013 and expiration date: September, 2018. Honey was obtained from a local bee keeper from Collo, East of Algeria.

Animals and Housing Conditions
The study was conducted on 9 healthy male, adult, Oryctolagus cuniculus local rabbits weighing 1500 to 1800 g at the beginning of experiment. The animals were kept in normal conditions of temperature and lighting (12 h light: 12 h dark) and given laboratory food and water, ad libitum during the study. The experimental protocol was approved by the scientific committee of the department of sciences of nature and life, faculty of sciences, University of august, 20th 1955, skikda, Algeria.

Experimental Design
Three days before burning, the animal backs were clipped and a depilatory cream was used to remove all the hairs from the rabbit's skin. On day zero, a local anesthesia was practiced for all rabbits using xylocaïn 2% (s/c) after disinfectant by surgical alcohol 70%. Then, four burns of identical size (22 mm in diameter) were created on the back of each animal, two cranially (left and right) and two caudally (left and right) ( Fig.  1), by a cylinder metal weighing 200 g immersed in prior in boiled water for 3 min and maintained on animal skin 15 sec (Djerrou et al., 2010).

Treatment and Assessment of Healing Process
The animals were randomly allocated into three groups, the first untreated was used as control (CRL group), in the second 3 rabbits (HON group), the burns were immediately covered by a thin layer of honey (≈1 mm), the third group was treated as HON group and then honey was covered by 0.5 g of Lawsonia inermis L. powder. All these drugs were applied topically slowly and they were repeated once daily until 24th day post burns for non healed burns. The clinical aspects of wounds and the different healing times were noted. Photographs were taken from the wounds on days 0, 8, 12 and 20.

Hair Growth Assessment
At 35 day post burns, the hair growth was compared in the different rabbits; the percentages of recovered areas were calculated and the lengths of back's new hair were compared with the lengths of hairs in the left and right normal sides of animals.

Statistical Analysis
The results were expressed as mean with their variance. One-way Analysis Of Variance (ANOVA) was used to compare the different group means. A value of p<0.05 was considered statistically significant.

Results
Generally, there was a favorable trend towards healing in different rabbits either treated or untreated. Body weight of the animals has recorded a significant decrease during the first 15 days after burns in different groups; noting a return to the original state that was obtained within 2 weeks. One rabbit from LI-HON group showed a dermal reaction following application of the depilatory and that has healed at the 6th day post burns (Fig. 2), the same rabbit also expressed a skin thickening 15 days after the beginning of the application of honey and henna mixture.

Healing Process
The results of healing process recorded in Table 1 and Fig. 3 showed that application of Lawsonia inermis and honey simultaneously stimulated significantly the healing process in its different stages compared to honey applied only or untreated animals. HON group has enregistred a significant reduction (p˂0.005) of necrosis duration compared to CRL group but no sgnificant differences were observed between these two groups in the processes of crust detachment or the total healing time. LI_HON group has recorded the best results compared to other groups; a short significant necrosis duration (LI_HON Vs CRL, p˂0.001; LI_Hon Vs HON, p˂0.05), precocious start of 1st crust detachment (LI_HON Vs CRL, p˂0.05; LI_Hon Vs HON, p˂0.001), a quick complete detachment of the first crust (LI_HON Vs CRL, p˃0.05; LI_Hon Vs HON, p˂0.05) and a significant reduction of healing time (LI_HON Vs CRL, p˂0.05; LI_Hon vs HON, p˂0.01).

Hair Growth
The results relative to hair growth registred in Table  2 and illustrated by Fig. 4 showed a real promoting activity of hair growth in LI-HON group in term of recovered surface percentage (LI_HON Vs CRL, p = 9,13038E -7 ; LI_Hon Vs HON, P = 1,70745E -7 ), the untreated rabbits have recorded a significant hair growth compared to HON group (p˂0.01), however, there was no significant difference between these two groups in term of new hair length (p˃0.05). In LI_HON group, the new hair length has reached a very significant difference compared to other groups (LI_HON Vs CRL, p˂0.01; LI_Hon Vs HON, p≤0.001).

Healing Process
The effect of honey applied alone was significant compared to control in the first 10 days of healing process with no infection of wounds. This period correspond to hemostasis and inflammatory phases (through 4 to 6 days post burns) and a part of the proliferative phase (which prolonged until 14th day) (Broughton et al., 2006). This result could be a consequence of honey pharmacological properties demonstrated in several studies. According to Namias (2003), honey has anti-inflammatory effect, promotes granulation tissue formation and exert antibacterial activity. The anti-inflammatory property may be associated with the antioxidant content of honey (Tanaka et al., 1995). The high osmolarity of honey has been considered a valuable tool in the management of sloughy and septic wounds; it produces a cleansing effect and naturally debrides non-viable tissue. A reduction but not significantly of time of first crusts detachment was recorded in honey group compared to control; this later has recorded a better healing time but not significantly than that of honey. Several studies have explained the effects of honey on granulation tissue formation and epithelialization by the generation of hydrogen peroxide (stimulation of angiogenesis), the growth of fibroblasts and the wound acidification. The nutrient content of honey may also stimulate growth because it has a wide range of amino acids, vitamins and trace elements (Reviewed by Molan, 1999). The addition of L. inermis on honey in the third group has resulted in very significant results in the different stages of wound healing. Phytochemical screening of henna plant has revealed the presence of numerous chemicals including alkaloids, tannins, flavonoids, steroids, glycosides, saponins etc. (Ibrahim et al., 2008;Arun et al., 2010). According to Muhammad and Muhammad (2005), the plant constituent are made up of mannite, tannic acid, mucilage and gallic acid, but the main constituent is 2hydroxynapthoquinone (lawsone), known to be the major bioactive constituent of this plant. In an in vitro study, the henna leaves extracts were able to inhibit the growth pattern of Aspergillus niger and Fusarium oxysporum, Streptococcus sp. and Staphylococcus aureus which are the primary invaders of burnt wounds. Inhibition of these microorganisms' growth suggests that henna may be valuable in the management of burnt wound infections (Muhammad and Muhammad, 2005). Chloroform extract of leaves of L. inemris had shown a highest antioxidant activity compared to α-tocopherol (Endrini et al., 2007). In addition, L. inermis L. extract exhibited absolute toxicity and showed broad fungitoxic spectrum when tested against 13 ring worm fungi (Singh and Pandey, 1989). A significant analgesic and antipyretic activities were also shown with leaves extract of this plant (Mohsin et al., 1989). In another study, isoplumbagin and lawsaritol, isolated from stem bark and root of L. inermis L. showed anti-inflammatory activity against Carrageenan induced paw edema in rats (Gupta et al., 1993). In a study of Nayak et al. (2007), the healing activity of L. inermis extract was compared with the control and reference standard animals; the results showed a positive effects on term of wound contraction, epithelialization period, skin breaking strength, granulation tissue weight and hydroxyproline content in this plant group. The authors have conducted a histological study which has showed increased and well organized bands of collagen, more fibroblasts and few inflammatory cells in henna group.

Hair Growth
Honey group has recorded a significant delay in hair growth, compared to control in term of percentage of recovered area, but no statistical significance was observed in comparing new hair length to normal hair length; this later may be due to individual characteristics and/or due to stress because these animals were manipulated more than the untreated animals. The real hair growth promoting activity recorded in LI_HON group is due to Lawsonia inermis powder that spreads, after it's sprinkling on the honey, in the whole surface of the shaved back. L. inermis has been cited as a growth accelerator and was used in an ancient Egyptian formula to cure the loss of hair. Henna has also been recognized to act as a very good conditioner to the hair (Ahmadian and Fakhree, 2009). Several histological and molecular studies have focused regeneration of skin and hair follicles after wounding. Ansell et al. (2011) have demonstrated an association between hair follicle cycling and wound healing. Chen et al. (2015) have shown that collagen VI is strongly deposited in hair follicles and is markedly up regulated by skin wounding; they have highlighted the essential relationships between extracellular matrix and hair follicle regeneration and suggested that collagen VI could be a potential therapeutic target for hair loss and other skin related diseases. These above described mechanisms of influence of the different hair-cycle stages on skin wound healing may be implicated in the interpretation of the present study results.

Conclusion
The mixture of Lawsonia inermis L. powder and honey stimulates burn wound healing process in its different stages. In addition to this, henna promotes hair growth. Further studies, including immunohistochemical investigations are required to confirm and elucidate the mechanism behind this promoting hair growth activity.