Breakdown hypothesis that this formulation could demonstrate a distinguished

Breakdown in normal anatomic structure and function of skin is named skin wound, which could occur through several causes including physical injuries, which leads to the opening and disruption of the skin(Lazarus et al., 1994). Wound healing is a dynamic process in which dermal and epidermal tissues regenerate as closely as possible to the normal status.  A sequence of events occurs to repair the damage following injury. These events have been classified into stages including inflammatory, proliferative and remodeling (Steidelman et al., 1998) .Cytokines are usually released in the inflammatory stage due to phagocytosis of bacterial pathogens leading to the migration of the cells involved in the proliferative cells. A subsequent of occurrences including angiogenesis, collagen deposition, granulation tissue formation, epithelialization and wound contraction take place upon inflammatory phase, which called proliferative stage(Midwood et al., 2004). In the final stage, collagen remodeling happens along tension lines(Esimone et al., 2008). Non-toxic, novel and cost-benefit therapeutic agents that contribute to increase healing rate, hasten epithelialization, inhibit bacterial infection, and support tissue remodeling have gained attention among researchers. Therefore, we herein attempted a hydrogel formulation containing 10% F. vaillantii total extract and applied it on an animal model of skin wound to verify the hypothesis that this formulation could demonstrate a distinguished treatment in healing wounds by providing enhanced tissue repairing.

Our results revealed that topical administration of our hydrogel formulation using HPMC 4000 cps (2.5%) on rat excision wound led to a significant acceleration of wound healing after 6, 10 and 14 days, which confirmed by the increased  wound contraction compared to negative control group. This enhanced potential of wound healing may be due to anti-inflammatory, antimicrobial and astringent properties of plant, which are well documented in the literatures (Bambal et al., 2011). In this regard, our literature survey found some phytochemicals containing flavonoids, alkaloids, tannins and saponins present in 10 % F. vaillantii total extract (Srivastava and Choudhary, 2014), which seems to be responsible for its wound contraction and enhanced rate of epithelialization 21 days post wound healing. This assumption supported by our previous study indicating a considerable amount of flavonoid in F. vaillantii total extract (Tabrizi et al., 2016). Hence the flavonoids are known to possess antioxidant effects, the wound healing activity of F. vaillantii extract may be attributed to this property in the inflammatory phase. Furthermore, the alkaloids present in F. vaillanti are also responsible for its antimicrobial property(Ivanov et al., 2014; Karou et al., 2006), which in turn leads to a better wound healing in the inflammatory phase.

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Hydrogels exhibited several advantages as a result of providing the required moist environment to the wound area and also acting as a suitable carrier for topical administration of substrates. Moreover, it causes a slow release of substances over the time. What this information brings out noticeably is that hydrogel formulation can be a suitable candidate to promote wound healing. Thus, we prepared a hydrogel formulation with 10% total extract of F. vaillantii using 2.5% HPMC 4000 cps displaying an optimum consistency and spreadability. Consequently, the proper hydrogel spreading would assist in uniform administration of the gel to the skin. Additionally, based on our results, our formulated herbal gel contributed to faster wound healing compared to negative control group. Surprisingly, collagen fiber thickness and hydroxyl proline content appeared to be more or less similar but basically higher in gel base than F. vaillantii gel formulation treated groups, which can be necessarily explained by the therapeutic effect of hydrogels alone. In addition, topical administration of F. vaillantii hydrogel was found to significantly increase the number of vessels as well as hair follicles in gel-treated compared to negative control wounds. Similar indications are also apparent from the study performed by Xiao-bo Wu et al, who found that  angiogenesis in granulation tissues results in improvement of circulation and subsequent providing oxygen and nutrients vital for healing procedure(Wu et al., 2012).

The establishment of an incision wound model needs to be further worked out in order to determine breaking strength confirming the wound healing activity of 10% total extract hydrogel of F. vaillantii. In our study, control rats exhibited wound breaking strength (W up to fmax) as 164.5±68.26 N/mm on the 10th post wound day, whereas gel base and hydrogel extract treated group displayed no significant breaking strength as 259.2±53.09 and 240.8±30.63 N /mm, respectively.  These data are in agreement with the results of our excision model and highlight the role of the gel base in collagen production, which leads to stabilization of fibers formation and subsequent stable intra and intermolecular crosslinks(Rahman et al., 2017; Udupa et al., 1995).