The effects of Eurycoma Longifolia Jack Tongkat Ali root extract hydrogel on histological and immunohistochemical parameters of wound healing

Abstract

Wound management is one of the significant health problems throughout the world. Medicinal plants have been used widely in wound management. Eurycoma longifolia Jack which is called Tongkat Ali (TA), is one of the Malaysian popular plants. Many studies have proved that TA has effective medicinal impacts. Our study aimed to investigate the possible effects of TA roots extract in the acceleration wound healing via enhancing wound contraction, epithelialization, histopathological parameters of wound healing and VEGF expression. TA roots were authenticated by microscopic techniques and extracted by ethanol and Soxhlet technique. In vitro cytotoxicity assay was conducted to determine the maximum safe dose for its topical application. TA extract was chemically analyzed by GC-MS to indicate its phytochemicals. TA roots extract was prepared in a hydrogel for in vivo healing studies. In vivo studies were conducted on 40 Sprague Dawley rats. The rats were divided into two batches; 5-days and 21-day application. The rats in each batch were arranged into 4 groups (n=5): untreated (-ve) control, Hydrocyn® aqua gel (+ve), vehicle (xanthan) and TA hydrogels. A full-thickness circular excisional wound (15×15) mm was created on the rat’s back. The wounded area was measured and photographed on days 3,6,9,12,15and 18 post wounding to determine the wound contraction percentage and epithelialization period for the rats of 21-day batch. For the histopathological studies, 5-days application batch was appointed to investigate the effect of TA hydrogel on wound healing parameters and VEGF expression by immunohistochemistry assay. 21-day application was appointed to investigate the effect of TA hydrogel on collagen formed in the healed skin by measuring the epidermal/dermal thicknesses and evaluating the fibres orientation. Crude sample presents the authentic microscopical features to that of E.l.J taproot. Extract weight was 45.3 gm and Ic50 was 118.5 μg/mL. TA extract contains several antioxidant/anti-inflammatory phytochemicals, which are necessary for wound healing according to GC-MS. The hydrogel was prepared using 2% XG and TA ethanol extract for wound application. From the in vivo studies, no irritation was reported from TA hydrogel and it showed significant raise in wound contraction percentage compared with the other groups on day 3. Although in the later healing stages and epithelialization, TA hydrogel did not show a statistical difference, yet it was comparable to a medically certified wound healing agent. Histologically, TA hydrogel group on day 5 showed a significant increase in fibroblast number and collagen density and it showed significant VEGF expression compared with the (-ve) group by IHC. On day 21, TA hydrogel improved collagen production through increasing the epidermis/dermis thicknesses compared with other experimental groups and the collagen fibres were oriented in a mixed pattern. These positive results might be attributed to the availability of phytochemicals in TA roots with antioxidant/anti-inflammatory effects which are the main mechanisms for an effective healing process. This is the first study that indicates the possibility of preparation of TA roots in a hydrogel and proved its positive impact on wound healing. TA hydrogel can be considered as a promising and effective wound healing agent.