The significant reduction in wound size after LJEE ointment treatment was correlated with the histopathological findings of increased epithelization activity, angiogenesis, granulation tissue formation, and finally remodeling of the extracellular matrix. Collagen not only confers strength and integrity to the tissue matrix but also plays an important role in homeostasis and epithelialization in the later stages of wound healing . Hydroxyproline is an uncommon amino acid present in the collagen fibers of granulation tissues. Biochemical analysis revealed increased hydroxyproline content, which is a reflection of increased cellular proliferation and therefore increased collagen synthesis, after LJEE ointment treatment . Increased hexosamine content reflects the stabilization of collagen molecules via enhanced electrostatic and ionic interactions . Hence, enhanced hydroxyproline and hexosamine synthesis provides strength to repaired tissue and stimulates healing. Significant increases in hydroxyproline and hexosamine content were observed in the wounds after LJEE ointment, and these findings were supported by the histopathological data. The potent wound-healing capacity of LJEE, as evidenced by the wound contraction and increased levels of biochemical parameters in healing tissue, has thus validated the ethnotherapeutic claim.
Open wounds are particularly prone to infection, especially by bacteria, and they also provide an entry point for organics that cause systemic infections. Infected wounds heal less rapidly, and infection often results in the formation of unpleasant exudates and the production of toxins concomitantly with the killing of regenerating cells . Wide ranges of antibiotics are presently being used to treat wound infections in humans . However, due to their adverse effects and the presence of antibiotic-resistant organisms, researchers are now investigating the extracts of biologically active compounds isolated from plant species that are used in herbal medicine . The use of LJEE to treat various skin infections is justified by this work, as LJEE exhibited commendable activity against all the organisms tested. The external application of LJEE on wounds prevented the invasion of microbes through the wound, resulting in protection of the wound against infection by various organisms.
Strong TNFα and IL-6 induction after cutaneous injury was observed as early as 12–24 h after wounding, and these components play a major role in the inflammatory phase of wound healing by enhancing angiogenesis . Our study revealed that TNFα and IL-6 levels were slightly lower at 24 h after wounding in LJEE ointment-treated animals. It is thus apparent that the LJEE ointment did not interfere with macrophage-derived proinflammatory cytokines during the first stage of healing. However, LJEE ointment treatment elevated IL-10 levels on days 1 and 9 after wounding. IL-10 is an anti-inflammatory cytokine produced by various cells including macrophages and T lymphocytes . IL-10 appears to influence the wound-healing environment by decreasing the expression of proinflammatory/profibrotic mediators, resulting in decreased recruitment of inflammatory cells to the wound . Treatment with LJEE ointment increased the serum IL-10 concentration and simultaneously downregulated TNFα and IL-6 expression, especially on day 9 after wounding. There are reports that TNFα inhibits collagen formation and hydroxyproline production, which are essential for the final part of the proliferative phase in wound healing . The findings suggested that LJEE regulates anti-inflammatory and proinflammatory cytokines and ultimately the systemic immune pathways associated with them, thus leading to cellular proliferation.
In the current Chinese Pharmacopoeia, chlorogenic acid is officially used as the indicator compound to characterize the quality of this herb . Considerable chlorogenic acid content has been detected in LJEE. It has been demonstrated that chlorogenic acid strongly inhibits the production of TNFα and IL-6 by human peripheral blood mononuclear cells stimulated with staphylococcal exotoxins . Chlorogenic acid inhibits the synthesis of other mediators such as IL-1, interferon-gamma, monocyte chemotactic protein-1, and macrophage inflammatory protein-1a . Additionally, chlorogenic acid has strong bacteriostatic activity . Hence, the synergistic effect of the antimicrobial and anti-inflammatory activities of LJEE accelerated the wound healing process.
Recent studies with other plant extracts revealed that phytochemical constituents such as flavonoids, triterpenoids, and tannins can promote the wound-healing process [26–28]. As L. japonica is being used and cultivated in more countries, its chemical components have been extensively studied. Essential oil, organic acids, flavones, saponins, iridoids, and inorganic elements were isolated and identified as the primary components . Among them, essential oil and chlorogenic acid have demonstrated pharmacological effects, and they are considered the active compounds of L. japonica.