Studies on Wound Healing and Antiulcer Activities of Extract of Aerial Parts of Phyllanthus niruri L. (Euphorbiaceae)

Problem statement: Phyllanthus niruri is widely used in many parts of the world to manage a wide range of diseases. As part of efforts t elucidate its pharmacological activities and hence medicinal potential, we studied the wound hea ling and antiulcer properties of extract of the aerial parts using experimentally-created wounds an d ulcers in rodents. Approach: Wound healing effect was assessed using excision and dead space w ound models while antiulcer activity was evaluated using indomethacin-, ethanol acidand co ld-restraint stress-induced ulcer models. Results: The extract (5,10%) significantly (p<0.05) reduced the wound diameter producing 90.9 and 93.7% wound contraction respectively on day 18 post wounding. It also reduced epithelialization time of excised wounds and increased the rate of wound c losure, with WC50 of 8.7%. The extract (400 mg kg) also significantly (p<0.05) increased the weight of granuloma tissue. In antiulcer studies, the extract significantly (p<0.05) inhibited the develo pment of ulcers induced by indomethacin and moderately inhibited ethanol acid-induced ulcer. It was, however, devoid of any such effect in coldrestraint stress-induced lesions at the doses used. Phytochemical analysis of the extract revealed the presence of alkaloids, saponins, tannins, flavonoid s, reducing sugar, carbohydrates and glycosides. Conclusion: These findings indicate that constituents of aeria l p rts of P. niruri possess wound healing and antiulcer properties.

In continuation of efforts to elucidate the pharmacological activities and hence medicinal potential of this plant, we studied the wound healing and antiulcer properties of extract of the aerial parts using experimentally created wounds and ulcers in rodents. The plants were thoroughly cleaned and the aerial parts cut into smaller pieces, sun-dried for 7 days and milled to coarse powder using a mechanical grinder. The powdered plant material (2 kg) was extracted with methanol by continuous extraction in a soxhlet. Concentration of the extract in a rotary evaporator under reduced pressure afforded 140.6 g of the methanol extract (ME; 7.03% w/w). Phytochemical analysis for identification of constituents using conventional methods [46,47] gave positive reactions for alkaloids, saponins, carbohydrates, reducing sugar, flavonoids, steroids, terpenoids, tannins, resins and glycosides.

Animals
Wound healing activity studies: The effect of ME on wound healing was studied in rodents using experimentally-created excision and dead space wounds.
Excision wound: Animals randomly placed into four groups (I-IV; n = 6) were anaesthetized with chloroform and excision wounds (2.5 cm diameter) created on the depilated dorsal thoracic region [48] . Groups I and II received ME (5 or 10%) ointment while III and IV were treated with neomycin-bacitracin (5%) or vehicle (ointment base) respectively. The ointments were topically applied once daily from day 1, after wound creation and continued until the wound healed. The wound diameter was measured on alternate days and the epithelialization period recorded at the end of the study. Wound contraction (%) was calculated using the relation: Where: W D0 = The wound diameter on day zero W Dt = The wound diameter on day t The time taken for 50% of wound closure (WC 50 ) was read off a plot of wound closure (%) against time (days).
Dead space wound: Dead space wounds were created by making a pouch through a small cut and implanting sterile cotton pellets (50 mg each) on either side of the axilla on the ventral surface of each rat. The animals were randomly divided into three groups (n = 6). Groups I and II received 200 or 400 mg kg −1 of ME suspended in DMSO (10%) respectively. Group III served as control and received the vehicle. Extract administration was done orally once a day from day 0-9. On day 10, the animals were sacrificed by overdose of chloroform anaesthesia and the pellets carefully removed and dried in an oven at 60°C to a constant weight and the weight recorded. The level of increase (%) in the weight of granuloma tissue formed was calculated relative to the control.
Antiulcer activity studies: The ulcer-healing or protective effect of the extract was studied in rodents using ulcers induced by different ulcerogens.
Indomethacin-induced ulcers: Animals were fasted for 18 h but allowed access to water only prior to the experiment and divided into 4 groups (n = 6). Groups I and II received 200 or 400 mg kg −1 of ME, while III and IV received cimetidine (32 mg kg −1 ) or the vehicle (10% v/v DMSO) respectively. Thirty minutes after oral administration of extract, ulcer was induced by oral administration of indomethacin (20 mg kg −1 ). After 7 h, the animals were scarified and the abdomen opened. The stomach was isolated and opened along the greater curvature and rinsed under a stream of water. Lesions on the gastric mucosa were observed with a hand lens (x10) [49] and scored 0-4 using an arbitrary scale where 0 = no lesions; 0.5 = hyperemia; 1 = one or two lesions; 2 = severe lesions; 3 = very severe lesions and 4 = mucosa full of lesions [50] .
Ethanol-acid induced ulcers: Animals were fasted, grouped and treated as above. Thirty minutes after oral administration of ME, ulcer was induced with oral administration of ethanol-acid (25 mL kg −1 of 0.3 M HCl in 60% ethanol) [51] . One hour later, the animals were sacrificed and their stomachs removed and cut along the greater curvature. The open stomach was rinsed in a stream of water and the lesions on the gastric mucosa observed and scored as described above.
Cold-restraint stress-induced ulcer: Acute, gastric lesions were induced by stress according to a modified method of Nagura and Bacchi [52,53] . Animals were fasted for 18 h, grouped and treated as above. Thirty minutes after oral administration of ME, ulcer was induced by immobilizing the animals in cylindrical cages vertically placed in bowls containing water to the level of the xiphoid process. The bowls were kept in a refrigerator maintained at 19°C. After 17 h, the animals were sacrificed by overdose of chloroform anaesthesia and their stomachs removed and opened along the greater curvature. The stomach was rinsed in a stream of water and the ulcer lesions observed and scored as described above.

Statistical analysis:
The data obtained was analyzed using One-way analysis of variance (ANOVA) and further subjected to LSD post hoc test for multiple comparisons. The results were presented as Mean ± SEM. Differences between means was accepted significant at p<0.05.

RESULTS
Effect of ME on excision wound: Topical application of ME caused a significant (p<0.05) concentrationrelated reduction in wound diameter ( Table 1). The ME also significantly (p<0.05) reduced the epithelialization period of excision wounds ( Table 2). On day 18, ME-treated rats showed 93.7% wound contraction compared to control rats (84.2%). The WC 50 values showed that ME (10%) produced a better wound closure rate (8.7 days) than cicatrin (9.0 days) (Fig. 1).

Effect of ME on dead space wound:
In the dead space wound model, oral administration of ME caused a significant (p<0.05) and dose-related increase in the weight of granuloma tissue ( Table 3).

Effect of ME on indomethacin-induced ulcer:
The ME significantly (p<0.05) inhibited the development of indomethacin-induced ulcer in a dose-related manner. The effect of the higher dose was comparable to that of cimetidine ( Table 4).

Effect of ME on ethanol acid-induced ulcer:
The ME (400 mg kg −1 ) moderately inhibited the development of ulcers induced by ethanol acid ( Table 4).
Effect of ME on cold-restraint stress-induced ulcer: Pretreatment with ME did not prevent the development of ulcers induced by cold restraint stress (Table 4).

DISCUSSION
Wounds occur when the continuity of the skin or mucous membrane is broken [54] . On the other hand, peptic ulcers, are benign lesions of gastric or duodenal mucosa which result from a shift in the balance between the aggressive (acid, pepsin, H. pylori) and protective (gastric mucus and bicarbonate secretion, prostaglandins) mechanisms. While wounds occur under acute conditions and are healed through a natural process that may be prolonged without treatment, peptic ulcers are amplified and sustained by their pathophysiological mechanisms. Whatever is the basic amplification mechanism of the disease process; treatment with wound-healing or anti-ulcer agents respectively stimulates or accelerates healing.
Experimental assessment of the wound healing and antiulcer activities of P. niruri extract showed increased rate of wound contraction and epithelialization, increased granuloma tissue formation and protection of the gastric mucosa from ulcerogenic effects of indomethacin and ethanol-acid in treated animals. Chronic topical application of the extract on excision wounds accelerated wound contraction and reduced epithelialization period in rats. Wound healing involves regeneration of specialized cells by proliferation of surviving cells and connective tissue response characterized by the formation of granulation tissue [55] . It is also characterized by haemostasis, reepithelialization and remodeling of the extracellular matrix. Epithelialization, which is the process of epithelial renewal after injury, involves the proliferation and migration of epithelial cells towards the centre of the wound while wound contraction is largely due to the action of myofibroblasts [56,57] . Thus, the effect of the extract on wound contraction and epithelialization suggest it may enhance epithelial cells migration and proliferation, as well as the formation, migration and action of myofibroblasts. On chronic oral administration, the extract enhanced granuloma tissue formation in dead space wounds. Granuloma tissue formed on an inert foreign body in a dead space comprises an accumulation of modified macrophages [55] and histological giant cells and undifferentiated connective tissue consisting largely of collagen [55,57,58] . Increase in granuloma tissue, in dead space wound, is associated with enhanced collagen maturation and increased protein content as well as angiogenesis [59][60][61] in the wound. These processes are indicators of new tissues generation and suggest that the extract may stimulate mechanisms associated with tissue regeneration. Closely related to this is the effect of growth factors secreted by macrophages on wounds. Macrophages secrete peptide growth factors that exert pro-healing effect by stimulating regeneration, fibroblast proliferation and activation and angiogenesis [55] . It is, therefore, likely that in addition to enhancing wound contraction and epithelialization, the extract may also stimulate processes associated with tissue regeneration.
In antiulcer studies, treatment with the extract protected rats from ulcers induced by indomethacin and ethanol-acid. These agents induce ulcers by different but related mechanisms that basically undermine gastric mucosal integrity and hence cytoprotection. Gastric mucosal damage caused by indomethacin and related non-steroidal anti-inflammatory drugs result from the inhibition of prostaglandins synthesis via the arachidonic pathway [62,63] . Prostaglandins serve protective functions in the stomach by maintaining gastric microcirculation [62,64] and causing gastric secretion of bicarbonate [65] and mucus [66] . Thus, the effect of the extract in this model suggests it may possess cytoprotective action probably by enhancing prostaglandins synthesis. The extract also mildly protected the gastric mucosa against ethanol-acid challenge. Ethanol-induced gastric mucosal lesions, predominant in the glandular part of the stomach, are caused by the direct toxic action of ethanol, reduction of the secretion of bicarbonate and depletion of gastric wall mucus [67,68] . Ethanol also reduces endogenous glutathione and prostaglandin levels, increases the release of histamine and influx of calcium ions [69] and stimulates the synthesis of leukotriene C 4 (LTC 4 ) [70][71][72] and oxygen free radicals [73] thus causing increased lipid peroxidation which damages cells and cell membranes [74] . Agents that enhance mucosal defensive factors inhibit ethanol-induced gastric mucosal lesions [75] . This is consistent with our earlier suggestion that the extract may owe its antiulcer effect to cytoprotection probably due to enhancement of prostaglandins synthesis. Although the precise mechanism of cytoprotection remains to be elucidated, the level of protection offered by the extract against indomethacin ulcers directly suggests it may enhance prostaglandins synthesis. This may be the reason for its ineffectiveness in protecting rats against cold-restraintstress ulcers where factors other than suppression of prostaglandin synthesis play major causative roles. Cold restraint ulcers are caused by factors such as increase in gastric motility and gastric acid secretion, decrease in pH [76] , vagal overactivity, mast cell degranulation, decrease in mucosal blood flow, gastric mucus and prostaglandins synthesis as well as generation of free radicals [77][78][79][80][81] . The extent of interaction of the extract with these mechanisms is not known, although the antioxidant [10] and radical scavenging [11] effects of this plant have been reported. Besides the likelihood of prostaglandin synthesis enhancement, it is unlikely that the extract interferes with any of the major processes responsible for ulcer induction in this model. As such, extract of aerial parts of this plant may not be effective in all cases of ulcer.

CONCLUSION
In conclusion, the results of this study indicate that aerial parts of P. niruri exhibit wound healing and antiulcer activities by accelerating wound closure and epithelialization and enhancing mucosal protection possibly by mobilization of endogenous prostaglandins. Although the extract may be effective in accelerating the healing of wounds, it may not be effective in all cases of ulcer.