The present study was undertaken to investigate the antioxidant and gastric cytoprotective prostaglandin properties of the aqueous roots extract of C. sieberiana, which we had previously demonstrated to possess anti-ulcerogenic properties. Gastric ulcer disease is a multi-factorial disease and among the various properties implicated in its pathophysiology is the role played by free radicals. Hence antioxidants which can scavenge free radicals are expected to heal or prevent gastric ulcers. Several methods developed to measure the efficiency of antioxidants focus on different mechanisms of the oxidant defence system. In most of the systems, irrespective of the stage in the oxidative chain in which the antioxidant action is measured, most non-enzymatic anti-oxidative activity is mediated by redox reactions. The redox potentials of compounds are related to their antioxidant activity against free radicals such as peroxyl or hydroxyl radicals, which have more positive redox potentials . As shown in Figure 1 the reducing power increased as the extract concentration increased indicating some compounds in the roots extract were both electron donors and could react with free radicals to converts them into more stable products to terminate radical chain reactions. This result indicates that although the reducing power of LAA was slightly higher it was not significantly different from that of C. sieberiana (p > 0.05). The significant reducing powers recoded therefore suggested that the roots extract of C. sieberiana would have significant antioxidant action which was subsequently confirmed by its scavenging action of free radicals generated by DPPH.
In solution, DPPH generates free radicals whose odd electrons become paired off in the presence of a hydrogen donor. The DPPH radical has been widely used to test the free radicals scavenging ability of various natural products and has been accepted as a model compound for free radicals originating in lipids . In DPPH assay the lower the IC50, the better it is able to scavenge the radicals, particularly peroxy radicals which are the propagators of the auto-oxidation of lipid molecules and thereby break the free radical chain reaction. Like the reducing power, at the IC50 of 0.075 ± 0.006 mg/mL, the DPPH scavenging activity of the crude extract was 63.2% that of LAA which indicates that the roots extract contains compounds that have strong antioxidant properties. Hydroxyl radical is the principal contributor for tissue injury. The deoxyribose method is used in determining the rate constants of reactions involving hydroxyl radicals. The reaction involves the incubation of a mixture of FeCl3-EDTA, H2O2 and ascorbate with deoxyribose in phosphate buffer (pH 7.4). The hydroxyl radicals generated from the mixture attack the deoxyribose and result in a series of reactions that cause the formation of MDA. A hydroxyl radical scavenger added to the reaction mixture will therefore compete with deoxyribose for the availability of hydroxyl radicals, thereby reducing the amount of MDA formed. In this study, the roots extract of C. sieberiana was able to achieve a 62% maximum scavenging activity indicating that it contains compounds that inhibit hydroxyl scavenging activity.
Lipid peroxidation has been implicated in the pathogenesis of various diseases including gastric ulcer disease in both humans  and experimental animals . It is well established that bio-enzymes are very much susceptible to lipid peroxides, which is considered to be the starting point of many degenerative processes. This result indicates that the antioxidant activity of C sieberiana is only about 17% that of LAA as shown by the peroxidation in the presence of 1 mM linoleic acid which was significantly lower compared with LAA. The chelating ability of the extract measures how effective the compounds in it can compete with ferrozine for Fe2+. The Fe2+-ferrozine complex has maximum absorbance at 562 nm and a large decrease in absorbance indicates strong chelating power. By forming a stable Fe2+ chelate, an extract with high chelating power reduces the free Fe2+ concentration thus decreasing the extent of Fenton reaction which is implicated in many diseases. The results indicate that the crude roots extract can chelate free Fe2+.
The observed antioxidant activity is significant because free radicals have been implicated to mediate in stress, NSAID and H. Pylori induced gastric ulcers [5, 29]. Neutrophil adherence to the endothelium of gastric microcirculation has also been shown to be critical in mucosal injury in animals and such adherence is believed to liberate oxygen radicals, resulting in the release of proteases and obstructing capillary blood flow or causing lipid peroxidation and damaging cell membranes . Therefore the reported anti-ulcer activity of the roots extract of C. sieberiana could be due in part to its strong reducing and antioxidant properties.
Phytochemical analysis detected alkaloids, saponins, anthraquinones, tannins and flavonoids in the C. sieberiana roots extract. TLC spots revealed by their reaction with ferric chloride and their fluorescence under UV light indicated that flavonol/flavonoid/flavone or related compounds with polyhydroxy and/or phenolic groups constituted major chemical substances in the roots extract. Evidence of the presence of flavonol/flavonoid/flavone or a related compound with polyhydroxy and/or phenolic groups is consistent with the significant antioxidant effect of the roots extract. Flavonoids or polyphenolic substances exert antioxidant actions by scavenging free radicals, chelating metal ions or inhibiting enzyme systems that generate free radicals. Several studies demonstrated that flavonoids from various plants are reportedly capable of preventing the occurrence of gastric ulcer. This may take place through an increase in the amounts of neutral glycoproteins and in prostaglandin concentrations, and inhibition of histamine secretion from mast cells by inhibition of histidine decarboxylase, thus reducing stimulation of H2 receptors, or by secretion of prostaglandin-like compounds . Another possible mechanism of action for inhibiting ulcer occurrences is by decreasing pepsin secretion and activity. In addition, several studies cited by Middleton and Kandaswami , indicate that certain flavonoids have anti-ulcer activity with some having direct mucosal protection activity similar to that of prostaglandins. This suggests that flavonoids and/or polyphenols may be responsible for the reported gastric mucus protection and anti-ulcer properties of the roots bark extract of C. sieberiana.
The major prostaglandins produced by human and rodent gastric mucosa are PGE2 and PGI2 which are vasodilators in the gastrointestinal mucosa . The vasodilatory properties of these two molecules may increase mucus production and reduce acid and pepsin levels in the stomach, thereby make an important contribution to gastric mucosal defence and facilitate the repair of pre-existing ulcers in the gastrointestinal mucosa [33, 34]. The present results show that the significant dose-dependent increase in gastric mucosal PGE2 and PGI2 (82.4% and 88.6% respectively in the high dose) in the absence of induced gastric ulcers is an indicator of anti-ulcerogenic activity of the roots bark extract. These results confirm an earlier study  on the anti-ulcerogenic properties of the roots bark extract which showed that its gastric cytoprotective effect in rats was reduced significantly by pre-treatment with indomethacin, an NSAID prostaglandin inhibitor.
The inflammatory process which occurs during gastric ulceration is also known to be a key component of mucosal defence against exogenous and endogenous factors. However, while the acute inflammatory response is aimed at reducing mucosal injury, some of the inflammatory mediators released increase the susceptibility of the stomach to damage induced by NSAIDs or other topical irritants, and thereby contributes to the generation of mucosal injury in certain circumstances . Several of these inflammatory mediators including histamine, tumor necrosis factor-α, platelet-activating factor, interleukin-1, interleukin-8 and leukotriene B4 have been shown to be down-regulated by endogenous generation of prostaglandins . In addition prostaglandins are also potent inhibitors of leukocyte adherence to the vascular endothelium which is a hallmark of inflammation such that leukocyte adherence that occurs within the gastrointestinal microcirculation following administration of an NSAID can be prevented by prostaglandin administration [35, 36]. It is imperative to note that activated inflammatory cells may also be potential sources of free radicals  which further aggravate the damaged gastric mucosa. This may be a potential mechanism of action of anti-oxidants as anti-ulcerogenic agents.
The roots extract did not show decline of sPLA2 protein; however, enzymatic activity of sPLA2 in serum was definitely inhibited. Thus, the impact may be on the enzyme-substrate level possibly by blunting sPLA2 interaction with its substrates rather than on the synthesis of sPLA2. It was expected that the stimulation of endogenous generation of gastric mucosal PGE2 and PGI2 will be coupled with an increase of arachidonic acid signaling as a result of stimulated sPLA2. The stimulation of endogenous generation of gastric mucosal PGE2 and PGI2 despite the inhibition of serum sPLA2 is interesting. An important clue may lie in the fact that the study measured sPLA2 in the serum and not in the stomach homogenate hence it is possible the extract is exerting its effect selectively at the two different locations due to the various isoforms of sPLA2 discovered [38, 39]. However, the inhibition of sPLA2 in the serum confirms an earlier report which suggests that the inhibition of serum sPLA2 by C. sieberiana roots bark extract may be partly responsible for its therapeutic use as an anti-asthmatic and anti-inflammatory agent . We are currently conducting studies into the effect of different fractionated portions of the extract on sPLA2 activity and expression in different tissues in a bid to elucidate its mechanism of action.
Despite these findings, inhibition of gastric mucosal sPLA2 activity may indicate anti-ulcerogenic abilities. Ischemic insult to intestinal mucosa causes excessive production of oxygen derived free radicals (ODFR) that may initiate a chain of reactions in membrane-bound lipids leading to lipid peroxidation and PLA2 activation [41, 42]. Inhibition of this cascade by scavenging ODFR and/or inhibiting sPLA2 could therefore be an effective tool to protect gastrointestinal mucosa against chemically induced lesions.