A study of antimicrobial activity, acute toxicity and cytoprotective effect of a polyherbal extract in a rat ethanol-HCl gastric ulcer model

The Haya people of Kagera region are endowed with a culture rich in traditional medicine practice owed to an extensive intercultural exchange among the diverse ethnic tribes of the Lake Victoria Basin [1]. In this series of reports combined documentation [1, 2] and evaluation to establish proof of the concept is being done. In previous reports a good corroboration of claims on antimicrobial activity has been established from laboratory tests [3–5] and using evidence from the literature which has indicated an average 20-30% agreement with the therapeutic claims by the Kagera traditional healers [1, 2].

One of the plants used in traditional medicine is Rhynchosia recinosa (A.Rich.) Bak. (Fabaceae) [Akashakanyaliyoya]. The aerial parts of this plant are pounded together with the stem barks of Ozoroa insignis Del. (Anacardiaceae) [Omkerenge], Maytenus senegalensis (Lam.) Excell. (Celastraceae) [Omurosho], Entada abyssinica Steud. ex A. Rich(Fabaceae) [Mwiganjula] and Lannea schimperi (Hochst.)Engl. (Anacardiaceae) [Omusangesange] and then boiled with water to make a decoction. The decoction is administered at a dose of one glass three times a day for the treatment of peptic ulcers. In this study a mixture of extracts of the five plants was evaluated for ability to offer protection against ethanol-HCl induced gastric ulceration [6–8], antibacterial activity against some Gram negative bacteria, acute toxicity in mice and brine shrimp toxicity.

Ethanol induced gastric ulceration is due to a number of mechanisms that include depletion of gastric mucus and breaking the mucosal barrier, back diffusion of acid, increased gastric mucosal permeability, leading to increased leakage of hydrogen ions from the lumen, and decreased transluminal electrical potential difference [18]. It also causes changes in mucosal blood flow, destruction of microvascular and nonvascular cells, mast cell degranulation, neutrophil mediated mucosal injury [18], and depletion of certain oxygen free radical scavengers [18]. Metabolism of ethanol also releases free radicals [19]. The proton pump inhibitor pantoprazole protected the rat gastric mucosa from insult by acidified ethanol suggesting role of gastric acid in the ethanol/HCl-induced gastric ulceration. There was a clear change in the gross appearance of the gastric mucosa as compared to the solvent treated group. The polyherbal extract, at the dose of 800 mg/kg body wt reduced the degree of gastric mucosal ulceration with an efficacy that was comparable to that of 40 mg/kg body wt pantoprazole. Phytochemical screening indicated that the polyherbal extract contained tannins and flavonoids, which are phenolic compounds. Plant phenolics are known to be antioxidants and free radical scavengers [20]. Terpenoids which have antioxidant activity were also present in the extract. Since ethanol-induced gastric ulceration causes neutrophil mediated mucosal injury through release of oxygen free radicals, proteases and lysosomal enzymes [19], the phytochemical results may suggest that the extract owe its anti-ulcerogenic effect partly due to the presence of these compounds which have antioxidant and free radical scavenging properties. A recent report indicates that a methanol extract of Entada abyssinica stem bark exhibited free radical scavenging activity and it was suggested that phenols, flavonoids, saponins and tannins identified in the extract and fractions were responsible for their antioxidant activity [21]. In the current results saponins were not detected in the E. abyssinica ethanol extract. Furthermore Entada abyssinica is reported to have anti-inflammatory activity and it inhibited acetic acid-induced vascular permeability in a dose-dependent manner in mice [22]. Inflammation is implicated in acidified ethanol-induced gastric ulceration [19]. This may explain the rationale for including E. abyssinica in the polyherbal preparation by traditional healers.

Some plants of the genus Maytenus are popular in Brazil as traditional antiulcer and anti-inflammatory remedies [23, 24]. Some of the species that already have proven antiulcer activity include Maytenus ilicifolia[18], Maytenus robusta[24], Maytenus obtusifolia[25], Maytenus aquifolium[26], and Maytenus truncata[27]. The hyroalcoholic extract of the aerial parts of Maytenus robusta, for example, reduced lesion index in a dose-dependent manner reaching maximum gastro-protection of 85.0 ± 9.2% at a dose of 500 mg/kg body wt as compared to 30 mg/kg body wt omeprazole which conferred 86.6 ±7.4% protection. The extract reduced the volume of gastric juice, total acidity and increased gastric pH [26]. These results are very similar to the results of this study and suggestion can be made here that the rationale for Maytenus senegalensis in this traditional antiulcer remedy is related to its potential to directly intercept the ulcerogenic mechanisms. Although it was not planned to measure volume of gastric juice in this study it was clearly observed that the stomachs of rats that were given the polyherbal extract had very little fluids, and in some cases, the stomachs had no fluids as opposed to the control ones from which we could draw up to 2 ml of gastric juice. Plants of this genus are also reported to have anti-inflammatory [23] and antioxidant activity [28], and these two processes are implicated in the mechanisms of acidified ethanol-induced gastric ulceration.

The phytochemical tests showed that Lannea schimperi, Ozoroa insignis and Rhynchosia recinosa also contained terpenoids and phenolics which are also likely to contribute to the anti-ulcerogenic activity of the polyherbal mixture.

In terms of meaningful clinical application the extract dose of 800 mg/kg boy wt is too large, so one can only speculate that the active components of the extract are compounds present in much lower concentrations. These compounds may be antioxidants and free radical scavengers, but given the fact that pantoprazole was equally effective these results still do not provide conclusive evidence for deducing the responsible anti-ulcerogenic mechanisms of the extract. More gastric ulcer models are, therefore, needed to determine the mechanisms of action of the extract and/or the active compound/s.

The most appropriate bacterium for this study was Helicobacter pylori, as this is the bacterium that is associated with peptic ulcer disease. Unavailability of this organism necessitated the use of other Gram negative bacteria; therefore the results reported in this study are only indicative rather than intended to imply possible anti-Helicobacter pylori activity. It is noted, however, that the individual plant extracts were not very active against the four Gram negative bacteria used but when their extracts were combined (C1) as used by traditional healers they showed higher activity. The different combinations were tested as an attempt to determine which combination would give the best antibacterial activity and probably determine the most important plant for use with R. recinosa. The results did not give an explicit clue on which plant is best for antibacterial activity when combined with R. recinosa. These results, however, show that the combination as used by traditional healers in which R. recinosa is used in combination with the other four plants is probably the best combination to achieve safety and efficacy.

Using the criteria suggested by Algiannis and colleagues [29], MICs up to 0.5 mg/ml represent strong activity; MICs between 0.6 and 1.5 mg/ml moderate activity, and MICs above 1.6 mg/ml weak activity. Based on these criteria it is evident that the combined extract exhibited weak to moderate antibacterial activity. These results support the observation reported earlier that multi-plant extracts are superior over single plant extracts and could be developed into more potent antimicrobials against resistant pathogens [30]. However, the antibacterial activity of the extracts was weak so unless actual testing is done using Helicobacter pylori it will be unrealistic to make any speculations regarding clinical efficacy.

This is not the first study to report the antibacterial activity of Entada abyssinica or Ozoroa insignis. Entada abyssinica has been reported to have antibacterial activity against Klebsiella pneumoniae, Salmonella typhi, Escherichia coli, Pseudomonas aeruginosa and other organisms [21]. Ozoroa insignis has also been reported to have antibacterial activity against Gram positive and Gram negative bacteria which include all the bacteria used in this study [31]. Similarly Maytenus senegalensis is reported to have antibacterial activity and the antibacterial compound, 3-oxo-friedelan-20α-oic acid, has been isolated from the root bark [32]. However, it is the first time the antibacterial activity of Rhynchosia recinosa and Lannea schimperi is being reported. The results (Table 3) show that the combinations C5 and C6 containing R.recinosa and L. schimperi with E.abyssinica and M.senegalensis, respectively were the least active. In fact C6 was less active than C5. It is evident that the reported results are inconclusive, and it is prudent that when Helicobacter pylori become available the effects of the extracts should be re-investigated.

Two preliminary explorative studies were done to investigate acute toxic effects of the plant extracts; acute toxicity in mice and the brine shrimp lethality test. Maytenus senegalensis and Rhynchosia recinosa extracts were well tolerated by mice up to 5000 mg/kg body wt. The ethanol extract of Maytenus obtusifolia which belongs to the same genus was earlier reported to exhibit low toxicity against brine shrimps and mice [25]. Information from the literature indicate that E. abyssinica has weak cytotoxic activity against some human cell lines, including human colon cancer cells [33] and is used traditionally for treatment of cancer else where in Tanzania [33]. However, both the brine shrimp results and acute toxicity in mice indicated a good safety margin of up to 2000 mg/kg body wt. The Lannea schimperi extract was also equally innocuous on acute administration. The Ozoroa insignis extract was the most toxic to both mice and brine shrimps. The literature indicate that Ozoroa insignis stem bark and root methanol extracts were cytotoxic against different human cell lines including liver, bladder and mammary cancer cell lines [34, 35] and has topoisomerase II inhibitory activity [36]. Despite the seemingly toxic profile of, in particular Ozoroa insignis, the polyherbal formulation showed very low acute toxicity in mice and in the brine shrimp lethality test. The combination of extracts of the five plant extracts was well tolerated during the whole 14 day observation period and at doses up to 5000 mg/kg body wt.

The combined extract of the five plants exhibited a dose-dependent cytoprotective activity in the rat ethanol-HCl gastric ulcer model. The extracts also exhibited weak antibacterial activity against four Gram negative bacteria and low acute toxicity in mice and brine shrimps. Although the results seem to support claims by traditional healers who use a decoction of the five plants for treatment of peptic ulcers, more models of gastric ulceration and proper animal toxicity studies are needed to validate possible clinical use of the polyherbal extract.