Antimicrobial and toxicological activities of five medicinal plant species from Cameroon Traditional Medicine

Infectious diseases are considered a major threat to human health, because of the unavailability of vaccines or limited chemotherapy. They account for approximately one half of all deaths in tropical countries [1]. Infectious diseases which ranked 5^th in 1981, became the 3^rd leading cause of death in 1992, with an increase of 58% [2]. Most of the current antibiotics have considerable limitations in terms of antimicrobial spectrum, side effects, and their widespread overuse has led to increasing clinical resistance of previously sensitive microorganisms and to the occurrence of uncommon infections [3]. Historically plants have provided a good source of anti-infective agents and the search for plants with antimicrobial activities has increasingly gained importance during recent years. With the advent of ever-increasing resistant bacteria and fungi strains, there has been a corresponding rise in the universal demand for natural antimicrobial therapeutics. Herbal medicines have widely been used and now form an integral part of the primary health care in many countries. They may constitute a reservoir of new antimicrobial substances to be discovered. Use of herbal medicines in Africa represents a long history of human interactions with the environment. Plants used for traditional medicine contain a wide range of substances that can be used to treat chronic as well as acute infectious diseases as stipulated by Diallo et al.[4]. In Cameroon, many plant species are used as traditional medicine to treat infectious diseases, and several interesting openings have originated for further inquiry following in vitro antimicrobial activity evaluation [5]. A previous epidemiological study on the characterization of urogenital tract microbial agents amongst women in the South West Region of Cameroon, revealed the presence of numerous multiresistant strains to currently used antibiotics in the treatment of urogenital tract infections [6]. Fighting local infections with natural products will be more advantageous and affordable to most patients. Our study was hence based on five selected medicinal plants described by Adjanohoun et al.[7] as traditional medication for various infectious ailments. The aim of this study was to evaluate the in vitro antimicrobial properties of methanolic, ethylacetate (EtOAc) and hexanic extracts of these five medicinal plants against eight Gram positive and Gram negative multiresistant bacteria species and two Candida isolates as well as to evaluate their in vivo acute toxicity on albino rats. The selection of these plants was based on their use in traditional medicine to cure current infectious diseases including urogenital tract infections [6].

Successful prediction of chemical compounds from plant material is largely dependent on the type of solvent used in the extraction procedure. The traditional healers or practitioners make use of water primarily as a solvent. In this study, extraction was done using organic solvent such as hexane, methanol and EtOAc. This is because these solvents are easily evaporated and permit an easier estimation of extract concentration, which is difficult to obtain with water as solvent. Results of the analysis of five medicinal plants extracts using these solvents showed that their chemical content differs depending on the nature of solvent used. Identical classes of chemical compounds are found in the hexanic, methanolic and EtOAc extracts of P. africana; however, flavonoids, polyphenols, cardiac glycosides and reducing sugars were mostly found in the methanolic and EtOAc fractions. Alkaloids were absent in the methanolic fraction and this could be justified by its poor polarity. Both Gram negative and Gram positive were sensitive to ethylacetate and methanolic fractions of the five medicinal plants rich in flavonoids, polyphenols and anthraquinons. This is probably due to their ability to complex with extracellular and soluble proteins and to complex with bacterial cell walls; more lipophilic flavonoids may also disrupt microbial membranes, [23, 24]. The presence of coumarins in the various fractions of P. muellerianus, H. latifolia, C. aralioides and P. africana, may explain their important inhibitory activity against Candida albicans and Candida krusei. This anticandidal activity of coumarins has been demonstrated in vitro and in vivo against vaginal candidiadis by Thornes [25]. Success of extracts from P. africana, C. aralioides and G. gregasius against both Gram positive and Gram negative agents is likely dependent on their content in alkaloids able to intercalate between DNA strands [26]. The presence of sterols and triterpens has been confirmed in another study carried out in Cameroon by Mbouangouere et al.[27] who isolated terpenes such as sitosterol, β-amyrin and eicosane; however they found no alkaloids in this plant. The absence of alkaloids in their study could be due to the fact that, their investigation was done on a defatted fraction of the methanol/CH₂Cl₂ extract. The hexanic fraction induced only mild activity compared to the ethylacetate and methanolic fractions to which all microbial strains tested were sensitive. This could indicate that important bioactive principles of Piptadeniastrum africana are of polar nature. Polar secondary metabolites like flavonoids, coumarins, anthraquinons, phenols and glycosides have been found to possess important antimicrobial properties [5, 28]. In accordance, Mbouangouere et al.[27] obtained inhibitory activity on Gram positive and Gram negative strains (Escherina coli, Bacillus subtilis, Shigella flexenari, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi) with ID varying between 9-16 mm when tested at 3 mg/ml. In the present study, we obtained a similar activity with P. africana against S. aureus, P. aeruginosa, C. albicans and C. krusei with ID varying between 14 and 26 mm. In a previous epidemiological study, these microorganisms were found to be highly resistance to ampicillin and augmentin [6]. With regards to these results we can deduce that this plant' extract, P. africana, is a good source of antimicrobial agents with interesting activity on multi resistant strains.

Looking at it toxicity parameters, no death of rats was neither recorded in the control nor in treatment groups during acute toxicity indicating an LD₅₀ > 5 g/Kg bw. From these results, it can be concluded that P. africana is not toxic with regard to the threshold of toxic substances (5 g/kg) as previously stipulated [15‐17].

Results of sera and liver biochemical parameters indicated that the variation of transaminase (ALT and AST) activities are associated with hepato-cellular damage, although marked increased in these parameters were only observed at a higher dosage (≥8 g/kg bw). The concurrent significant increase of ALT and AST activities in sera and decrease ALT and AST activities in hepatocytes after treatment of the male and female rats above 8 g/kg bw clearly indicate leakages of liver cell contents into the blood stream. The creatinine and urea levels of treated rats increased significantly as compared to the control only at doses above 12 g/kg bw. This result confirms that the rats' kidneys were affected only when administered high quantity of the extract (≥8 g/kg bw). The variation of other parameters such as glutathione and malondialdehyde was also significant only at large doses (≥8 g/kg bw) indicating that lipid peroxidation started earlier and resulted in oxidative stress as reflected by the increased GSH depletion and MDA levels in rats hepatocytes.

The antimicrobial property of Hileria latifolia has not been revealed before; only few studies have indicated the presence of alkaloids and flavonoids in Ivory Coast [29]. The species investigated in this study shows that it is instead rich in triterpens, sterols and coumarins, in addition to saponins, glycosides and reducing sugars found in the methanolic fraction of the plant. The important antimicrobial property of the plant in the ethylacetate fraction may be due to the presence of these metabolites [30‐34]. Previous studies had shown that this plant when administered orally at a low dosage in rats is less toxic [35].

Mild antibacterial and antifungal activities (on Candida albicans and Candida krusei) of Gladiolus gregasius observed in this study are in contrast with previous work done by Assob et al.[36] who found that the bulb extract was fungicidal with no antibacterial activity. The difference may be due to harvesting periods and solvent used in the extraction of the plant [37]. The hydroethanolic extract of the plant was found not to induce toxicity in rats [36].

An important antimicrobial activity was observed with the three fractions of Phyllantus muellerianus against Gram positive and Gram negative bacteria species and as well as against Candida species. These results are in concordance with the work carried out by Doughari and Sunday [38] who obtained similar activity against some pathogenic bacteria. However, the species tested in this study seem to be more active considering its inhibitory properties (Highest ID = 30 mm, MIC = 0.15, vs. ID = 18 mm, MIC = 625 μg/ml in their study) and to possess a larger spectrum of activity against tested microorganisms. The phytochemical screening showed almost the same chemical composition (tannins, flavonoids, polyphenols, saponins, alkaloids, and anthraquinones), except that alkaloids were absent in our study. Another study revealed the antitimicrobial properties of the methanolic extract of the stem bark of Phyllanthus muellerianus against Clostridium sporogenes, Streptococcus mutans and Streptococcus pyogenes[39]. P. muellerianus presented no adverse effect on the liver and kidney functions when administered at a low dosage (4 g/kg bw) to male and female rats in this study. Based on this, it can be concluded that P. muellerianus is not toxic, taking into consideration the threshold of toxic substances (5 g/kg) as earlier stipulated [16‐18]. No previous toxicological evaluation has been carried out on this plant. But, works done on Phyllantus sp. have revealed their hepatoprotective, antiulcerative, gastroprotective and antiviral properties which may probably justify the innocuity of the plant [40‐43]. The occurrence of decreased levels of GSH and increased MDA respectively when administered at elevated dosage (8 and 12 g/kg bw respectively) shows that this extract contains bioactive hepatoprotective agents whose efficacy is only exceeded when high concentration of the extract is absorbed. The liver injuries due to this extract is minimized and may be due to the enhancement of hepatic glutathione regeneration capacity and the decreased level of lipid peroxidation, particularly when moderate doses of the extract are absorbed (4 g/kg bw). This protective effect is also associated with the presence of flavonoids and triterpens in the stem bark of the plant conferring it, an important antioxidant property [44]. Experiments conducted at the Niwa Institute of Immunology in Japan have shown flavonoids in E. officinalis to be potent scavenger of free radicals [45] and triterpenoids to play an essential role in the protection of the liver against free radicals. Triterpenoids were shown to be able to protect mice against carbon tetrachloride (CCl4)-induced hepatotoxicity, and inhibit lipid peroxidation in rat liver microsomes [46‐48].

In general, the highest antimicrobial activities were obtained with Phyllanthus muellerianus and Pitadeniastum africana extracts. However, all the five medicinal plants, Pitadeniastum africana, Cissus aralioides, Hileria latifolia, Phyllantus muellerianus and Gladiolus gregasius tested are antibacterial and anticandidal agents and could be used in the treatment of various urogenital and gastrointestinal ailments caused by multiresistant microbial agents. It is also clear from this study that, the antimicrobial activity of these five medicinal was mainly found in the methanolic and ethylacetate fractions. The antimicrobial activities of Cissus aralioides and Hilaria latifolia have not been studied before. This study has also revealed that Piptadeniastrum africana and Phyllantus muellierianus are not toxic at therapeutic doses with good antimicrobial properties. This study is an important step towards clinical evaluation in order to produce improved phytomedicine in the treatment of multiresitant microbial infections. Alongside further chemical analysis involving CG-MS, HPLC-MS or ESI-MS analysis will enhance our comprehension on the nature of the chemical compounds contain in the extracts.