Full Length Research Paper
ABSTRACT
The aim of this study was to determine in vitro anti-Salmonella activity of extracts of five selected Kenyan medicinal plants against Salmonella ser. Typhi and Salmonella ser. Typhimurium. The extracts from Tithonia diversifolia, Warburgia ugandesis, Croton megalocarpus, Carissa edulis and Launae cornuta plants traditionally used in treatment of typhoid fever were screened for anti-Salmonella activity using disc diffusion and microdilution techniques. The results from the present study have shown that out of thirty six extracts investigated, only nine extracts from T. diversifolia and W. ugendensis showed activity against Salmonella ser. Typhi and Salmonella ser. Typhimurium at 1000 mg/ml. The inhibition zone of ethyl acetate, hexane and methanol extracts of T. diversifolia leaves, ethyl acetate and hexane extracts of T. diversifolia flowers, ethyl acetate and hexane extracts of W. ugandensis stem barks, ethyl acetate and hexane extract of W. ugandensis roots ranged from 8 to 18.5 ± 0 mm. These results were comparable with those of ciprofloxacin (19.67 to 26 mm) and chloramphenicol (6.67 to 24.33 mm). The minimum inhibitory concentration (MIC) of the active extracts were in the range of 0.031 to 15.63 mg/ml which compared very well with ciprofloxacin (0.015 to 0.02) and chloramphenicol (0.022 to 0.03 mg/ml). Extracts with anti-Salmonella activity can be used to source antibiotic substances useful in the treatment of typhoid fever. The study provides the scientific basis for the traditional application against typhoid fever.
Key words: Anti-Salmonella activity, medicinal plant extracts, minimum inhibitory concentration, disc diffusion technique, microdilution technique, Salmonella strains, typhoid.
INTRODUCTION
Salmonella serotype Typhimurium (S. ser. Typhimurium), is a Gram-negative bacterial pathogen that infects humans and animals, causing significant morbidity and mortality worldwide (Fink and Cookson, 2007). It is an obligate intracellular bacterial pathogen that causes gastroenteritis in millions of people worldwide each year (Grassl et al., 2008). For instance, the Centre for Disease Control (CDC) estimates that there are nearly 1.4 million food-borne Salmonella infections annually in the USA (Mead et al., 1999). Various strategies have been employed in the treatment and management of Salmonella infection. Fluoroquinolones and tetracyclines are most commonly used to treat Salmonella infections. However, Salmonella strains resistant to these antibiotics have been reported in Korea and other countries (Choi et al., 2005; Stevenson et al., 2007). One major concern to public health has been the global dissemination of S. typhimurium Definitive Type 104, which is resistant to cotrimoxazole, nalidixic acid and ampicilin (Perron et al., 2008; Kariuki et al., 2010). The rise in antibiotic-resistant strains has led to increased interest in use of plant materials to develop new effective drugs. According to World Health Organization (WHO) more than 80% of world’s population relies on traditional medicine for their primary healthcare, majority of who use plant active principles (Gupta et al., 2005). A wide variety of plants are used in Africa for treatment of fever, dysentry, cholera, diarrhoea and other infections typical of the tropical countries (Ayogu and Amadi, 2009; Ajayi and Akintola, 2010). For instance, traditional practitioners in Nigeria use herbal preparations to treat microbial infections such as typhoid and paratyphoid infections (Iroha et al., 2010).
Plants used in this study have traditionally been associated with disease curative and preventive practices in many countries for a long time. Garcia and Delgado (2006) have reported that Tithornia diversifolia has promising medicinal value. Skin products formulated from T. diversifolia extracts have been shown to have antimicrobial properties (Kareru et al., 2010).
In Ethiopia Warburgia ugandensis extracts are used to treat malaria, tuberculosis, bronchitis, pneumonia, hepatitis, tapeworm, gonorrhea and asthma (Wube et al., 2010; Were et al., 2010; Opiyo et al., 2011). The decoction from Croton megalocarpus bark is used as a remedy for worms and whooping cough. Grounded roots are used for syphilis, anthrax, and snakebites treatment (Kabir et al., 2005). Different communities in Africa use parts of Carissa edulis to alleviate pain, treat venereal diseases, glandular inflammation, induce abortion and restore virility (Githiori et al., 2004). In Kenya and Tanzania decoction from Launae cornuta roots is used as a remedy for cough, typhus fever and measles (Schippers, 2004).
This study investigated the anti-Salmonella activities of T. diversifolia, W. ugandensis, C. megalocarpus, C. edulis and Launae cornuta. Clinical isolates of S.ser.Typhi (ATCC 13347), S.ser.Typhi (ATCC 43579), S.enterica (ATCC 2162) and S. ser. Typhimurium (ATCC 1408) were used in the study.
MATERIALS AND METHODS
Salmonella strains
Clinical samples of S.ser.Typhi (ATCC 13347), S.ser.Typhi (ATCC 43579), S.enterica (ATCC 2162) and S. ser. Typhimurium (ATCC 1408) were provided by the Centre of Microbiology Research, Kenya Medical Research Institute (CMR-KEMRI) for this study.
Plant
The five plants selected for this study were collected in Nyamira County as indicated in Table 1. The plants were authenticated at Jomo Kenyatta University of Agriculture and Technology, Botany Department.
Experimental design
The nine plant parts obtained from 5 selected medicinal plants indicated in Table 1 were extracted using four solvent systems namely; hexane, ethyl acetate, methanol and water. The extracts obtained were subjected to standard phytochemical analyses as described by Jigna et al. (2006). In addition, the extracts were screened for anti-Salmonella activity against four clinical isolates. Samples in triplicate were subjected to disc diffusion and microdilition in duplicates to confirm anti-Salmonella activity.
Preparation of plant materials for extraction
The plant materials were washed under running tap water and left to drain off. The plant parts were chopped into small pieces. The dried pieces were ground into powder and their weights recorded.
Extraction techniques
The nine plant materials were extracted using selected solvents. Each plant material was extracted sequentially using hexane, ethyl acetate and methanol in the order of increasing polarity. Single extraction was carried out using water for each of the nine plant materials.
Preparation of hexane extract
Approximately 500 g of each plant powder was soaked separately in 1500 ml of hexane. The contents were kept for 3 days away from direct sunlight, undisturbed, then filtered through sterile filter paper. The filtrate was concentrated at 38.5 to 42°C.
Preparation of ethyl acetate extract
The hexane residues were re-soaked in 1500 ml of ethyl acetate. The contents were kept for 5 days away from direct sunlight, undisturbed and afterward filtered. The filtrate was concentrated at 38.5 to 42°C.
Preparation of methanol extract
The ethyl acetate residues were re-soaked in 1500 ml of methanol and kept for 36 h away from direct sunlight undisturbed. After filtration, the filtrate was concentrated at 65°C. The extracts were stored at 4°C until used.
Preparation of aqueous extract
Five hundred grams (500 g) of each of the powdered plant materials was weighed and soaked separately in 1500 ml of distilled water. The contents were warmed in a water bath for 2 h at 60°C, then left to stand at room temperature for 10 h, undisturbed. They were subsequently sterile filtered and filtrate freeze dried to powder. The powder were weighed and stored until used.
Determination of phytochemical constituents
The freshly prepared extracts were subjected to standard phytochemical analyses for tannins, alkaloids, terpenoids, flavanoids, glycosides, steroids and saponin as described by Jigna et al. (2006).
Controls
Water and dimethyl sulfoxide (DMSO) were used as negative controls. Ciprofloxacin and chloramphenicol (Transchem pharmaceutical Ltd, Kenya) were used as positive controls.
Disc diffusion assay
Circular paper discs (6mm diameter) were placed on Muller Hinton media inoculated with Salmonella strains. Sterile paper discs were dampened with 10 µl of plant extracts at 1000 mg/ml. The loaded disc was placed on the surface of the medium, the compound was allowed to diffuse for 5 min and plates were incubated for 24 h at 37°C. Discs containing ciprofloxacin and chloramphenicol were used as positive controls. Discs loaded with DMSO and water served as negative controls. The assays were performed in triplicate. Anti-Salmonella activity was evaluated by measuring diameter of the inhibition zone.
Determination of minimum inhibitory concentration (MIC) values
The MIC values were determined using microdilution assay as described by Eloff (1998). Ciprofloxacin and chloramphenicol were used as positive controls and DMSO was used as negative control. Plant extracts were tested against Salmonella strains with varying concentration ranging from 62.5 to 0.0305 mg/ml. Briefly, 100 μl of sterile distilled water was added to each well of 96-well microtitre plates (SIGMA Aldrich, German) followed by the addition of 100 μl of 62.5 mg/ml and thereafter serially diluted plant extracts. Then 100 μl of Salmonella strains were added to each micro well to give a final volume of 200. The prepared plates were sealed to avoid drying and incubated overnight at 37°C. After overnight incubation, 50 μl of 5 mg/ml 2, 3, 5 triphenyltetrazolium chloride (SIGMA Aldrich, German) was added to the wells and incubated overnight. The pink colour was indicative of bacterial growth while lack of color was linked to growth inhibition. The MIC was defined as the lowest concentration of plant extract that completely suppress the growth of Salmonella strains.
Statistical analysis
Anti-Salmonella activity was determined from means of triplicates in zones of inhibition and duplicates in MICs. Collected data was analysed statistically using one way ANOVA (SAS, Version 9.0). Difference in values at P < 0.0001 were considered statistically significant.
RESULTS
Out of 36 plant extracts screened using disc diffusion assay, only nine extracts inhibited the growth of clinical Salmonella organisms at 1000 mg/ml. Extracts of hexane (flowers), ethyl acetate (leaves) and methanol (leaves) extracts from T. diversifolia were active against S.ser.Typhi ATCC 13347, S.ser.Typhi ATCC 43579, S.enterica ATCC 2162 and S.ser.Typhimurium ATCC 1408. Extracts of hexane (leaves) and ethyl acetate (flowers) from T. diversifolia inhibited growth of S.ser.Typhi ATCC13347. As was observed with ciprofloxacin and chloramphenicol controls, extracts of hexane and ethyl acetate (roots and stem bark) from W. ugandensis inhibited growth of all the tested Salmonella organisms. Extracts of methanol (leaves) from T. diversifolia were also observed to inhibit all the clinical isolates at 8 to 12 mm. The zones of inhibition for the active extracts are shown in Table 2.
The MIC values of the nine plant extracts was evaluated and shown to range from 0.031 to 15.63 mg/ml. The MICs of hexane extracts from T. diversifolia leaves and flowers ranged from 0.24 to 1.95 mg/ml and 0.12 to 3.91 mg/ml, respectively. The MICs of hexane extracts from W. ugandensis roots and stem bark ranged from 0.031 to 3.91 mg/ml and 0.031 to 0.488 mg/ml, respectively. Table 3 shows MICs of the nine plant extracts and controls. It is evident from these results that W. ugandensis extracts were the most active against all the Salmonella strains tested.
Table 4 illustrates anti-Salmonella activity values obtained from disc diffusion and microdilution methods. The nine plant extracts showed different value of anti-Salmonella activity against test strains. Ethyl acetate extract of W. ugandensis stem bark gave the lowest MIC value of 0.031 mg/ml against S.ser.Typhi ATCC 13347, S.ser.Typhi ATCC 43579, and S.ser.Typhimurium ATCC 1408 with zones of inhibition of 6, 7 and 7.33 mm, respectively (Table 4). The extract showed MIC of 0.061 mg/ml against S.enterica ATCC 2162 with inhibition zone of 6 mm. The T. diversifolia extracts had anti-Salmonella activity against all the tested clinical isolates. Methanol extract of the leaves showed activity with MIC values of 0.031, 0.24, 0.448 and 0.98 mg/ml against S.ser.Typhi ATCC 43579, S. ser. Typhi ATCC 13347, S. ser. Typhimurium ATCC 1408 and S. enterica ATCC 2162 with inhibition zones of 11.5, 11, 11.67 and 7.33, respectively.
The nine active plant extracts were screened further to study the presence of medicinally active phytochemicals in leaves, stem barks, roots and flowers. Phytochemical analysis revealed presence of alkaloids, saponin, tannins, flavanoids, steroids, terpenoids and glycosides (Table 5). Steroids were detected in all the extracts. Flavonoids and tannins were absent in hexane extracts of T. diversifolia flower and W. ugandensis stem bark. Terpenoids were found in extracts of hexane and ethyl acetate from T. diversifolia flower, W. ugandensis root and W. ugandensis stem bark. Extracts of hexane (T. diversifolia flower) and (W. ugandensis stem bark) lacked alkaloids. Glycosides were detected in extracts of hexane and ethyl acetate (T. diversifolia leaf) and ethyl acetate (W. ugandensis stem bark). Saponins were detected only in extracts of methanol from T. diversifolia leaf (Table 5).
DISCUSSION
In the present study, the extracts from 5 medicinal plants were screened for activity against clinical Salmonella strains. Of the extracts tested, both ethyl acetate and hexane extracts of T. diversifolia and W. ugandensis exhibited activity against all four Salmonella strains tested in this study. Methanol extracts of T. diversifolia leaf also inhibited all the clinical isolates tested.
T. diversifolia plant extracts exhibited different zones of inhibition against the isolates. The ethyl acetate flower and hexane leaf extracts of T. diversifolia gave zones of inhibition at 18.5 ± 5 mm and 17.67 ± 2 mm, respectively (Table 2). This compared well with ciprofloxacin which gave zone of inhibition of 19.67 mm and therefore no significant difference in activity (p < 0.0001). Methanol extract of T. diversifolia leaf also showed anti-Salmonella activity against test isolates. The observed anti salmonella activity of T. diversifolia extracts agrees with the finding of Ogunfolakan et al. (2010), on broad spectrum antimicrobial activity. Kareru et al. (2010) has reported that soap made from leaf extract of T. diversifolia was effective against E. coli.
The phytochemicals and secondary metabolites from plants possess antimicrobial activity (Srikumar et al., 2007). Phytochemical analysis demonstrated the presence of alkaloids, tannin, flavonoids, terpenoids steroids and glycosides in the active extracts. Ethyl acetate flower extracts exhibited the highest anti-Salmonella activity. This is due to the difference in the type and concentrations of secondary metabolites in different plant parts (Srikumar et al., 2007) and may be contributing to the observed differences in anti-Salmonela activity. The results of this study show that extract with high content of steroids contribute to significant inhibition of Salmonella growth. This finding agrees with Ashok and Vijayalakshmi (2013) who have demonstrated that sterols from Vitis vinifera seed exhibited antibacterial activity. The sterols could be interacting with the bacterial cell wall and membrane ultimately leading to pore formation and disrupting bacterial membrane integrity (Devjani and Barkha, 2011). Odeyemi et al. (2014) has also reported that T. diversifolia leaf, flower and roots extracts have antibacterial activity due to the presence of metabolic toxins such as flavonoids, steroids and alkaloids.
Hexane extracts of W. ugandensis roots and stem barks showed inhibition zones of 14 and 11 mm, respectively against S.ser.Typhi ATCC 13347. Hexane extract of W. ugandensis roots and chloramphenicol showed inhibition zones of 10.67 and 8.67 mm against S. ser. Typhimurium ATCC 1408, respectively (Table 2). This was significantly lower than that of ciprofloxacin (19.67 mm). The observed anti-Salmonella activity of W. ugandensis is however supported by Yibeltal et al. (2013) who demonstrated activity of crude and semi-purified fractions of W. ugandensis against Shigella boydii and Staphylococcous aureus. Studies carried out by Olila et al. (2001) on aqueous extracts of W. ugandensis stem bark showed activity against both Escherischia coli and S. aureus in agar well assays but not in disc diffusion assay. The anti-Salmonella activity of W. ugandensis observed in our present study could be attributed to several secondary metabolites, among them steroids.
The extracts of three plants namely Croton megalocarpus, Croton edulis and Lactoria cornuta had no anti-Salmonella activity for the extracts (Table 2). Their anti-Salmonella activity values were not significantly different with those of negative controls (p < 0.0001). The lack of anti-Salmonella activity in these plants may not necessarily imply the same in vivo since compounds may either act as pro-drug which must undergo metabolic changes to achieve the required activity. Besides, the presence of bioactive compounds depends on many factors such as the season, age, intra-species variation, part of the plant collected, soil and climate (Gessier et al., 1995).
The MIC values of the nine active plant extracts was determined. These extracts were selected because of their appreciable anti-Salmonella performance determined by disc diffusion. The active extracts against Salmonella strains were W. ugandensis bark, W. ugandensis root, T. diversifolia leaf and T. diversifolia flower. The ethyl acetate extract of W. ugandensis stem bark showed anti-Salmonella activity among the extracts tested. The extract had MIC value of 0.031 mgl/ml against S.ser.Typhi ATCC 13347, S.ser.Typhi ATCC 43579, and S.ser.Typhimurium ATCC 1408. It showed MIC value of 0.061 mg/ml against S. enterica ATCC 2162. Hexane extract of W. ugandensis root showed MIC value of 0.031 mg/ml against Salmonella strains tested except S. enterica ATCC 2162 which was inhibited at 3.91 mg/ml. The extracts of hexane (stem bark) and ethyl acetate (root) from W. ugandensis showed anti-Salmonella activity against all strains tested (Table 3). Anti-Salmonella activity of W. ugandensis extracts compared well with ciprofloxacin and chloramphenicol. In a study carried out by Yibeltal et al. (2013), on antimicrobial activity of crude extracts of W. ugandensis against E. coli and P. aeruginosa demonstrated MIC values of 1.75 mg/ml. These results compared well with those of our study, which were in the range of 0.031 to 3.91 mg/ml (Table 3).
The methanol extract of T. diversifolia leaf had MIC values ranging from 0.031 to 0.98 mg/ml. The extract gave MIC values of 0.031, 0.24, 0.98 and 0.488 against S.ser.Typhi ATCC 43579, S.ser.Typhi ATCC 13347, S.ser.Typhimurium ATCC 1408 and S. enterica ATCC 2162, respectively (Table 3). Methanol and ethyl acetate extracts of T. diversifolia exhibited MIC values of 0.031 mg/ml each against S.ser.Typhi ATCC 43579 and S.enterica ATCC 2162, respectively. These values were not significantly different from ethyl acetate extracts of W.ugandensis (p < 0.0001). It was noted in our study that clinical Salmonella strains were sensitive to all T. diversifolia extracts at different MIC values (Table 3). Our present study has demonstrated lower MIC values for T. diversifolia extracts against Salmonella strains than what Ogundare (2007) reported. According to their report, MIC values of chloroform and methanol extracts of T. diversifolia were 6.25 and 3.125 mg/ml, respectively against S. typhi. The two extracts however gave MIC values of 6.25 mg/ml each against P. aeruginosa.
It was noted from this study that plant extracts tested by microdilution technique showed higher anti-Salmonella activity compared to values obtained from disc diffusion technique. W. ugandensis extracts showed lower MIC values when determined by microdilution method than by disc diffusion method. Olila et al. (2001) has reported that the paper disc retains the active component and does not allow it to diffuse into Muller Hinton agar. The paper disc is composed of cellulose [b-(1-4) linked glucose monomers]. The many free hydroxyls groups present on each glucose residues renders the surface of hydrophilic (Burgess et al., 1999). Thus, if natural products were cationic, they would be expected to adsorb to the surface of the disc and not diffuse into the medium. Consequently, a cationic polar compound displays a good antibacterial activity, but which is therefore not noticeably antibacterial by paper disc diffusion (Cleidson et al., 2007).
Most of the antibiotics used nowadays have lost their effectiveness due to development of resistant genes in microbes (Davis, 1994; Service, 1995). The antibiotics are sometimes associated with side effects such as hypersensitivity, immune suppression and allergic reaction (Ahmad et al., 1998). More interest is being shown in developing alternative antimicrobial drugs for the treatment of infectious diseases without side effects (Berahou et al., 2007; Salomao et al., 2008). The results of our present study demonstrates anti-Salmonella activity of W. ugandensis and T diversifolia that compared well with ciprofloxacin and chloramphenicol. The results obtained from the nine active plant extracts tested are encouraging. Further work is in progress to isolate and identify the bioactive compound(s) that could be used in the development of safer and cost effective alternative drugs for typhoid fever.
CONCLUSION
The results of our study showed anti-Salmonella activity in extracts from W. ugandensis and T. diversifolia plants. This activity compared well with that of ciprofloxacin and chloramphenicol. The study provides the basis for use of these plants in the development of drugs for management of typhoid fever.
ACKNOWLEDGEMENTS
The authors would like to thank the Deutscher Akademischer Austauschdienst (DAAD) for their financial support, Centre of Microbiology Research-Kenya Medical Research Institute (CMR-KEMRI) for providing Salmonella isolates and microbiology laboratory, and Jomo Kenyatta University of Agriculture and Technology (JKUAT) for providing Laboratory facilities.
CONFLICT OF INTEREST
The author(s) have not declared any conflict of interests.
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