Phytochemical screening and antibacterial activity of medicinal plants used to treat typhoid fever in Bamboutos division , West Cameroon

Tsobou Roger, Mapongmetsem Pierre-Marie, Voukeng Kenfack Igor, Van Damme Patrick Department of Plant Biology, University of Dschang, P.O.Box 67 Dschang, Cameroon; Department of Biological Sciences, University of Ngaoundéré, P.O.Box 454 Ngaoundéré, Cameroon. Department of Plant Production, Laboratory of Tropical and Subtropical Agriculture and Ethnobotany, Department of Biological Sciences, University of Ngaoundéré, P.O.Box 454 Ngaoundéré, Cameroon. Department of Biochemistry, Faculty of Science, University of Dschang; Dschang, Cameroon.


INTRODUCTION
Knowledge on plant uses is the result of many years of man's interaction and selection on the most desirable, the most vigorous and the most successful plant present in the immediate environment at a given time (Rindos, 1984).According to the World Health Organization, 80% of people in developing countries still depend on local medicinal plants to fulfill their primary health needs (WHO, 2002).Besides that, there is a global consensus on the benefits of phytopharmacy and at present medicinal plants occupy a key position in plant research and medicine.In many African countries, such as Ghana, Mali, Nigeria and Zambia, the first line of treatment for 60% of the .The problem has become endemic in many developing countries, causing enormous childhood morbidity and high cost of treatment (Leume, 1999).Multidrug resistant Salmonella species are being increasingly reported from the developed world.There is therefore, the need for efficient and safe vaccine which can be used as a preventive public health tool (Leume, 1999).
Thus, the resistances of Salmonella to these antibiotics couple with the high cost of treatment have prompted the present study on plant used for the treatment of typhoid fever.Furthermore, typhoid fever is a systemic infection caused by the bacterium Salmonella enterica subspecies enterica serotype typhi, which is acquired by ingestion of contaminated food and water (Iroha et al., 2010).Much more, in 2009, Zofou et al., report that this disease remains the second cause of mortality in the Bamboutos Division after malaria.In the same way, the report provided by the NGO "Action for the formation and the natural stock management", indicated that, 2715 cases of typhoid fever and 1998 cases of diarrheas and gastro-enteritis were recorded in 2010 in this Division.In the same line, Nanfack et al., in 2014 have found that typhoid fever was the hydrous widespread disease in this locality, follow by dysentery and gastro-enteritis.Besides, each year the disease affects at least 12.6 million people world-wide, most of them resides in the developing countries of Southeast Asia and Africa (WHO, 2003).
Typhoid fever is uncommon in industrialized regions such as the USA, Canada, Europe, Australia and Japan and new cases of the disease in these countries are related to a journey in the developing countries (Papadimitropoulos et al., 2004).Mortality rates associated with this disease vary form one region to other, with highest reported from Indonesia, Nigeria and India (Miller et al., 1994).
The prevalence of this disease and the increasing prices of medicine have resulted in the demand for discovery of less expensive but more potent sources of drugs.Plants are one of the best sources of potent drugs.In fact, medicinal plants represent a rich source form which antimicrobial agents may be obtained (Kubmarawa et al., 2007).
The traditional knowledge on medicinal plants that is inherent within local communities is a very important source of information that continually provides the present-day herbal remedies (Shahid-ud-Daula & Basher, 2009).In recent years, researches in various countries have been directed towards discovering the medicinal uses of plants through phytochemical and antimicrobial screening (Hashim et al., 2010).
Nevertheless, the present investigation represents a preliminary screening of some medicinal plants used against typhoid fever in Bamboutos division.
These medicinal plants have been claimed by traditional medical practitioners in this locality to be effective when used for the treatment of typhoid fever.However, no detailed reports on anti-typhoid activity of these plants exist in literature in Cameroon; therefore, the present work investigates phytochemical compounds and antibacterial activity against Salmonella species.

Study area
The Bamboutos Division is located in the western highlands and extends between 5-6° N and between 9-11°E (Figure 1).It's one of the eight divisions which forms the West Region of Cameroon and is bordered to the north by Mezam Division, to the south by Mifi and Menoua divisions, and to the west by Noun Division.The Bamboutos division covers an area of 1155 km² which represents 8.31% of the total area of the West Region of Cameroon.Located on the eastern slope of the Bambouto Mountain from which it is named, Bamboutos Division is characterized by the great diversity of its relief, climate, vegetation, and soils.Mount Bambouto is the third highest mountain in Cameroon (2740 m) after Mount Cameroon (Fako) (4100 m) and Mount Oku (3008 m).The main occupation in the community is farming.There are two seasons: the dry season from November to March and the rainy season from March to October.The climate is subtropical with an annual rainfall estimated at 1621.5 mm and a mean annual temperature of 24-29°5C (MINEF, 1999).They have four main languages used in the community (Ngomba'a, Ngiembon, Ngombalé and Megaka).

Collection of ethnomedical information
An ethnobotanical survey was carried out in the Bamboutos Division January-November 2009.Traditional healers and elderly persons were the target key informants in the study, and the selection process was based on the knowledge base of informants, experience, and current practices in ethnobotany medicine of the target individual.The traditional healers were interviewed using semi-structured questionnaires as described by Martin (1995) and open-ended conversations.Trips were made to the sites where traditional healers usually go to harvest plants.The interviews and discussions were carried out in the local language for each of the villages visited.Since the author is a native of the division, data on the local names of the plants, the plant parts used, mode of usage and administration, and mode of preparation were easily obtained.Selected respondents were well-known in the community due to their long practice in providing services related to traditional health care.The age of the respondents ranged between 40 and 95 years (29 were between 40-50, whereas 18 were between 51-60 and 23 were above 60 years old).Forty of them were traditional healers, whereas the rest were elders who had gained knowledge on medicinal uses of plants from their parents and relatives who were historically using the plants.

Collection of plant samples
Fresh plant (s) parts that were found to be used by traditional healers and elderly people for treatment of typhoid fever after carrying out the survey by use of questionnaires were collected from Bamboutos division as shown in Figure 1.The taxonomic identities of these plants were confirmed by the senior taxonomist at the Yaoundé National Herbarium and the voucher specimens were kept in the Department of Plant Biology, University of Dschang.

Microbial strains
Fresh clinical strains of Salmonella typhi ATCC 6539, S. paratyphi A and S. paratyphi B. were obtained from the Microbiology Laboratory, Department of Biochemistry, University of Dschang.All the strains were stored at 4°C temperature until use.

Preparation of plant extracts
Fresh plant material were washed with tap water, air dried at room temperature for 15-30 days, and then homogenized to fine powder.A sample (200 g) of each powdered plant material was soaked in ethanol (500 ml) for 48 h with constant stirring.The suspension was filtered through Whatman filter paper N° 1.The filtrate was concentrated under vacuum using a rota-vapor to obtain the dry ethanol extract and stored at 4°C until further use.These extracts were used both for the phytochemical screening of secondary metabolites and antibacterial activity.

Antibacterial assay Determination of diameter inhibition
The media were prepared according to the manufacturers' standard, 38 g/1000ml of distilled water.The ethanolic extracts were dissolved in 10% dimethylsulpoxide (DMSO) and further diluted to obtain different concentrations (160 mg/ml, 80 mg/ml and 40 mg/ml).Negative control used was DMSO.Ciprofloxacin was used as positive reference standard having a concentration of 10 µl/ml for all bacterial strains.
The organisms were maintained on nutrient agar plates and were revived for bioassay by subculturing in fresh nutrient agar for 24 h before being used.The agar wells diffusion method described by Kuete, (2010), was adopted.Briefly, nutrient agar was prepared by autoclaving and allowed to cool to 40-50°C before seeded with the test organism (in sterile petri-dishes of 90 mm diameter).The seeded plates were allowed to set and cylindrical plugs were removed from the agar plates by means of a cork borer to produce wells of approximately 6 mm diameter.The wells were equidistant from each other and the edge of the plate (Washington, 1995).The wells were then filled with 50 µl of each ethanolic extract at a concentration of 160 mg/ml, 80 mg/ml and 40 mg/ml.Also, concentration of 10 µg/ml of ciprofloxacin and 200 µg/ml DMSO were introduced into other wells as positive and negative controls, respectively with the aid of micropipette.The plates were incubated at 37°C for 24 h.antibacterial activity was determined by measuring the zone of inhibition surrounding the well.The zones of inhibition were then measured, recorded and compared with standard control, Ciprofloxacin (10 µg/ml).The assays were carried out under aseptic conditions.All tests were performed in triplicate.

Determination of MIC and MBC Preparation of the solutions of extracts
The stock ethanolic extracts solutions were prepared by dissolving 59 mg of each extracts in 200 µl of 10% dimethylsulphoxide (DMSO) and mixed with 1600 µl of Mueller Hinton broth, to give the final concentration of 8192 µg/ml and serially diluted two fold to obtain the concentration ranges from 128-8192 µg/ml for the extract and 2-256 µg/ml for ciprofloxacin.100 µl of each concentration was added in 96 wells plates containing 100 µl of MHB and inoculums standardized at 1,5x 10 8 CFU/ml.Negative growth controls were included in every test.The plates were covered with a sterile plate sealer, then agitated to mix the contents of the wells using a plate shaker and incubated at 37°C for 24 h.The MIC of each sample was detected following addition (40 µl) of 2% p-iodonitrotetrazolium chloride and incubation at 37°C for 30 minutes (Pettit et al., 2005).Viable microorganisms reduced the yellow dye to a pink colour.MIC was defined as the lowest sample concentration that prevented this change and exhibited complete inhibition of bacterial growth.For the determination of MBC, a portion of liquid (50 µl) from each well that showed no change in color was placed on MHB (150 µl each new 96 wells plates) and incubated at 37°C for 24 h.The lowest concentration that yielded no growth after this subculturing was taken as the MBC.The MBC/MIC was calculated to determine if the extract is bactericidal or bacteriostatic, according to Noumedem et al., (2013).

STATISTICAL ANALYSIS
Data were expressed as mean ±standard deviation.The data obtained were subjected to ANOVA test to determine whether there was significant difference in zone of inhibition between extract concentrations and also antibiotic used.

RESULTS
Seventy informants (traditional healers and elderly persons) were interviewed on how they used plants to treat typhoid fever.Fifty-nine species, distributed among 56 genera and 33 families were recorded (Tsobou et al., 2013).Thirty-eight of the 59 species recorded were selected for this study (Table 1).Various parts were harvested depending on the parts the informants used in the treatment of typhoid fever.The leaves, barks, whole plant, roots, fruits and stem were the ones that were harvested, but the part that is used most was found to be the leaves, followed by stem bark.The most common mode of preparation was decoction.The species name, family, vernacular name, ethnomedical uses elsewhere, plant part used, biological activity and chemical studies of the 38 selected species are presented in Table 1.However, Bidens pilosa was the most cited plant in our study.The only mode of administration was oral.

Phytochemical screening
Phytochemical screening of collected plant species has been carried out following the methods reported in literature and the results have been reported in table 2. The result of the phytochemical analysis showed that, these plants extracts are rich in at least one of the following class of compounds: triterpenes, flavonoids, alkaloids, polyphenols, sterols and saponins.All these group of compounds were found in Annona muricata, Picralima nitida, Laggera alata, Spathodea campanulata, Cordia platythyrsa, Carica papaya, Terminalia glaucescens, Pseudarthria confertiflora, Senna alata and Musa paradisiaca.Triterpenes were found in all plant extracts except Gossypium barbadense and Dioscorea dumetorium.While alkaloids have been found to be absent in 18 species (i.e.Pseudospondias microcarpa, Stereospermum acuminatissimum, Cupressus lusitanica, Entada abyssinica, Theobroma cacao and Mitracarpus villosus).Polyphenols have been found to be present in thirty seven species.Flavonoids are generally present in most of the 38 plants except in Dracaena deisteliana, Pteridium aquilinum, Dioscorea dumetorum, Ocimum gratissimum and Paullinia pinnata.Sterols and saponins are present in 36 and 23 plants species respectively.

Antibacterial activity
The results of the antibacterial screening of the ethanolic extract on the test strains are shown in table 2. All the thirty eight (38) plants tested showed activity against S. typhi.While 23 and 24 plant species did not showed any activity against S. paratyphi A and S. paratyphi B respectively.The zone of inhibition increased with increasing concentration of extract in wells.This showed that the concentration influence the activity.The highest zone of inhibition were obtained from Senna alata against Salmonella paratyphi A, S. paratyphi B and S. typhi with diameter of 24, 22.5 and 20.5 mm respectively at 160 mg/ml.
Among 38 plants tested Pseudarthria confertiflora, Terminalia glaucescens, Senna alata, Dacryodes edulis and Stereospermum acuminatissimum were found to be the most effective against S. typhi.Senna alata and Rauvolfia vomitoria were found to be the most sensitive against Salmonella paratyphi A. Similarly, Senna alata, Pseudarthria confertiflora and Rauvolfia vomitoria were found to be the most active against S. paratyphi B. The inhibition zone (7.5 -24 mm) of each extract on all the bacterial used was less than the zone of inhibition caused by ciprofloxacin (Table 2).1997).Diabetes, liver diseases, malaria (Brandao et al., 1997).

L (D)
Hepatoprotective (Pieme et al., 2006).Antimicrobial (Awal et al., 2004) When analyzing carefully the MIC and MBC results for each plant samples, it can be noted that MBC/MIC ratios lower than 4 were obtained for Carica papaya, Pseudarthria confertiflora, Moringa oleifera and Harungana madagascariensis, thus considered to have bactericidal activities (Noumedem et al., 2013).To the best of our knowledge, the activity of Pseudarthria confertiflora on the microorganisms studied in the present work is being reported for the first time.et al., 2005).Dysentery, diarrhoea (Dalziel, 1937).

DISCUSSION
The high diversity of species used for treatment of typhoid fever in the study area, is an indication of the importance of medicinal plants.The reliance on traditional medicine may be due to the perceived potency of the plants.High consensus among users in different countries reflects the significance of medicinal plants to the people (Cotton, 1996).It was clear that the informants harvest the bark, roots, stem, fruits and leaves; but the part that is used most was found to be the leaves.Leaves as frequently used organ in traditional herbal drugs is also reported elsewhere (Muthu et al., 2006 ;Panghal et al., 2010).In addition to this, leaves are the main photosynthetic organs in plants and are considered to be the natural pharmacy for synthesis of many active constituents those are pharmacologically more active against certain diseases (Passalacqua et al., 2007).In general the use of leaves as the chosen plant part is a more sustainable practice as opposed to where roots and/or the bark are used.The prevalence in the use of leaves for preparation of traditional herbal remedies has been reported in other studies (Muthu et al. 2006;Focho et al. 2009;Panghal et al. 2010;Amri & Kisangau 2012).This practice helps to increase the chances of species survival and enhances the sustainable management of plants, as long as only an appreciable amount of leaves is harvested (Abede & Ayehu, 1993;Tadesse et al. 2005).
It was not surprising that decoction was the preferred preparation method.Decoction is known to be an effective extraction method compared to maceration in cold water, since boiling facilitates the extraction of constituents and also kills microorganisms potentially associated with the harvested plants.Plant parts were generally prepared using water as the solvent, likely because water is a readily available and cheap solvent and provides good solubility of the active components.Oral administration was the only mode of dispensing of herbal medicines against typhoid fever.This is because the causal bacterium is located in the intestinal tract.This mode of administration of herbal medicine was also reported elsewhere (Kamatenesi & Oryem-Origa, 2006;Bhattarai et al. 2010).
Bidens pilosa is the most commonly used plant for treatment by informant.Several studies document good antimicrobial effects of its extract (Rojas et al., 2006;Deba et al., 2008).
Phytochemical screening conducted on the plant extracts (Table 2) revealed the presence of compounds which are known to exhibit biological as well as physiological activities (Sofowora, 1993).These are alkaloids, flavonoids, polyphenols, saponins, sterols and triterpens.The presence of all the secondary metabolites in Annona muricata, Laggera alata, Spathodea campanulata, Cordia platythirsa, Carica papaya, Terminalia glaucescens and Pseudarthria confertiflora, is an indication that these plants are of pharmacological importance (Adebayo & Ishola, 2009), and also justified their potential use as drug in the study area.Almost all the plants contained triterpenes except in Dioscorea dumetorum and Gossypium barbadense.Triterpenes have been found to be useful in the prevention and therapy of several diseases, including cancer.Triterpenoids are also known to possess antimicrobial, antifungal, antiparasitic, antiviral, antiallergenic, antispasmodic, antihyperglycemic, antiinflammatory and immunomodulatory properties (Wagner & Elmadfa, 2003).The presence of this compound probably justified the use of the selected plants for the treatment of typhoid fever.However, the absence of this constituent in the leaves of Gossypium barbadense did not corroborates the findings of Charu et al., (2012).
Furthermore, most of these plants contained phenolic compounds (Polyphenols and flavonoids) which are one of the largest and most ubiquitous groups of plant metabolites (Singh et al., 2007).They possess biological properties such as antiapoptosis, antiaging, anticarcinogenesis, antiinflammation, antiatherosclerosis, cardiovascular protection, improvement of endothelial function, as well as inhibition of angiogenesis and cell proliferation activities (Han et al., 2007).Flavonoids are hydroxylated phenolic substances known to be synthesized by plants in response to microbial infection and they have been found to be antimicrobial substances against wide range of microorganisms in vitro.Their activity is probably due to their ability to complex with extracellular and soluble proteins and to complex with bacterial cell wall (Marjorie, 1996).In addition, Polyphenols have been reported to exhibit antibacterial activities with distinguished characteristics in their reactivity with proteins related polyamides polymers (Haslam, 1996).Sterols present in most of our plant sample, have been reported to have antibacterial properties (Raquel, 2007) and they are very important compounds especially due to their relationship with compounds such as sex hormones (Okwu, 2001).
The absence of alkaloids and saponins in Moringa oleifera in the present work is in contrast with the opinion of Gills (1992) and Kasolo et al., (2010) who noted that saponins and alkaloids are two of the active constituents.For instance, alkaloids are nitrogen-containing naturally occurring compound, commonly found to have antimicrobial properties due to their ability to intercalate with DNA of the microorganisms.Also, the presence of saponin in Annona muricata is in agreement with previous findings of Yusha 'a et al. (2011).
The activity of plant extracts against bacteria have been studied for years, but in more intensified way during the last three decades.During this period, numerous antimicrobial screening evaluations have been published based on the traditional Chinese, African and Asian uses of plant-based drugs (Suffredim et al., 2004).In the present study, the results of antibacterial property of the 38 plant extracts (table 2) against tested organisms varied depending on bacteria tested and concentration as previously reported by (Ravikumar et al., 2007;Rajesk et al., 2010).However, 38, 15 and 14 plants shows inhibition diameter against Salmonella typhi, S. paratyphi B and S. paratyphi A respectively.Nevertheless, negative results obtain with some of the plants extracts tested do not indicate the absence of bioactive constituents, nor that the plant is inactive.Active compound(s) may be present in insufficient quantities in the extracts to show activity with the dose levels employed (Taylor et al., 2001).Lack of the activity can thus only be proven by using large dose (Farnsworth, 1993), if the active principle is present in high enough quantities, there could be other constituents exerting antagonistic effects or negating the positive effects of the bioactive agents (Jager et al., 1999).
It was noticed that Senna alata, showing the highest antibacterial activity on both pathogens with zone of inhibition of 24, 22.5, 20.5 mm on the Salmonella paratyphi A, S. paratyphi B and S. typhi respectively at 160 mg/ml.This could probably have been due to the fact that the rate of the active ingredients or constituents in the plant materials is higher compared to the other plants used in this research (Kunle & Egharevba, 2009).Preliminary phytochemical analysis of S. alata leaves showed that, they possess polyphenols, triterpenes and saponins.Phytoconstituents such as polyphenols, triterpenes and saponins have been reported to inhibit bacterial growth and to be protective to plants against bacterial and fungal infections (Mather & Gonzalel, 1982;Okwute, 1992).The presence of saponins in this plant must have exhibited direct antibacterial activity and suppression of bacterial virulence resulting to the antimicrobial activity seen in this study (Gills, 1992).This also corroborate with the report of Owoyale et al., (2005) who documented on the plant Senna alata to be effective in treatment of fungal and bacterial diseases.
In local area, these plants are being used in the treatment of typhoid fever, diarrhea, dysentery etc.So, the uses of these plants as antibacterial agent are justified; since they showed good inhibition zones.Demonstration of low MIC (128 µg/ml) especially by Vitex doniana (table 2) is an indication that the phytoconstituents of the plant have therapeutic potential.Similar report revealed the antimicrobial potency of Vitex doniana with MIC range from 0.4-128 µg/ml (Iroha et al., 2010).It is important to highlight that Vitex doniana has been thoroughly studied phytochemically and its antimicrobial activity has been evaluated (Kubmarawa et al., 2007;Iroha et al., 2010).In this study, its antibacterial activity was demonstrated.It is no surprising the activity shown by this plant, since other species of the same genus (V.negundo) have shown to have antibacterial, anti-inflammatory and anti-fungal activity (Rusia & Srivastava, 1998;Dharmasiri et al., 2003;Panda et al., 2009).
Based on literature on these 38 plants, there is no biological and chemical on Pseudarthria confertiflora.Several species of the genus Pseudarthria (P.viscida and P. hookeri) has been studied and it has been demonstrated that they presented immunomodulatory and anti-fungal activity (Clarkson et al., 2004;Deepa et al., 2004;Mathew & Sasikumar, 2007;Vijayabaskaran et al., 2010), but the particular species Pseudarthria confertiflora has never been studied before and in this work, its antibacterial activity was demonstrated.The activity of this plant against salmonella typhi, S. paratyphi A and B has added one more plant that can offer alternative medicare to diseases caused by these microorganisms.
According to Noumedem et al. (2013), a sample is bactericidal when the ratio MBC/MIC ≤ 4 and bacteriostatic when this ratio is >4.It therefore appeared that bactericidal effects were obtained with the extracts from Carica papaya, Pseudarthria confertiflora, Moringa oleifera and Harungana madagascariensis.The result of antibacterial of Vitex doniana, Rauvolfia vomitoria, Senna alata, Terminalia glaucescens Bidens pilosa, Harungana madagascariensis Gardenia ternifolia and Lantana camara is highly encouraging and gives new lead plants for isolation and characterization of the active compounds.It is probably these active chemical constituents present in these plants which are responsible for the treatment of various bacterial infections caused by Salmonella sp.

CONCLUSION
The result of the present study offers a scientific basic for the use of the majority of tested plants in the treatment of typhoid fever in Bamboutos division.These plants species can be regarded as promising resources for anti-typhoid drugs.It seems that further investigations are necessary in order to draw solid conclusions.

Table 1 :
Information about medicinal plants recorded and tested in this study.

Table 2 .
. The lowest MIC values 128 µg/ml was exhibited by the extract of Vitex doniana against Salmonella paratyphi A; followed by the extract of Rauvolfia vomitoria, Senna alata and Terminalia glaucescens with MIC values of 512 µg/ml each on S. paratyphi A, while S. paratyphi B was most sensitive on Senna alata and Terminalia glaucescens extract with a MIC of 512 µg/ml.Gardenia ternifolia and Lantana camara were most effective against Salmonella typhi with MIC values of 512 µg/ml.Annona muricata, Senna alata, Terminalia glaucescens, Paullinia pinnata and Vitex doniana inhibited the growth of all the bacteria tested.The results of table 2 showed detectable MBC values for some of the studied plants on the tested microbial strains.

Table 2 :
Screening of 38 plant species for potential antibacterial activity against some Salmonella species.