Science Publications

Sign Up
Sign In
Submit
Journal of Biomedical and Life Sciences
Cite This Article Share Download PDF XML
  1. Home
  2. Journals
  3. Journal of Biomedical and Life Sciences
  4. Archive
  5. Volume 2, Issue 1, 2022
  6. Article
Article Open Access December 08, 2022

Antibacterial Activity of Phyllanthus Amarus (Schum and Thonn) Extract Against Salmonella Typhi Causative Agent of Typhoid Fever

Christiana Peprah Dabanka 1,* and Richard Otchere 2
1
Department of Science, St. Louis College of Education, Kumasi, Ashanti Region, Ghana
2
Department of Science, Wesley College of Education, Kumasi, Ashanti Region, Ghana
Publihed in: Journal of Biomedical and Life Sciences (Volume 2, Issue 1, 2022)
Page(s): 7-19
DOI: 10.31586/jbls.2022.561
Received
March 20, 2022
Revised
July 28, 2022
Accepted
September 16, 2022
Published
December 08, 2022
Keywords
Phyllanthus Amarus; Salmonella Typhi; Extract; Zone Of Inhibition
Creative Commons

This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.
Copyright: Copyright © The Author(s), 2022. Published by Scientific Publications

Abstract

The study was conducted to assess the antibacterial activity of Phyllanthus amarus (Schum and Thonn) extract against Salmonella typhi causative agent of typhoid fever at the laboratories of the Departments of Chemistry and Theoretical and Applied Biology of the College of Science, Kwame Nkrumah University of Science and Technology, Kumasi. The objectives were to determine the highest yield of crude extract of P. amarus using different proportions of water to ethanol and to determine the sensitivity of Salmonella typhi to these. Three different extraction procedures were carried out. In the first procedure, seven extraction setups each containing different proportions of the two extract (water and ethanol) were used with 10g of the plant sample. In the second procedure, eight setups were used for the two solvents. Ten grams of both fresh and dry plant sample were extracted in two different 200ml of water and in another two different 200ml of water; 20g of both fresh and dry plant sample were again extracted. The same procedure was repeated using ethanol as the solvent. In the third procedure, 10g each of fresh plant sample were boiled in 100ml and 200ml of water for 30 minutes. A sensitivity test to determine the zones of inhibition for the various plant extracts was done on Salmonella typhi isolated from human. Results from the crude yield of P. amarus using water only had the highest crude yield of 2.57g, followed by ethanol only which was 2.52g. The sensitivity studies conducted on the fresh P. amarus indicated that aqueous extract of P. amarus inhibited S. typhi to a zone of 5.00mm in 10g/200ml and 7.17mm in 20g/200ml. Ethanol extract also recorded an inhibition zone of 2.67mm and 5.33mm in 10g/200ml and 20g/200ml respectively. Again, sensitivity studies using dry P. amarus samples showed that the aqueous extracts recorded a zone of inhibition of 7.33mm in 10g/200ml and 13.50mm in 20g/200ml. Also ethanol extracts also recorded an inhibition zone of 6.83mm in 10g/200ml and 10.50mm in 20g/200ml. Significant differences were observed among the extracts and the control in both 10g/200ml and 20g/200ml concentrations (P<0.05). Aqueous and ethanol extracts of P. amarus proved inhibitory to S. typhi.

1. Introduction

Medicinal plants have been used for the treatment of several human diseases over the century and have been very important in the health care delivery of every nation at one stage or the other [1]. Recent research has focused on natural plant product as alternatives to the existing drugs for disease remedy in developing countries [2]. Plant derived medicines have been part of traditional health care in most parts of the world for ages and there is increasing interest in them as sources of agents to fight microbial diseases [3, 4, 5].

The development of multiple antibiotic resistant organisms has constituted a global problem as far as treatment of some infectious diseases is concerned. Typhoid fever caused by Salmonella typhi has since 1989 developed simultaneously, resistance to conventional antibiotics of choice in several endemic areas [6].

The vehicles of transmission of this etiologic agent are mainly food and water. Several other disease-causing organisms of medical importance have also developed resistance to these conventional antibiotics.

Infectious diseases still remain an important cause of morbidity and mortality in man, especially in developing countries. Today, in Africa, many resort to the use of locally made herbal medicines prepared as infusions in hot water, decoction in cold water, concoction with food and as tinctures with alcohol as an alternative therapy for bacterial infections [7]. Plant parts such as the roots, leaves, shoots, barks, fruit peels, immature and unripe fruits have been used in most herbal preparations.

According to George and Pamplona-Roger, the therapeutic value of some common plants such as Anthocleista vogelii Planch, Morinda lucida, Triplochiton scleroxylon, Alchornea cordifolia, Cassia sieberiana, Mangifera indica, Anacardium occidentale, Nauclea latifolia, Daniela oliveri,Citrus paradise, Ananas sativus and Carica papaya have been used in the treatment of various ailments including enteric fever, diarrhoea, dysentery, malaria, common cold, convulsion, yellow fever, jaundice, dental caries, intestinal parasites, gastroenteritis, bacterial, viral and protozoan diseases [8]. Antiseptic, diuretic, antibacterial and anti-inflammatory properties have equally been reported [9].

Herbal medicine is readily available in our diverse vegetation, cheap and above all carries the potential for introducing new templates into modern medicine [10]. In many parts of the world, including Ghana, herbal medicine practitioners are still consulted as a first choice in the treatment of ailments, due to the fact that traditional medicine blends readily with the socio-cultural life of the people, and the fact that orthodox medicine are more expensive to procure and some orthodox pharmaceutical preparations are many times faked [11]. The use of water as the sole solvent yielded the highest crude extract of 2.57g. Ethanol only also yielded the second highest crude extract of 2.52g. A fraction of water/ethanol (3:2) yielded 2.37g of crude extract. Also a fraction of water/ethanol (7:3) yielded 2.21g of crude extract. 2.14g of crude extract was recorded by a fraction of water/ethanol (9:1) while a fraction of water/ethanol (4:1) yielded 2.01g of crude extract.

There is a vast array of medicinal plants used singly or in combination with other medicinal plants that confer synergistic effect in the treatments of various ailments.

The study was conducted to assess an extract against Salmonella typhi causative agent of typhoid fever at the laboratories of the Departments of Chemistry and Theoretical and Applied Biology of the College of Science, Kwame Nkrumah University of Science and Technology, Kumasi.

A Completely Randomised Design with three replications was used.

The study was guided by these research questions:

  1. Determine the yield of the crude extract of Phyllanthus amarus using different proportions of water: ethanol
  2. Determine the sensitivity of Salmonella typhi to the various concentrations of the crude extract of P. amarus
1.1. Description of Phyllantus Amarus

Phyllantus amarus is a small erect tropical herb that grows to a height of 10-60cm. It is an annual and widespread throughout the tropics and subtropics. It is found in sandy regions as a weed in cultivated and wastelands. The plant is a common tropical weed that grows well in moist, shady and sunny places [12]. Some common names of Phyllanthus amarus in North, Central and South America are black catnip, carry-me seed, egg woman, hurricane weed, quinine creole, quinine weed, seed-under-leaf, stone breaker among others [13]. The name ‘Phyllanthus’ means “leaf and flower” since both the flower as well as the fruit seems to come one with the leaf [14].

P. amarus is a member of the Euphorbiaceae family (Spurge family), with over 6500 species in 300 genera. Euphorbiaceae family consist of upright or prostrate herbs or shrubs, often with milky acrid juice [15]. And is mainly a pan-tropical family with some species either more or less temperate. Numerous species of this family are native to North, Central and South America [16]. The plants are monoecious or monogamous. It has smooth cylindrical stem (1.5-2mm) thick and deciduous horizontal branch lets (4-12cm) long and 0.5cm thick, with 15 to 30 leaves. The leaves are simple, alternate or opposite and leathery, and borne on petioles 0.3 to 0.5 mm long. The flowers are very small and diclinous, often in clusters borne in greenish cup-shaped structures with glands. The fruit is a three-lobed capsule containing six seeds and extends from the cup with a long stalk pendant about 1-2mm [15]. The seeds are triangular (like an orange segment), light brown, 1mm long, with 5-6 ribs on the back and the seeds are hurled away when the fruits burst open [13].

Figure 1
Phyllantus amarus
1.2. Pathology of Salmonella typhi

Salmonella typhi has killed over 600,000 people annually all over the world [17]. It is a deadly bacterial disease that causes typhoid fever and is transmitted through food and water. It has become an epidemic in South Asian countries where sanitation is lacking. S. typhi usually invades the surface of the intestine in humans, but have developed and adapted to grow into the deeper tissues of the spleen, liver, and the bone marrow. Symptoms most characterized by this disease often include a sudden onset of a high fever, a headache, and nausea. Other common symptoms include loss of appetite, diarrhoea, and enlargement of the spleen (depending on where it is located). S. typhi involves colonization of the reticuloendothelial system. Some individuals who are infected with S. typhi become life-long carriers that serve as the reservoir for these pathogens. S typhi has an endotoxin (which is typical of Gram negative organisms), as well as the VI antigen, which increases virulence. It also produces a protein called invasion that allows non-phagocytic cells to take up the bacterium and allows it to live intra cellular. S. typhi is a strong pathogen for humans due to its resistance to the innate immune response system [17].

Recently, strains of MDR (multi-drug resistant) Salmonella have been identified and grouped together in a single haplotype named H58. It has been found that these strains are now resistant to nalidixic acid and have reduced susceptibility to fluoroquinolones. This strain has been recently found in Morocco, which shows that the MDR strain has reached as far as Africa [18].

1.3. Current Research on Salmonella typhi

Much research is going on since the global emergence of multi drug resistant strains. In India, samples of 21 Salmonella typhi strains were tested for their vulnerability to antimicrobial agents. Three different antibiotics were tested; chloramphenicol (256mg/l), trimethoprim (64mg/l), and amoxicillin (>128mg/l). Eleven of the S. typhi strains were resistance to chloramphenicol, trimethoprim and amoxicillin. Four of the isolates were resistant to all of them except for amoxicillin. Six of the isolates were completely sensitive to all of the antimicrobial agents tested. All the S. typhi isolates were susceptible to cephalosporin agents, gentamicin, amoxicillin plus clavulanic acid, and imipenem. The genes responsible for the resistance of the antibiotics included chloramphenicol acetyltransferase type I, dihydrofolate reductase type VII, and TEM-1 β-lactamase. Pulsed-field gel electrophoresis analysis of XbaI-generated genomic restriction fragments identified a single distinct profile (18 DNA fragments) for all of the resistant isolates. After comparing this, six different profiles were recognized among the sensitive isolates. It was found that a single strain containing a plasmid having multi drug-resistance has emerged in the S. typhi population and has been able to adapt and survive the antibiotics as they are introduced into clinical medicine [19].

With the current spread of Salmonella, researchers are looking for easier ways to detect typhoid fever in order to better treat patients. Another project has to do with dipstick assay which detects antibodies and analyses the effect of typhoid fever in patients. It found specific IgM antibodies on patients in 43.5%, 92.9%, and 100% for samples collected 4-6 days, 6-9 days, and greater than 9 days after the onset of fever, respectively, the number of antibodies increasing during the length of the duration. Testing of serum samples from culture negative patients with a clinical diagnosis of typhoid fever resulted in staining of the dipstick in 4.3% of the samples collected on the day of admission and in 76.6% one week later. This shows the late development of antibodies in the blood for a large number of patients. The advantages of the dipstick assay are that the result can be obtained on the same day allowing a prompt treatment. No special laboratory equipment is really needed to perform the assay and one would only need a small amount of serum. What makes it even better is that the simplicity of the assay would allow it to be used in places that lack laboratory facilities, such as third world countries that lack modern facilities and where disease is running high [20].

More people have taken a greater interest in Salmonella typhi since the decreasing effects of antibiotics. In 2006, more research was done in order to find the global gene expression by Salmonella typhi during infection. Global expression profiles of S. typhi grown in vitro and within macrophages at different time points were obtained and studied. Virulence factors, such as the SPI-1- and SPI-2-encoded type III secretion systems, were found as expected during infection by Salmonella. The results concluded that S. typhi had an increased expression of genes encoding resistance to antimicrobial peptides, which used the glyoxylate bypass for fatty acid utilization, and did not induce the SOS response or the oxidative stress response. It was also found that genes coding for the flagella apparatus, chemo taxis, and iron transport systems were down-regulated in vivo. This experiment allowed a better understanding of Salmonella and found a safer and more effective way to determine the bacterial transcriptome in vivo. This could possibly lead to the investigation of transcriptional profiles of other bacterial pathogens without the need to recover much bacterial mRNA from the host [21].

Typhoid fever is an acute illness associated with fever that is most often caused by the Salmonella typhi bacteria. It can also be caused by Salmonella paratyphi, a related bacterium that usually leads to a less severe illness. The bacteria are deposited in water or food by a human carrier and are then spread to other people in the area. Typhoid fever is rare in industrial countries but continues to be a significant public-health issue in developing countries [22, 23].

1. Materials and Methods

The study was conducted at the laboratories of the Departments of Chemistry and Theoretical and Applied Biology, College of Science, Kwame Nkrumah University of Science and Technology, Kumasi.

2.1. Source of Plant Materials

The plant used for the study was Phyllanthus amarus. Plant samples were collected from Kwame Nkrumah University of Science and Technology, Kumasi campus and Saint Louis College of Education campus, all in the Kumasi Metropolis. It was observed that the plant was shade loving and grew best in moist areas and sandy soils, especially at the edges of concrete floors. Identification of plant sample collected was done at the Department of Theoretical and Applied Biology, Kwame Nkrumah University of Science and Technology, Kumasi.

2.2. Preparation of Plant Samples

Plant materials collected were washed under running tap water and were allowed to drain before air drying under shade for two weeks. The roots were separated from the leaves and the stem, because that is what is traditionally done. The leaves together with the stem and the small branches were then grinded mechanically with mortar and pestle.

2.3. Extraction Procedure

Three different extraction procedures were carried out. The first extraction process consisted of using different proportions of the two main extraction solvents namely water and ethanol. The ratios of water to ethanol used for the proportions are presented in Table 1.

Table 1
Proportions of water to ethanol used in extraction

Ten grams (10g) of the P. amarus plant samples was placed into each of the seven flasks containing the different proportions of the two extraction solvents. The flasks were then connected to a setup consisting of Soxhlet Extractor Apparatus and a condenser. Power was applied for the extraction process to begin.

In the second extraction process, eight extraction setups were used for the two solvent (water and ethanol), four for each solvent. For water extraction, 10g of both fresh and dry P. amarus plant samples were extracted in 200ml of water and another 20g of both fresh and dry P. amarus plant sample was also extracted in 200ml of water. The same procedure was repeated for ethanol extraction.

In the third extraction process, 10g of fresh P. amarus plant sample was boiled in 100ml and 200ml of water for 30minutes.

2.4. Isolation of Salmonella From Human Faeces
2.4.1. Preparation of Buffered Peptone Water

Twenty grams (20g) of powdered peptone water was weighed and dissolved in 1 litre of distilled water in an Erlenmeyer flask. The solution was thoroughly mixed before distributing into universal bottles. The universal bottles with their contents were sterilized by autoclaving at 121°C for 15 minutes.

2.4.2. Preparation of Non-Selective Pre-Enrichment Medium:

Twenty-five grams (25g) of faeces was weighed and placed into an Erlenmeyer flask. 225ml of buffered peptone water was added to the Erlenmeyer flask to obtain 1 part sample and 9 parts buffered peptone water. This was thoroughly mixed and incubated at 370 C overnight (16-20 hours).

2.4.3. Preparation of Selective Enrichment Medium
2.4.3.1. Selenite broth preparation

Twenty-three grams (23g) of selenite broth powder was suspended in 1 litre of distilled water. The suspension was thoroughly mixed before heating to boiling point. The resulting solution was then distributed into universal bottles using syringe.1ml of the non-selective pre-enrichment medium was transferred with a pipette into 10ml of the selenite broth and incubated at 44°C for 48 hours.

2.4.3.2. SS agar preparation

Fifty-two grams (52g) of SS agar powder were suspended into one litre of distilled water. The solution was boiled until completely dissolve. 10ml of the resulting solution was poured unto sterile petri dishes (plates) and allowed to solidify.

2.4.3.3. Inoculation of Salmonella on Selective Agar Plates

Using an inoculating loop, streaks from the selenite broth were made on the solidified SS agar on the plate. The inoculated plates were then incubated at 37 C overnight (18-24 hours). Black colonies on the SS agar were seen after 48 hours confirming the presences of Salmonella typhi.

2.4.3.4. Storing of Salmonella typhi

Black colony of Salmonella typhi identified was washed with 10ml distilled water before adding to a selective SS agar medium in a universal bottle. This was incubated at 37°C for 48hours before storing in a refrigerator for future use.

2.5. Experimental Design

A Completely Randomised Design with three replications was used. Three sets of experiment were carried out. In the first experimental setup, different ratios of the extraction solvent were used to determine which combination gives the highest plant extract. In the second experimental setup, sensitivity of S. typhi to fresh plant extract was investigated. The treatments used consisted of (i) boiled plant sample in water, (ii) crude extract of plant with water, (iii) crude extract of plant with ethanol and (iv) a control treatment consisting of a standard antibiotic (Chloramphenicol tablet).

In the third experiment, sensitivity of S. typhi to dry plant sample was investigated. The treatments used consisted of (i) crude extract of plant with water, (ii) crude extract of plant with ethanol and (iii) a control treatment consisting of a standard antibiotic (Chloramphenicol tablet).

2.6. Sensitivity Test

To determine the effect of the plant extracts and chloramphenicol on the test organisms, a disc diffusion technique using the Kirby- Bauer method was applied in testing pure cultures of the test organism for their antimicrobial sensitivities based on zones of inhibition on agar plates [46]. In this method, circular discs from filter paper were sterilized in a hot air oven for 1hour. The discs were then impregnated by soaking with each plant extract and air-dried for a few minutes. 1ml of Salmonella typhi culture suspension was placed onto solidified SS agar plates using a sterile micropipette. A glass spreader was used to evenly distribute the test suspension on the SS agar. A sensitivity disc of each plant extract and the standard chloramphenicol disc of 10µg potency were aseptically transferred onto the SS agar plates using a sterilized forceps. The plates were labelled accordingly. The plates were then incubated at 37°C for 24-48 hours. The plates were later observed for zones of inhibition.

2.7. Statistical Analysis

The data collected were subjected to analysis of variance (ANOVA) using Statistic 9 statistical package. Differences in means were done using the least significant difference (LSD) at P=0.05. The results were expressed as mean ± standard error of mean (S.E.M.).

3. Results

3.1. Yield of Crude Extract of Phyllanthus Amarus

Table 2 indicates the yield of crude plant extract from different fractions of extraction solvent. Using water as the sole solvent yielded the highest crude extract of 2.57g. Ethanol only also yielded the second highest crude extract of 2.52g. A fraction of water/ethanol (3:2) yielded 2.37g of crude extract. Also a fraction of water/ethanol (7:3) yielded 2.21g of crude extract. 2.14g of crude extract was recorded by a fraction of water/ethanol (9:1) while a fraction of water/ethanol (4:1) yielded 2.01g of crude extract. However, a fraction of water/ethanol (1:1) yielded the lowest crude extract of 1.05g.

Table 2
Yield of crude plant extract from different fractions of extraction solvent.

Sensitivity of Fresh Phyllanthus Amarus to Salmonella Typhi: Sensitivity of S. typhi to fresh P. amarus sample in 10g/200ml is presented in Table 3. The results showed that water extract of fresh P. amarus was able to suppress activities of S. typhi and recorded the largest zone of inhibition of 5.00mm. This was followed by ethanol extract of fresh P. amarus which recorded an inhibition zone of 2.67mm. However, boiling of plant sample in water and the standard antibiotic, chloramphenicol recorded the smallest zone of inhibition of 1.83mm each. Statistically, there were significant differences observed among the extraction methods and the standard antibiotic (P<0.05).

Table 3
Sensitivity of S. typhi to fresh P. amarus sample in 10g/200ml

Table 4 shows the sensitivity of S. typhi to fresh P. amarus sample in 20g/200ml. The results of the experiment showed that S. typhi was very sensitive to the water extract of P. amarus with the largest inhibition zone of 7.17mm. The ethanol extracts recorded the second largest zone of inhibition of 5.33mm. The standard antibiotic, chloramphenicol had an inhibition zone of 5.00mm. However, boiling of fresh P. amarus in water recorded the smallest zone of inhibition of 3.33mm. There were significant differences observed among the different extraction methods and the standard antibiotic (P<0.05).

Table 4
Sensitivity of S. typhi to fresh P. amarus extract in 20g/200ml
3.2. Sensitivity Of Dry Phyllanthus Amarus to Salmonella Typhi

Sensitivity of S.typhi to dry P. amarus in 10g/200ml is presented in Table 5. The results showed that activities of S. typhi were inhibited by water extract of dry P. amarus with the largest zone of inhibition of 7.33mm. Ethanol extract of dry P. amarus was able to suppress activities of S. typhi to a zone of 6.83mm. The control treatment, chloramphenicol however, recorded the smallest zone of inhibition of 1.00mm. There were significant differences observed among the extraction methods and the control (P<0.05).

Table 5
Sensitivity of S. typhi to dry P. amarus extract in 10g/200ml

Table 6 depicts the sensitivity of S .typhi to dry P. amarus in 20g/200ml. The results showed that water extract of dry P. amarus suppressed activities of S. typhi and recorded the largest zone of inhibition of 13.50mm. Again, activities of S. typhi were inhibited by ethanol extract of dry P. amarus and recorded an inhibition zone of 10.50mm. However, chloramphenicol recorded the small zone of inhibition of 6.33mm. There were significant differences observed among the extraction methods and the control (P<0.05).

Table 6
Sensitivity of S. typhi to dry P. amarus extract in 20g/200ml

4. Discussion

4.1. Sensitivity of Phyllanthus Amarus to Salmonella Typhi

The sensitivity tests of Phyllanthus amarus to Salmonella typhi showed that both aqueous and ethanolic extracts of the plant material showed antimicrobial activity on the test organism. The test pathogen, S. typhi was more susceptible to the aqueous extract of P. amarus than the ethanol extract at all concentrations. The highest susceptibility was recorded with the dry plant aqueous extract at 20g/200ml, followed by the ethanol extract at 20g/200ml. The susceptibility of the test inoculum to the extract of the fresh and dry plant material increased with increasing concentration of the extract. The antibacterial efficacy of the plant extracts were very high compared to the low activities recorded with chloramphenicol. Chlorampenicol showed different sensitivity to the test organism, this indicates that it still has the potential of curing typhoid fever. But it was realized that, the sensitivity increased with increasing concentration. According to Oluduro and Omoboye in 2010, the antibacterial activities of most plant extracts are concentration dependent as zone of growth inhibition increased with increasing concentration of the extracts [7]. Ekwenye and Elegalam and Azu and Onyeagha reported that the efficacy of most plant extracts is concentration dependent [24, 25].

Assessing the quality of Phyllanthus amarus leaves extract for its hypolipidemic activity found the presence of four phyto-constituent namely alkaloids, flavonoids, saponins and tannins in the plant sample [26]. Flavonoids, tannins, alkaloids, steroids, terpenoids, saponins and glycosides were also obtained by Obianime and Uche, 2009 in their comparative study of the methanol extract of P. amarus leaves [27]. The presence of phytochemicals in plant extracts is a function of their antimicrobial activities against the test pathogen as they play important roles in bioactivity of medicinal plants [7]. They further explained that phytochemicals exert antimicrobial activity through different mechanisms. Chonoko and Rufai in 2011 also indicated that there was a link between the antibacterial activity exhibited by the plant extracts to the presence of steroids flavonoids, tannins, alkaloids and saponins [28].

Tannins, for example, act by iron deprivation, hydrogen binding or specific interactions with vital proteins such as enzymes in microbial cells [29, 30]. Herbs that have tannins as their component are astringent in nature and are used for the treatment of gastrointestinal disorders such as diarrhoea and dysentery; [31, 32]. Saponinis believed to be responsible for numerous pharmacological properties [33] and has been shown to have immense significance as anti hypercholesterol, hypotensive and cardiac depressant properties [34]. This perhaps justifies the already locally established function of the plant in the treatment and management of hypertensions. Alkaloids on the other hand are detoxifying and have antihypertensive properties; [35, 36]. It’s toxicity against cells of foreign organisms has been reported by Akinpelu and others [30]. Waterman 1992 reported that alkaloids and flavonoids were useful as antimicrobial, anti-inflammatory and anti-oxidant agents [37]. Okwu and Josiah in 2006 also indicated that flavonoids are antioxidants [38]; hence, Adeneye and others in 2006b reported that the flavonoids contributed to the antioxidant activity of the P. amarus plant [39].

The choice of antimicrobial drugs in the absence of susceptibility information is often influenced by the signs and symptoms of disease, site of infection, and history of illness including patient’s age. In many medical set-ups in the developing nations where laboratory facilities are inadequate, broad spectrum antibiotics are often used for suspected bacterial infections. These practices are not without danger which could worsen the disease prognosis. Intensive use of antibiotics often results in the development of resistant strains creating a problem in the treatment of infectious diseases [40].

Harvey and others in 2006 reported chloramphenicol as the drug of choice in the management of typhoid fever although its toxicity is well known. According to them, higher doses of chloramphenicol may not be favourable to the host because its toxicity is known to precipitate some serious adverse reactions [41]. Cunha in 2001, reiterated that intake of antibiotics are associated with side effects [42]. The increasing emergence of antibiotic-resistant strains to traditional antimicrobials makes it necessary that sensitivity tests should be carried out prior to initiation of antibiotic therapy [43]. Disk diffusion and subsequent Minimum Inhibition Concentration (MIC) and Minimum Bactericidal Concentration (MBC) tests have remain the most commonly used methods to test organisms’ susceptibility to antibiotics although they were outside the scope of the present study [44].

The present work has provided the basis for selecting plant extracts that are likely to be effective against Salmonella typhi. The problem of increasing emergence of resistance to antibacterial drugs makes these studies unavoidable, and equally highlights the need for periodic review of antimicrobial activities of common antibiotic drugs available on the market since chloramphenicol was found to be less effective at a lower concentration or dosage than at a higher one. Moreover, medicinal plants often have fewer side effects, better patient tolerance, relatively less expensive, acceptance due to long history of use and being renewable in nature [45].

Generally, the present study has shown that all the different concentrations of the plant extract tested possess a measure of antimicrobial properties and is concentration dependent. The plant material examined in the present study have been in used in Ghana in the preparation of decoction and concoction for the treatment of typhoid fever caused by the test pathogen particularly, when modern drugs of choice failed in achieving the therapy.

This study has confirmed the antibacterial potentials of P. amarus, thus supporting their folklore application as a medical remedy for typhoid fever. Hence, supports the usefulness of the plant in the treatment of other ailments caused by microorganisms.

4.2. Yield of Crude Extract of Phyllanthus Amarus

The use of water as the sole solvent yielded the highest crude extract of 2.57g. Ethanol only also yielded the second highest crude extract of 2.52g. A fraction of water/ethanol (3:2) yielded 2.37g of crude extract. Also a fraction of water/ethanol (7:3) yielded 2.21g of crude extract. 2.14g of crude extract was recorded by a fraction of water/ethanol (9:1) while a fraction of water/ethanol (4:1) yielded 2.01g of crude extract. However, a fraction of water/ethanol (1:1) yielded the lowest crude extract of 1.05g. Comparing the use of the different fractions of water to ethanol, it was identified that there was no relation between the ratio of water to ethanol used and the yield of the crude plant extract. Factors like age of the plant and the polarity of the solvent used may affect the yield. Water and ethanol seems to be a good solvent for this plant which supports the use of water and alcohol as traditional solvent.

5. Conclusions

Generally, water was the best solvent used for the crude extraction of Phyllanthus amarus since it gave the highest crude extract. Ethanol gave the second highest yield of crude extract of P. amarus. The different proportions of water to ethanol gave different yields of the crude plant which has no relation. The proportion of 1:1 of water to ethanol gave the least yield.

The water extract of fresh P. amarus showed the largest and significant zone of inhibition of 5.00mm and 7.17mm in 10g/200ml and 20g/200ml concentration respectively compared to the other extracts antibacterial activity on S. typhi investigated in the study.

The ability of the dry P. amarus extract showing sensitivity to S. typhi was observed in the water extract which also showed the largest and significant zone of inhibition of 7.33mm and 13.50mm in both 10g/200ml and 20g/200ml concentration respectively.

However, the ethanolic extracts of both the fresh and dry P. amarus in 10g/200ml and 20g/200ml concentration also showed significant sensitivity on the test organism. Chloramphenicol which was the control showed different sensitivity on the test organism.

The present study showed that fresh and dry extracts of Phyllanthus amaru showed antimicrobial activity on S. typhi, hence this provides a scientific basis that reflects the idea of traditional healers for using this plant for curing of typhoid fever as well as other ailments.

In conclusion, the results of the present study support the folkloric usage of the plant and suggest that both the water and ethanolic plant extracts possess compounds with antimicrobial properties that can be further explored for antimicrobial activity. This antibacterial study of the plant extracts demonstrated that folk medicine can be as effective as modern medicine to combat pathogenic microorganisms. The use of the plants in folk medicine suggests that they represent an economic and safe alternative to treat infectious diseases.

6. Recommendation

Based on the findings from this study, it is recommended that:

  1. Further studies should be conducted to determine the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Phyllanthus amarus against Salmonella typhi because of the antibacterial activity exhibited by the fresh and dry plant extracts.
  2. Evaluation of the phytochemical properties and toxicological studies of Phyllanthus amarus must be carried out to ascertain its relative safety as a possible antimicrobial agent.

Authors Contribution

Conceptualization, C,P.D. and R.O; methodology, C.P.D . and R. O.; validation, R. O, C.P.D. formal analysis, C.P.D.; investigation, C.P.D.; resources, R. O.; data curation, R.O; writing—original draft preparation, C.P.D.; writing—review and editing,C.P.D.; visualization, R.O; supervision, R. O.; project administration, C. P.D and R.O. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

Data is available on request from the corresponding author.

Acknowledgments

We acknowledge Dr. Anthony Bordoh for his input and suggestions.

Conflicts of Interest

The authors declare no conflict of interest. No funders had any role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

  1. Oluma, H.O, Umoh, E.U., Onekutu, A., & Okolo, J. (2004). Antibacterial potentials of eight medicinal plants from the lower Benue valley of Nigeria against Salmonella typhi. Journal of Botany 17: 1-11.
  2. Aiyegoro, O.A, Akinpelu, D.A., & Okoh, A.I. (2007). In Vitro antibacterial potentials of the stem bark of Redwater Tree (Erythrophleum suaveolena). Journal of Biological Sciences 7(7): 1233-1238.[CrossRef]
  3. Mohana, D.C, Satish, S., & Raveesha, K.A. (2008). Antibacterial evaluation of some plant extracts against some human pathogenic bacteria. Advances in biological Research 2(3-4): 49-55.
  4. Ghaleb, M.A., Bassam, A. A., & Kamel, M.A. (2009). In Vitro activity of certain drugs in combination with plant extracts against Staphylococcus aureus infections. African Journal of Biotechnology8 (17): 4239-4241.
  5. Ajayi, A.O., & Akintola, T.A. (2010). Evaluation of antibacterial activity of some medicinal plants on common enteric food-borne pathogens.African Journal of Microbiology Research 4(4): 314-316.
  6. Greenwood, D., Slack, R.C., & Peutherer, J.F. (2009). Salmonella Food Poisoning and enteric fever in Medical Microbiology [16th edition]. Churchill Livingstone, Edinburgh. Pp 250-259.
  7. Oluduro, A., & Omoboye, O. (2010). In Vitro Antibacterial Potentials and Synergistic Effect of South-Western Nigerian Plant Parts Used in Folklore Remedy for Salmonella typhi infection. Nature and Science 8(9): 52-59.
  8. George, D., & Pamplona-Roger, M.D. (1998). Encyclopedia of Medicinal Plants, Part 2. Safeliz S.L. Madrid, Spain. Pp. 267, 425, 435, 710.
  9. Alanis, A.D., Calzada, F., Cervantes, J.A., Torres, J., & Ceballos, G.M. (2005). Antimicrobial properties of some plants used in Mexican traditional medicine for the treatment of gastrointestinal disorders. Journal of Ethnopharmacology 100: 153-157.[CrossRef] [PubMed]
  10. Akinyemi, K.O., Smith, S.I., Oyefolu, A. O., & Coker, A.O. (2005). Multidrug resistance in Salmonella enterica serovar typhi isolated from patients with typhoid fever complications in Lagos, Nigeria. Public Health119: 321-327.[CrossRef] [PubMed]
  11. Amuse, A.M., Nwodo, F.O.C., & Yusuf, G.O. (2011). A comparative study of the antibacterial activity of aqueous ethanol and chloroform extracts of some selected medicinal plants used in Igalaland of Nigeria. Der Pharmacia Sinica 2(1): 222-227.
  12. Nanden-Amattaram, T. 1998. Medicinale Planten: tips en simpele recepten voor eengoede gezondheid. “Medicinal plants and simple recipes for a good health.”Paramaribo-Suriname.p.18.
  13. Morton, J.F. (1981). Atlas of Medicinal Plants of Middle America.Library of Congress cataloging in Publication Data. Thomas books. p 1420.
  14. Cabieses, F. (1993), Apuntes de medicina traditional. La racionalizacion de lo irracional. “Notes of traditional medicine.”Consejo Nacional de Ciencia y Technologia CONCYTEC LimaPeru.P p 414.
  15. Lewis, H.W. and Elvin-Lewis, M.P.H. (1977). Medical Botany Plants Affecting Man’sHealth.A Wiley-Interscience Publication.John Wiley & Sons, New York-London-Sydney-Toronto.p. 515.
  16. Unander, D.W., Webster, G.L., & Blumberg, B.S. (1995). Usage and bioassays inPhyllanthus (Euphorbiaceae). IV. Clustering of antiviral uses and other effects.Journal of Ethnopharmacology45: 1-18.[CrossRef] [PubMed]
  17. Falkow, S., Monack, D.M., & Mueller, A. (2004). Persistent bacterial infections: the interface of the pathogen and the host immune system. Nature Review of Micribiology2: 747-765.[CrossRef] [PubMed]
  18. Ojcius, D. (2007). In the News.Nature Reviews Microbiology5: 10-11.[CrossRef]
  19. Philippa, M.A., Shanahan, J., Mary, V., Thomson, C.J., & Sebastian, G.B. (1998). Molecular analysis of and identification of antibiotic resistance genes in clinical isolates of Salmonella typhi from India.American Society for Microbiology36: 1595–1600.[CrossRef] [PubMed]
  20. Hatta, M., Gorris, M.G., Heerkens, E., Gooskens J., & Smits, H.L. (2002). Simple dipstick assay for the detection of Salmonella typhi-specific IgM antibodies and the evolution of the immune response in patients with typhoid fever.American Journal of Tropical Medicine and Hygiene66: 416-421[CrossRef] [PubMed]
  21. Faucher, S.P., Porwollik, S., Dozois, C., McClelland, M., & Daigle, F. (2006). Transcriptome of Salmonella enterica serovar typhi within macrophages revealed through the selective capture of transcribed sequences. The National Academy of Sciences of the USA 103: 1906-1911.[CrossRef] [PubMed]
  22. Brusch, J.L. (2011). Typhoid fever [online]. Available from: http://emedicine. medscape.com/article/231135-overview. [Access date: 21/09/ 2011].
  23. Balentine, J.R. (2011). Tytphoid fever [online]. Available from: http://www. medicinenet.com/typhoid_fever/article.htm. [Accessed date: 21/09/2011].
  24. Ekwenye, U.N., & Elegalam, N.N. (2005). Antibacterial activity of ginger (Zingiber officinales Roscoe) and garlic (Allium sativum L) extracts on Escherichia coli and Salmonella typhi. Journal of Molecular Medicine and Advanced Science 1(4): 41-416.
  25. Azu, N.C., & Onyeagba, R.A. (2007). Antimicrobial properties of extracts of Allium cepa (onions) and Zingiber officinale (ginger) on Escherichia coli, and Bacillus subtilis. The Internet Journal of Tropical Medicine.3 (2): 277-286.
  26. Umbare, R. P., Mate1, G. S., Jawalkar, D.V., Patil, S. M., & Dongare, S. S. (2009). Quality evaluation of Phyllanthus amarus (Schumach) leaves extract for its hypolipidemic activity. Biology and Medicine1 (4): 28-33.
  27. Obianime A.W., & Uche F.I. (2009). The comparative effects of methanol extract of Phyllanthus amarus leaves and Vitamin E on the Sperm parameters of Male guinea pigs. Journal of Applied Sciences and Environmental Management 13(1): 37-41.[CrossRef]
  28. Chonoko, U. G., & Rufai, A. B. (2011).Phytochemical screening and antibacterial activity of Cucurbita pepo (pumpkin) against Staphylococcus aureus and Salmonella typhi. Bayero Journal of Pure and Applied Sciences 4(1): 145 – 147.[CrossRef]
  29. Scalbert, A. (1991). Antimicrobial Properties of tannins.Phytochemistry. 30: 3875-3883.[CrossRef]
  30. Akinpelu, D.A., Aiyegoro, D.A., & Okoh, A.I. (2008). In Vitro antimicrobial and Phytochemical Properties of Crude extract of Stem bark of Afzelia africana (Smith). African Journal of Biotechnology 7(20): 3662-3667.
  31. Dharmananda, S. (2003). Golinuts and the uses of tannins in Chinese Medicine.In proceedings of Institute for Traditional Medicine Portland, Oregon, USA, 25 September, 2003.
  32. Asquith, T.N., & Butler, L.G. (1986). Interaction of condensed tannins with selected proteins.Phytochemistry25 (7): 1591-1593.[CrossRef]
  33. Estrada, A., Katselis, G.S., Laarveid, B. and Bari, B. (2000). Isolation and evaluation of immunological adjuvant activities of saponins from Polygaja senega.L. Comparative Immunology. Microbial Infectious Disease 23: 27-43.[CrossRef] [PubMed]
  34. Price, K.R., Johnson, T.I., & Fenwick, G.R. (1987).The Chemistry and Biological Significance of Saponins in Food and feeding stuffs.Critical Reviews in Food Science and Nutrition26:22-48.[CrossRef] [PubMed]
  35. Trease, G.E., & Evans, W.C. (1978).Pharmacology (11th Edition). Bailliere Tindall Ltd, London. p. 784.
  36. Zee-cheng, R.K. (1997). Anticancer research on Loranthaceae plants. Drugs Future 22(5): 515-530.
  37. Waterman, P. H. (1992).Searching for bioactive compounds various strategies.Journal of National Products53 (1): 13-22.[CrossRef]
  38. Okwu, D.E., & Josiah, C. (2006).Evaluation of the chemical composition of two Nigerian Medicinal plants.African Journal of Biotechnolgy5 (4): 357-361.
  39. Adeneye, A. A., Benebo, A. S., & Agbaje, E. O. (2006b). Protective effect of the Aqueous Leaf and seed Extract of Phyllanthus amarus on Alcohol-induced hepatotoxity in rats. West Africa Journal of Pharmacology and Drug Research 22&23: 42-50.[CrossRef]
  40. Sydney, S., Lacy, R.W. and Bakhtiar, M. (1980). In: The Betalactam antibiotics Penicillin and Cephalosporin in perspective, Hodder and stongton, London, p. 224.
  41. Harvey, R. A., Champe, P.C., Howland, R. D., & Mycek M. J. (2006). Lippincott’s Illustrated Reviews Pharmacology (3rd edition). Lippincott Williams & Wilkins, Baltimore. Pp. 341-395.
  42. Cunha, B. A. (2001). Antibiotics side effects. Medical Clinics of North America85:149-185.[CrossRef] [PubMed]
  43. Madukosiri, C.H., Edike, T., & Ghandi, E. O. (2009). Comparative Studies of Susceptibility of Salmonella typhi to Antibiotics and some Plant Extracts.Nigerian Journal of Biochemistry and Molecular Biology24 (1):16-21.
  44. Nester, E.W., Anderson, D.G., Roberts, C.E., Pearsall, N.N., & Nester, M. T. (2004). Microbiology: A human perspective (4th edition) McGraw-Hill, New York. Pp. 817.
  45. Vermani, K., & Garg, S. (2002). Herbal medicine for sexually transmitted diseases and AIDS. Journal of Ethnopharmacology, 80: 49-66.[CrossRef] [PubMed]
  46. Presscott, L.M., Harley, J.P. and Klein, D.A. (2005). Microbiology. (6th ed.) Boston: McGraw-Hill.
Article metrics
Views
575
Downloads
283

Cite This Article

APA Style
Dabanka, C. P. , & Otchere, R. (2022). Antibacterial Activity of Phyllanthus Amarus (Schum and Thonn) Extract Against Salmonella Typhi Causative Agent of Typhoid Fever. Journal of Biomedical and Life Sciences, 2(1), 7-19. https://doi.org/10.31586/jbls.2022.561
ACS Style
Dabanka, C. P. ; Otchere, R. Antibacterial Activity of Phyllanthus Amarus (Schum and Thonn) Extract Against Salmonella Typhi Causative Agent of Typhoid Fever. Journal of Biomedical and Life Sciences 2022 2(1), 7-19. https://doi.org/10.31586/jbls.2022.561
Chicago/Turabian Style
Dabanka, Christiana Peprah, and Richard Otchere. 2022. "Antibacterial Activity of Phyllanthus Amarus (Schum and Thonn) Extract Against Salmonella Typhi Causative Agent of Typhoid Fever". Journal of Biomedical and Life Sciences 2, no. 1: 7-19. https://doi.org/10.31586/jbls.2022.561
AMA Style
Dabanka CP, Otchere R. Antibacterial Activity of Phyllanthus Amarus (Schum and Thonn) Extract Against Salmonella Typhi Causative Agent of Typhoid Fever. Journal of Biomedical and Life Sciences. 2022; 2(1):7-19. https://doi.org/10.31586/jbls.2022.561 
@Article{jbls561,
AUTHOR = {Dabanka, Christiana Peprah and Otchere, Richard},
TITLE = {Antibacterial Activity of Phyllanthus Amarus (Schum and Thonn) Extract Against Salmonella Typhi Causative Agent of Typhoid Fever},
JOURNAL = {Journal of Biomedical and Life Sciences},
VOLUME = {2},
YEAR = {2022},
NUMBER = {1},
PAGES = {7-19},
URL = {https://www.scipublications.com/journal/index.php/JBLS/article/view/561},
ISSN = {2771-2303},
DOI = {10.31586/jbls.2022.561},
ABSTRACT = {The study was conducted to assess the antibacterial activity of Phyllanthus amarus (Schum and Thonn) extract against Salmonella typhi causative agent of typhoid fever at the laboratories of the Departments of Chemistry and Theoretical and Applied Biology of the College of Science, Kwame Nkrumah University of Science and Technology, Kumasi. The objectives were to determine the highest yield of crude extract of P. amarus using different proportions of water to ethanol and to determine the sensitivity of Salmonella typhi to these. Three different extraction procedures were carried out. In the first procedure, seven extraction setups each containing different proportions of the two extract (water and ethanol) were used with 10g of the plant sample. In the second procedure, eight setups were used for the two solvents. Ten grams of both fresh and dry plant sample were extracted in two different 200ml of water and in another two different 200ml of water; 20g of both fresh and dry plant sample were again extracted. The same procedure was repeated using ethanol as the solvent. In the third procedure, 10g each of fresh plant sample were boiled in 100ml and 200ml of water for 30 minutes. A sensitivity test to determine the zones of inhibition for the various plant extracts was done on Salmonella typhi isolated from human. Results from the crude yield of P. amarus using water only had the highest crude yield of 2.57g, followed by ethanol only which was 2.52g. The sensitivity studies conducted on the fresh P. amarus indicated that aqueous extract of P. amarus inhibited S. typhi to a zone of 5.00mm in 10g/200ml and 7.17mm in 20g/200ml. Ethanol extract also recorded an inhibition zone of 2.67mm and 5.33mm in 10g/200ml and 20g/200ml respectively. Again, sensitivity studies using dry P. amarus samples showed that the aqueous extracts recorded a zone of inhibition of 7.33mm in 10g/200ml and 13.50mm in 20g/200ml. Also ethanol extracts also recorded an inhibition zone of 6.83mm in 10g/200ml and 10.50mm in 20g/200ml. Significant differences were observed among the extracts and the control in both 10g/200ml and 20g/200ml concentrations (P<0.05). Aqueous and ethanol extracts of P. amarus proved inhibitory to S. typhi.},
}
%0 Journal Article
%A Dabanka, Christiana Peprah
%A Otchere, Richard
%D 2022
%J Journal of Biomedical and Life Sciences

%@ 2771-2303
%V 2
%N 1
%P 7-19

%T Antibacterial Activity of Phyllanthus Amarus (Schum and Thonn) Extract Against Salmonella Typhi Causative Agent of Typhoid Fever
%M doi:10.31586/jbls.2022.561
%U https://www.scipublications.com/journal/index.php/JBLS/article/view/561

TY  - JOUR
AU  - Dabanka, Christiana Peprah
AU  - Otchere, Richard
TI  - Antibacterial Activity of Phyllanthus Amarus (Schum and Thonn) Extract Against Salmonella Typhi Causative Agent of Typhoid Fever
T2  - Journal of Biomedical and Life Sciences
PY  - 2022
VL  - 2
IS  - 1
SN  - 2771-2303
SP  - 7
EP  - 19
UR  - https://www.scipublications.com/journal/index.php/JBLS/article/view/561
AB  - The study was conducted to assess the antibacterial activity of Phyllanthus amarus (Schum and Thonn) extract against Salmonella typhi causative agent of typhoid fever at the laboratories of the Departments of Chemistry and Theoretical and Applied Biology of the College of Science, Kwame Nkrumah University of Science and Technology, Kumasi. The objectives were to determine the highest yield of crude extract of P. amarus using different proportions of water to ethanol and to determine the sensitivity of Salmonella typhi to these. Three different extraction procedures were carried out. In the first procedure, seven extraction setups each containing different proportions of the two extract (water and ethanol) were used with 10g of the plant sample. In the second procedure, eight setups were used for the two solvents. Ten grams of both fresh and dry plant sample were extracted in two different 200ml of water and in another two different 200ml of water; 20g of both fresh and dry plant sample were again extracted. The same procedure was repeated using ethanol as the solvent. In the third procedure, 10g each of fresh plant sample were boiled in 100ml and 200ml of water for 30 minutes. A sensitivity test to determine the zones of inhibition for the various plant extracts was done on Salmonella typhi isolated from human. Results from the crude yield of P. amarus using water only had the highest crude yield of 2.57g, followed by ethanol only which was 2.52g. The sensitivity studies conducted on the fresh P. amarus indicated that aqueous extract of P. amarus inhibited S. typhi to a zone of 5.00mm in 10g/200ml and 7.17mm in 20g/200ml. Ethanol extract also recorded an inhibition zone of 2.67mm and 5.33mm in 10g/200ml and 20g/200ml respectively. Again, sensitivity studies using dry P. amarus samples showed that the aqueous extracts recorded a zone of inhibition of 7.33mm in 10g/200ml and 13.50mm in 20g/200ml. Also ethanol extracts also recorded an inhibition zone of 6.83mm in 10g/200ml and 10.50mm in 20g/200ml. Significant differences were observed among the extracts and the control in both 10g/200ml and 20g/200ml concentrations (P<0.05). Aqueous and ethanol extracts of P. amarus proved inhibitory to S. typhi.
DO  - Antibacterial Activity of Phyllanthus Amarus (Schum and Thonn) Extract Against Salmonella Typhi Causative Agent of Typhoid Fever
TI  - 10.31586/jbls.2022.561
ER  -
  1. Oluma, H.O, Umoh, E.U., Onekutu, A., & Okolo, J. (2004). Antibacterial potentials of eight medicinal plants from the lower Benue valley of Nigeria against Salmonella typhi. Journal of Botany 17: 1-11.
  2. Aiyegoro, O.A, Akinpelu, D.A., & Okoh, A.I. (2007). In Vitro antibacterial potentials of the stem bark of Redwater Tree (Erythrophleum suaveolena). Journal of Biological Sciences 7(7): 1233-1238.[CrossRef]
  3. Mohana, D.C, Satish, S., & Raveesha, K.A. (2008). Antibacterial evaluation of some plant extracts against some human pathogenic bacteria. Advances in biological Research 2(3-4): 49-55.
  4. Ghaleb, M.A., Bassam, A. A., & Kamel, M.A. (2009). In Vitro activity of certain drugs in combination with plant extracts against Staphylococcus aureus infections. African Journal of Biotechnology8 (17): 4239-4241.
  5. Ajayi, A.O., & Akintola, T.A. (2010). Evaluation of antibacterial activity of some medicinal plants on common enteric food-borne pathogens.African Journal of Microbiology Research 4(4): 314-316.
  6. Greenwood, D., Slack, R.C., & Peutherer, J.F. (2009). Salmonella Food Poisoning and enteric fever in Medical Microbiology [16th edition]. Churchill Livingstone, Edinburgh. Pp 250-259.
  7. Oluduro, A., & Omoboye, O. (2010). In Vitro Antibacterial Potentials and Synergistic Effect of South-Western Nigerian Plant Parts Used in Folklore Remedy for Salmonella typhi infection. Nature and Science 8(9): 52-59.
  8. George, D., & Pamplona-Roger, M.D. (1998). Encyclopedia of Medicinal Plants, Part 2. Safeliz S.L. Madrid, Spain. Pp. 267, 425, 435, 710.
  9. Alanis, A.D., Calzada, F., Cervantes, J.A., Torres, J., & Ceballos, G.M. (2005). Antimicrobial properties of some plants used in Mexican traditional medicine for the treatment of gastrointestinal disorders. Journal of Ethnopharmacology 100: 153-157.[CrossRef] [PubMed]
  10. Akinyemi, K.O., Smith, S.I., Oyefolu, A. O., & Coker, A.O. (2005). Multidrug resistance in Salmonella enterica serovar typhi isolated from patients with typhoid fever complications in Lagos, Nigeria. Public Health119: 321-327.[CrossRef] [PubMed]
  11. Amuse, A.M., Nwodo, F.O.C., & Yusuf, G.O. (2011). A comparative study of the antibacterial activity of aqueous ethanol and chloroform extracts of some selected medicinal plants used in Igalaland of Nigeria. Der Pharmacia Sinica 2(1): 222-227.
  12. Nanden-Amattaram, T. 1998. Medicinale Planten: tips en simpele recepten voor eengoede gezondheid. “Medicinal plants and simple recipes for a good health.”Paramaribo-Suriname.p.18.
  13. Morton, J.F. (1981). Atlas of Medicinal Plants of Middle America.Library of Congress cataloging in Publication Data. Thomas books. p 1420.
  14. Cabieses, F. (1993), Apuntes de medicina traditional. La racionalizacion de lo irracional. “Notes of traditional medicine.”Consejo Nacional de Ciencia y Technologia CONCYTEC LimaPeru.P p 414.
  15. Lewis, H.W. and Elvin-Lewis, M.P.H. (1977). Medical Botany Plants Affecting Man’sHealth.A Wiley-Interscience Publication.John Wiley & Sons, New York-London-Sydney-Toronto.p. 515.
  16. Unander, D.W., Webster, G.L., & Blumberg, B.S. (1995). Usage and bioassays inPhyllanthus (Euphorbiaceae). IV. Clustering of antiviral uses and other effects.Journal of Ethnopharmacology45: 1-18.[CrossRef] [PubMed]
  17. Falkow, S., Monack, D.M., & Mueller, A. (2004). Persistent bacterial infections: the interface of the pathogen and the host immune system. Nature Review of Micribiology2: 747-765.[CrossRef] [PubMed]
  18. Ojcius, D. (2007). In the News.Nature Reviews Microbiology5: 10-11.[CrossRef]
  19. Philippa, M.A., Shanahan, J., Mary, V., Thomson, C.J., & Sebastian, G.B. (1998). Molecular analysis of and identification of antibiotic resistance genes in clinical isolates of Salmonella typhi from India.American Society for Microbiology36: 1595–1600.[CrossRef] [PubMed]
  20. Hatta, M., Gorris, M.G., Heerkens, E., Gooskens J., & Smits, H.L. (2002). Simple dipstick assay for the detection of Salmonella typhi-specific IgM antibodies and the evolution of the immune response in patients with typhoid fever.American Journal of Tropical Medicine and Hygiene66: 416-421[CrossRef] [PubMed]
  21. Faucher, S.P., Porwollik, S., Dozois, C., McClelland, M., & Daigle, F. (2006). Transcriptome of Salmonella enterica serovar typhi within macrophages revealed through the selective capture of transcribed sequences. The National Academy of Sciences of the USA 103: 1906-1911.[CrossRef] [PubMed]
  22. Brusch, J.L. (2011). Typhoid fever [online]. Available from: http://emedicine. medscape.com/article/231135-overview. [Access date: 21/09/ 2011].
  23. Balentine, J.R. (2011). Tytphoid fever [online]. Available from: http://www. medicinenet.com/typhoid_fever/article.htm. [Accessed date: 21/09/2011].
  24. Ekwenye, U.N., & Elegalam, N.N. (2005). Antibacterial activity of ginger (Zingiber officinales Roscoe) and garlic (Allium sativum L) extracts on Escherichia coli and Salmonella typhi. Journal of Molecular Medicine and Advanced Science 1(4): 41-416.
  25. Azu, N.C., & Onyeagba, R.A. (2007). Antimicrobial properties of extracts of Allium cepa (onions) and Zingiber officinale (ginger) on Escherichia coli, and Bacillus subtilis. The Internet Journal of Tropical Medicine.3 (2): 277-286.
  26. Umbare, R. P., Mate1, G. S., Jawalkar, D.V., Patil, S. M., & Dongare, S. S. (2009). Quality evaluation of Phyllanthus amarus (Schumach) leaves extract for its hypolipidemic activity. Biology and Medicine1 (4): 28-33.
  27. Obianime A.W., & Uche F.I. (2009). The comparative effects of methanol extract of Phyllanthus amarus leaves and Vitamin E on the Sperm parameters of Male guinea pigs. Journal of Applied Sciences and Environmental Management 13(1): 37-41.[CrossRef]
  28. Chonoko, U. G., & Rufai, A. B. (2011).Phytochemical screening and antibacterial activity of Cucurbita pepo (pumpkin) against Staphylococcus aureus and Salmonella typhi. Bayero Journal of Pure and Applied Sciences 4(1): 145 – 147.[CrossRef]
  29. Scalbert, A. (1991). Antimicrobial Properties of tannins.Phytochemistry. 30: 3875-3883.[CrossRef]
  30. Akinpelu, D.A., Aiyegoro, D.A., & Okoh, A.I. (2008). In Vitro antimicrobial and Phytochemical Properties of Crude extract of Stem bark of Afzelia africana (Smith). African Journal of Biotechnology 7(20): 3662-3667.
  31. Dharmananda, S. (2003). Golinuts and the uses of tannins in Chinese Medicine.In proceedings of Institute for Traditional Medicine Portland, Oregon, USA, 25 September, 2003.
  32. Asquith, T.N., & Butler, L.G. (1986). Interaction of condensed tannins with selected proteins.Phytochemistry25 (7): 1591-1593.[CrossRef]
  33. Estrada, A., Katselis, G.S., Laarveid, B. and Bari, B. (2000). Isolation and evaluation of immunological adjuvant activities of saponins from Polygaja senega.L. Comparative Immunology. Microbial Infectious Disease 23: 27-43.[CrossRef] [PubMed]
  34. Price, K.R., Johnson, T.I., & Fenwick, G.R. (1987).The Chemistry and Biological Significance of Saponins in Food and feeding stuffs.Critical Reviews in Food Science and Nutrition26:22-48.[CrossRef] [PubMed]
  35. Trease, G.E., & Evans, W.C. (1978).Pharmacology (11th Edition). Bailliere Tindall Ltd, London. p. 784.
  36. Zee-cheng, R.K. (1997). Anticancer research on Loranthaceae plants. Drugs Future 22(5): 515-530.
  37. Waterman, P. H. (1992).Searching for bioactive compounds various strategies.Journal of National Products53 (1): 13-22.[CrossRef]
  38. Okwu, D.E., & Josiah, C. (2006).Evaluation of the chemical composition of two Nigerian Medicinal plants.African Journal of Biotechnolgy5 (4): 357-361.
  39. Adeneye, A. A., Benebo, A. S., & Agbaje, E. O. (2006b). Protective effect of the Aqueous Leaf and seed Extract of Phyllanthus amarus on Alcohol-induced hepatotoxity in rats. West Africa Journal of Pharmacology and Drug Research 22&23: 42-50.[CrossRef]
  40. Sydney, S., Lacy, R.W. and Bakhtiar, M. (1980). In: The Betalactam antibiotics Penicillin and Cephalosporin in perspective, Hodder and stongton, London, p. 224.
  41. Harvey, R. A., Champe, P.C., Howland, R. D., & Mycek M. J. (2006). Lippincott’s Illustrated Reviews Pharmacology (3rd edition). Lippincott Williams & Wilkins, Baltimore. Pp. 341-395.
  42. Cunha, B. A. (2001). Antibiotics side effects. Medical Clinics of North America85:149-185.[CrossRef] [PubMed]
  43. Madukosiri, C.H., Edike, T., & Ghandi, E. O. (2009). Comparative Studies of Susceptibility of Salmonella typhi to Antibiotics and some Plant Extracts.Nigerian Journal of Biochemistry and Molecular Biology24 (1):16-21.
  44. Nester, E.W., Anderson, D.G., Roberts, C.E., Pearsall, N.N., & Nester, M. T. (2004). Microbiology: A human perspective (4th edition) McGraw-Hill, New York. Pp. 817.
  45. Vermani, K., & Garg, S. (2002). Herbal medicine for sexually transmitted diseases and AIDS. Journal of Ethnopharmacology, 80: 49-66.[CrossRef] [PubMed]
  46. Presscott, L.M., Harley, J.P. and Klein, D.A. (2005). Microbiology. (6th ed.) Boston: McGraw-Hill.