Anti-typhoidal and Toxicity Effect of the Combined Extracts of Monetes kerstingii, Mitragyna inermis and Boswellia dalzielii

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1 International Journal of Microbiology and Application 2016; 3(2): ISSN: X (Print); ISSN: (Online) Anti-typhoidal and Toxicity Effect of the Combined Extracts of Monetes kerstingii, Mitragyna inermis and Boswellia dalzielii Zumbes Hosea Jwan 1, Nanfa Ponlir 1, Dabo Diana Adah 2, Azi Hezikiah Yusuf 1, David Veronica Ekpiwre 1, Anejo-Okopi Joseph 1, * 1 Department of Microbiology, Faculty of Natural Sciences, University of Jos, Jos, Nigeria 2 Department of General Health Services, Plateau State College of Health Technology, Zawan, Jos, Nigeria address josephokopi@yahoo.com (Anejo-Okopi J.) * Corresponding author To cite this article Zumbes Hosea Jwan, Nanfa Ponlir, Dabo Diana Adah, Azi Hezikiah Yusuf, David Veronica Ekpiwre, Anejo-Okopi Joseph. Anti-typhoidal and Toxicity Effect of the Combined Extracts of Monetes kerstingii, Mitragyna inermis and Boswellia dalzielii. International Journal of Microbiology and Application. Vol. 3, No. 2, 2016, pp Received: June 16, 2016; Accepted: June 27, 2016; Published: July 29, 2016 Abstract The combined extracts of Monetes kerstingii flower, Mitragyna inermis root and Boswellia dalzielii bark were screened for their antimicrobial activity on Salmonella Typhi and Salmonella Paratyphi. These combined plants were also screened for their phytochemical properties as well as toxicity profile on Mice. The antimicrobial susceptibility pattern of the combined extracts together with the MIC, were determined using agar well diffusion and broth dilution method respectively. The susceptibility of the test organisms to the plants extracts was dose dependent. The ethanolic extract had stronger antimicrobial activity with zone of inhibition of 20mm against S. Paratyphi than the aqueous extract, which had 17mm. Meanwhile, the aqueous extract exhibited stronger antibacterial activity of 20mm against S. Typhi when compared with 18mm of the ethanolic extract. Both organisms had MICs of 12.5mg/ml for ethanolic extract. S. Typhi and S. Paratyphi had MICs of 12.5mg/ml and 25mg/ml respectively for aqueous extract. The MBC of S. Typhi was found to be 100mg/ml for ethanolic extract. S. Paratyphi had MBC of 200mg/ml for ethanolic extract. The MBC of the aqueous extract for S. Typhi and S. Paratyphi were 200mg/ml and 400mg/ml respectively. The phytochemical screening revealed the presence of tannins, flavonoids, saponins, cardiac glycosides, resins, alkaloids, carbohydrates, anthraquinones, steroids and terpenoids. The combined extract was non-toxic orally, but moderately toxic intraperitoneally with an LD 50 of mg/kg body weight in Mice. Thus the traditional claims of the oral use of these combined plants extracts in the treatment of typhoid fever is therefore justified. Keywords Anti-typhoidal, Toxicity, Extracts, Monetes kerstingii, Mitragyna inermis and Boswellia dalzielii 1. Introduction Herbs are plants or plant parts valued for their medicinal, aromatic or savoury qualities. The application of herbal plants as traditional health remedies is the most popular for over 80% of the world population in Asia, Latin America and Africa, and had reported to have minimal side effects [1]. The increasing cost of conventional drugs has resulted to a high participation in the use of plants as alternative for combating a wide range of ailments, particularly in the resource poor communities of the African continent [2]. Although, several synthetic drugs are wide available, attention is currently being focused on the use of plants and plant products in the prevention of specific diseases because of several side effects associated with the use of synthetic drugs [2]. Typhoid fever is one of the common causes of morbidity and mortality throughout the world, with approximately 15% case fatality rate. Annually, it is estimated that there are about 16,000,000 typhoid cases

2 7 Zumbes Hosea Jwan et al.: Anti-typhoidal and Toxicity Effect of the Combined Extracts of Monetes kerstingii, Mitragyna inermis and Boswellia dalzielii world-wide; with 600,000 deaths resulting from these cases [3]. Enteric fever is particularly a major public problem in Nigeria, being a developing country. Salmonella Typhi and Salmonella Paratyphi have devised several mechanisms of resistance as mediated by plasmid to antibiotics frequently used on them. This menace prompted the search and subsequent testing of these alleged holistic medicinal plants for their antityphoidal activity against S. Typhi and S. Paratyphi in vitro and in vivo. Monetes kerstingii is a shrub or a tree with an open, rounded crown. It is called Hantsi in Hausa. Decoctions of the bark and leafy twigs are taken to treat dysentery and diarrhoea. A decoction of root and leafy twigs is used to treat jaundice [4] Mitragyna inermis exists as a shrub or tree with a dense, wide crown. The plant is known as Giyayya in Hausa; Okobo in Yoruba; and Koli in Fulfulde. M. inermis leaf extracts are used in Ivorian traditional medicine to treat diabetes [5]. The leaf, stem bark and root extracts of the plant has been commonly used in Nigerian traditional medicine to treat diverse medical conditions, including longterm ailments such as diabetes and epilepsy [6]. Boswellia dalzielii is a tree of the savannah forest recognizable by its papery bark peeling off in a ragged manner. It is commonly called Frankincense tree. The tree is called Hano or Arrarabi in Hausa; and Mangalede in Fulfulde. The decoction of the stem bark is used to treat rheumatism, septic sores, venereal and gastrointestinal diseases [7], [8]. These three plants have been used in combinations by Fulani herbalists around Jos, and some inhabitants of Langtang North LGA in the treatment of typhoid fever in Plateau state. The aim of the present study was to evaluate the antityphoidal activity, phytochemical composition and toxicity profile of the combined extracts. 2. Materials and Method 2.1. Collection and Identification of the Plant Materials The flowers of Monetes kerstingii, roots of Mitragyna inermis, and stem barks of Boswellia dalzielii were collected from Toro Local government Area of Bauchi state. The samples together with the fresh whole plants were taken to the herbarium unit of Federal College of Forestry, Jos for authentication and voucher number Preparation of the Plant Extracts The roots and stem barks were washed with several volumes of clean water. They were then cut into different pieces. The three samples- root, stem bark and flower were air-dried under a shade. The dried parts were later pulverized separately into powdered form using a mortar and pestle. The powdered parts were extracted using a modified method of Timothy et al. (2015). In this case, 200g of each of the powdered materials were weighed and macerated separately with 2000ml of distilled water and 2000ml of 70% ethanol. Each of the mixtures was properly agitated and kept undisturbed for 24 hours. These were further filtered using a white muslin cloth and Whatman No _ 1 filter paper. The filtrates were evaporated in a rotary evaporator at 60 C (aqueous) and 50 C (ethanol). The resultant extracts were scraped and stored in labeled sterile sample containers. The extracts obtained in each case were called aqueous extract and ethanolic extract Determination of Phytochemicals The active pharmacological components of the combined extracts that actually effect positive response in antityphoidal therapy were determined for both ethanolic and aqueous extracts, using standard qualitative procedures as previously described [9]. These include: test for cardiac glycosides, test for resins, test for flavonoids, test for saponins, test for anthraquinones, test for alkaloids, test for steroids and terpenoids, test for carbohydrates and test for tannins Preparation of Plant Extracts Concentration The three plant extracts were mixed in equal proportion to form the combined extracts. Then 4g was weighed from the aqueous combined extracts and dissolved in 5ml of sterile distilled water to produce a concentration of 800mg/ml. Similarly, 4g from the ethanolic combined extracts was weighed and dissolved in 5ml of Dimethylsulfo-oxide (DMSO). The stock solution was used to prepare the following concentrations through two-fold dilutions: 400mg/ml, 200mg/ml, 100mg/ml, 50mg/ml, 25mg/ml, 12.5mg/ml and 6.25mg/ml. The dilutions were labelled appropriately Collection, Preparation and Identification of Isolates The test organisms for this work were clinical isolates of S. Typhi and S. Paratyphi, which were collected from Central Diagnostic Laboratory of National Veterinary Research Institute, Vom. The procedure for culture standardization as described by the Clinical and Laboratory Standards Institute was used [10]. In this case, fresh cultures of each of the test organisms were made from the pure cultures on Mueller- Hinton agar slants. Several colonies of each of the bacterial isolates were picked into 2ml of Peptone water, and incubated for 20 minutes at 37 C. The peptone water tube was vortexed to create a smooth suspension. The turbidity of actively growing bacterial suspension was adjusted to match the turbidity standard of 0.5 McFarland units. This was done by either adding more organisms or diluting with more sterile peptone water as the case may be. The standardized suspension was obtained as it matched 0.5 McFarland standards, which is equivalent to CFU/ml. The suspension was used within 15 minutes of preparation. The identification of bacterial isolates was confirmed by Gram staining and biochemical procedures such as triple sugar ion medium test (TSIA), motility test, oxidase test and citrate

3 International Journal of Microbiology and Application 2016; 3(2): utilization test Plant Extract Activity Assay The agar well diffusion method as previously described by Lino and Deogracious was used [11]. Mueller-Hinton agar was prepared, dried, and a standardized culture of each test bacterium was spread separately on the agar using a sterile cotton swab. They were allowed to dry, and a sterile cork borer of 6.0mm was used to bore 9 wells in each inoculated plate. 7 wells were filled to the brim with the combined extracts. The other 2 wells left were filled with ciprofloxacin (as positive control) and sterile distilled water (as negative control). The plates were then incubated at 37 C for 24 hours. Zones of inhibition 7mm were considered positive [12]. The MIC of the potent extracts was determined according to the technique as described by Junaid et al. [13]. Mueller-Hinton broth was prepared and dispensed into sterile tubes, and doubling dilution was done. Then Standardized suspensions of the test organisms was inoculated into the two-fold dilution tubes. The tubes were all incubated at 37 C for 24 hours. The least concentration that inhibited the growth of the test organism was taken as the MIC. Two loopful of broth culture were taken from the tubes that showed no growth in the MIC determination, and were inoculated onto Mueller-Hinton agar plate. The plates were incubated at 37 C for 24 hours. The least concentration that did not show any growth after the incubation was therefore regarded as the MBC Acute Toxicity of the Aqueous Extract The Mean Lethal Dose (LD 50 ) of the combined extracts was determined using modified Lorke s method (1983), in Mice through oral and parenteral route. 16 Mice were divided into 4 groups. The 1st group was treated with 10mg/kg, 100mg/kg, 1000mg/kg and 2000mg/kg of the combined extracts orally. The same dose was administered to the 2nd group, but in this case intraperitoneally. The doses for the 3rd and 4th groups depend on the reactions obtained from group1 and 2. Base on the results of group3 and 4, the LD 50 was calculated. Which is the geometric mean of the highest nonlethal dose (with no deaths) and the lowest lethal dose (where deaths occurred) (Lorke, 1983). 3. Results Table 1. Susceptibility of the Test Organisms to Aqueous and Ethanolic Extracts. Zones of inhibition (mm) of the various concentrations (mg/ml) The results of antibacterial susceptibility of the ethanolic and aqueous extracts were presented in table 1. The ethanolic extract had higher antibacterial activity of 20mm against S. paratyphi than S. Typhi, which had 18mm. Meanwhile, the aqueous extract exerted more antibacterial activity of 20mm against S. Typhi, as compared with17mm on S. paratyphi. The combined extracts showed activity on S. paratyphi at concentrations 25mg/ml. Whereas, the activity against S. Typhi was at concentrations 50mg/ml. The MIC of the combined extracts for ethanolic was 12.5mg/ml for both organisms tested. These were presented in table 2. While the MIC of the combined extracts for aqueous was found to be 12.5mg/ml for S. Typhi, and 25mg/ml for S. paratyphi as presented in table 3. The MBC of the combined extracts against S. Typhi for ethanolic was found to be 100mg/ml. Meanwhile, the MBC for the ethanolic extract against S. paratyphi was 200mg/ml. These were presented in table 4. The MBC for the aqueous extract against S. Typhi and S. paratyphi as presented in table 5, were 200mg/ml and 400mg/ml respectively. The results of the acute toxicity as presented in table 6, showed that the combined extracts had an LD 50 of mg/kg intraperitoneally, but non-toxic orally. The synopsis of the qualitative phytochemical screening carried out on the combined extract for both aqueous and ethanolic was shown in table 7. This revealed the presence of tannins, flavonoids, saponins, cardiac glycosides, resins, alkaloids, carbohydrates, anthraquinones, steroids and terpenoids. Cipro A E A E A E A E A E A E A E 50mg/ml SDW S. Paratyphi S. Typhi Key: = No zone of inhibition, A = Aqueous extract, E = Ethanolic extract, Cipro = Ciprofloxacin (positive control), SDW = Sterile distilled water (negative control) Table 2. The Minimum Inhibitory Concentration (MIC) of the Ethanolic Extract. S. Typhi S. Paratyphi MIC (mg/ml)

4 9 Zumbes Hosea Jwan et al.: Anti-typhoidal and Toxicity Effect of the Combined Extracts of Monetes kerstingii, Mitragyna inermis and Boswellia dalzielii Table 3. The Minimum Inhibitory Concentration (MIC) of the Aqueous Extract. S. Typhi S. Paratyphi Table 4. The Minimum Bactericidal Concentration (MBC) of the Ethanolic Extract. S. Typhi S. Paratyphi Table 5. The Minimum Bactericidal Concentration (MBC) of the Aqueous Extract. S. Typhi S. Paratyphi Table 6. Acute Toxicity of the Combined Aqueous extract. MIC (mg/ml) MBC (mg/ml) MBC (mg/ml) PHASE 1 (Orally) PHASE 2 (Intraperitoneally) Doses (mg/kg) Result Remark Result Remark 10 No death 4/4 No death 4/4 100 No death 4/4 No death 4/ No death 4/4 No death 4/ No death 4/4 No death 4/4 PHASE 3 (Orally) PHASE 4(Intraperitoneally) 3000 No death 4/4 No death 4/ No death 4/4 No death 4/ No death 4/4 Death ¼ 6000 Death 1/4 Death ¼ LD 50 Nil mg/kg Key: 4/4 = All survived; 1/4=One survived Table 7. Phytochemical Screening of the combined extracts. Constituent Aqueous extract Ethanolic extract Tannins Flavonoids Saponins Alkaloids Cardiac glycosides Anthraquinones Resins ++ + Steroids and terpenoids Carbohydrates Key: - = Not detected; + = Present in trace amount; ++ = Present in moderate amount; +++ = Present in large amount 4. Discussion The results from susceptibility test as determined by agar well diffusion showed that the combined extracts had greater activity against the causative agents of enteric fever (i.e S. Typhi and S. paratyphi). This is because, the highest zones of inhibition obtained for S. Typhi and S. paratyphi were 20mm each. According to earlier reported study, zones of inhibition 7mm are considered positive [12]. The ethanolic extract had pronounced activity than the aqueous extract on S. paratyphi. Whereas, the aqueous extract had greater activity on S. Typhi than the ethanolic counterpart. The activity was concentration dependent. There was no observed antibacterial activity with negative control agent (sterile distilled water). This implies that the tested organisms were susceptible to the plant extracts. The MIC for both organisms was 12.5mg/ml for the ethanolic extract. For the aqueous extract however, S. Typhi had lower MIC of 25mg/ml than S. Paratyphi, which had 25mg/ml. The bactericidal activity was more pronounced on S. Typhi than on S. Paratyphi for both ethanolic and aqueous extract. The results obtained so far from the antibacterial assay suggested that the combined extracts is both bacteriostatic and bactericidal, depending on its concentration.

5 International Journal of Microbiology and Application 2016; 3(2): The preliminary phytochemical screening revealed the presence of tannins, flavonoids, cardiac glycosides, resins, alkaloids, anthraquinones, carbohydrates,steroids and terpenoids. It is probably that the antityphoidal properties allotted to these combined extracts are related to these bioactive compounds. In addition, tannins have been reported to prevent the development of microorganisms by precipitating microbial protein and making nutritional protein unavailable for them [14]. The presence of flavonoids and tannins may also be responsible for the antibacterial properties as they do play a major role in bacteria growth inhibition (Wunwisa and Areeya, 2005). Individual plants showed less or no activity against the tested organisms as compared to the combined one. Abdulazeez et al reported that, B. dalzielli exhibited no activity against S. Typhi even up to 80mg/ml [15]. The results of acute toxicity study showed that the aqueous extract of the combined plants was non-toxic orally. However, it was found to be relatively less toxic in Mice intraperitoneally with lethal dose that can kill 50% of the animals (LD50) being equal to mg/kg. And it is assumed that LD 50 values 4000mg/kg but 5000mg/kg are moderately toxic [16]. Reported from the acute toxicity of Mitragyna inermis by suggested that the plant was relatively less toxic in the tested animals [6]. Another study corroborated on the acute toxicity of Boswellia dalzielii as non-toxic in the experimental animals [15]. 5. Conclusion The pronounced result obtained from the antimicrobial studies, together with the MIC and MBC values against the test organisms were all promising. Moreover, the combined extracts were non-toxic orally in the tested animals. Thus, this study provides a scientific rational for the traditional oral use of these combined plants extracts in the treatment and management of typhoid fever by the inhabitants of Langtang North and Fulanis in Plateau state. References [1] Doughari JH. Antibacterial activity of Tamrindus indica Linn. Tropical Journal of Pharmaceutical Research, 2006; 5: [2] Timothy SY, Helga BI, Bomai HI, Musa AH. Acute and Subchronic Toxicity Study of the Aqueous and Ethanolic Extracts of Mitragyna inermis bark in Albino rats. International Journal of Pharmacology and Toxicology, 2015; 5(1): [3] World Health Organization. Communicable Disease Surveillance and Response, Vaccines and Biologicals: The diagnosis, treatment and prevention of typhoid fever. Background Document. WHO, 2003, V&B/ [4] Yahaya O, Yabefa AJ and Usman B. Phytochemical Screening and Antibacterial Activity of Combretum glutinosum Extract against Some Human Pathogens. British Journal of Pharmacology an d Toxicology, 2012; 3(5): [5] Konkon NG, Adjoungouna AL, Manda P, Simaga DN, Guessan KE, Kone BD. Toxicological and phytochemical screening of Mitragyna inermis (Wild) O Ktze (Rubiaceae), antidiabetic plant. Journal of Medicinal Plant Research, 2008; 2(10): [6] Timothy SY, Goji SY, AbdussalamB, Mava Y, and Galadima IH. Antibacterial and phytochemical screening of the ethanolic leaf extract of azadirachta indica (neem) (meliaceae). International Journal of Applied Biology and Pharmaceutical Technology, 2011; 2(3): 1-6. [7] Burkill HM. Useful plants of West Tropical Africa: Cassia species. Fioterapia, 1985; 61: [8] Evans WC. Trease and Evans Pharmacognosy; 13th Ed; ECBS/Baillere Tindal, United Kingdom, 1989; [9] Trease GE, Evans WC. Pharmacognosy. 15 th edition, Bailliere Tindal, London: 2002; pp [10] Clinical Laboratory Standards Institute, CLSI.(2006). Performance standards for antimicrobial disc susceptibility tests. Approved standard. 9th ed. CLSI document M2-A9, Wayne, Pa; [11] Lino A, Deogracious, O. The In vitro antimicrobial activity of Annona senegalensis, Securidacca longipendiculata and Steganotaenia araliaca: Uganda Medicinal plants. African Health Sciences, 2006; 6(1): [12] Mbah JM, Ngemenya AL, Babaiaka SB, Nubed LN, Nyongdela KD, Lemuth, ND, Efange SMN. Bioassay-guided discovery of antibacterial agents: In vitro screening of Peromia vulcania, Peperomia fernandopoioana and Scleria striatinux. Annals of Clinical Microbiology and Antimicrobials, 2012; 11: 10. [13] Junaid SA, Olabode AO. Onwuliri FC, Okwori AEJ, Agina SE. The antimicrobial potency and Synergistic Effect of certain plant extracts against Food-borne Diarrheagenic Bacteria. International Journal of Biomedical and Pharmaceutical Sciences, 2006; 2: [14] Fluck H. Medicinal plants and their uses. W. Feulshams and Co. Ltd. New York, 1973; pp [15] Abdulazeez AT, Kabele-Toge B, Lawal M, Abubakar G. Phytochemical, Antibacterial and Toxicological Studies of Aqueous Stem Bark Extract of Boswellia dalzielli. African Journal of Pharmaceutical Research and Development, 2013; 5(1): [16] Lorke D. A new Approach to Practical Acute Toxicity Testing. Archieve of Toxicology, 1983; 53: [17] Wakirwa JH, Yawate UE, Zakama SG, Muazu J. Madu SJ. Phytochemical and Antibacterial Screening of the Methanol Leaf Extract of Mitragyna Inermi. International Journal of Pharmaceutical Research and Innovation,2013; 6: 1-6.

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