In ternationa l Scholars Journa ls African Journal of Food Science Research ISSN 2375-0723 Vol. 6 (7), pp. 347-351, July, 2018. Available online at www.internationalscholarsjournals.org © International Scholars Journals Author(s) retain the copyright of this article. Full Length Research Paper A study of the scientific basis for the traditional use of medicinal plants among the local people in Nigeria Nwinyi, Obinna C. 1 *, Chinedu, Nwodo S. 1 , Ajani, Olayinka O. 2 , Ikpo Chinwe.O 2 and Ogunniran, Kehinde O. 2 1 Department of Biological Sciences, College of Science and Technology, Covenant University. KM 10 Idioroko Road, Canaanland, PMB 1023 Ota, Ogun State, Nigeria. 2 Department of Chemistry, College of Science and Technology, Covenant University, Km 10 Idiroko Road, PMB 1023, Ota, Ogun State, Nigeria. Accepted 11 January, 2018 The upsurge in the prevalence of side effects of many synthetic antimicrobial agents and incidence of multidrug resistant bacteria has spurred scientists on the research for plant based antimicrobial of therapeutic potentials. Ocimum gratissimum and Piper guineense present such potential of high medicinal value. These plants are used in Nigeria traditionally as condiments and for treatment of various ailments such as pyorrhea, dysentery and bronchitis. Aqueous and ethanol extracts of O. gratissimum and P. guineense leaves were screened for antibacterial activity against Escherichia coli and Staphylococcus aureus. Both extracts were found to exhibit selective inhibition against the isolates. The diameter zones of inhibition exhibited by the extracts were between 2 + 0.01 – 10 + 0.10 The minimum inhibitory concentration (MIC) determined by the agar diffusion method was between 10.00 and 2.50 mg/ml -1 . Ethanol extracts showed more inhibitory effect compared to the aqueous extracts. Results obtained show that the extracts of O. gratissimum and P. guineense possess some level of antibacterial activities against E. coli and S. aureus. Key words: Ocimum gratissimum, Piper guineense, antibacterial activity, minimum inhibitory concentration (MIC), Escherichia coli, Staphylococcus aureus. INTRODUCTION There is growing interest in exploiting plants for medicinal purposes especially in Africa. This stems from the fact that microorganisms are developing resistance to many drugs and as such created situation where some of the common and less expensive antimicrobial agents are loosing effectiveness (Montefore et al., 1989). Herbal medicine which uses medicinal plants primarily presents as an alternative to such situation (Sofowora, 1993). The- se medicinal plants have immensely contributed to the development of human health and welfare. Concomitant- ly, there is an increase in data and huge patronage to *Corresponding author. E-Mail: nwinyiobinna@gmail.com. herbal products round the world (Elsenberg et al., 1990; Omoseyindemi, 2003). Medicinal plants such as Ocimum gratissimum and Piper guineense have been asserted to provide various culinary and medicinal properties. These medicinal properties exert bacteriostatic and bacteriocidal effects on some bacteria. These effects have been attri- buted to the peptides, alkaloids, essential oils, phenols and flavonols which are major components in these pla- nts (Okigbo and Igwe, 2007). O. gratissimum belongs to the family leguminocaeae, commonly known as “alfava- ca”. It is naturally used in the treatment of different disea- ses which includes: upper respiratory tract infections, dia- rrhea, headache, conjunctivitis, skin disease, pneumonia tooth and gum disorder, fever and as mosquito repellants Nwinyi et. al. 348 (Onajobi, 1986; Ilori et al., 1996; Okigbo and Mneka, 2006). O. gratissimum is found in the tropical and warm temperature regions such as India and Nigeria (Okigbo and Ogbonnaya, 2006). Some of the vernacular names in Nigeria include: (Ncho-anwu, Ahuji) Igbo, (Efinrin,) Yoru- ba, (Aramogbo) Edo and (Daidoya) Hausa (Effraim et al., 2000). O. gratissimum has been described to have other species in the flora of tropical West Africa. These include: Ocimum viride Linn, Ocimum suave Linn, Ocimum basili- cum Linn and Ocimum canum Sims. Mshana et al. (2000) reported their numerous medical uses. The Ocimum oil has been described to be active again- st several species of bacteria and fungi. These include Listeria monocytogenes, Shigella, Salmonella and Prot- eus, for fungi Trichophyton rubrum, Trichophyton menta- grophytes, Cryptococcus neoformans, Penicillum islandi cum, and Candida albicans (Begum et al., 1993; Nwosu and Okafor, 1995; Akinyemi et al., 2004; Janine de Aqui- no Lemos et al., 2005; Lopez et al., 2005). From recent findings, O. gratissimum has proved to be useful in the medication for people living with Human Immuno defici- ency Virus (HIV), and Acquired Immuno Deficiency Synd- rome virus AIDs (Elujoba, 2000). In Congo, O. gratissimum decoction is used for gonorr- heal infection, vaginal douches for metritis and vaginitis and used in treatment of mental illness (Abdulrahma, 1992). P. guineense commonly referred to as African black pepper or Ashanti pepper is very similar to Piper nigrum which is the true pepper of commerce from which black and white peppers are processed (Isawumi, 1984). P. guineense belongs to the family piperaceae. It has more than 700 species throughout the tropical and subtropical regions of the world. It is known with different vernacular names in Nigeria: Igbo (Uziza), and Yoruba (iyere). P. guineense has culinary, medicinal, cosmetic and insecti- cidal uses (Dalziel, 1955; Okwute, 1992). P. guineense insecticidal activity against Zonocerus variegatus is attri- butable to the piperine-amide composed by the plant. The leaves are considered aperitive, carminative and eu- peptic. They are also used for the treatment of cough, bronchitis, intestinal diseases and rheumatism (Sumathy- kutty et al., 1999). Escherichia coli and Staphylococcus aureus are organi- sms associated with the gastrointestinal tract of man and animals. They occur in other environment. S. aureus is among the invasive gram positive known as pyogenic cocci implicated in several diseases of human. It has been found to be normal flora of upper respiratory tract and vagina . S. aureus has been known to produce heat stable toxins which are implicated in illnesses and stom- ach upset (CDC, 1989; Mosset et al., 1990). From litera- tures, S. aureus had shown to be very resistant to a wide variety of antibiotics (Nwinyi et al., 2008). E. coli, a facul- tative anaerobe of wide distribution in the environment, has been implicated in the cause of urinary tract infec- tions, meningitis, sepsis, wound infections, noscomial pneumonia and arthritis. A subgroup enterohemorrhagic E. coli (EHEC) can cause severe potentially fatal illness known as hemorrhagic colitis with symptoms of blood diarrhea and severe abdominal pain (Dolore et al., 2001). Thus, this study seeks to investigate the scientific basis for the traditional use, of these medicinal plants among the local people in Nigeria, for treating ailments associa- ted with E. coli and S. aureus infections. MATERIALS AND METHODS Collection of plant material Fresh samples of O. gratissimum leaves were collected from a for- est at Alor town in Anambra State between the hours of 6-8 am at a prevailing temperature of about 28+ 2 o C while the fresh leaves of P. guineense were bought from traditional healers at a local market in Awka, Anambra State. All the collections were done in the month of May. The plants were identified and authenticated at the Herbarium of the Department of Biological Sciences, Nnamdi Azikiwe Univer- sity, Awka, Anambra State. A voucher specimen was deposited at the Herbarium for reference purposes. Preparation of extracts Fresh leaves of O. gratissimum and P. guineense were thoroughly washed using tap water and rinsed with distilled water. The leaves were dried for 5 min in an oven at 60 o C to stop enzyme activity (Effraim et al., 2000). They were then air dried to a constant weight and milled to a fine powder with the aid of a Binatone blender (Mod- el BLG-401). Two solvent were used for the preparation of the ext- racts, namely distilled deionized water and ethanol 60% conc. The aqueous extract was prepared by weighing out (250 g) of the milled powdered leaves of O. gratissimum and P. guineense and adding in 200 ml of distilled deionized water respectively in a 500 mL beaker and stirring vigorously with a glass rod. The combination was allo- wed to settle for 3 h using the infusion method. The extracts were then filtered using Whatman no.1 filter paper. The ethanol extracts were obtained by weighing out same fraction 250 g of the different plants and wrapping it in Whatman no.1 filter paper and placed in the holding chamber of the soxhlet extractor. About 500 mL of the 60% ethanol was used as solvent for the extraction of the leaves using the reflux method for a period of 48 h. This was carried out exhaustively. From literature, Soxhlet extraction has been found to give a higher yield of the extracts. The extracts were then concen- trated by evaporating to dryness using rotary evaporator at a tem- perature 40 o C. A dark- green colored mass for P. guineense and O. gratissimum were obtained and stored in airtight bottles at 4 o C in a refrigerator until ready for use. The stored extract was reconstituted using the corresponding solvent to obtain extracts of several con- centrations 10.0, 5.0, 2.5, 1.25 mg/ml -1 and stored at 4 o C prior to determination of the minimum inhibitory concentration. Preparation of test organisms Clinical isolate of S. aureus was obtained from Glanson labora- tories, Awka, Anambra State, Nigeria while the strain of E. coli was obtained from a culture collection centre at Sammlung von Microor- ganismen (DSM) Germany. The isolates were tested for viability by resuscitating the organisms in buffered peptone broth, after which it was sub- cultured into nutrient agar medium and incubated at 37 o C for 24 h. The isolates were sub- cultured in a nutrient broth at 37 o C for 8 h prior to antibacterial testing. Table 1. Antibacterial activity of the aqueous extracts of Ocimum gratissimum and Piper guineense against Escherichia coli and Staphylococcus aureus. Plant Test organism Mean zones of Inhibition (mm) Control (water) 10.0 5.0, 2.5, 1.25 Ocimum gratissimum Escherichia coli - 3+ 0.09 - - - Staphylococcus aureus - 7.5+ 0.10 - - - Piper guineense Escherichia coli - 2 + 0.07 - - - Staphylococcus aureus - 4+ 0.10 - - - (-), No inhibition of growth; (10.0, 5.0, 2.5, 1.25) mg/ml -1 , different concentrations. Table 2. Antibacterial activity of the ethanol extracts of Ocimum gratissimum and Piper guineense against Escherichia coli and Staphylococcus aureus. Plant Test organism Mean zones of Inhibition (mm) Control (ethanol) 10.0, 5.0, 2.5, 1.25 Ocimum gratissimum Escherichia coli - 5+ 0.09 - - - Staphylococcus aureus - 8 + 0.11 7.5+ 0.11 6 + 0.06 - Piper guineense Escherichia coli - 10 + 0.10 8+ 0.10 7+ 0.11 - Staphylococcus aureus - 2+ 0.01 - - - (-), No inhibition of growth; (10.0, 5.0, 2.5, 1.25 mg/ml -1 ), different concentration. Determination of antibacterial activity of the aqueous and ethanolic extracts Agar well diffusion technique as described by Adeniyi et al. (1996) was used to determine the antibacterial activity of the extracts. An 18 mL of Mueller Hinton agar plates (MHA Difco, France) were see- ded with 2 mL of an overnight broth culture of each bacterial isolate (equivalent to 10 7 – 10 8 CFU mL -1 ) in sterile Petri-dish .The seeded plates were allowed to set after a uniform distribution of the bacte-rial isolate following slow rotation of the Petri dish . A standard sterile cork borer of 8 mm diameter was used to cut uniform wells on the surface of the agar. The wells filled with 2 mL of each extracts were with the aid of a sterile Pasteur pipette. The dishes were allowed to stand for 45 min at room temperature to allow proper diffusion of the extract to occur. The control experi- ments were setup with 2 mL of ethanol (60%) and distilled deioni- zed water which served as controls were also put in separate wells. All the plates were incubated at 37 o C for 24 h. The assay was conducted at regular intervals of 24 h until marked decline in the potency of the extracts to inhibit the growth of the test organisms was noticed. Zones of clearance round each well means inhibition and the diameter of such zones was measured in millimeter (mm). The minimum inhibitory concentration (MIC) in mg/ml was determi- ned by comparing the different concentration of the extracts that have different zones of inhibition and then selecting the lowest con- centration of each extract (Agaetmor, 2009). RESULTS Antibacterial activity of the extracts (aqueous and ethanol) extracts. The results of the antibacterial activity of the extracts against the test organisms namely E. coli, S. aureus are shown in Tables 1 and 2. The mean zones of inhibition of growth of the isolates are a function of relative antibacte- rial activity of the extracts. The extracts showed selective levels of activities against the isolates. The highest gro- wth inhibitory activity was obtained from the ethanol extr- acts. Ethanol and distilled deionized water served as con- trol and were inactive against both test organisms. The minimum inhibitory concentration of the aqueous and ethanol extracts Results of the minimum inhibitory concentration (MIC) are summarized in Table 3. Generally, less values were obt- ained for the aqueous extracts. Higher concentrations of the O. gratissimum extract were needed to inhibit E. coli when compared to S. aureus. The MIC of the aqueous extract of O. gratissimum was 10.00 and 10.00 mg ml -1 for E. coli and S. aureus, resp- ectively whereas that of the ethanol extract ranged bet- ween 10.00 and 2.50 mg ml -1 for E. coli and S. aureus, respectively. DISCUSSION From this investigation, the aqueous extracts of P. guine- ense and O. gratissimum showed minimal antibacterial activity against the isolates at 10 mg/ml -1 concentrations. This observed difference between these plants extracts may be due to insolubility of active compounds in water or the presence of inhibitors to the antimicrobial compo- nents (Okigbo and Ogbonnanya, 2006). Amadioha and Obi (1999), Okigbo and Ajale (2005a), Okigbo et al. 349 Afr. J. Food Sci. Res. Nwinyi et. al. 350 Table 3. Minimum inhibitory concentration (MIC) of the aqueous and ethanol extracts of Ocimum gratissimum and Piper guineense. Test organism Ocimum gratissimum Piper guineense Aqueous extract Ethanol extract (mg Aqueous extract Ethanol extract (mg (mg ml -1 ) ml -1 ) (mg ml -1 ) ml -1 ) Escherichia coli 10.00 10.00 10.00 2.50 Staphylococcus aureus 10.00 2.50 10.00 10.00 (2005b) reported that inactivity of plant extracts may be due to age of plant, extracting solvent, method of extrac- tion and time of harvesting of plant materials. Conversely, the ethanol extracts of P. guineense and O. gratissimum showed a concentration dependent gradient decrease in the level of inhibition against isolates. From the results there is variation in the degrees of antibacterial activities of the extracts on the isolates. The variation is presumed to be due to different active compounds present in these plants. Ethanol extracts of O. gratissimum showed more antibacterial activity against S. aureus than E. coli. This result is in agreement to Agatemor (2009) where it was reported that gram negative bacteria are more resistant than gram positive bacteria to the essential oil which are antimicrobial agents. Nweze et al. (2004) reported phyto- chemical screening of O. gratissimum in the presence of alkaloids, tannins, glycoside, saponin, resins, cardiac gly- coside, steroidal terpens and flavonoids. Flavonoids are reported to exhibit antioxidant activity (Ramanthan, et al., 1989) and are effective scavengers of superoxide anions (Robak and Gryglewski, 1988).Thus this can significantly affect the cell wall of S. aureus which invariably may lead to the collapse of the cell wall and overall, affect the enti- re mechanism of the organism. E. coli a gram negative organism contains a high level of lipid material. These materials are thought to make a substantial contribution to the mechanism whereby inju- rious chemicals are prevented from reaching their sites of action within the cell. P. guineense ethanol extract sho- wed significant inhibition to E. coli due to high content of essential oils such as monoterpenes and sesquiterpene and minimal effect was observed on S. aureus. This result agrees to Obafemi et al. (2006) where sesqui- terpene was tested against S. aureus. From this study, it was observed that ethanol extracts exhibited high inhibitory activity on the test organisms. This can be deduced to the ability of ethanol to extract more of the essential oils and secondary plant metabo- lites which are believed to exert antibacterial activity on the test organisms. 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