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Phytochemical screening and in vitro antimicrobial activities of Mimosa invisa Mart. leaves and stems

Phytochemical screening and in vitro antimicrobial activities of Mimosa invisa Mart. leaves and... Abstract Preliminary phytochemical screening was carried out and in vitro antimicrobial activities of leaves and stem of Mimosa invisa Mart. against some clinical pathogens were tested using standard techniques. Significance was measured using Duncan’s multiple range test. The inhibitory actions of the leaf and stem extracts increased with concentration. The rates of inhibition of the two extracts were low and high against Salmonella typhi and Aspergillus flavus respectively. The phytochemicals present in the plant parts had antimicrobial properties and reveal that M. invisa may have potential in pharmaceutical applications. Mimosa invisa, alkaloid, antimicrobial actions, zone of inhibition, Salmonella typhi, Aspergillus flavus Introduction The leaves, stems, flowers, fruits, seeds, roots, rhizomes and bark of some plants may be used for medicinal purposes. Many plant and herb species have unrealized antimicrobial and antiviral features (Shelef, 1983; Zaika, 1988) and may thus be a source of phyto-antimicrobial agents (Das et al., 1999). Sieradzki et al. (1999) stated that the emergence of bacterial strains with reduced vulnerability to antibiotics and the increasingly wide spread of multidrug resistant strains of bacteria would require the search for new infection fighting strategies. The properties of medicinal plants are of great benefit (Finar, 1986; Harborne, 1998). Antibacterial compounds extracted from plants have drawn much attention and could be used as an alternative strategy to prevent the spread of diseases. Flavonoids have been shown to inhibit topoisomerase enzymes. Alkaloids are of great benefit to pharmaceutical industries including as anti-malarial agents (for example, quinine) (Kittakoop, Mahidol, Ruchirawat, 2014). It has been recorded that tannin has antiviral, antibacterial and antiparasitic properties (Lui et al., 2004; Njoku and Obi, 2009). Saponins have been implicated as bioactive antibacterial agents (Mandel, Babu, Mandal, 2005). Anthraquinones are used to relieve constipation (Kittakoop, Mahidol, Ruchirawat, 2014). However, there is no information on the antimicrobial actions of phytochemicals present in M. invisa. Therefore, the objectives of this work were to determine the phytochemicals present in the plant as well as to assess their antimicrobial activity against selected microorganisms that cause ailments in humans: Escherichia coli, a facultative anaerobic, gram-negative, rod-shaped bacterium that is abundant in the human gut (OED, 2005); Salmonella typhi, a gram-negative bacterium that causes typhoid fever, a disease that has afflicted humans throughout history and continues to be a public health concern (Deng et al., 2003); Klebsiella pneumoniae, a gram-negative nonmotile, lactose-fermenting, facultative anaerobic, rod-shaped bacterium that is enclosed in a capsule and causes destructive changes to human and animal lungs if aspirated (inhaled), particularly to the alveoli (in the lungs), resulting in bloody sputum (Ryan and Ray, 2004); and Aspergillus flavus, a saprotrophic and pathogenic (Masayuki and Katsuyai, 2010) fungus with a cosmopolitan distribution (Ramírez-Camejo et al., 2012). Materials and methods Collection of sample The leaves and stem of mature M. invisa were collected from a roadside at Idi-Oshe, opposite IITA Ibadan, Oyo State, Nigeria, in August 2016. The analyses were then carried out at the Department of Microbiology in IITA, Ibadan. The plant was identified by Mr. Kayode, a Plant Taxonomist at IITA Ibadan. Preparation of samples for analyses The fresh specimens were cleaned with clean water and oven dried at a temperature of 60°C for 72 h. The samples were separated by hand and were ground using a grinding machine (SPM-5, China), then placed in an air-tight container. Extraction of plant materials Methanol extraction An aqueous decoction of the plant was prepared by soaking 500 g of the powdered samples of leaves and stem each in 2.5 L of methanol. The whole setup was left at room temperature for 72 hours. The extract was then concentrated using a rotary evaporator (RE-52A, China) and the solvent allowed to evaporate. The concentrated extract was stored in an air-tight container in a cold store at a regulated temperature of 20°C until it was required for analyses. Preliminary phytochemical screening The methods and tests used in the phytochemical screening of the plant extract were described by Ezeabara and Okonkwo (2016). The sign ‘+’ denotes presence. Quantitative phytochemical screening Alkaloids and flavonoids were determined using the alkaline precipitation gravimetric method and gravimetric method of Harborne (1973), respectively. The method used to determine saponins was described by the AOAC (2000). The Folin-Dennis colorimetric method was used to determine the tannin content of the sample (Kirk and Sawyer, 1998). Anthraquinone was determined by the spectrophotometric method used by Ezeabara and Okonkwo (2016). Microbial analysis Isolation of the test organisms Using a wire loop, colonies of the test organisms (Escherichia coli, Salmonella typhi, Klebsiella pneumoniae and Aspergillus flavus) were collected from pure cultures kept at the Soil Microbiology Unit, IITA Ibadan, Oyo State, Nigeria. Antimicrobial test procedures Preparation of stock solution Stock solutions of the two methanolic extracts of leaves and stem were prepared by weighing 2.0 g of each methanolic extract using electronic scales. This was then dissolved completely in 2.0 ml of dimethyl sulphoxide (DMSO) in a sterile test tube, giving a stock solution concentration of 1000 mg/ml per extract. The stock solution was then labelled and stored at room temperature until needed for use. Determination of inhibitory activity The inhibitory activity of the methanolic extract of the leaves and stems of M. invisa, antibiotics (positive control), and dimethyl sulphoxide (negative control) was determined using the disc diffusion method in 5 mm discs. The concentrations used were as follows: 1000 mg/ml of the methanolic extract, 100 mg/ml of antibiotic (Streptomycin) and 1000 mg/ml of DMSO. After the discs had absorbed the leaf and stem extract, the antibiotic or the control DMSO, the discs were removed, dried and placed on media on which the test microorganisms were freshly inoculated, then incubated at 37°C for 24 hours for bacteria and 25°C for 72 hours for fungi. Antimicrobial activity was determined after 24 hours (for bacteria) of incubation and 72 hours (for fungi) of incubation by measuring the zone of inhibition around each paper disc in millimetres (mm) (Reynolds, 2003). Determination of minimum inhibitory concentration The minimum inhibitory concentrations (MIC) of the absolute (stock) concentrations were determined using the agar well diffusion method; five sterile plates were prepared, and nutrient broth was poured into each of the plates and then allowed to dry. Using standardized inoculums (106 cfu/ml), a loop full of the different test organisms was streaked onto each of the five plates when dry. Then, 10 holes were dug using an agar borer for the varying concentrations of methanolic extracts of leaves and stem. A ruler was used to separate the holes for each of the methanolic extracts. Varying concentrations of the different extracts were made by serial dilution using DMSO as the diluent. The absolute/stock concentrations of extracts used were 1000 mg/ml (equivalent to 100%). Five test tubes per extract were prepared in a test tube rack. The dilutions were as follows: 50% (500 mg/ml), 25% (250 mg/ml), 12.5% (125 mg/ml) and 6.25% (62.5 mg/ml) in each of the test tubes. DMSO served as the negative control. A measured 50 μL volume of each dilution was added aseptically into the holes seeded with the test organisms in the nutrient agar plate using a syringe and placed in an incubator at 35°C for 24 hours. MIC was considered as the lowest concentration of methanolic extract showing a clear zone of inhibition (Thongson et al., 2004). Determination of minimum bactericidal/fungicidal concentration The plates with the MIC were incubated for a further 24 hours at 35°C to test which organism would grow on the zones of inhibition. Those plates on which organisms were completely killed after 24 hours, and clear zones remained, were referred to as bactericidal for bacteria and fungicidal for fungi (Espinel-Ingroff, 2002). Statistical analysis The test was carried out using a completely randomized design. Analysis of Variance (ANOVA) using SPSS version 21 was employed to analyse the data collected in the study. The Duncan’s multiple range test (DMRT) was used to test the difference among treatment means with more than two levels. All statistical analyses were carried out at 5% level of significance. The data were expressed as the mean (±standard deviation) of triplicate determinations. Results and discussion The result of the qualitative phytochemical analysis of the leaf and stem extracts of M. invisa demonstrated that alkaloids, anthraquinones, saponins, tannins and flavonoids were present (Table 1). Moreover, the quantitative analysis revealed that the leaf had the largest concentration of alkaloids among the plant parts, at 0.16 ± 0.11 mg/100 g, and anthraquinones at 0.88 ± 0.06 mg/100 g, whereas the highest concentrations of saponins at 16.81 ± 5.12 mg/100 g, tannins at 0.15 ± 0.04 mg/100 g and flavonoids at 0.48 ± 0.41 mg/100 g occurred in the stem (Table 2). These correspond largely with the findings of Ezeabara and Mbah (2016), who reported a wide range of chemical compounds in the leaves, stem and root of M. invisa and M. pudica. Stated that M. invisa is traditionally used in the treatment of diabetes. In addition, this species has been reported to have potent hypoglycaemic effects in alloxan-induced diabetic mice. The medicinal properties of this plant are likely to be a result of the wide range of biological activities of the phytochemicals. The pharmacological activities of alkaloids are varied, and include anti-malarial (e.g. quinine) and antiasthma activity (e.g. homoharringtonine) (Kittakoop, Mahidol, Ruchirawat, 2014). Due to saponins’ immune-enhancing properties they are used as immunological adjuvants in the formulation of vaccines (Francis et al., 2002). Tannins may be used medicinally in antidiarrheal, haemostatic and antihaemorrhoidal compounds (Praveen and Kumud, 2012). Kanokmedhakul, Kanokmedhakul, Phatchana (2005) reported that anthraquinones isolated from Primatomeris fragrans showed both antifungal and antituberculosis activities. Table 1. Qualitative phytochemical composition of the leaf and stem extracts of Mimosa invisa Phytochemicals . Plant part . Leaf . Stem . Alkaloid + + Saponin + + Flavonoid + + Phenol + + Tannin + + Anthraquinone + + Phytochemicals . Plant part . Leaf . Stem . Alkaloid + + Saponin + + Flavonoid + + Phenol + + Tannin + + Anthraquinone + + Open in new tab Table 1. Qualitative phytochemical composition of the leaf and stem extracts of Mimosa invisa Phytochemicals . Plant part . Leaf . Stem . Alkaloid + + Saponin + + Flavonoid + + Phenol + + Tannin + + Anthraquinone + + Phytochemicals . Plant part . Leaf . Stem . Alkaloid + + Saponin + + Flavonoid + + Phenol + + Tannin + + Anthraquinone + + Open in new tab Table 2. Mean quantitative phytochemical composition of the leaf and stem of M. invisa composition (mg/100 g) Plant part . Saponin . Alkaloid . Tannin . Anthraquinone . Flavonoid . Stem 16.81 ± 5.12b 0.04 ± 0.01a 0.15 ± 0.04b 0.79 ± 0.09a 0.48 ± 0.41b Leaf 8.64 ± 1.47a 0.16 ± 0.11b 0.10 ± 0.00a 0.88 ± 0.06b 0.38 ± 0.32a p-value 0.19 0.13 0.28 0.08 0.01 Plant part . Saponin . Alkaloid . Tannin . Anthraquinone . Flavonoid . Stem 16.81 ± 5.12b 0.04 ± 0.01a 0.15 ± 0.04b 0.79 ± 0.09a 0.48 ± 0.41b Leaf 8.64 ± 1.47a 0.16 ± 0.11b 0.10 ± 0.00a 0.88 ± 0.06b 0.38 ± 0.32a p-value 0.19 0.13 0.28 0.08 0.01 Results are in mean ± std error of duplicate determinations. The same letter in a column means that the respective means were not significantly different. Bold values indicates that p<0.05. Open in new tab Table 2. Mean quantitative phytochemical composition of the leaf and stem of M. invisa composition (mg/100 g) Plant part . Saponin . Alkaloid . Tannin . Anthraquinone . Flavonoid . Stem 16.81 ± 5.12b 0.04 ± 0.01a 0.15 ± 0.04b 0.79 ± 0.09a 0.48 ± 0.41b Leaf 8.64 ± 1.47a 0.16 ± 0.11b 0.10 ± 0.00a 0.88 ± 0.06b 0.38 ± 0.32a p-value 0.19 0.13 0.28 0.08 0.01 Plant part . Saponin . Alkaloid . Tannin . Anthraquinone . Flavonoid . Stem 16.81 ± 5.12b 0.04 ± 0.01a 0.15 ± 0.04b 0.79 ± 0.09a 0.48 ± 0.41b Leaf 8.64 ± 1.47a 0.16 ± 0.11b 0.10 ± 0.00a 0.88 ± 0.06b 0.38 ± 0.32a p-value 0.19 0.13 0.28 0.08 0.01 Results are in mean ± std error of duplicate determinations. The same letter in a column means that the respective means were not significantly different. Bold values indicates that p<0.05. Open in new tab The antimicrobial results of the methanolic extracts of leaves and stem of M. invisa indicated that the plant showed antimicrobial activity against the tested microorganisms at five concentrations: 62.5, 125, 250, 500 and 1000 mg/ml (Tables 3–7). At 62.5 mg/ml, the stem extract showed greater inhibition of Salmonella typhi (0.50 ± 0.710 mm) and Aspergillus flavus (1.80 ± 0.35 mm) than the leaf extract (undetectable mm and 0.80 ± 1.06 mm respectively) (Table 3); at 125 mg/ml the stem extract showed higher inhibition of S. typhi (1.50 ± 0.71 mm), Klebsiella pneumonia (1.00 ± 1.41 mm) and A. flavus (3.50 ± 0.71 mm) than the leaf extract (undetectable mm, undetectable mm and 2.30 ± 1.06 mm, respectively) (Table 4); at 250 mg/ml the stem extract showed higher inhibition than the leaf extract of all the test organisms, including S. typhi (2.50 ± 0.71 mm), Escherichia coli (2.50 ± 0.71 mm), K. pneumonia (3.00 ± 1.41 mm) and A. flavus (5.50 ± 0.71 mm) (Table 5); at 500 mg/ml the stem extract showed higher inhibition than the leaf extract of all the test organisms, including S. typhi (3.50 ± 0.71 mm), E. coli (4.50 ± 0.71 mm), K. pneumonia (4.50 ± 0.71 mm) and A. flavus (7.50 ± 0.71 mm) (Table 6); and at 1000 mg/ml concentration the stem extract showed higher inhibition than the leaf extract of all the test organisms, including S. typhi (4.50 ± 0.71 mm), E. coli (6.50 ± 0.71 mm), K. pneumoniae (6.50 ± 0.71 mm) and A. flavus (9.50 ± 0.71 mm) (Table 7). The stem extract showed the highest inhibitory activity against A. flavus at all concentrations and the lowest activity against S. typhi and E. coli (Fig. 1) while the leaf extract showed the highest inhibitory activity against A. flavus at all concentrations and the lowest activity against S. typhi and E. coli (Fig. 2). Table 3. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 62.5 mg/ml concentration Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 0.50 ± 0.71a 0.00 ± 0.00a 0.00 ± 0.00a 1.80 ± 0.35a Leaf 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.80 ± 1.06a Drug 12.00 ± 0.00b 11.00 ± 0.00b 11.00 ± 0.00b 8.50 ± 0.71b Control 0.00 ± 0.000a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.01 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 0.50 ± 0.71a 0.00 ± 0.00a 0.00 ± 0.00a 1.80 ± 0.35a Leaf 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.80 ± 1.06a Drug 12.00 ± 0.00b 11.00 ± 0.00b 11.00 ± 0.00b 8.50 ± 0.71b Control 0.00 ± 0.000a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.01 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05). Open in new tab Table 3. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 62.5 mg/ml concentration Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 0.50 ± 0.71a 0.00 ± 0.00a 0.00 ± 0.00a 1.80 ± 0.35a Leaf 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.80 ± 1.06a Drug 12.00 ± 0.00b 11.00 ± 0.00b 11.00 ± 0.00b 8.50 ± 0.71b Control 0.00 ± 0.000a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.01 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 0.50 ± 0.71a 0.00 ± 0.00a 0.00 ± 0.00a 1.80 ± 0.35a Leaf 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.80 ± 1.06a Drug 12.00 ± 0.00b 11.00 ± 0.00b 11.00 ± 0.00b 8.50 ± 0.71b Control 0.00 ± 0.000a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.01 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05). Open in new tab Table 4. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 125 mg/ml concentration Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 1.50 ± 0.71b 0.50 ± 0.71a 1. 00 ± 1.41a 3.50 ± 0.71b Leaf 0.00 ± 0.00a 0.50 ± 0.71a 0.00 ± 0.00a 2.30 ± 1.06b Drug 12. 00 ± 0.00c 11. 00 ± 0.00b 11. 00 ± 0.00b 6.50 ± 0.71c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.01 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 1.50 ± 0.71b 0.50 ± 0.71a 1. 00 ± 1.41a 3.50 ± 0.71b Leaf 0.00 ± 0.00a 0.50 ± 0.71a 0.00 ± 0.00a 2.30 ± 1.06b Drug 12. 00 ± 0.00c 11. 00 ± 0.00b 11. 00 ± 0.00b 6.50 ± 0.71c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.01 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05). Open in new tab Table 4. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 125 mg/ml concentration Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 1.50 ± 0.71b 0.50 ± 0.71a 1. 00 ± 1.41a 3.50 ± 0.71b Leaf 0.00 ± 0.00a 0.50 ± 0.71a 0.00 ± 0.00a 2.30 ± 1.06b Drug 12. 00 ± 0.00c 11. 00 ± 0.00b 11. 00 ± 0.00b 6.50 ± 0.71c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.01 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 1.50 ± 0.71b 0.50 ± 0.71a 1. 00 ± 1.41a 3.50 ± 0.71b Leaf 0.00 ± 0.00a 0.50 ± 0.71a 0.00 ± 0.00a 2.30 ± 1.06b Drug 12. 00 ± 0.00c 11. 00 ± 0.00b 11. 00 ± 0.00b 6.50 ± 0.71c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.01 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05). Open in new tab Table 5. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 250 mg/ml concentration Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 2.50 ± 0.71b 2.50 ± 0.71b 3. 00 ± 1.41b 5.50 ± 0.71b Leaf 0.50 ± 0.71a 1. 00 ± 1.41a 1.80 ± 0.35a 4. 00 ± 1.41b Drug 12. 00 ± 0.00c 11. 00 ± 0.00c 11. 00 ± 0.00c 8.50 ± 0.71c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.03 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 2.50 ± 0.71b 2.50 ± 0.71b 3. 00 ± 1.41b 5.50 ± 0.71b Leaf 0.50 ± 0.71a 1. 00 ± 1.41a 1.80 ± 0.35a 4. 00 ± 1.41b Drug 12. 00 ± 0.00c 11. 00 ± 0.00c 11. 00 ± 0.00c 8.50 ± 0.71c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.03 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05). Open in new tab Table 5. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 250 mg/ml concentration Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 2.50 ± 0.71b 2.50 ± 0.71b 3. 00 ± 1.41b 5.50 ± 0.71b Leaf 0.50 ± 0.71a 1. 00 ± 1.41a 1.80 ± 0.35a 4. 00 ± 1.41b Drug 12. 00 ± 0.00c 11. 00 ± 0.00c 11. 00 ± 0.00c 8.50 ± 0.71c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.03 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 2.50 ± 0.71b 2.50 ± 0.71b 3. 00 ± 1.41b 5.50 ± 0.71b Leaf 0.50 ± 0.71a 1. 00 ± 1.41a 1.80 ± 0.35a 4. 00 ± 1.41b Drug 12. 00 ± 0.00c 11. 00 ± 0.00c 11. 00 ± 0.00c 8.50 ± 0.71c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.03 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05). Open in new tab Table 6. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 500 mg/ml concentration Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 3.50 ± 0.71c 4.50 ± 0.71b 4.50 ± 0.71b 7.50 ± 0.71c Leaf 1.50 ± 0.71b 1.50 ± 2.12a 3.50 ± 0.71b 6.00 ± 1.41b Drug 12. 00 ± 0.00d 11. 00 ± 0.00c 11. 00 ± 0.00c 8.50 ± 0.07c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.02 0.00 0.02 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 3.50 ± 0.71c 4.50 ± 0.71b 4.50 ± 0.71b 7.50 ± 0.71c Leaf 1.50 ± 0.71b 1.50 ± 2.12a 3.50 ± 0.71b 6.00 ± 1.41b Drug 12. 00 ± 0.00d 11. 00 ± 0.00c 11. 00 ± 0.00c 8.50 ± 0.07c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.02 0.00 0.02 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05) Open in new tab Table 6. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 500 mg/ml concentration Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 3.50 ± 0.71c 4.50 ± 0.71b 4.50 ± 0.71b 7.50 ± 0.71c Leaf 1.50 ± 0.71b 1.50 ± 2.12a 3.50 ± 0.71b 6.00 ± 1.41b Drug 12. 00 ± 0.00d 11. 00 ± 0.00c 11. 00 ± 0.00c 8.50 ± 0.07c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.02 0.00 0.02 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 3.50 ± 0.71c 4.50 ± 0.71b 4.50 ± 0.71b 7.50 ± 0.71c Leaf 1.50 ± 0.71b 1.50 ± 2.12a 3.50 ± 0.71b 6.00 ± 1.41b Drug 12. 00 ± 0.00d 11. 00 ± 0.00c 11. 00 ± 0.00c 8.50 ± 0.07c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.02 0.00 0.02 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05) Open in new tab Table 7. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 1000 mg/ml Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 4.50 ± 0.71c 6.50 ± 0.71c 6.50 ± 0.71b 9.50 ± 0.71a Leaf 2.50 ± 0.71b 3. 00 ± 1.41b 5.50 ± 0.71b 8. 00 ± 1.41a Drug 12. 00 ± 0.00d 11. 00 ± 0.00d 11. 00 ± 0.00c 8.50 ± 0.71b Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.01 0.00 0.01 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 4.50 ± 0.71c 6.50 ± 0.71c 6.50 ± 0.71b 9.50 ± 0.71a Leaf 2.50 ± 0.71b 3. 00 ± 1.41b 5.50 ± 0.71b 8. 00 ± 1.41a Drug 12. 00 ± 0.00d 11. 00 ± 0.00d 11. 00 ± 0.00c 8.50 ± 0.71b Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.01 0.00 0.01 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05). Open in new tab Table 7. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 1000 mg/ml Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 4.50 ± 0.71c 6.50 ± 0.71c 6.50 ± 0.71b 9.50 ± 0.71a Leaf 2.50 ± 0.71b 3. 00 ± 1.41b 5.50 ± 0.71b 8. 00 ± 1.41a Drug 12. 00 ± 0.00d 11. 00 ± 0.00d 11. 00 ± 0.00c 8.50 ± 0.71b Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.01 0.00 0.01 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 4.50 ± 0.71c 6.50 ± 0.71c 6.50 ± 0.71b 9.50 ± 0.71a Leaf 2.50 ± 0.71b 3. 00 ± 1.41b 5.50 ± 0.71b 8. 00 ± 1.41a Drug 12. 00 ± 0.00d 11. 00 ± 0.00d 11. 00 ± 0.00c 8.50 ± 0.71b Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.01 0.00 0.01 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05). Open in new tab Figure 1. Open in new tabDownload slide Zone of inhibition (mm) of bacterial pathogens by stem extract of Mimosa invisa. Figure 2. Open in new tabDownload slide Zone of inhibition (mm) of bacterial pathogens by leaf extract of Mimosa invisa. In summary, the stem and leaf extract of M. invisa inhibited the human pathogens tested here. This is probably a result of the synergistic actions of the active components present in the plant parts. This implies that the leaf and stem extracts from M. invisa may have potential as a source of raw material for the pharmaceutical industry and in modern medicine for the treatment of human diseases. Gastroenteritis, typhoid fever, dysentery, cholera, and urinary tract infections have been associated with E. coli (Orji, Ezenwaje, Anyaegbunam, 2006). Salmonella typhi is the causative agent for paratyphoid fever, a mild form of enteric fever (Ekhaise and Anyansi, 2005), and K. pneumonia has been identified to account for up to 55% of nosocomial infections in some parts of Nigeria (Ajayi and Akonai, 2005). Aspergillosis in immunocompromised individuals is caused by A. flavus, an opportunistic human and animal pathogen (Amanike, Nancy, Keller, 2011). The stem extract of M. invisa was found to have higher inhibitory activity against all test organisms than the leaf extract. Akinsinde and Olukoya (1995) reported that the stem of this plant is used traditionally in the treatment of leprosy, dysentery, vaginal and uterine complaints, inflammations, burning sensation, asthma, leucoderma, and fatigue and blood diseases. The findings of this study support these uses. It is also reported to be of great importance in controlling diarrhoea (Athisaara), amoebic dysentery (Raktaatisaara), bleeding piles and urinary infections (Valsala, 2000). The inhibitory activities of leaf and stem extracts increased with concentration. It has been extensively reported that more active ingredients in plants are dissolved in solution at higher concentrations (Amadioha, 2000; Onifade, 2002; Okigbo and Ogbonnaya, 2006). Meanwhile, comparison of the inhibitory activity of the antibiotic (Streptomycin) and plant extract revealed that the antibiotic showed higher inhibition than the plant extract of all test organisms with the exception of A. flavus at 1000 mg/ml of stem extract. This result therefore shows that the stem extract of M. invisa has greater antifungal action at higher concentrations than Streptomycin, which is a synthetic drug. All test pathogens were found to vary in their susceptibility to the plant extract and antibiotic. Aspergillus flavus was more susceptible to the plant extracts while S. typhi and E. coli were less susceptible. In addition, A. flavus was least susceptible to the drug whereas S. typhi was the most susceptible. Ogu et al. (2012) stated that such differences are due to the fact that bacterial pathogens like S. typhi and E. coli develop resistance to inhibition caused by plant extracts (Valsala, 2000). Conclusion and recommendations This study reports the antimicrobial properties of the leaves and stem of M. invisa. The stem extract had greater antifungal and antibacterial properties than the leaf extract at all concentrations tested. Hence, the stem extract is recommended for potential use in the treatment of ailments caused by A. flavus, S. typhi, E. coli and K. pneumoniae. However, further clinical testing of M. invisa is required to determine its potential for treatment of infectious diseases in humans. Acknowledgements I acknowledge with warm hearted appreciation my supervisor, Dr. Mrs. C.A. Ezeabara for her advice and guidance. I wish to express my profound gratitude to my parents Mr. and Mrs. Chukwudi Onwumbiko for their support, advice, love and provisions in the course of this work. Author’s biography H.C.C. graduated from Department of Botany, Nnamdi Azikiwe University, Awka, Nigeria in 2017. His interest lies in Phytomedicine. He performed the experiments and has primary responsibility for the final content. C.A.E. designed and supervised the study, assisted in the research and writing of the paper. References Ajayi , A. O. and Akonai , K. A. ( 2005 ) Distribution pattern of enteric organisms in the Lagos Lagoon , African Journal of Biomedicinal Resources , 8 ( 3 ), 163 – 168 . Google Scholar OpenURL Placeholder Text WorldCat Akinsinde , K. A. and Olukoya , D. K. ( 1995 ) Vibriocidal activities of some local herbs , Journal of Diarrhoeal Disease Research , 13 ( 2 ), 127 – 129 . Google Scholar OpenURL Placeholder Text WorldCat Amadioha , A. C. ( 2000 ) Control of anthracnose disease of cowpea by Cympogonoi stratus and Ocimumgratissimum , ActaPhytopathological Entomology Hung Kun , 34 , 83 – 89 . Google Scholar OpenURL Placeholder Text WorldCat Amanike , S. , Nancy , P. and Keller , C. ( 2011 ) Aspergillusflavus , Annual Review of Phytopathology , 49 , 107 – 133 . Google Scholar Crossref Search ADS PubMed WorldCat Association of Official Analytical Chemists . ( 2000 ) Official Method of Analysis , Association of Official Analytical Chemists , Washington D.C , p. 44 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Das , A. and Singh , G. P. ( 1999 ) Effect of different levels of berseem (Trifolium alexandrinum) supplementation of wheat straw on some physical factors regulating intake and digestion , Animal Feed science technology , 81 ( 1–2 ), 133 – 149 . Google Scholar Crossref Search ADS WorldCat Deng , W. , Liou , S. R., Plunkett , G. , III et al. . ( 2003 ) Comparative genomics of Salmonella enterica and Serovar typhi Strains Ty2 and CT18 , Journal of Bacteriology , 185 , 2330 – 2337 . Google Scholar Crossref Search ADS PubMed WorldCat Ekhaise , F. O. and Anyansi , C. C. ( 2005 ) Influence of brewery effluent discharge on the microbiological and physicochemical quality of Ikpoba River, Nigeria , African Journal of Biotechnology , 4 ( 10 ), 1062 – 1065 . Google Scholar OpenURL Placeholder Text WorldCat Espinel-Ingroff , A. ( 2002 ) E-test method for testing susceptibilities of Aspergillus spp. to the new triazolesvoriconazole and posaconazole and to established antifungal agents: comparison with NCCLS broth microdilution method , Nature , 40 , 2101 – 2107 . Google Scholar OpenURL Placeholder Text WorldCat Ezeabara , C. A. and Mbah , E. U. ( 2016 ) Comparative phytochemical and proximate investigations of leaf, root and stem of M. invisa Mart. and M. pudica L , Journal of Pharma Science , 1 , 1 . Google Scholar OpenURL Placeholder Text WorldCat Ezeabara , C. A. and Okonkwo , E. E. ( 2016 ) Comparison of phytochemical and proximate components of leaf, root and stem of croton hirtusl’herit and croton lobatuslinn , Journal of Medical and Health Research , 1 ( 2 ), 23 – 33 . Google Scholar OpenURL Placeholder Text WorldCat Finar , I. L. ( 1986 ) Stereo Chemistry and the Chemistry of Natural Products , Longman , London , p. 213 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Francis , G. , Kerem , Z., Makkar , H. P. S. et al. . ( 2002 ) The biological action of saponins in animal systems: a review , British Journal of Nutrition , 88 , 587 – 605 . Google Scholar Crossref Search ADS PubMed WorldCat Harborne , J. B. ( 1973 ) Phytochemical Methods; a Guide to Modern Techniques of Plant Analysis , Chapmann and Hall Publishers , London , p. 456 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Harborne , J. B. ( 1998 ) Phytochemical Methods: A Guide to Modern Techniques of Plant Analyses , Chapmann and Hall Publishers , London , p. 342 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Kanokmedhakul , K. , Kanokmedhakul , S. and Phatchana , R. ( 2005 ) Biological activity of anthraquinones and triterpenoids from Prismatomerisfragrans , Journal of Ethnopharmacology , 100 ( 3 ), 284 – 288 . Google Scholar Crossref Search ADS PubMed WorldCat Kirk , H. and Sawyer , R. ( 1998 ) Frait Pearson Chemical Analysis of Food . (ed.) Edinburgh , Longman Scientific and Technical , pp. 211 – 212 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Kittakoop , P. , Mahidol , C. and Ruchirawat , S. ( 2014 ) Alkaloids as important scaffolds in therapeutic drugs for the treatments of cancer, tuberculosis, and smoking cessation , Curr Top Medical Chemistry , 14 ( 2 ), 239 – 252 . Google Scholar Crossref Search ADS WorldCat Lui , L. , Liu , S. W., Jiang , S. B. et al. . ( 2004 ) Tannin inhibits HIV-1 entry by targeting gp41 , Acta Pharmacology Sinica , 25 ( 2 ), 213 – 218 . Google Scholar OpenURL Placeholder Text WorldCat Mandel , P. , Babu , S. P. and Mandal , N. C. ( 2005 ) Antimicrobial activity of saponins from Acacin auriculiformis , Fitoterapia , 76 ( 5 ), 462 – 465 . Google Scholar Crossref Search ADS PubMed WorldCat Masayuki , M. and Katsuyai , G. ( 2010 ) Aspergillus: Molecular Biology and Genomics , Horizon Scientific Press, UK , 157 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Njoku , O. V. and Obi , C. ( 2009 ) Phytochemical constituents of some selected medicinal plants , African Journal of Pure and Applied Chemistry , 3 ( 11 ), 228 – 233 . Google Scholar OpenURL Placeholder Text WorldCat Ogu , G. I. , Tanimowo , W. O., Nwachukwu , P. U. et al. . ( 2012 ) Antimicrobial and phytochemical evaluation of the leaf, stem bark and root extracts of CyathulaprostrataL. Blume against some human pathogens , Journal of Intercultutural Ethnopharmacognosis , 1 , 35 – 43 . Google Scholar Crossref Search ADS WorldCat Okigbo , R. N. and Ogbonnaya , O. U. ( 2006 ) Antifungal effects of two tropical plant extracts (Ocimumgratissimumand Afromaomummelegueta) on postharvest yam (Dioscoreaspp.) rot , African Journal of Biotechnology , 5 ( 9 ), 727 – 731 . Google Scholar OpenURL Placeholder Text WorldCat Onifade , A. K. ( 2002 ) Antifungal effect of AzadirachtaindicaA. Joss extracts on Collectotricum lindemathianum , Global Journal of Pure and Applied Science , 6 ( 3 ), 423 – 428 . Google Scholar OpenURL Placeholder Text WorldCat Orji , M. U. , Ezenwaje , E. E. and Anyaegbunam , B. C. ( 2006 ) Spatial appraisal of shallow well water pollution in Awka, Nigeria , Nigerian Journal of Microbiology , 20 ( 3 ), 1384 – 1389 . Google Scholar OpenURL Placeholder Text WorldCat Oxford English Dictionary . ( 2005 ) Coli , Oxford University Press, UK , p. 34 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Praveen , K. and Kumud , U. ( 2012 ) Tannins are astringent , Journal of Pharmacognosy and Phytochemistry , 1 ( 3 ), 8192 . Google Scholar OpenURL Placeholder Text WorldCat Ramírez-Camejo , L. A. , Zuluaga-Montero , A., Lázaro-Escudero , M. A. et al. . ( 2012 ) Phylogeography of the cosmopolitan fungus Aspergillusflavus: Is everything everywhere? Fungal Biology , 116 ( 3 ), 452 – 463 . Google Scholar Crossref Search ADS PubMed WorldCat Reynolds , D. ( 2003 ) Recruitment of through 319-phosphorylated Ndd1p to the FHA domain of Fkh2p requires CLB kinase activity: a mechanism for CLB cluster gene activation , Genes Development , 17 ( 14 ), 1789 – 1802 . Google Scholar Crossref Search ADS PubMed WorldCat Ryan , K. J. and Ray , C. G. ( 2004 ) Sherris Medical Microbiology (ed.) McGraw Hill, USA . ISBN 0-8385-8529-9. Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Shelef , L. A. ( 1983 ) Antimicrobial effects of spices—U S Forest Service 2008 ‘M. pudica’, Usambara invasive plants , Journal of Food Safety , 6 , 29 – 44 . Google Scholar Crossref Search ADS WorldCat Sieradzki , K. , Robert , R. B., Haber , S. W. et al. . ( 1999 ) The development of vancomycin resistance in a patient with methicillin-resistant Staphylococcus aureus infection , National England Medicine , 340 ( 7 ), 517 – 523 . Google Scholar Crossref Search ADS WorldCat Thongson , C. , Davidson , P. M., Mahakarnchanakul , W. et al. . ( 2004 ) Antimicrobial activity of ultrasound-assisted solvent-extracted spices , Letters in Applied Microbiology , 39 , 401 – 406 . Google Scholar Crossref Search ADS PubMed WorldCat Valsala , S. ( 2000 ) Estrogenic and antiestrogenic activities of M. pudica on Rattusnorvegicus , Journal of Ecotoxicology , 10 ( 1 ), 25 – 29 . Google Scholar OpenURL Placeholder Text WorldCat Zaika , L. L. ( 1988 ) Spices and herbs: their antimicrobial activity and its determination , Journal of Food Safety , 9 , 97 – 118 . Google Scholar Crossref Search ADS WorldCat Author notes Supervisor: Dr. Mrs. Chinelo A. Ezeabara, Department of Botany, Nnamdi Azikiwe University, P.M.B. 5025 Awka, Nigeria. © The Author(s) 2018. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com © The Author(s) 2018. Published by Oxford University Press. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BioScience Horizons Oxford University Press

Phytochemical screening and in vitro antimicrobial activities of Mimosa invisa Mart. leaves and stems

Phytochemical screening and in vitro antimicrobial activities of Mimosa invisa Mart. leaves and stems

BioScience Horizons , Volume 11 – Jan 1, 2018

Abstract

Abstract Preliminary phytochemical screening was carried out and in vitro antimicrobial activities of leaves and stem of Mimosa invisa Mart. against some clinical pathogens were tested using standard techniques. Significance was measured using Duncan’s multiple range test. The inhibitory actions of the leaf and stem extracts increased with concentration. The rates of inhibition of the two extracts were low and high against Salmonella typhi and Aspergillus flavus respectively. The phytochemicals present in the plant parts had antimicrobial properties and reveal that M. invisa may have potential in pharmaceutical applications. Mimosa invisa, alkaloid, antimicrobial actions, zone of inhibition, Salmonella typhi, Aspergillus flavus Introduction The leaves, stems, flowers, fruits, seeds, roots, rhizomes and bark of some plants may be used for medicinal purposes. Many plant and herb species have unrealized antimicrobial and antiviral features (Shelef, 1983; Zaika, 1988) and may thus be a source of phyto-antimicrobial agents (Das et al., 1999). Sieradzki et al. (1999) stated that the emergence of bacterial strains with reduced vulnerability to antibiotics and the increasingly wide spread of multidrug resistant strains of bacteria would require the search for new infection fighting strategies. The properties of medicinal plants are of great benefit (Finar, 1986; Harborne, 1998). Antibacterial compounds extracted from plants have drawn much attention and could be used as an alternative strategy to prevent the spread of diseases. Flavonoids have been shown to inhibit topoisomerase enzymes. Alkaloids are of great benefit to pharmaceutical industries including as anti-malarial agents (for example, quinine) (Kittakoop, Mahidol, Ruchirawat, 2014). It has been recorded that tannin has antiviral, antibacterial and antiparasitic properties (Lui et al., 2004; Njoku and Obi, 2009). Saponins have been implicated as bioactive antibacterial agents (Mandel, Babu, Mandal, 2005). Anthraquinones are used to relieve constipation (Kittakoop, Mahidol, Ruchirawat, 2014). However, there is no information on the antimicrobial actions of phytochemicals present in M. invisa. Therefore, the objectives of this work were to determine the phytochemicals present in the plant as well as to assess their antimicrobial activity against selected microorganisms that cause ailments in humans: Escherichia coli, a facultative anaerobic, gram-negative, rod-shaped bacterium that is abundant in the human gut (OED, 2005); Salmonella typhi, a gram-negative bacterium that causes typhoid fever, a disease that has afflicted humans throughout history and continues to be a public health concern (Deng et al., 2003); Klebsiella pneumoniae, a gram-negative nonmotile, lactose-fermenting, facultative anaerobic, rod-shaped bacterium that is enclosed in a capsule and causes destructive changes to human and animal lungs if aspirated (inhaled), particularly to the alveoli (in the lungs), resulting in bloody sputum (Ryan and Ray, 2004); and Aspergillus flavus, a saprotrophic and pathogenic (Masayuki and Katsuyai, 2010) fungus with a cosmopolitan distribution (Ramírez-Camejo et al., 2012). Materials and methods Collection of sample The leaves and stem of mature M. invisa were collected from a roadside at Idi-Oshe, opposite IITA Ibadan, Oyo State, Nigeria, in August 2016. The analyses were then carried out at the Department of Microbiology in IITA, Ibadan. The plant was identified by Mr. Kayode, a Plant Taxonomist at IITA Ibadan. Preparation of samples for analyses The fresh specimens were cleaned with clean water and oven dried at a temperature of 60°C for 72 h. The samples were separated by hand and were ground using a grinding machine (SPM-5, China), then placed in an air-tight container. Extraction of plant materials Methanol extraction An aqueous decoction of the plant was prepared by soaking 500 g of the powdered samples of leaves and stem each in 2.5 L of methanol. The whole setup was left at room temperature for 72 hours. The extract was then concentrated using a rotary evaporator (RE-52A, China) and the solvent allowed to evaporate. The concentrated extract was stored in an air-tight container in a cold store at a regulated temperature of 20°C until it was required for analyses. Preliminary phytochemical screening The methods and tests used in the phytochemical screening of the plant extract were described by Ezeabara and Okonkwo (2016). The sign ‘+’ denotes presence. Quantitative phytochemical screening Alkaloids and flavonoids were determined using the alkaline precipitation gravimetric method and gravimetric method of Harborne (1973), respectively. The method used to determine saponins was described by the AOAC (2000). The Folin-Dennis colorimetric method was used to determine the tannin content of the sample (Kirk and Sawyer, 1998). Anthraquinone was determined by the spectrophotometric method used by Ezeabara and Okonkwo (2016). Microbial analysis Isolation of the test organisms Using a wire loop, colonies of the test organisms (Escherichia coli, Salmonella typhi, Klebsiella pneumoniae and Aspergillus flavus) were collected from pure cultures kept at the Soil Microbiology Unit, IITA Ibadan, Oyo State, Nigeria. Antimicrobial test procedures Preparation of stock solution Stock solutions of the two methanolic extracts of leaves and stem were prepared by weighing 2.0 g of each methanolic extract using electronic scales. This was then dissolved completely in 2.0 ml of dimethyl sulphoxide (DMSO) in a sterile test tube, giving a stock solution concentration of 1000 mg/ml per extract. The stock solution was then labelled and stored at room temperature until needed for use. Determination of inhibitory activity The inhibitory activity of the methanolic extract of the leaves and stems of M. invisa, antibiotics (positive control), and dimethyl sulphoxide (negative control) was determined using the disc diffusion method in 5 mm discs. The concentrations used were as follows: 1000 mg/ml of the methanolic extract, 100 mg/ml of antibiotic (Streptomycin) and 1000 mg/ml of DMSO. After the discs had absorbed the leaf and stem extract, the antibiotic or the control DMSO, the discs were removed, dried and placed on media on which the test microorganisms were freshly inoculated, then incubated at 37°C for 24 hours for bacteria and 25°C for 72 hours for fungi. Antimicrobial activity was determined after 24 hours (for bacteria) of incubation and 72 hours (for fungi) of incubation by measuring the zone of inhibition around each paper disc in millimetres (mm) (Reynolds, 2003). Determination of minimum inhibitory concentration The minimum inhibitory concentrations (MIC) of the absolute (stock) concentrations were determined using the agar well diffusion method; five sterile plates were prepared, and nutrient broth was poured into each of the plates and then allowed to dry. Using standardized inoculums (106 cfu/ml), a loop full of the different test organisms was streaked onto each of the five plates when dry. Then, 10 holes were dug using an agar borer for the varying concentrations of methanolic extracts of leaves and stem. A ruler was used to separate the holes for each of the methanolic extracts. Varying concentrations of the different extracts were made by serial dilution using DMSO as the diluent. The absolute/stock concentrations of extracts used were 1000 mg/ml (equivalent to 100%). Five test tubes per extract were prepared in a test tube rack. The dilutions were as follows: 50% (500 mg/ml), 25% (250 mg/ml), 12.5% (125 mg/ml) and 6.25% (62.5 mg/ml) in each of the test tubes. DMSO served as the negative control. A measured 50 μL volume of each dilution was added aseptically into the holes seeded with the test organisms in the nutrient agar plate using a syringe and placed in an incubator at 35°C for 24 hours. MIC was considered as the lowest concentration of methanolic extract showing a clear zone of inhibition (Thongson et al., 2004). Determination of minimum bactericidal/fungicidal concentration The plates with the MIC were incubated for a further 24 hours at 35°C to test which organism would grow on the zones of inhibition. Those plates on which organisms were completely killed after 24 hours, and clear zones remained, were referred to as bactericidal for bacteria and fungicidal for fungi (Espinel-Ingroff, 2002). Statistical analysis The test was carried out using a completely randomized design. Analysis of Variance (ANOVA) using SPSS version 21 was employed to analyse the data collected in the study. The Duncan’s multiple range test (DMRT) was used to test the difference among treatment means with more than two levels. All statistical analyses were carried out at 5% level of significance. The data were expressed as the mean (±standard deviation) of triplicate determinations. Results and discussion The result of the qualitative phytochemical analysis of the leaf and stem extracts of M. invisa demonstrated that alkaloids, anthraquinones, saponins, tannins and flavonoids were present (Table 1). Moreover, the quantitative analysis revealed that the leaf had the largest concentration of alkaloids among the plant parts, at 0.16 ± 0.11 mg/100 g, and anthraquinones at 0.88 ± 0.06 mg/100 g, whereas the highest concentrations of saponins at 16.81 ± 5.12 mg/100 g, tannins at 0.15 ± 0.04 mg/100 g and flavonoids at 0.48 ± 0.41 mg/100 g occurred in the stem (Table 2). These correspond largely with the findings of Ezeabara and Mbah (2016), who reported a wide range of chemical compounds in the leaves, stem and root of M. invisa and M. pudica. Stated that M. invisa is traditionally used in the treatment of diabetes. In addition, this species has been reported to have potent hypoglycaemic effects in alloxan-induced diabetic mice. The medicinal properties of this plant are likely to be a result of the wide range of biological activities of the phytochemicals. The pharmacological activities of alkaloids are varied, and include anti-malarial (e.g. quinine) and antiasthma activity (e.g. homoharringtonine) (Kittakoop, Mahidol, Ruchirawat, 2014). Due to saponins’ immune-enhancing properties they are used as immunological adjuvants in the formulation of vaccines (Francis et al., 2002). Tannins may be used medicinally in antidiarrheal, haemostatic and antihaemorrhoidal compounds (Praveen and Kumud, 2012). Kanokmedhakul, Kanokmedhakul, Phatchana (2005) reported that anthraquinones isolated from Primatomeris fragrans showed both antifungal and antituberculosis activities. Table 1. Qualitative phytochemical composition of the leaf and stem extracts of Mimosa invisa Phytochemicals . Plant part . Leaf . Stem . Alkaloid + + Saponin + + Flavonoid + + Phenol + + Tannin + + Anthraquinone + + Phytochemicals . Plant part . Leaf . Stem . Alkaloid + + Saponin + + Flavonoid + + Phenol + + Tannin + + Anthraquinone + + Open in new tab Table 1. Qualitative phytochemical composition of the leaf and stem extracts of Mimosa invisa Phytochemicals . Plant part . Leaf . Stem . Alkaloid + + Saponin + + Flavonoid + + Phenol + + Tannin + + Anthraquinone + + Phytochemicals . Plant part . Leaf . Stem . Alkaloid + + Saponin + + Flavonoid + + Phenol + + Tannin + + Anthraquinone + + Open in new tab Table 2. Mean quantitative phytochemical composition of the leaf and stem of M. invisa composition (mg/100 g) Plant part . Saponin . Alkaloid . Tannin . Anthraquinone . Flavonoid . Stem 16.81 ± 5.12b 0.04 ± 0.01a 0.15 ± 0.04b 0.79 ± 0.09a 0.48 ± 0.41b Leaf 8.64 ± 1.47a 0.16 ± 0.11b 0.10 ± 0.00a 0.88 ± 0.06b 0.38 ± 0.32a p-value 0.19 0.13 0.28 0.08 0.01 Plant part . Saponin . Alkaloid . Tannin . Anthraquinone . Flavonoid . Stem 16.81 ± 5.12b 0.04 ± 0.01a 0.15 ± 0.04b 0.79 ± 0.09a 0.48 ± 0.41b Leaf 8.64 ± 1.47a 0.16 ± 0.11b 0.10 ± 0.00a 0.88 ± 0.06b 0.38 ± 0.32a p-value 0.19 0.13 0.28 0.08 0.01 Results are in mean ± std error of duplicate determinations. The same letter in a column means that the respective means were not significantly different. Bold values indicates that p<0.05. Open in new tab Table 2. Mean quantitative phytochemical composition of the leaf and stem of M. invisa composition (mg/100 g) Plant part . Saponin . Alkaloid . Tannin . Anthraquinone . Flavonoid . Stem 16.81 ± 5.12b 0.04 ± 0.01a 0.15 ± 0.04b 0.79 ± 0.09a 0.48 ± 0.41b Leaf 8.64 ± 1.47a 0.16 ± 0.11b 0.10 ± 0.00a 0.88 ± 0.06b 0.38 ± 0.32a p-value 0.19 0.13 0.28 0.08 0.01 Plant part . Saponin . Alkaloid . Tannin . Anthraquinone . Flavonoid . Stem 16.81 ± 5.12b 0.04 ± 0.01a 0.15 ± 0.04b 0.79 ± 0.09a 0.48 ± 0.41b Leaf 8.64 ± 1.47a 0.16 ± 0.11b 0.10 ± 0.00a 0.88 ± 0.06b 0.38 ± 0.32a p-value 0.19 0.13 0.28 0.08 0.01 Results are in mean ± std error of duplicate determinations. The same letter in a column means that the respective means were not significantly different. Bold values indicates that p<0.05. Open in new tab The antimicrobial results of the methanolic extracts of leaves and stem of M. invisa indicated that the plant showed antimicrobial activity against the tested microorganisms at five concentrations: 62.5, 125, 250, 500 and 1000 mg/ml (Tables 3–7). At 62.5 mg/ml, the stem extract showed greater inhibition of Salmonella typhi (0.50 ± 0.710 mm) and Aspergillus flavus (1.80 ± 0.35 mm) than the leaf extract (undetectable mm and 0.80 ± 1.06 mm respectively) (Table 3); at 125 mg/ml the stem extract showed higher inhibition of S. typhi (1.50 ± 0.71 mm), Klebsiella pneumonia (1.00 ± 1.41 mm) and A. flavus (3.50 ± 0.71 mm) than the leaf extract (undetectable mm, undetectable mm and 2.30 ± 1.06 mm, respectively) (Table 4); at 250 mg/ml the stem extract showed higher inhibition than the leaf extract of all the test organisms, including S. typhi (2.50 ± 0.71 mm), Escherichia coli (2.50 ± 0.71 mm), K. pneumonia (3.00 ± 1.41 mm) and A. flavus (5.50 ± 0.71 mm) (Table 5); at 500 mg/ml the stem extract showed higher inhibition than the leaf extract of all the test organisms, including S. typhi (3.50 ± 0.71 mm), E. coli (4.50 ± 0.71 mm), K. pneumonia (4.50 ± 0.71 mm) and A. flavus (7.50 ± 0.71 mm) (Table 6); and at 1000 mg/ml concentration the stem extract showed higher inhibition than the leaf extract of all the test organisms, including S. typhi (4.50 ± 0.71 mm), E. coli (6.50 ± 0.71 mm), K. pneumoniae (6.50 ± 0.71 mm) and A. flavus (9.50 ± 0.71 mm) (Table 7). The stem extract showed the highest inhibitory activity against A. flavus at all concentrations and the lowest activity against S. typhi and E. coli (Fig. 1) while the leaf extract showed the highest inhibitory activity against A. flavus at all concentrations and the lowest activity against S. typhi and E. coli (Fig. 2). Table 3. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 62.5 mg/ml concentration Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 0.50 ± 0.71a 0.00 ± 0.00a 0.00 ± 0.00a 1.80 ± 0.35a Leaf 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.80 ± 1.06a Drug 12.00 ± 0.00b 11.00 ± 0.00b 11.00 ± 0.00b 8.50 ± 0.71b Control 0.00 ± 0.000a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.01 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 0.50 ± 0.71a 0.00 ± 0.00a 0.00 ± 0.00a 1.80 ± 0.35a Leaf 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.80 ± 1.06a Drug 12.00 ± 0.00b 11.00 ± 0.00b 11.00 ± 0.00b 8.50 ± 0.71b Control 0.00 ± 0.000a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.01 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05). Open in new tab Table 3. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 62.5 mg/ml concentration Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 0.50 ± 0.71a 0.00 ± 0.00a 0.00 ± 0.00a 1.80 ± 0.35a Leaf 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.80 ± 1.06a Drug 12.00 ± 0.00b 11.00 ± 0.00b 11.00 ± 0.00b 8.50 ± 0.71b Control 0.00 ± 0.000a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.01 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 0.50 ± 0.71a 0.00 ± 0.00a 0.00 ± 0.00a 1.80 ± 0.35a Leaf 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.80 ± 1.06a Drug 12.00 ± 0.00b 11.00 ± 0.00b 11.00 ± 0.00b 8.50 ± 0.71b Control 0.00 ± 0.000a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.01 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05). Open in new tab Table 4. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 125 mg/ml concentration Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 1.50 ± 0.71b 0.50 ± 0.71a 1. 00 ± 1.41a 3.50 ± 0.71b Leaf 0.00 ± 0.00a 0.50 ± 0.71a 0.00 ± 0.00a 2.30 ± 1.06b Drug 12. 00 ± 0.00c 11. 00 ± 0.00b 11. 00 ± 0.00b 6.50 ± 0.71c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.01 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 1.50 ± 0.71b 0.50 ± 0.71a 1. 00 ± 1.41a 3.50 ± 0.71b Leaf 0.00 ± 0.00a 0.50 ± 0.71a 0.00 ± 0.00a 2.30 ± 1.06b Drug 12. 00 ± 0.00c 11. 00 ± 0.00b 11. 00 ± 0.00b 6.50 ± 0.71c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.01 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05). Open in new tab Table 4. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 125 mg/ml concentration Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 1.50 ± 0.71b 0.50 ± 0.71a 1. 00 ± 1.41a 3.50 ± 0.71b Leaf 0.00 ± 0.00a 0.50 ± 0.71a 0.00 ± 0.00a 2.30 ± 1.06b Drug 12. 00 ± 0.00c 11. 00 ± 0.00b 11. 00 ± 0.00b 6.50 ± 0.71c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.01 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 1.50 ± 0.71b 0.50 ± 0.71a 1. 00 ± 1.41a 3.50 ± 0.71b Leaf 0.00 ± 0.00a 0.50 ± 0.71a 0.00 ± 0.00a 2.30 ± 1.06b Drug 12. 00 ± 0.00c 11. 00 ± 0.00b 11. 00 ± 0.00b 6.50 ± 0.71c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.01 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05). Open in new tab Table 5. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 250 mg/ml concentration Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 2.50 ± 0.71b 2.50 ± 0.71b 3. 00 ± 1.41b 5.50 ± 0.71b Leaf 0.50 ± 0.71a 1. 00 ± 1.41a 1.80 ± 0.35a 4. 00 ± 1.41b Drug 12. 00 ± 0.00c 11. 00 ± 0.00c 11. 00 ± 0.00c 8.50 ± 0.71c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.03 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 2.50 ± 0.71b 2.50 ± 0.71b 3. 00 ± 1.41b 5.50 ± 0.71b Leaf 0.50 ± 0.71a 1. 00 ± 1.41a 1.80 ± 0.35a 4. 00 ± 1.41b Drug 12. 00 ± 0.00c 11. 00 ± 0.00c 11. 00 ± 0.00c 8.50 ± 0.71c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.03 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05). Open in new tab Table 5. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 250 mg/ml concentration Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 2.50 ± 0.71b 2.50 ± 0.71b 3. 00 ± 1.41b 5.50 ± 0.71b Leaf 0.50 ± 0.71a 1. 00 ± 1.41a 1.80 ± 0.35a 4. 00 ± 1.41b Drug 12. 00 ± 0.00c 11. 00 ± 0.00c 11. 00 ± 0.00c 8.50 ± 0.71c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.03 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 2.50 ± 0.71b 2.50 ± 0.71b 3. 00 ± 1.41b 5.50 ± 0.71b Leaf 0.50 ± 0.71a 1. 00 ± 1.41a 1.80 ± 0.35a 4. 00 ± 1.41b Drug 12. 00 ± 0.00c 11. 00 ± 0.00c 11. 00 ± 0.00c 8.50 ± 0.71c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.03 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05). Open in new tab Table 6. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 500 mg/ml concentration Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 3.50 ± 0.71c 4.50 ± 0.71b 4.50 ± 0.71b 7.50 ± 0.71c Leaf 1.50 ± 0.71b 1.50 ± 2.12a 3.50 ± 0.71b 6.00 ± 1.41b Drug 12. 00 ± 0.00d 11. 00 ± 0.00c 11. 00 ± 0.00c 8.50 ± 0.07c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.02 0.00 0.02 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 3.50 ± 0.71c 4.50 ± 0.71b 4.50 ± 0.71b 7.50 ± 0.71c Leaf 1.50 ± 0.71b 1.50 ± 2.12a 3.50 ± 0.71b 6.00 ± 1.41b Drug 12. 00 ± 0.00d 11. 00 ± 0.00c 11. 00 ± 0.00c 8.50 ± 0.07c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.02 0.00 0.02 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05) Open in new tab Table 6. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 500 mg/ml concentration Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 3.50 ± 0.71c 4.50 ± 0.71b 4.50 ± 0.71b 7.50 ± 0.71c Leaf 1.50 ± 0.71b 1.50 ± 2.12a 3.50 ± 0.71b 6.00 ± 1.41b Drug 12. 00 ± 0.00d 11. 00 ± 0.00c 11. 00 ± 0.00c 8.50 ± 0.07c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.02 0.00 0.02 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 3.50 ± 0.71c 4.50 ± 0.71b 4.50 ± 0.71b 7.50 ± 0.71c Leaf 1.50 ± 0.71b 1.50 ± 2.12a 3.50 ± 0.71b 6.00 ± 1.41b Drug 12. 00 ± 0.00d 11. 00 ± 0.00c 11. 00 ± 0.00c 8.50 ± 0.07c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.02 0.00 0.02 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05) Open in new tab Table 7. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 1000 mg/ml Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 4.50 ± 0.71c 6.50 ± 0.71c 6.50 ± 0.71b 9.50 ± 0.71a Leaf 2.50 ± 0.71b 3. 00 ± 1.41b 5.50 ± 0.71b 8. 00 ± 1.41a Drug 12. 00 ± 0.00d 11. 00 ± 0.00d 11. 00 ± 0.00c 8.50 ± 0.71b Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.01 0.00 0.01 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 4.50 ± 0.71c 6.50 ± 0.71c 6.50 ± 0.71b 9.50 ± 0.71a Leaf 2.50 ± 0.71b 3. 00 ± 1.41b 5.50 ± 0.71b 8. 00 ± 1.41a Drug 12. 00 ± 0.00d 11. 00 ± 0.00d 11. 00 ± 0.00c 8.50 ± 0.71b Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.01 0.00 0.01 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05). Open in new tab Table 7. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 1000 mg/ml Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 4.50 ± 0.71c 6.50 ± 0.71c 6.50 ± 0.71b 9.50 ± 0.71a Leaf 2.50 ± 0.71b 3. 00 ± 1.41b 5.50 ± 0.71b 8. 00 ± 1.41a Drug 12. 00 ± 0.00d 11. 00 ± 0.00d 11. 00 ± 0.00c 8.50 ± 0.71b Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.01 0.00 0.01 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 4.50 ± 0.71c 6.50 ± 0.71c 6.50 ± 0.71b 9.50 ± 0.71a Leaf 2.50 ± 0.71b 3. 00 ± 1.41b 5.50 ± 0.71b 8. 00 ± 1.41a Drug 12. 00 ± 0.00d 11. 00 ± 0.00d 11. 00 ± 0.00c 8.50 ± 0.71b Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.01 0.00 0.01 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05). Open in new tab Figure 1. Open in new tabDownload slide Zone of inhibition (mm) of bacterial pathogens by stem extract of Mimosa invisa. Figure 2. Open in new tabDownload slide Zone of inhibition (mm) of bacterial pathogens by leaf extract of Mimosa invisa. In summary, the stem and leaf extract of M. invisa inhibited the human pathogens tested here. This is probably a result of the synergistic actions of the active components present in the plant parts. This implies that the leaf and stem extracts from M. invisa may have potential as a source of raw material for the pharmaceutical industry and in modern medicine for the treatment of human diseases. Gastroenteritis, typhoid fever, dysentery, cholera, and urinary tract infections have been associated with E. coli (Orji, Ezenwaje, Anyaegbunam, 2006). Salmonella typhi is the causative agent for paratyphoid fever, a mild form of enteric fever (Ekhaise and Anyansi, 2005), and K. pneumonia has been identified to account for up to 55% of nosocomial infections in some parts of Nigeria (Ajayi and Akonai, 2005). Aspergillosis in immunocompromised individuals is caused by A. flavus, an opportunistic human and animal pathogen (Amanike, Nancy, Keller, 2011). The stem extract of M. invisa was found to have higher inhibitory activity against all test organisms than the leaf extract. Akinsinde and Olukoya (1995) reported that the stem of this plant is used traditionally in the treatment of leprosy, dysentery, vaginal and uterine complaints, inflammations, burning sensation, asthma, leucoderma, and fatigue and blood diseases. The findings of this study support these uses. It is also reported to be of great importance in controlling diarrhoea (Athisaara), amoebic dysentery (Raktaatisaara), bleeding piles and urinary infections (Valsala, 2000). The inhibitory activities of leaf and stem extracts increased with concentration. It has been extensively reported that more active ingredients in plants are dissolved in solution at higher concentrations (Amadioha, 2000; Onifade, 2002; Okigbo and Ogbonnaya, 2006). Meanwhile, comparison of the inhibitory activity of the antibiotic (Streptomycin) and plant extract revealed that the antibiotic showed higher inhibition than the plant extract of all test organisms with the exception of A. flavus at 1000 mg/ml of stem extract. This result therefore shows that the stem extract of M. invisa has greater antifungal action at higher concentrations than Streptomycin, which is a synthetic drug. All test pathogens were found to vary in their susceptibility to the plant extract and antibiotic. Aspergillus flavus was more susceptible to the plant extracts while S. typhi and E. coli were less susceptible. In addition, A. flavus was least susceptible to the drug whereas S. typhi was the most susceptible. Ogu et al. (2012) stated that such differences are due to the fact that bacterial pathogens like S. typhi and E. coli develop resistance to inhibition caused by plant extracts (Valsala, 2000). Conclusion and recommendations This study reports the antimicrobial properties of the leaves and stem of M. invisa. The stem extract had greater antifungal and antibacterial properties than the leaf extract at all concentrations tested. Hence, the stem extract is recommended for potential use in the treatment of ailments caused by A. flavus, S. typhi, E. coli and K. pneumoniae. However, further clinical testing of M. invisa is required to determine its potential for treatment of infectious diseases in humans. Acknowledgements I acknowledge with warm hearted appreciation my supervisor, Dr. Mrs. C.A. Ezeabara for her advice and guidance. I wish to express my profound gratitude to my parents Mr. and Mrs. Chukwudi Onwumbiko for their support, advice, love and provisions in the course of this work. Author’s biography H.C.C. graduated from Department of Botany, Nnamdi Azikiwe University, Awka, Nigeria in 2017. His interest lies in Phytomedicine. He performed the experiments and has primary responsibility for the final content. C.A.E. designed and supervised the study, assisted in the research and writing of the paper. References Ajayi , A. O. and Akonai , K. A. ( 2005 ) Distribution pattern of enteric organisms in the Lagos Lagoon , African Journal of Biomedicinal Resources , 8 ( 3 ), 163 – 168 . Google Scholar OpenURL Placeholder Text WorldCat Akinsinde , K. A. and Olukoya , D. K. ( 1995 ) Vibriocidal activities of some local herbs , Journal of Diarrhoeal Disease Research , 13 ( 2 ), 127 – 129 . Google Scholar OpenURL Placeholder Text WorldCat Amadioha , A. C. ( 2000 ) Control of anthracnose disease of cowpea by Cympogonoi stratus and Ocimumgratissimum , ActaPhytopathological Entomology Hung Kun , 34 , 83 – 89 . Google Scholar OpenURL Placeholder Text WorldCat Amanike , S. , Nancy , P. and Keller , C. ( 2011 ) Aspergillusflavus , Annual Review of Phytopathology , 49 , 107 – 133 . Google Scholar Crossref Search ADS PubMed WorldCat Association of Official Analytical Chemists . ( 2000 ) Official Method of Analysis , Association of Official Analytical Chemists , Washington D.C , p. 44 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Das , A. and Singh , G. P. ( 1999 ) Effect of different levels of berseem (Trifolium alexandrinum) supplementation of wheat straw on some physical factors regulating intake and digestion , Animal Feed science technology , 81 ( 1–2 ), 133 – 149 . Google Scholar Crossref Search ADS WorldCat Deng , W. , Liou , S. R., Plunkett , G. , III et al. . ( 2003 ) Comparative genomics of Salmonella enterica and Serovar typhi Strains Ty2 and CT18 , Journal of Bacteriology , 185 , 2330 – 2337 . Google Scholar Crossref Search ADS PubMed WorldCat Ekhaise , F. O. and Anyansi , C. C. ( 2005 ) Influence of brewery effluent discharge on the microbiological and physicochemical quality of Ikpoba River, Nigeria , African Journal of Biotechnology , 4 ( 10 ), 1062 – 1065 . Google Scholar OpenURL Placeholder Text WorldCat Espinel-Ingroff , A. ( 2002 ) E-test method for testing susceptibilities of Aspergillus spp. to the new triazolesvoriconazole and posaconazole and to established antifungal agents: comparison with NCCLS broth microdilution method , Nature , 40 , 2101 – 2107 . Google Scholar OpenURL Placeholder Text WorldCat Ezeabara , C. A. and Mbah , E. U. ( 2016 ) Comparative phytochemical and proximate investigations of leaf, root and stem of M. invisa Mart. and M. pudica L , Journal of Pharma Science , 1 , 1 . Google Scholar OpenURL Placeholder Text WorldCat Ezeabara , C. A. and Okonkwo , E. E. ( 2016 ) Comparison of phytochemical and proximate components of leaf, root and stem of croton hirtusl’herit and croton lobatuslinn , Journal of Medical and Health Research , 1 ( 2 ), 23 – 33 . Google Scholar OpenURL Placeholder Text WorldCat Finar , I. L. ( 1986 ) Stereo Chemistry and the Chemistry of Natural Products , Longman , London , p. 213 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Francis , G. , Kerem , Z., Makkar , H. P. S. et al. . ( 2002 ) The biological action of saponins in animal systems: a review , British Journal of Nutrition , 88 , 587 – 605 . Google Scholar Crossref Search ADS PubMed WorldCat Harborne , J. B. ( 1973 ) Phytochemical Methods; a Guide to Modern Techniques of Plant Analysis , Chapmann and Hall Publishers , London , p. 456 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Harborne , J. B. ( 1998 ) Phytochemical Methods: A Guide to Modern Techniques of Plant Analyses , Chapmann and Hall Publishers , London , p. 342 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Kanokmedhakul , K. , Kanokmedhakul , S. and Phatchana , R. ( 2005 ) Biological activity of anthraquinones and triterpenoids from Prismatomerisfragrans , Journal of Ethnopharmacology , 100 ( 3 ), 284 – 288 . Google Scholar Crossref Search ADS PubMed WorldCat Kirk , H. and Sawyer , R. ( 1998 ) Frait Pearson Chemical Analysis of Food . (ed.) Edinburgh , Longman Scientific and Technical , pp. 211 – 212 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Kittakoop , P. , Mahidol , C. and Ruchirawat , S. ( 2014 ) Alkaloids as important scaffolds in therapeutic drugs for the treatments of cancer, tuberculosis, and smoking cessation , Curr Top Medical Chemistry , 14 ( 2 ), 239 – 252 . Google Scholar Crossref Search ADS WorldCat Lui , L. , Liu , S. W., Jiang , S. B. et al. . ( 2004 ) Tannin inhibits HIV-1 entry by targeting gp41 , Acta Pharmacology Sinica , 25 ( 2 ), 213 – 218 . Google Scholar OpenURL Placeholder Text WorldCat Mandel , P. , Babu , S. P. and Mandal , N. C. ( 2005 ) Antimicrobial activity of saponins from Acacin auriculiformis , Fitoterapia , 76 ( 5 ), 462 – 465 . Google Scholar Crossref Search ADS PubMed WorldCat Masayuki , M. and Katsuyai , G. ( 2010 ) Aspergillus: Molecular Biology and Genomics , Horizon Scientific Press, UK , 157 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Njoku , O. V. and Obi , C. ( 2009 ) Phytochemical constituents of some selected medicinal plants , African Journal of Pure and Applied Chemistry , 3 ( 11 ), 228 – 233 . Google Scholar OpenURL Placeholder Text WorldCat Ogu , G. I. , Tanimowo , W. O., Nwachukwu , P. U. et al. . ( 2012 ) Antimicrobial and phytochemical evaluation of the leaf, stem bark and root extracts of CyathulaprostrataL. Blume against some human pathogens , Journal of Intercultutural Ethnopharmacognosis , 1 , 35 – 43 . Google Scholar Crossref Search ADS WorldCat Okigbo , R. N. and Ogbonnaya , O. U. ( 2006 ) Antifungal effects of two tropical plant extracts (Ocimumgratissimumand Afromaomummelegueta) on postharvest yam (Dioscoreaspp.) rot , African Journal of Biotechnology , 5 ( 9 ), 727 – 731 . Google Scholar OpenURL Placeholder Text WorldCat Onifade , A. K. ( 2002 ) Antifungal effect of AzadirachtaindicaA. Joss extracts on Collectotricum lindemathianum , Global Journal of Pure and Applied Science , 6 ( 3 ), 423 – 428 . Google Scholar OpenURL Placeholder Text WorldCat Orji , M. U. , Ezenwaje , E. E. and Anyaegbunam , B. C. ( 2006 ) Spatial appraisal of shallow well water pollution in Awka, Nigeria , Nigerian Journal of Microbiology , 20 ( 3 ), 1384 – 1389 . Google Scholar OpenURL Placeholder Text WorldCat Oxford English Dictionary . ( 2005 ) Coli , Oxford University Press, UK , p. 34 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Praveen , K. and Kumud , U. ( 2012 ) Tannins are astringent , Journal of Pharmacognosy and Phytochemistry , 1 ( 3 ), 8192 . Google Scholar OpenURL Placeholder Text WorldCat Ramírez-Camejo , L. A. , Zuluaga-Montero , A., Lázaro-Escudero , M. A. et al. . ( 2012 ) Phylogeography of the cosmopolitan fungus Aspergillusflavus: Is everything everywhere? Fungal Biology , 116 ( 3 ), 452 – 463 . Google Scholar Crossref Search ADS PubMed WorldCat Reynolds , D. ( 2003 ) Recruitment of through 319-phosphorylated Ndd1p to the FHA domain of Fkh2p requires CLB kinase activity: a mechanism for CLB cluster gene activation , Genes Development , 17 ( 14 ), 1789 – 1802 . Google Scholar Crossref Search ADS PubMed WorldCat Ryan , K. J. and Ray , C. G. ( 2004 ) Sherris Medical Microbiology (ed.) McGraw Hill, USA . ISBN 0-8385-8529-9. Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Shelef , L. A. ( 1983 ) Antimicrobial effects of spices—U S Forest Service 2008 ‘M. pudica’, Usambara invasive plants , Journal of Food Safety , 6 , 29 – 44 . Google Scholar Crossref Search ADS WorldCat Sieradzki , K. , Robert , R. B., Haber , S. W. et al. . ( 1999 ) The development of vancomycin resistance in a patient with methicillin-resistant Staphylococcus aureus infection , National England Medicine , 340 ( 7 ), 517 – 523 . Google Scholar Crossref Search ADS WorldCat Thongson , C. , Davidson , P. M., Mahakarnchanakul , W. et al. . ( 2004 ) Antimicrobial activity of ultrasound-assisted solvent-extracted spices , Letters in Applied Microbiology , 39 , 401 – 406 . Google Scholar Crossref Search ADS PubMed WorldCat Valsala , S. ( 2000 ) Estrogenic and antiestrogenic activities of M. pudica on Rattusnorvegicus , Journal of Ecotoxicology , 10 ( 1 ), 25 – 29 . Google Scholar OpenURL Placeholder Text WorldCat Zaika , L. L. ( 1988 ) Spices and herbs: their antimicrobial activity and its determination , Journal of Food Safety , 9 , 97 – 118 . Google Scholar Crossref Search ADS WorldCat Author notes Supervisor: Dr. Mrs. Chinelo A. Ezeabara, Department of Botany, Nnamdi Azikiwe University, P.M.B. 5025 Awka, Nigeria. © The Author(s) 2018. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com © The Author(s) 2018. Published by Oxford University Press.

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Abstract

Abstract Preliminary phytochemical screening was carried out and in vitro antimicrobial activities of leaves and stem of Mimosa invisa Mart. against some clinical pathogens were tested using standard techniques. Significance was measured using Duncan’s multiple range test. The inhibitory actions of the leaf and stem extracts increased with concentration. The rates of inhibition of the two extracts were low and high against Salmonella typhi and Aspergillus flavus respectively. The phytochemicals present in the plant parts had antimicrobial properties and reveal that M. invisa may have potential in pharmaceutical applications. Mimosa invisa, alkaloid, antimicrobial actions, zone of inhibition, Salmonella typhi, Aspergillus flavus Introduction The leaves, stems, flowers, fruits, seeds, roots, rhizomes and bark of some plants may be used for medicinal purposes. Many plant and herb species have unrealized antimicrobial and antiviral features (Shelef, 1983; Zaika, 1988) and may thus be a source of phyto-antimicrobial agents (Das et al., 1999). Sieradzki et al. (1999) stated that the emergence of bacterial strains with reduced vulnerability to antibiotics and the increasingly wide spread of multidrug resistant strains of bacteria would require the search for new infection fighting strategies. The properties of medicinal plants are of great benefit (Finar, 1986; Harborne, 1998). Antibacterial compounds extracted from plants have drawn much attention and could be used as an alternative strategy to prevent the spread of diseases. Flavonoids have been shown to inhibit topoisomerase enzymes. Alkaloids are of great benefit to pharmaceutical industries including as anti-malarial agents (for example, quinine) (Kittakoop, Mahidol, Ruchirawat, 2014). It has been recorded that tannin has antiviral, antibacterial and antiparasitic properties (Lui et al., 2004; Njoku and Obi, 2009). Saponins have been implicated as bioactive antibacterial agents (Mandel, Babu, Mandal, 2005). Anthraquinones are used to relieve constipation (Kittakoop, Mahidol, Ruchirawat, 2014). However, there is no information on the antimicrobial actions of phytochemicals present in M. invisa. Therefore, the objectives of this work were to determine the phytochemicals present in the plant as well as to assess their antimicrobial activity against selected microorganisms that cause ailments in humans: Escherichia coli, a facultative anaerobic, gram-negative, rod-shaped bacterium that is abundant in the human gut (OED, 2005); Salmonella typhi, a gram-negative bacterium that causes typhoid fever, a disease that has afflicted humans throughout history and continues to be a public health concern (Deng et al., 2003); Klebsiella pneumoniae, a gram-negative nonmotile, lactose-fermenting, facultative anaerobic, rod-shaped bacterium that is enclosed in a capsule and causes destructive changes to human and animal lungs if aspirated (inhaled), particularly to the alveoli (in the lungs), resulting in bloody sputum (Ryan and Ray, 2004); and Aspergillus flavus, a saprotrophic and pathogenic (Masayuki and Katsuyai, 2010) fungus with a cosmopolitan distribution (Ramírez-Camejo et al., 2012). Materials and methods Collection of sample The leaves and stem of mature M. invisa were collected from a roadside at Idi-Oshe, opposite IITA Ibadan, Oyo State, Nigeria, in August 2016. The analyses were then carried out at the Department of Microbiology in IITA, Ibadan. The plant was identified by Mr. Kayode, a Plant Taxonomist at IITA Ibadan. Preparation of samples for analyses The fresh specimens were cleaned with clean water and oven dried at a temperature of 60°C for 72 h. The samples were separated by hand and were ground using a grinding machine (SPM-5, China), then placed in an air-tight container. Extraction of plant materials Methanol extraction An aqueous decoction of the plant was prepared by soaking 500 g of the powdered samples of leaves and stem each in 2.5 L of methanol. The whole setup was left at room temperature for 72 hours. The extract was then concentrated using a rotary evaporator (RE-52A, China) and the solvent allowed to evaporate. The concentrated extract was stored in an air-tight container in a cold store at a regulated temperature of 20°C until it was required for analyses. Preliminary phytochemical screening The methods and tests used in the phytochemical screening of the plant extract were described by Ezeabara and Okonkwo (2016). The sign ‘+’ denotes presence. Quantitative phytochemical screening Alkaloids and flavonoids were determined using the alkaline precipitation gravimetric method and gravimetric method of Harborne (1973), respectively. The method used to determine saponins was described by the AOAC (2000). The Folin-Dennis colorimetric method was used to determine the tannin content of the sample (Kirk and Sawyer, 1998). Anthraquinone was determined by the spectrophotometric method used by Ezeabara and Okonkwo (2016). Microbial analysis Isolation of the test organisms Using a wire loop, colonies of the test organisms (Escherichia coli, Salmonella typhi, Klebsiella pneumoniae and Aspergillus flavus) were collected from pure cultures kept at the Soil Microbiology Unit, IITA Ibadan, Oyo State, Nigeria. Antimicrobial test procedures Preparation of stock solution Stock solutions of the two methanolic extracts of leaves and stem were prepared by weighing 2.0 g of each methanolic extract using electronic scales. This was then dissolved completely in 2.0 ml of dimethyl sulphoxide (DMSO) in a sterile test tube, giving a stock solution concentration of 1000 mg/ml per extract. The stock solution was then labelled and stored at room temperature until needed for use. Determination of inhibitory activity The inhibitory activity of the methanolic extract of the leaves and stems of M. invisa, antibiotics (positive control), and dimethyl sulphoxide (negative control) was determined using the disc diffusion method in 5 mm discs. The concentrations used were as follows: 1000 mg/ml of the methanolic extract, 100 mg/ml of antibiotic (Streptomycin) and 1000 mg/ml of DMSO. After the discs had absorbed the leaf and stem extract, the antibiotic or the control DMSO, the discs were removed, dried and placed on media on which the test microorganisms were freshly inoculated, then incubated at 37°C for 24 hours for bacteria and 25°C for 72 hours for fungi. Antimicrobial activity was determined after 24 hours (for bacteria) of incubation and 72 hours (for fungi) of incubation by measuring the zone of inhibition around each paper disc in millimetres (mm) (Reynolds, 2003). Determination of minimum inhibitory concentration The minimum inhibitory concentrations (MIC) of the absolute (stock) concentrations were determined using the agar well diffusion method; five sterile plates were prepared, and nutrient broth was poured into each of the plates and then allowed to dry. Using standardized inoculums (106 cfu/ml), a loop full of the different test organisms was streaked onto each of the five plates when dry. Then, 10 holes were dug using an agar borer for the varying concentrations of methanolic extracts of leaves and stem. A ruler was used to separate the holes for each of the methanolic extracts. Varying concentrations of the different extracts were made by serial dilution using DMSO as the diluent. The absolute/stock concentrations of extracts used were 1000 mg/ml (equivalent to 100%). Five test tubes per extract were prepared in a test tube rack. The dilutions were as follows: 50% (500 mg/ml), 25% (250 mg/ml), 12.5% (125 mg/ml) and 6.25% (62.5 mg/ml) in each of the test tubes. DMSO served as the negative control. A measured 50 μL volume of each dilution was added aseptically into the holes seeded with the test organisms in the nutrient agar plate using a syringe and placed in an incubator at 35°C for 24 hours. MIC was considered as the lowest concentration of methanolic extract showing a clear zone of inhibition (Thongson et al., 2004). Determination of minimum bactericidal/fungicidal concentration The plates with the MIC were incubated for a further 24 hours at 35°C to test which organism would grow on the zones of inhibition. Those plates on which organisms were completely killed after 24 hours, and clear zones remained, were referred to as bactericidal for bacteria and fungicidal for fungi (Espinel-Ingroff, 2002). Statistical analysis The test was carried out using a completely randomized design. Analysis of Variance (ANOVA) using SPSS version 21 was employed to analyse the data collected in the study. The Duncan’s multiple range test (DMRT) was used to test the difference among treatment means with more than two levels. All statistical analyses were carried out at 5% level of significance. The data were expressed as the mean (±standard deviation) of triplicate determinations. Results and discussion The result of the qualitative phytochemical analysis of the leaf and stem extracts of M. invisa demonstrated that alkaloids, anthraquinones, saponins, tannins and flavonoids were present (Table 1). Moreover, the quantitative analysis revealed that the leaf had the largest concentration of alkaloids among the plant parts, at 0.16 ± 0.11 mg/100 g, and anthraquinones at 0.88 ± 0.06 mg/100 g, whereas the highest concentrations of saponins at 16.81 ± 5.12 mg/100 g, tannins at 0.15 ± 0.04 mg/100 g and flavonoids at 0.48 ± 0.41 mg/100 g occurred in the stem (Table 2). These correspond largely with the findings of Ezeabara and Mbah (2016), who reported a wide range of chemical compounds in the leaves, stem and root of M. invisa and M. pudica. Stated that M. invisa is traditionally used in the treatment of diabetes. In addition, this species has been reported to have potent hypoglycaemic effects in alloxan-induced diabetic mice. The medicinal properties of this plant are likely to be a result of the wide range of biological activities of the phytochemicals. The pharmacological activities of alkaloids are varied, and include anti-malarial (e.g. quinine) and antiasthma activity (e.g. homoharringtonine) (Kittakoop, Mahidol, Ruchirawat, 2014). Due to saponins’ immune-enhancing properties they are used as immunological adjuvants in the formulation of vaccines (Francis et al., 2002). Tannins may be used medicinally in antidiarrheal, haemostatic and antihaemorrhoidal compounds (Praveen and Kumud, 2012). Kanokmedhakul, Kanokmedhakul, Phatchana (2005) reported that anthraquinones isolated from Primatomeris fragrans showed both antifungal and antituberculosis activities. Table 1. Qualitative phytochemical composition of the leaf and stem extracts of Mimosa invisa Phytochemicals . Plant part . Leaf . Stem . Alkaloid + + Saponin + + Flavonoid + + Phenol + + Tannin + + Anthraquinone + + Phytochemicals . Plant part . Leaf . Stem . Alkaloid + + Saponin + + Flavonoid + + Phenol + + Tannin + + Anthraquinone + + Open in new tab Table 1. Qualitative phytochemical composition of the leaf and stem extracts of Mimosa invisa Phytochemicals . Plant part . Leaf . Stem . Alkaloid + + Saponin + + Flavonoid + + Phenol + + Tannin + + Anthraquinone + + Phytochemicals . Plant part . Leaf . Stem . Alkaloid + + Saponin + + Flavonoid + + Phenol + + Tannin + + Anthraquinone + + Open in new tab Table 2. Mean quantitative phytochemical composition of the leaf and stem of M. invisa composition (mg/100 g) Plant part . Saponin . Alkaloid . Tannin . Anthraquinone . Flavonoid . Stem 16.81 ± 5.12b 0.04 ± 0.01a 0.15 ± 0.04b 0.79 ± 0.09a 0.48 ± 0.41b Leaf 8.64 ± 1.47a 0.16 ± 0.11b 0.10 ± 0.00a 0.88 ± 0.06b 0.38 ± 0.32a p-value 0.19 0.13 0.28 0.08 0.01 Plant part . Saponin . Alkaloid . Tannin . Anthraquinone . Flavonoid . Stem 16.81 ± 5.12b 0.04 ± 0.01a 0.15 ± 0.04b 0.79 ± 0.09a 0.48 ± 0.41b Leaf 8.64 ± 1.47a 0.16 ± 0.11b 0.10 ± 0.00a 0.88 ± 0.06b 0.38 ± 0.32a p-value 0.19 0.13 0.28 0.08 0.01 Results are in mean ± std error of duplicate determinations. The same letter in a column means that the respective means were not significantly different. Bold values indicates that p<0.05. Open in new tab Table 2. Mean quantitative phytochemical composition of the leaf and stem of M. invisa composition (mg/100 g) Plant part . Saponin . Alkaloid . Tannin . Anthraquinone . Flavonoid . Stem 16.81 ± 5.12b 0.04 ± 0.01a 0.15 ± 0.04b 0.79 ± 0.09a 0.48 ± 0.41b Leaf 8.64 ± 1.47a 0.16 ± 0.11b 0.10 ± 0.00a 0.88 ± 0.06b 0.38 ± 0.32a p-value 0.19 0.13 0.28 0.08 0.01 Plant part . Saponin . Alkaloid . Tannin . Anthraquinone . Flavonoid . Stem 16.81 ± 5.12b 0.04 ± 0.01a 0.15 ± 0.04b 0.79 ± 0.09a 0.48 ± 0.41b Leaf 8.64 ± 1.47a 0.16 ± 0.11b 0.10 ± 0.00a 0.88 ± 0.06b 0.38 ± 0.32a p-value 0.19 0.13 0.28 0.08 0.01 Results are in mean ± std error of duplicate determinations. The same letter in a column means that the respective means were not significantly different. Bold values indicates that p<0.05. Open in new tab The antimicrobial results of the methanolic extracts of leaves and stem of M. invisa indicated that the plant showed antimicrobial activity against the tested microorganisms at five concentrations: 62.5, 125, 250, 500 and 1000 mg/ml (Tables 3–7). At 62.5 mg/ml, the stem extract showed greater inhibition of Salmonella typhi (0.50 ± 0.710 mm) and Aspergillus flavus (1.80 ± 0.35 mm) than the leaf extract (undetectable mm and 0.80 ± 1.06 mm respectively) (Table 3); at 125 mg/ml the stem extract showed higher inhibition of S. typhi (1.50 ± 0.71 mm), Klebsiella pneumonia (1.00 ± 1.41 mm) and A. flavus (3.50 ± 0.71 mm) than the leaf extract (undetectable mm, undetectable mm and 2.30 ± 1.06 mm, respectively) (Table 4); at 250 mg/ml the stem extract showed higher inhibition than the leaf extract of all the test organisms, including S. typhi (2.50 ± 0.71 mm), Escherichia coli (2.50 ± 0.71 mm), K. pneumonia (3.00 ± 1.41 mm) and A. flavus (5.50 ± 0.71 mm) (Table 5); at 500 mg/ml the stem extract showed higher inhibition than the leaf extract of all the test organisms, including S. typhi (3.50 ± 0.71 mm), E. coli (4.50 ± 0.71 mm), K. pneumonia (4.50 ± 0.71 mm) and A. flavus (7.50 ± 0.71 mm) (Table 6); and at 1000 mg/ml concentration the stem extract showed higher inhibition than the leaf extract of all the test organisms, including S. typhi (4.50 ± 0.71 mm), E. coli (6.50 ± 0.71 mm), K. pneumoniae (6.50 ± 0.71 mm) and A. flavus (9.50 ± 0.71 mm) (Table 7). The stem extract showed the highest inhibitory activity against A. flavus at all concentrations and the lowest activity against S. typhi and E. coli (Fig. 1) while the leaf extract showed the highest inhibitory activity against A. flavus at all concentrations and the lowest activity against S. typhi and E. coli (Fig. 2). Table 3. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 62.5 mg/ml concentration Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 0.50 ± 0.71a 0.00 ± 0.00a 0.00 ± 0.00a 1.80 ± 0.35a Leaf 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.80 ± 1.06a Drug 12.00 ± 0.00b 11.00 ± 0.00b 11.00 ± 0.00b 8.50 ± 0.71b Control 0.00 ± 0.000a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.01 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 0.50 ± 0.71a 0.00 ± 0.00a 0.00 ± 0.00a 1.80 ± 0.35a Leaf 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.80 ± 1.06a Drug 12.00 ± 0.00b 11.00 ± 0.00b 11.00 ± 0.00b 8.50 ± 0.71b Control 0.00 ± 0.000a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.01 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05). Open in new tab Table 3. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 62.5 mg/ml concentration Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 0.50 ± 0.71a 0.00 ± 0.00a 0.00 ± 0.00a 1.80 ± 0.35a Leaf 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.80 ± 1.06a Drug 12.00 ± 0.00b 11.00 ± 0.00b 11.00 ± 0.00b 8.50 ± 0.71b Control 0.00 ± 0.000a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.01 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 0.50 ± 0.71a 0.00 ± 0.00a 0.00 ± 0.00a 1.80 ± 0.35a Leaf 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.80 ± 1.06a Drug 12.00 ± 0.00b 11.00 ± 0.00b 11.00 ± 0.00b 8.50 ± 0.71b Control 0.00 ± 0.000a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.01 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05). Open in new tab Table 4. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 125 mg/ml concentration Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 1.50 ± 0.71b 0.50 ± 0.71a 1. 00 ± 1.41a 3.50 ± 0.71b Leaf 0.00 ± 0.00a 0.50 ± 0.71a 0.00 ± 0.00a 2.30 ± 1.06b Drug 12. 00 ± 0.00c 11. 00 ± 0.00b 11. 00 ± 0.00b 6.50 ± 0.71c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.01 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 1.50 ± 0.71b 0.50 ± 0.71a 1. 00 ± 1.41a 3.50 ± 0.71b Leaf 0.00 ± 0.00a 0.50 ± 0.71a 0.00 ± 0.00a 2.30 ± 1.06b Drug 12. 00 ± 0.00c 11. 00 ± 0.00b 11. 00 ± 0.00b 6.50 ± 0.71c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.01 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05). Open in new tab Table 4. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 125 mg/ml concentration Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 1.50 ± 0.71b 0.50 ± 0.71a 1. 00 ± 1.41a 3.50 ± 0.71b Leaf 0.00 ± 0.00a 0.50 ± 0.71a 0.00 ± 0.00a 2.30 ± 1.06b Drug 12. 00 ± 0.00c 11. 00 ± 0.00b 11. 00 ± 0.00b 6.50 ± 0.71c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.01 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 1.50 ± 0.71b 0.50 ± 0.71a 1. 00 ± 1.41a 3.50 ± 0.71b Leaf 0.00 ± 0.00a 0.50 ± 0.71a 0.00 ± 0.00a 2.30 ± 1.06b Drug 12. 00 ± 0.00c 11. 00 ± 0.00b 11. 00 ± 0.00b 6.50 ± 0.71c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.01 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05). Open in new tab Table 5. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 250 mg/ml concentration Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 2.50 ± 0.71b 2.50 ± 0.71b 3. 00 ± 1.41b 5.50 ± 0.71b Leaf 0.50 ± 0.71a 1. 00 ± 1.41a 1.80 ± 0.35a 4. 00 ± 1.41b Drug 12. 00 ± 0.00c 11. 00 ± 0.00c 11. 00 ± 0.00c 8.50 ± 0.71c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.03 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 2.50 ± 0.71b 2.50 ± 0.71b 3. 00 ± 1.41b 5.50 ± 0.71b Leaf 0.50 ± 0.71a 1. 00 ± 1.41a 1.80 ± 0.35a 4. 00 ± 1.41b Drug 12. 00 ± 0.00c 11. 00 ± 0.00c 11. 00 ± 0.00c 8.50 ± 0.71c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.03 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05). Open in new tab Table 5. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 250 mg/ml concentration Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 2.50 ± 0.71b 2.50 ± 0.71b 3. 00 ± 1.41b 5.50 ± 0.71b Leaf 0.50 ± 0.71a 1. 00 ± 1.41a 1.80 ± 0.35a 4. 00 ± 1.41b Drug 12. 00 ± 0.00c 11. 00 ± 0.00c 11. 00 ± 0.00c 8.50 ± 0.71c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.03 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 2.50 ± 0.71b 2.50 ± 0.71b 3. 00 ± 1.41b 5.50 ± 0.71b Leaf 0.50 ± 0.71a 1. 00 ± 1.41a 1.80 ± 0.35a 4. 00 ± 1.41b Drug 12. 00 ± 0.00c 11. 00 ± 0.00c 11. 00 ± 0.00c 8.50 ± 0.71c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.00 0.00 0.03 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05). Open in new tab Table 6. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 500 mg/ml concentration Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 3.50 ± 0.71c 4.50 ± 0.71b 4.50 ± 0.71b 7.50 ± 0.71c Leaf 1.50 ± 0.71b 1.50 ± 2.12a 3.50 ± 0.71b 6.00 ± 1.41b Drug 12. 00 ± 0.00d 11. 00 ± 0.00c 11. 00 ± 0.00c 8.50 ± 0.07c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.02 0.00 0.02 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 3.50 ± 0.71c 4.50 ± 0.71b 4.50 ± 0.71b 7.50 ± 0.71c Leaf 1.50 ± 0.71b 1.50 ± 2.12a 3.50 ± 0.71b 6.00 ± 1.41b Drug 12. 00 ± 0.00d 11. 00 ± 0.00c 11. 00 ± 0.00c 8.50 ± 0.07c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.02 0.00 0.02 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05) Open in new tab Table 6. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 500 mg/ml concentration Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 3.50 ± 0.71c 4.50 ± 0.71b 4.50 ± 0.71b 7.50 ± 0.71c Leaf 1.50 ± 0.71b 1.50 ± 2.12a 3.50 ± 0.71b 6.00 ± 1.41b Drug 12. 00 ± 0.00d 11. 00 ± 0.00c 11. 00 ± 0.00c 8.50 ± 0.07c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.02 0.00 0.02 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 3.50 ± 0.71c 4.50 ± 0.71b 4.50 ± 0.71b 7.50 ± 0.71c Leaf 1.50 ± 0.71b 1.50 ± 2.12a 3.50 ± 0.71b 6.00 ± 1.41b Drug 12. 00 ± 0.00d 11. 00 ± 0.00c 11. 00 ± 0.00c 8.50 ± 0.07c Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.02 0.00 0.02 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05) Open in new tab Table 7. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 1000 mg/ml Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 4.50 ± 0.71c 6.50 ± 0.71c 6.50 ± 0.71b 9.50 ± 0.71a Leaf 2.50 ± 0.71b 3. 00 ± 1.41b 5.50 ± 0.71b 8. 00 ± 1.41a Drug 12. 00 ± 0.00d 11. 00 ± 0.00d 11. 00 ± 0.00c 8.50 ± 0.71b Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.01 0.00 0.01 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 4.50 ± 0.71c 6.50 ± 0.71c 6.50 ± 0.71b 9.50 ± 0.71a Leaf 2.50 ± 0.71b 3. 00 ± 1.41b 5.50 ± 0.71b 8. 00 ± 1.41a Drug 12. 00 ± 0.00d 11. 00 ± 0.00d 11. 00 ± 0.00c 8.50 ± 0.71b Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.01 0.00 0.01 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05). Open in new tab Table 7. Zone of inhibition (mm) of test organisms by the stem and leaf extracts of Mimosa invisa at 1000 mg/ml Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 4.50 ± 0.71c 6.50 ± 0.71c 6.50 ± 0.71b 9.50 ± 0.71a Leaf 2.50 ± 0.71b 3. 00 ± 1.41b 5.50 ± 0.71b 8. 00 ± 1.41a Drug 12. 00 ± 0.00d 11. 00 ± 0.00d 11. 00 ± 0.00c 8.50 ± 0.71b Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.01 0.00 0.01 Plant part . Salmonella typhi . Escherichia coli . Klebsiella pneumonia . Aspergillus flavus . Stem 4.50 ± 0.71c 6.50 ± 0.71c 6.50 ± 0.71b 9.50 ± 0.71a Leaf 2.50 ± 0.71b 3. 00 ± 1.41b 5.50 ± 0.71b 8. 00 ± 1.41a Drug 12. 00 ± 0.00d 11. 00 ± 0.00d 11. 00 ± 0.00c 8.50 ± 0.71b Control 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a p-value 0.00 0.01 0.00 0.01 Results are in mean ± standard deviation. Means with the same letter in a column are not significantly different (p > 0.05). Open in new tab Figure 1. Open in new tabDownload slide Zone of inhibition (mm) of bacterial pathogens by stem extract of Mimosa invisa. Figure 2. Open in new tabDownload slide Zone of inhibition (mm) of bacterial pathogens by leaf extract of Mimosa invisa. In summary, the stem and leaf extract of M. invisa inhibited the human pathogens tested here. This is probably a result of the synergistic actions of the active components present in the plant parts. This implies that the leaf and stem extracts from M. invisa may have potential as a source of raw material for the pharmaceutical industry and in modern medicine for the treatment of human diseases. Gastroenteritis, typhoid fever, dysentery, cholera, and urinary tract infections have been associated with E. coli (Orji, Ezenwaje, Anyaegbunam, 2006). Salmonella typhi is the causative agent for paratyphoid fever, a mild form of enteric fever (Ekhaise and Anyansi, 2005), and K. pneumonia has been identified to account for up to 55% of nosocomial infections in some parts of Nigeria (Ajayi and Akonai, 2005). Aspergillosis in immunocompromised individuals is caused by A. flavus, an opportunistic human and animal pathogen (Amanike, Nancy, Keller, 2011). The stem extract of M. invisa was found to have higher inhibitory activity against all test organisms than the leaf extract. Akinsinde and Olukoya (1995) reported that the stem of this plant is used traditionally in the treatment of leprosy, dysentery, vaginal and uterine complaints, inflammations, burning sensation, asthma, leucoderma, and fatigue and blood diseases. The findings of this study support these uses. It is also reported to be of great importance in controlling diarrhoea (Athisaara), amoebic dysentery (Raktaatisaara), bleeding piles and urinary infections (Valsala, 2000). The inhibitory activities of leaf and stem extracts increased with concentration. It has been extensively reported that more active ingredients in plants are dissolved in solution at higher concentrations (Amadioha, 2000; Onifade, 2002; Okigbo and Ogbonnaya, 2006). Meanwhile, comparison of the inhibitory activity of the antibiotic (Streptomycin) and plant extract revealed that the antibiotic showed higher inhibition than the plant extract of all test organisms with the exception of A. flavus at 1000 mg/ml of stem extract. This result therefore shows that the stem extract of M. invisa has greater antifungal action at higher concentrations than Streptomycin, which is a synthetic drug. All test pathogens were found to vary in their susceptibility to the plant extract and antibiotic. Aspergillus flavus was more susceptible to the plant extracts while S. typhi and E. coli were less susceptible. In addition, A. flavus was least susceptible to the drug whereas S. typhi was the most susceptible. Ogu et al. (2012) stated that such differences are due to the fact that bacterial pathogens like S. typhi and E. coli develop resistance to inhibition caused by plant extracts (Valsala, 2000). Conclusion and recommendations This study reports the antimicrobial properties of the leaves and stem of M. invisa. The stem extract had greater antifungal and antibacterial properties than the leaf extract at all concentrations tested. Hence, the stem extract is recommended for potential use in the treatment of ailments caused by A. flavus, S. typhi, E. coli and K. pneumoniae. However, further clinical testing of M. invisa is required to determine its potential for treatment of infectious diseases in humans. Acknowledgements I acknowledge with warm hearted appreciation my supervisor, Dr. Mrs. C.A. Ezeabara for her advice and guidance. I wish to express my profound gratitude to my parents Mr. and Mrs. Chukwudi Onwumbiko for their support, advice, love and provisions in the course of this work. Author’s biography H.C.C. graduated from Department of Botany, Nnamdi Azikiwe University, Awka, Nigeria in 2017. His interest lies in Phytomedicine. He performed the experiments and has primary responsibility for the final content. C.A.E. designed and supervised the study, assisted in the research and writing of the paper. References Ajayi , A. O. and Akonai , K. A. ( 2005 ) Distribution pattern of enteric organisms in the Lagos Lagoon , African Journal of Biomedicinal Resources , 8 ( 3 ), 163 – 168 . Google Scholar OpenURL Placeholder Text WorldCat Akinsinde , K. A. and Olukoya , D. K. ( 1995 ) Vibriocidal activities of some local herbs , Journal of Diarrhoeal Disease Research , 13 ( 2 ), 127 – 129 . Google Scholar OpenURL Placeholder Text WorldCat Amadioha , A. C. ( 2000 ) Control of anthracnose disease of cowpea by Cympogonoi stratus and Ocimumgratissimum , ActaPhytopathological Entomology Hung Kun , 34 , 83 – 89 . Google Scholar OpenURL Placeholder Text WorldCat Amanike , S. , Nancy , P. and Keller , C. ( 2011 ) Aspergillusflavus , Annual Review of Phytopathology , 49 , 107 – 133 . Google Scholar Crossref Search ADS PubMed WorldCat Association of Official Analytical Chemists . ( 2000 ) Official Method of Analysis , Association of Official Analytical Chemists , Washington D.C , p. 44 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Das , A. and Singh , G. P. ( 1999 ) Effect of different levels of berseem (Trifolium alexandrinum) supplementation of wheat straw on some physical factors regulating intake and digestion , Animal Feed science technology , 81 ( 1–2 ), 133 – 149 . Google Scholar Crossref Search ADS WorldCat Deng , W. , Liou , S. R., Plunkett , G. , III et al. . ( 2003 ) Comparative genomics of Salmonella enterica and Serovar typhi Strains Ty2 and CT18 , Journal of Bacteriology , 185 , 2330 – 2337 . Google Scholar Crossref Search ADS PubMed WorldCat Ekhaise , F. O. and Anyansi , C. C. ( 2005 ) Influence of brewery effluent discharge on the microbiological and physicochemical quality of Ikpoba River, Nigeria , African Journal of Biotechnology , 4 ( 10 ), 1062 – 1065 . Google Scholar OpenURL Placeholder Text WorldCat Espinel-Ingroff , A. ( 2002 ) E-test method for testing susceptibilities of Aspergillus spp. to the new triazolesvoriconazole and posaconazole and to established antifungal agents: comparison with NCCLS broth microdilution method , Nature , 40 , 2101 – 2107 . Google Scholar OpenURL Placeholder Text WorldCat Ezeabara , C. A. and Mbah , E. U. ( 2016 ) Comparative phytochemical and proximate investigations of leaf, root and stem of M. invisa Mart. and M. pudica L , Journal of Pharma Science , 1 , 1 . Google Scholar OpenURL Placeholder Text WorldCat Ezeabara , C. A. and Okonkwo , E. E. ( 2016 ) Comparison of phytochemical and proximate components of leaf, root and stem of croton hirtusl’herit and croton lobatuslinn , Journal of Medical and Health Research , 1 ( 2 ), 23 – 33 . Google Scholar OpenURL Placeholder Text WorldCat Finar , I. L. ( 1986 ) Stereo Chemistry and the Chemistry of Natural Products , Longman , London , p. 213 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Francis , G. , Kerem , Z., Makkar , H. P. S. et al. . ( 2002 ) The biological action of saponins in animal systems: a review , British Journal of Nutrition , 88 , 587 – 605 . Google Scholar Crossref Search ADS PubMed WorldCat Harborne , J. B. ( 1973 ) Phytochemical Methods; a Guide to Modern Techniques of Plant Analysis , Chapmann and Hall Publishers , London , p. 456 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Harborne , J. B. ( 1998 ) Phytochemical Methods: A Guide to Modern Techniques of Plant Analyses , Chapmann and Hall Publishers , London , p. 342 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Kanokmedhakul , K. , Kanokmedhakul , S. and Phatchana , R. ( 2005 ) Biological activity of anthraquinones and triterpenoids from Prismatomerisfragrans , Journal of Ethnopharmacology , 100 ( 3 ), 284 – 288 . Google Scholar Crossref Search ADS PubMed WorldCat Kirk , H. and Sawyer , R. ( 1998 ) Frait Pearson Chemical Analysis of Food . (ed.) Edinburgh , Longman Scientific and Technical , pp. 211 – 212 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Kittakoop , P. , Mahidol , C. and Ruchirawat , S. ( 2014 ) Alkaloids as important scaffolds in therapeutic drugs for the treatments of cancer, tuberculosis, and smoking cessation , Curr Top Medical Chemistry , 14 ( 2 ), 239 – 252 . Google Scholar Crossref Search ADS WorldCat Lui , L. , Liu , S. W., Jiang , S. B. et al. . ( 2004 ) Tannin inhibits HIV-1 entry by targeting gp41 , Acta Pharmacology Sinica , 25 ( 2 ), 213 – 218 . Google Scholar OpenURL Placeholder Text WorldCat Mandel , P. , Babu , S. P. and Mandal , N. C. ( 2005 ) Antimicrobial activity of saponins from Acacin auriculiformis , Fitoterapia , 76 ( 5 ), 462 – 465 . Google Scholar Crossref Search ADS PubMed WorldCat Masayuki , M. and Katsuyai , G. ( 2010 ) Aspergillus: Molecular Biology and Genomics , Horizon Scientific Press, UK , 157 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Njoku , O. V. and Obi , C. ( 2009 ) Phytochemical constituents of some selected medicinal plants , African Journal of Pure and Applied Chemistry , 3 ( 11 ), 228 – 233 . Google Scholar OpenURL Placeholder Text WorldCat Ogu , G. I. , Tanimowo , W. O., Nwachukwu , P. U. et al. . ( 2012 ) Antimicrobial and phytochemical evaluation of the leaf, stem bark and root extracts of CyathulaprostrataL. Blume against some human pathogens , Journal of Intercultutural Ethnopharmacognosis , 1 , 35 – 43 . Google Scholar Crossref Search ADS WorldCat Okigbo , R. N. and Ogbonnaya , O. U. ( 2006 ) Antifungal effects of two tropical plant extracts (Ocimumgratissimumand Afromaomummelegueta) on postharvest yam (Dioscoreaspp.) rot , African Journal of Biotechnology , 5 ( 9 ), 727 – 731 . Google Scholar OpenURL Placeholder Text WorldCat Onifade , A. K. ( 2002 ) Antifungal effect of AzadirachtaindicaA. Joss extracts on Collectotricum lindemathianum , Global Journal of Pure and Applied Science , 6 ( 3 ), 423 – 428 . Google Scholar OpenURL Placeholder Text WorldCat Orji , M. U. , Ezenwaje , E. E. and Anyaegbunam , B. C. ( 2006 ) Spatial appraisal of shallow well water pollution in Awka, Nigeria , Nigerian Journal of Microbiology , 20 ( 3 ), 1384 – 1389 . Google Scholar OpenURL Placeholder Text WorldCat Oxford English Dictionary . ( 2005 ) Coli , Oxford University Press, UK , p. 34 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Praveen , K. and Kumud , U. ( 2012 ) Tannins are astringent , Journal of Pharmacognosy and Phytochemistry , 1 ( 3 ), 8192 . Google Scholar OpenURL Placeholder Text WorldCat Ramírez-Camejo , L. A. , Zuluaga-Montero , A., Lázaro-Escudero , M. A. et al. . ( 2012 ) Phylogeography of the cosmopolitan fungus Aspergillusflavus: Is everything everywhere? Fungal Biology , 116 ( 3 ), 452 – 463 . Google Scholar Crossref Search ADS PubMed WorldCat Reynolds , D. ( 2003 ) Recruitment of through 319-phosphorylated Ndd1p to the FHA domain of Fkh2p requires CLB kinase activity: a mechanism for CLB cluster gene activation , Genes Development , 17 ( 14 ), 1789 – 1802 . Google Scholar Crossref Search ADS PubMed WorldCat Ryan , K. J. and Ray , C. G. ( 2004 ) Sherris Medical Microbiology (ed.) McGraw Hill, USA . ISBN 0-8385-8529-9. Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Shelef , L. A. ( 1983 ) Antimicrobial effects of spices—U S Forest Service 2008 ‘M. pudica’, Usambara invasive plants , Journal of Food Safety , 6 , 29 – 44 . Google Scholar Crossref Search ADS WorldCat Sieradzki , K. , Robert , R. B., Haber , S. W. et al. . ( 1999 ) The development of vancomycin resistance in a patient with methicillin-resistant Staphylococcus aureus infection , National England Medicine , 340 ( 7 ), 517 – 523 . Google Scholar Crossref Search ADS WorldCat Thongson , C. , Davidson , P. M., Mahakarnchanakul , W. et al. . ( 2004 ) Antimicrobial activity of ultrasound-assisted solvent-extracted spices , Letters in Applied Microbiology , 39 , 401 – 406 . Google Scholar Crossref Search ADS PubMed WorldCat Valsala , S. ( 2000 ) Estrogenic and antiestrogenic activities of M. pudica on Rattusnorvegicus , Journal of Ecotoxicology , 10 ( 1 ), 25 – 29 . Google Scholar OpenURL Placeholder Text WorldCat Zaika , L. L. ( 1988 ) Spices and herbs: their antimicrobial activity and its determination , Journal of Food Safety , 9 , 97 – 118 . Google Scholar Crossref Search ADS WorldCat Author notes Supervisor: Dr. Mrs. Chinelo A. Ezeabara, Department of Botany, Nnamdi Azikiwe University, P.M.B. 5025 Awka, Nigeria. © The Author(s) 2018. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com © The Author(s) 2018. Published by Oxford University Press.

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BioScience HorizonsOxford University Press

Published: Jan 1, 2018

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