Samples of M. indica were collected from Barako in Gokana of Rivers State, Nigeria. Barako is located at latitude 4˚40'5"N, and longitude 4˚43'5" East. The plant samples (leaf, root, and bark) were collected by plucking the leaves, scrapping off the bark, and digging the soil off the root before cutting the exposed roots. The samples were immediately transported in plastic bags to the Department of Medical Laboratory Science Laboratory of Rivers State University, Port Harcourt for processing.

The samples were washed thoroughly to remove dirt and further sterilized to destroy any contaminating microorganisms. The fresh extracts were prepared by grinding 600 g of leaves, roots, and bark in a sterile mortar. Each sample was soaked in 200 ml of absolute ethanol for 72 h at room temperature in an enclosed container to extract the phytochemical components. The samples were first filtered through a sterilized wire net to sift the debris from the solution. The solution was finally filtered through a sterile no.1 Whatman filter paper. The filtrate was evaporated to dryness in a water bath at 80˚C. The dried samples were put in specimen bottles and reconstituted with 200 ml of sterile distilled water. The samples were stored in a refrigerator at 4˚C until further use. To prepare the dry extracts, 600 g of leaves, roots, and bark dried in an incubated at 25˚C for four (4) weeks. The samples were then grounded in a sterile mortar and the procedure for the extraction of fresh samples was followed. The combination of the extract before efficacy testing was in the ratio of 1:1.

Microorganisms used for the experiment were E. coli ATCC 252922 and S. aureus ATCC 29213 which were identified using PCR amplification of the 16S rRNA at Lahor Research Laboratories, Benin, Edo State, Nigeria. The organisms were stored in 10% glycerol and kept at −20˚C.

Tryptic Soy Agar (TSA), Mueller-Hinton agar (MHA) and Tryptic Soy Broth (TSB) were prepared according to the manufacturer’s instruction and sterilized by autoclaving at 15 psi or 121˚C for 15 minutes. The solid media were allowed to cool to about 50˚C, aseptically poured into a sterile Petri dish and allowed to solidify at room temperature before storage at 4˚C for subsequent use. The TSB was stored at room temperature.

The MIC assay of the extracts was performed using the broth dilution method in the TSB as described by the Clinical and Laboratory Standard Institute (CLSI) [13]. The 3-(4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide (MTT) was used as an indicator as it is utilized by physiologically viable bacteria. The MIC was read as the lowest concentration of the extract that resulted in the production of color change. The study performed a negative control in 100 µl of 0.4% dimethylsulfoxide (DMSO).

The antibacterial susceptibility of the bacterial isolates was determined using agar well diffusion assay. The overnight cultures of E. coli and S. aureus were diluted to produce an optical density (OD) of 0.5 at 590 nm and 100 µl of the diluted overnight cultures are spread on MHA. Sterilized Pasteur pipettes were used to make wells 6 mm in diameter. 100 µl of extracts (C₁ = 1.5 g/ml, C₂ = 3 g/ml) were added into the wells and incubated at 37˚C for 24 h in an upright position. The zones of clearance around the wells were measured in millimeters. Gentamycin [10 µl] and vancomycin [30 µl] disks were used as controls for both E. coli and S. aureus respectively.

The data obtained in this study were represented as mean ± SD (standard deviation) for n = 3. The statistical analysis used was a t-test for comparisons between two variables and ANOVA for more than two variables. The post analysis was performed by ANOVA. P-value was considered to be statistically significant at P < 0.05.

Table 2 represents the comparison of the antibacterial efficacies of fresh and dry single-component of M. indica on E. coli. The higher concentrations (C₂ = 3.0 g/ml) of the dry extract of root and bark were significantly (represented by the superscript c) higher than the lower concentrations (C₁ = 1.5 g/ml) but the difference was not significant (P > 0.05) in the dry leaf extract. Also, the higher concentrations of the fresh leaf, root, and bark extracts were significantly (P < 0.05) higher than the lower concentrations. A comparative analysis of the dry and fresh extracts between corresponding concentrations indicates that the fresh leaf, root, and bark extracts were significantly (P values: leaf; C₁ = 0.0533, C₂ = 0.0050; root C₁ = 0.0080, C₂ = 0.0327; bark C₁ = 0.0249) effective than the dry extracts except for the higher concentration of the bark extract which is not significant (P = 0.1481).

Table 3 represents the comparison of the antibacterial efficacies of fresh and dry single-component of M. indica on S. aureus. The higher concentrations (C₂ = 3.0 g/ml) of the dry and extracts of leaf, root, bark were significantly (represented by the superscript c) higher than the lower concentrations (C₁ = 1.5 g/ml). Relatively comparison of the dry and fresh extracts between corresponding concentrations indicates that the fresh leaf, root and bark extracts were significantly (P values: leaf; C₁ = 0.0111, C₂ = 0.0257; root C₂ = 0.0325; bark C₁ = 0.0088, C₂ = 0.0002) effective than the dry extracts except for the lower concentration of the root extract which is not significant (P = 0.1890).

Key: C₀—gentamycin [10 µl], C₁—1.5 g of extract, C₁—3.0 g of extract. Superscripts a, b, c show significant comparison (P < 0.05) between C₀ vs C₁, C₀ vs C₂, and C₁ vs C₂ respectively.

Key: C₀—Vancomycin, C₁—1.5 g of extract, C₁—3.0 g of extract. Superscripts a, b, c show significant comparison (P < 0.05) between C₀ vs C₁, C₀ vs C₂, and C₁ vs C₂ respectively.

Table 4 represents the comparison of the antibacterial efficacies of fresh and dry two mixed components of M. indica on E. coli. The higher concentrations (C₂ = 3.0 g/ml) of the fresh and dry combined extracts of leaf, root, and bark were significantly ([P < 0.05] represented by the superscript c) higher than the lower concentrations (C₁ = 1.5 g/ml). The comparative evaluation shows that all the dry extracts exhibited higher efficacies than the fresh extracts for both matching concentrations with significant differences seen in C₁ and C₂ (P = 0.0021 and 0.0020 respectively) for only root + bark and C₂ for leaf + root and leaf + bark (P = 0.0070 and 0.0241).

Table 5 displays the assessment of the antibacterial efficacies of fresh and dry two mixed components of M. indica on S. aureus. All the higher concentrations (C₂ = 3.0 g/ml) of the double-combined fresh and dry combined extracts of leaf, root, and bark were significantly ([P < 0.05] represented by the superscript c) higher than the lower concentrations (C₁ = 1.5 g/ml). A comparative analysis shows that just about all the dry extracts exhibited higher efficacies than the fresh extracts for both matching concentrations with significant changes observed in C₁ and C₂for only root + bark (P = 0.0101 and 0.0424 respectively), C₂ for leaf + root and leaf + bark (P = 0.0019) and C₁ for leaf + bark (P = 0.0295). The exception to this rule was the C₁ of the leaf + root which had non-significantly (P = 0.2051) lower efficacy of the dry compared to the fresh extract.

Key: C₀—gentamycin [10 µl], C₁—1.5 g of extract, C₁—3.0 g of extract. Superscripts a, b, c show significant comparison (P < 0.05) between C₀ vs C₁, C₀ vs C₂ and C₁ vs C₂ respectively.

Key: C₀—Vancomycin, C₁—1.5 g of extract, C₁—3.0 g of extract. Superscripts a, b, c show significant comparison (P < 0.05) between C₀ vs C₁, C₀ vs C₂, and C₁ vs C₂ respectively.

Figure 2, Figure 3 display the antibacterial efficacies of fresh and dry three-mixed components of M. indica on E. coli and S. aureus respectively. Figure 2 shows that the higher concentration (C₂ = 3.0 g/ml) of the three-combined fresh extracts of leaf, root, and bark was significantly ([P < 0.05] represented by the superscript c) higher than the lower concentration (C₁ = 1.5 g/ml) while the variations in that of the dry extract were not significant (P > 0.05). A comparative analysis for both matching concentrations shows that the fresh extract exhibited higher efficacies than the dry extract with significant changes observed in C₂ of leaf + root + bark (P = 0.0101), while the C₁ of the fresh extract had non-significantly (P = 0.1012) higher antimicrobial efficacy compared to the dry extract.

Again, Figure 3 shows that the higher concentration (C₂ = 3.0 g/ml) of the three-combined fresh extracts of leaf, root, and bark was higher than the lower concentration (C₁ = 1.5 g/ml) but not significantly (P > 0.05) while the variations in that of the dry extract were significant (P < 0.05). A comparative analysis for both identical concentrations shows that the fresh extract exhibited higher efficacies than the dry extract with significant changes observed in both C₁ and C₂ of leaf + root + bark (P = 0.0044 and 0.0307).