Monodora myristica (ehuru) was purchased from Onitsha main market and identified by a plant taxonomist Mrs Anthonia, Emezie at Pharmacognosy department, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Agulu.

Ethical approval was obtained from Nnamdi Azikiwe University Teaching Hospital Ethics Committee (NAUTHEC), Nnewi, Anambra State.

The pods of M. myristica were opened up and the grain weighing 750 g was collected, air dried for some days, crushed with the mechanical/electrical grinder, and sieved to obtain a fine specimen of the sample of about 719 g.

Using soxhlet extraction method, some 719 g of the ground material was extracted with 75% ethanol over a period of time of 72 hours to generate the ethanolic extract. Concentration was done using HH-S water bath and approximately 292.30 g of ethanol extract was obtained and stored in a 50 ml conical flask laced with an aluminium foil and stored a refrigerator of 4˚C.

The phyto screening and quantitative analysis of the extract were conducted to reveal the constituents of the extract by using standard phytochemical method as indicated by Evans and Sofowora [12][13].

The antimicrobial tests was done using clinical isolates of Bacillus subtilis,Escherichia coli,Staphylococcus aureus, and Pseudomonas aeruginosa. All the organisms were obtained in Pharmaceutical Microbiology and Biotechnology laboratory at Nnamdi Azikiwe University, Awka, Nigeria. All the microbial isolates were grown in their respective selective media and cultured in the axenic culture.

Culture media used were nutrient broth, and Mueller-Hinton Agar (Oxoid Limited, England).

The formation of culture media was done in keeping with the strategy of the manufacturer. After the autoclaving, it was allowed to cool to a temperature of 50˚C and added immediately to a flat bottom petri dish in a horizontal plane. Surgical tools called Mc Farland 0.5 turbidity prepared standard, sodium hypochlorite solution, 0.5.

Distilled water 1 ltr

Distilled water 1 ltr

Broth Nutrient broth (13 g/L) was prepared according to the specification given by the manufacturer. Nutrient agar (28 g/L) was prepared in the same way. Mueller-Hinton agar (38 g/L) was prepared as well.

The weight of nutrient agar powder was 8.4 grams which was dissolved in distilled water 300 ml that had been mixed with that and then the solutions, 20 ml apiece were put into McCartney bottles and steamed in autoclave at a temperature of 121˚C for 15 minutes.

The nutrient broth powder weighing 3.9 gram was dissolved in 300ml of distilled water and mixed and then resorted into 20ml of every solution was collected in McCartney bottles that were subjected to autoclave at 121˚C 15 minutes.

The weight of Mueller Hinton powder (8.4) was weighed, dissolved in 300 ml of distilled water and subsequently homogenised prior to addition of the test solution, 20 ml, in different McCartney bottles and be sterilised by autoclave using 121˚C at most before adding 20 ml of the extracts [14].

Cultures of test organism (bacteria) which consisted of gram positive (S. aureus and B. subtilis) and gram negative (P. aeruginosa and E. coli) were taken in-vitro and then used in the antimicrobial test. Clinical isolates: Ensured that all the lab isolates belonged to the laboratory of Pharma advisor microbiology and biotechnology, faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka. They were incubated in 48 hours at 4˚C in refrigerator.

12 mice of mixed sex weighing 21 - 28 g were purchased from the department of Pharmaceutical Science animal house and were kept in the cage of animal house Pharmacology department, Faculty of Pharmaceutical sciences, Nnamdi Azikiwe University, Agulu. They were then used for the acute toxicity study of seed extract of M. myristica. The animals were provided with vital feed that was palletized commercial rat feed (palletized feed) along with the tap water.

It was the Lorke method [15] for carrying out acute Toxicity of M. myristica seed. The experiment was conducted in two steps. During the first phase of the research 9 mice were randomised into three different groups of three mice each and treated to receive 10, 100 and 1000 mg/kg. body weight of the M. myristica extracts by suprolipidious technique that is oral lavage using sterile syringe. The signs of toxicity were noted in the mice which included salivation, entire body stretching, weakness, drowsy, restrained breathing, coma and death in the initial two hours, periodically after another six hours and eventually after twenty four hours. The second phase of the study would represent the second group of 3 mice randomised into three groups with the result of the first phase determining the administered body weight (kg) of the M. myristica seed extract 1600, 2900 and 5000 mg/kg each. These were monitored to find out indications of toxicity and mortality as in phase one and the number of deaths registered.

The aim of this paper was to examine the ethanol extract of Monodora myristica (seed) in terms of its antibacterial systems. The extract of Monodora myristica was obtained as a seed extract and bacteria that were tested (S. aureus, B. subtilis, P. aeruginosa and E. coli) were also performed against the bacterial using agar well diffusion assay. This phytochemical screening showed Saponin, Glycoside and Protein. The highest dose of 5000 mg/kg of seed extract was not lethal to the mice, there was no death in them by the use of Lorke method of determination of oral LD₅₀ in rat. It worked only with B. subtilis and P. aeruginosa, had the minimum inhibitory concentration of 50 mg/ml product and 100 mg/ml product of the seed extract. Going further, the extract of the seed was found to be inactive toE.coliand S.aureus. Presence of the standard (gentamicin 30 ug/ mL) has shown no reaction with the seed extracts, where, however, presence of standard (gentamicin concentration of 10 ug/ml) was very high when compared to the seed extract against various concentration against S. aureus,P. aeruginosa, an activity close to that of the seed extracts was present against E. coli(p = 0.008). Conventional chloramphenicol was statistically not significant in P. aeruginosaand B. subtilis on (p > 0.05). Comparison of the effect of the seed extract on the test organism and the standard Amoxicillin revealed that the differences between the two were great in favor of the standard Amoxicillin (p < 0.05). The research was shown to be successful with the consequences that ethanol possessed antibacterial properties.

Diffusion Agar well method; 800mg of the extract was weighed and dissolved into 2 ml of DMSO and final resultant concentration was termed as 400 mg/ml which at 45˚C was deemed as stock and then 2 times serial dilution was performed each on a test tube with 2 ml of DMSO up to 200 mg/ml, and down to 50 mg/ml, 25 mg/ml and 12.5 mg/ml [16] at 37˚C, the antimicrobial was done in the extract by quantifying the zone of hindrance round any wells (except the well diameter) with aid of sterile syringe and pre-diffusion using 25 to 30 minutes and incubating them, the seed extracts were investigated on all bacteria. The bacterial against positive control was 10 ug/ml of gentamicin, Amoxicillin 10 ug/ml, and Chloramphenicol 30 ug/ml. The diameter of the inhibition radii against the test organisms has assumed its concentration strength and was determined and inserted into further analysis. Any deliberation of all the tests was carried out in duplicate of the bacteria pathogens as well as the antimicrobial performance in terms of mean diameter clear zone (mm) at plant extracts.

The MIC of it was determined by agar dilution method. The extract of 4000 mg/ml was drawn and 1-fold serial dilution in the test tube coated with 2 ml of each of the segmented concentration to acquire the end results of 2000 mg/ml, 1000 mg/ml, 500 mg/ml, 250 mg/ml and 125 mg/ml. To speed up the study it was done in serial dilution of 1ml of the respective concentrations of the extract solution and 19 ml of molten Mueller Hinton agar were used to dilute the 1 ml of extract solution, giving the resultant concentrations of 400 mg/ml, 200 mg/ml, 100 mg/ml, 50 mg/ml, 25 mg/ml and 12.5 mg/ml. Upon continuous dilution from 4000 mg/ml after adding the extract solution of 19 ml of molten Mueller Hinton agar and 1 ml of nutmeg extract making 20 ml as a diluent reduces the concentration from 4000 mg/ml, 2000 mg/ml, 1000 mg/ml, 500 mg/ml, 250 mg/ml and 125 mg/ml to 400 mg/ml, 200 mg/ml, 100 mg/ml 50 mg/ml, 25 mg/ml and 12.5, Which had been stored in the sterile Petri-dish and allowed to solidify. Streaking was carried out followed by incubation of colonies on the agar within 24 hours at 37˚C. The plates were then checked and a result of (+) or (−) was recorded on respective plates as presence and absence of growth respectively.

The results were represented by use of mean and standard deviation. To conduct the statistical analysis, one way analysis of variance (ANOVA) was run and conducted in SPSS (version 20) statistical programme. The obtained results were considered to be significant at P 0.05 or less.

As observed in the phytochemical screening (Qualitative), the seed extracts of M. myristica presented alkaloids, saponin, flavonoid, tannins, steroid and terpenoid (Table 1). The traces with low intensity of the presence exhibited the presence of Flavonoids, saponins, and Terpenoids over the other phytoconstituents demonstrated in the ethanol extract.

Table 2 indicated that the plant extract was not toxic to the rat even at considerable dosage of 5000 mg/kg. The model utilised by Lorke [15] was utilized and the experiment was conducted in two stages with varying number of rats. During the phase one, no signs of toxicity were recorded at 10 mg/kg, 100 mg/kg and 1000 mg/kg. Phase two toxicity; two of the three mice each dose 1600, 2900 and 5000 mg/kg, of the drug have not been recorded.

Table 1. Phytochemical screening of monodora myristica (ehuru) seed extracts.

KEY: + = Trace/mildly present, − = Absent.

Table 2. Tabular result toxicity study of M. myristica ethanol seed extract.

Note: Phase 1 = 3 is the number of mice; Phase 2 = 1 is the number of mice; LD₅₀ > 5000 mg/kg.

Table 3 compared mean IZDs of the various concentrations of the extracts on the organisms and found this to be significantly different (p = 0.000). The IZDs of 400, 200, and 100 mg of concentration on P. aeruginosa and B. subtilis were also staticstically significant (p = 0.053) yet were also significantly different compared to that of S. aureus and E. coli (p = 0.000). B. subtilis had the only significant activity with the extract at 50 mg concentration (p = 0.000).

Table 4 applied the Post Hoc Tukey test to establish the differences in the mean of IZDs obtained using gentamicin at 10 ug/mL of concentration and plant extracts at a different concentration form. The activity using normal gentamicin was very high as compared to extract in different concentrations in the case of S. aureus, E. coli, P. aeruginosa and B. subtilis.

(P < 0.05). See Table 3.

Table 3. Comparative potency evaluation of plant extracts against Control (Gentamicin) on Test Organisms.

F = ANOVA value. P < 0.05.

Table 4. Post-hoc (Tukey) multiple comparisons of extracts against control (Gentamycin) against test organisms.

As seen in Table 5 and Table 6, the various concentrations of the plant extract failed to effectively show any activity on S. aureus and E. coli, however the concentrations were active on P. aeruginosa and B. subtilis. In S. aureus with the standard (30 mg of Chloramphenicol). Another significant activity in comparison with those of the extracts was seen on E. coli (p = 0.008). As it was noted, the standard was statistically non-significant (p > 0.05) on P. aeruginosa and B. subtilis.

Table 5. Comparative potency evaluation of plant extracts against control (Chloramphenicol) on test organisms.

F = ANOVA value. p < 0.05.

Table 6. Post hoc Tukey multiple comparison of plant extract against control (chloramphenicol) on test organisms.

In the same way, Table 7 and Table 8 below showed the action of a normal Amoxicillin (10 µg/mL) against the diverse organisms used in a study. Along with the extracts, no activity was demonstrated in S. aureus and E. coli, but activities and was demonstrated in P. aeruginosa and B. subtilis at concentrations of 100 mg and 50 mg respectively. The comparison between the IZDs of the standard and the test organism showed that significant difference ranged against the standard (p < 0.05).

Table 7. Comparative potency evaluation of plant extracts against control (Amoxicillin) on test organisms.

F = ANOVA value. p< 0.05.

Table 8. Post-hoc (Tukey) multiple comparisons of extracts against antibiotic control (Amoxicillin) on test organisms.

As indicated in Table 9 below, the extract was significantly active, MIC was established of the plant extract on the chosen pathogenic bacteria. Table 9 showed sensitivity to staph. aureusand E. coli at high concentration i.e. 400 mg/ml to a diameter of 8 mm whilst P. aureuginosa and B. subtilis showed no growth at varying concentration i.e. 50 mg/ml to 400 mg/ml to diameter of 8 mm.

Table 9. Minimum inhibitory concentration of the ehuru extract against test organisms.

Key: − = no growth; + = growth.