Preliminary Phytochemistry, Antimicrobial Properties and Acute Toxicity of Stachytarpheta jamaicensis (L.) Vahl. Leaves

MATERIALS AND METHODS

Sample Collection
S. jamaicensis was collected in the month of March around Ugbowo area in Benin-City, Edo State, Nigeria from a growing site. It was identified properly by comparing with the leaves, inflorescence and stems in standard text (Akobundun and Agyakwa, 1998) and further substantiated by Prof. M. Idu of Botany Department, University of Benin. The leaves were plucked from the stem using knife and shears, they were cleaned of debris and dried in an oven at 40°C for 48 h then grinded.

Extraction of Plant Material for Chemical Analysis:
Twenty gram of grinded plant sample was dissolved in 200 mL of water and allowed to boil for 30 min. The sample was left to cool and filtered with Whatman No. 1 filter paper.

Water and ethanol were used for extraction to obtain samples for the antimicrobial and acute toxicity tests. One hundred gram of leaves was boiled by decoction in 1000 mL of water for 30 min and 150 g of leaves was soaked in 1000 mL of ethanol for 48 h, then filtered. The filtrates were placed into evaporators to drive off the solvents. The pastes formed were kept in two containers and labelled thus: WL-Water extract from leaves and AL-Alcoholic extract from leaves. Tests for alkaloids, saponins, tannins, flavonoids and anthraquinones, were according to procedures outlined by Trease and Evans (1996).

Determination of Antimicrobial Activity
The organisms used for study were B. subtillis, E. coli, C. albicans, S. aureus, P. aureginosa, P. vulgaris, K. arogene, P. mirabilis and high resistance standard strain of Escherichia coli (J62k₁₂). The gutter and punch hole methods were applied.

Gutter Method
This was used to determine preliminary activities of the extracts on the organisms. Sterilized nutrient agar was poured into four Petri dishes and allowed to set. The plates were partitioned into four segments using a marker pen. With the aid of a sterilized loop, each organism was streaked across a segment on the agar surface. A sterilized spatula was then used to cut out a gutter across the streaks. The extracts were poured into the gutters and the Petri dishes kept in an incubator for 24 h at 37°C to allow the organisms grow. Clearance of streak growth from the gutter margins indicated inhibitory activity of the extract on the organism.

Punch Hole Method
Punch hole method (Stoke, 1975) was used to measure the zone of inhibition. Eight Petri dishes were poured with already sterilized nutrient agar to the level of obtaining a standard well and allowed to set. The organisms, dissolved in nutrient broth were poured into set Petri dishes and uniform distribution was ensured. Sterile cork borer of 10 mm in diameter was used to punch holes in the agar. Each of the holes (numbering 2) were filled with extracts 1000 mg mL^-1 and kept in an incubator for 24 h at 37°C for the organisms to grow.

The active extracts showed zones of inhibition which were measured using meter rule by measuring 2 points across the zone and the average diameter was taken.

Minimum Inhibitory Concentration (MIC)
Agar plates were prepared and two for each of the extracts were flooded the with same organism. Two holes were punched in each plate and filled with 0.2 mL of extracts of different dilutions.

Double dilution of the extract was carried out. Double strength nutrient broth of 5 mL was pipette into universal bottles and each were labeled N, 2, 4, 8. Using a sterile graduated pipette, 5 mL of the extract was measured into the bottle labeled 2 and mixed. Same was done for 4 and 8 using fresh pipettes. In another bottle, broth only was put in without the extract, this served as the control. The plates were incubated at 37°C for 24 h. The order of concentration were N-1000, 2-500, 4-250 and 8-125 mg mL^-1.

Acute Toxicity Test
Twenty male wistar rats, five weeks old, were obtained from a single source to reduce the influence of variablity. They and were fed with mouse cubes and had unrestricted access to water.

The animals were divided into groups of five each and the groups labeled as I, II, III and IV respectively. All animals of the respective groups were fasted overnight before proceeding with the experiment.

Doses of 1, 2 and 4 g kg^-1 of aqueous extract of the leaves were administered intraperitoneally to the animals in group I, II and III respectively and 0.5 mL of Normal saline was administered to the animals in group IV which served as control.

The animals were observed for physical signs of toxicity such as weight change and mortality observed for 24 h and beyond but not more than 48 h. Mortality was calculated as percentage death of the animals used.

RESULTS

Phytochemical Analysis
Results from alkaloidal test proved negative. The presence of frothing when filtrate was shaken was a preliminary evidence of the presence of saponins which was confirmed after it was mixed with sulphuric acid and with 90% ethanol added, frothing disappeared. The test for tannins gave a bluish precipitate which confirmed its presence. Anthraquinones was absent. Flavonoids was confirmed present by the change from colourless to yellow coloration on addition of hydrochloric acid (Table 1).

Antimicrobial Activity
Table 2 shows that WL was active on all organisms while AL was also active on all organisms but slight activity on S. aureus and P. vulgaris. From Table 3, inhibition zones measurement for WL revealed highest diameter of 18 mm for P. mirabilis and lowest diameter of 12 mm for P. aureginosa while the AL showed higher inhibition zone for P. aureginosa than P. mirabilis. Table 4 shows minimum inhibition concentration of extracts were obtained at high concentrations, while the resistant E. coli, J62k₁₂, was not inhibited by any of the extracts.

Acute Toxicity
This is presented in three sections.

Table 1:	Summary of phytochemical analyses of S. jamaicensis leaves
+: Indicates present, -: Indicates absent

Table 2:	Antimicrobial activity of extracts of S. jamaicensis leaves using Gutter method
++: Indicates presence of inhibition, -: Indicates absence of inhibition, +: Indicates minute presence of inhibition, WL = Water extract of leaves, AL = Alcohol extract of leaves

Table 3:	Zone of inhibition diameter (mm) produced by extracts of S. jamaicensis leaves using Punch hole method
-: Shows absence, WL = Water extract of leaves, AL = Alcohol extract of leaves

Table 4:	Minimum Inhibition Concentration (MIC) of extracts of S. jamaicensis leaves on the microorganisms
-: Zone of inhibition absent, +: Zone of inhibition present, WL = Water extract of leaves, AL = Alcohol extract of leaves

Table 5:	Weekly weights (g) of wistar rats treated intraperitoneally with aqueous extract of S. jamaicensis leaves
Values with same superscripts mean no significant differences in weights

Table 6:	Mortality rates of wistar rats due to intraperitoneal administration of aqueous extract S. jamaicensis at different does

Physical Characteristics/observation of Experimental Rats
There was no noticeable difference in feeding habit throughout the 3 weeks. There was no obvious hair loss in all the rats and eye colour remained normal throughout period.

Body-weight
The average weights in groups I, II and III were taken and no significant weight loss (p>0.005) was observed between the initial and final periods (Table 5).

Mortality
None of the rat observed died during the period of this experiment (Table 6).

DISCUSSION

The preliminary phytochemical investigation carried out on S. jamaicensis showed that it consists of metabolites such as saponins, tannins and flavonoids. These metabolites have been shown to be responsible for the therapeutic activity of plants (Trease and Evans, 1999; Rabe and Vanstoden, 2000). The tannins have been traditionally used on inflammed surfaces of mouth and treatment of catarrh, wounds, hemorrhoids and diarrhea and as antidote in heavy metal poisoning (Sodipo et al., 1991). Tannins have been reported to inhibit growth of micro-organisms by precipitating microbial protein and making nutritional proteins unavailable to them (Ogunleye and Ibitoye, 2003). Saponins are special class of glycosides that have shown to be an antifugal agent (Sodipo et al., 1991). The class of alkaloid is among the powerful poisons known (Fluck, 1973). Plant phenolics especially flavonoids have health promoting properties (Rauha et al., 2000). The absence of alkaloids in this plant maybe responsible for it’s non-toxicity.

The antimicrobial results from the aqueous extract (WL) showed activity on B. subtillis, E. coli, C. albicans, S. aureus, P. aureginosa, P. vulgaris, K. arogenes and P. mirabilis. Minimum Inhibitory Concentration (MIC) was attained at 0.25 g mL^-1 for E. coli and 0.5 g mL^-1 for P. aureginosa.

The result from the alcoholic extract (AL) showed antimicrobial activity on B. subtillis, E. coli, C. albicans, P. aureginosa and P. mirabilis, while at very high concentration. The Minimum Inhibitory Concentration (MIC) was obtained at 0.5 g mL^-1 for P. aureginosa and 0.25 g mL^-1 for P. mirabilis.

Ataman et al. (2006) reported that rat showed variations in physical signs/body appearance and mild histhopathologic lesions such as congesting fatty tissue changes and necrosis in selective tissues such as liver, blood vessels, kidney, lungs and testis, but the brain, eyes, intestine and heart tissues were essential indicators to the toxicity of drugs due either to dosage or duration of use.

The results of the present research showed that there was no mortality, no changes in eye colour, no loss of hair and no significant weight loss (p>0.005) in the wistar rats even at 4 g kg^-1 dose. Food intake remained normal. All these show that the plant extracts were non-toxic. This research is similar to that reported by Taylor (2006) stating that S. jamaicensis had no toxicity even at 2 g kg^-1 body weight by intraperitoneal administration.

CONCLUSIONS

The results obtained from phytochemical analysis has shown the presence of some secondary metabolites which proves that the plant is of great medicinal values. The plant being resistant to some strains of clinical microorganisms especially the fungi and bacteria at high concentration ascertain that it is a possible antibiotic which should be administered high doses.

Finally, its having no toxicity at high doses shows that it is relatively safe for therapeutic cure of diseases and could be potentially beneficial to human. Thus more investigation should be carried out scientifically to confirm and prove the efficacy of this plant as well as further studies on its chronic toxicity effects.