INTRODUCTION:

The used of plants and herbs in the treatment of pathogenic diseases in most of the developing world. Researchers around the world have been engaged in the findings and formulation of new drugs; this is as a result of every day challenge of drug resistivity by microorganisms (Maureer-Gimes et al., 1996). Incidents due to drug resistant microorganisms and the emergence of unknown disease causing microbes, posed enormous global public health concern (Iwu, 1993).This resistance can be due to inappropriate administration of drugs commonly used on the treatment of these infectious diseases (Afolayan and Aliero, 2006). Furthermore some antibiotics have serious undesirable side effect which limit their application, so there is serious need to develop new antimicrobial agents that are very effective with minimal unwanted side effect and higher plants represent a potential source of novel antibiotic prototypes (Afolayan, 2003).

In recent years, there has been a gradual revival of interest in the use of medicinal plants in developing countries because herbal medicines have been reported safe and without any adverse side effect especially when compared with synthetic drugs (Ezekiel et al., 2009). Also, there has been little or no report of any form of microbial resistance during the use and administration of herbal medicines (Stephen et al., 2009). More importantly in Africa, particularly West Africa, new drugs are often beyond the reach of the poor such that up to 80% of the population use medicinal plants as remedy against infections and diseases (Kirby, 1996; Hostettmann and Maston, 2002).

The used of medicine medicinal plants all over the world predates the introduction of antibiotics and other modern drugs. There is a need for everyday research on the development and formulation of new drugs to counterfeit/curtail resistant of pathogenic microorganisms to antibiotics (Hammuel et al; 2011). Apart from employing the medicinal plants (herbs) for treatment of pathogenic disease it can be used to treat various ailments that are not directly caused by microorganisms such as jaundice, fever, rheumatism, epilepsy (Igbinosa, et al; 2009). The world health organization has recorded over 20,000 species of the plant Acalypha with medicinal properties providing treatment for such complaints as pneumonia, ulcers, diarrhoea, bronchitis, cold and diseases of the respiratory tract (Flavia et al ; 2088).

Acalypha plant is a genus of the family Euphorbiaceae and grows as an annual bedding plant (Oladunmoye, 2006). There are about 570 species, a large proportion of which are weeds that is open to forest zones of Nigeria and it is widely spread across tropics of Africa for example the macrophylla, hoffamanii, godseffiana, macafeeana, hispida, marginata and racemosa are peculiar cultivars within Nigeria (Oladunmoye, 2006; Yusha’u et al., 2008; Iniaghe et al., 2009). Euphorbiaceae species are large, fast-growing, evergreen shrub provides a continuous splash of colour in the landscape with the bronze red to muted red, 4 to 8 inch long, heart-shaped leaves available in varying mottled combinations of green, purple, yellow, orange, pink, or white, depending upon cultivar (Gilman, 1999). Others are ornamental plants or called as horticultural shrubs (Iniaghe et al; 2009). Some of the species Acalypha are known to be used in traditional medicine. Musa et al; 2000 reported the medicinal important of the A. racemosa to have exhibited the antibacterial activity. Iniaghe et al; 2009 also reported the used of the decocted leave of the plant by traditional practitioners in Ilorin metropolis, Nigeria to treat liver disorders and other disease condition which resulted in jaundice. The aqueous extract of the leaves of A. racemosa has also been reported to have hepaprotective effect in caborn tetrachloride induced liver of rats (Iniaghe et al; 2008).

MATERIALS AND METHOD:

Preparation of the extract:

Fresh leaves of A. racemosa, was collected from parish house Zaria and was identified by the department of Botany Ahmadu Bello University, Zaria, Nigeria.

The collected leaves were dried at 40^oC using Gallenhamp drying cabinet. The dried leaves were grinded into powdered form using mortar and pestle. 300gm of the powdered sample was dissolved in 400ml each of distilled water and methanol, the process was allowed to stay for 2 days to ensure proper extraction. The mixture was then filtered using Whatman No. 1 filter paper. The solvents were evaporated using water bath at temperature of 50^oC in order to concentrate the extract.

The phytochemical screening of the plant extracts:

Conventional standard protocols (Odebiyi & Sofowora, 1978; Trease & Evans, 1983) for detecting the presence of different chemical constituents in the plant extract were employed. The tests for the secondary metabolites viz. tannins, alkaloids, saponins, glycosides, flavonoids, cardiac glycoside, phlabatannins, and steroids were carried out.

Test for tannins (FeCl₃ test):

About 0.5g of the dried powdered sample was dissolved in 20ml of distilled water in a test tube, boiled and filtered. To the filtrate few drops of 1.0% Iron II chloride solution was added and observed for a blue-green precipitate.

Test for flavonoid:

The test was conducted in two different ways or methods:

To 1.0ml of the extract 1.0ml of 10% lead acetate was added, and observed for yellow precipitation.

And to 1.0ml of the extract of the plant 1.0ml of dilute NaOH was added and observed for precipitation.

Test for alkaloids:

Two different reagents (Wagner’s and Mayer’s reagents) were used to ascertain the presence of alkaloids in the sample:

Two (2.0) ml of the mixed and shook with 10.0ml of 2% HCl on a steam bath and filtered. The filtrate was divided into two equal portions. To the first portion Wagner’s reagent was added in drops and observed for a brown precipitate. To the second portion of the filtrate, Mayer’s reagent was added also in drops and observed for white to yellow or creamy with precipitate.

Test for saponins (frothing test):

1.0ml of extract was boiled with 5.0ml of distilled water for five (5) minutes and decanted while still hot. Then was filtered, and 1.0ml of the filtrate was diluted with 4.0ml of distilled water and was shook vigorously. It was observed on standing for stable froth.

Test for glycoside (fecl₃test):

To 5.0ml of the extract in a test tube 2.5ml of dilute H₂SO₄ acid was added and boiled in water bath for 15 minutes. It was cooled and neutralized with 20% KOH solution.5ml of a mixture of Fehling’s solution A and B were added, boiled and observed for reddish brown colour at the interphase.

Test for cardiac glycoside:

To 1.0ml of the extract 8% of methanol was added and mixed with 1.0ml of 2% solution 3, 5-dinitrobenzoic acid in 95% alcohol. The solution was made alkaline with 5% NaOH. It was observed for violet colour which faded through reddish brown to brownish yellow.

The test organisms:

The test organisms for the study are bacteria which include the Bacillus subtilis, Pseudomonas aeruginosa, Proteus mirabilis, Escherichia coli and Klebsiella pneumoniae. These organisms were isolated from patients in the Department of Microbiology, Ahmadu Bello University Teaching Hospital (ABUTH), Zaria, Nigeria. They were transported in slants to National Research Institute for Chemical Technology, Zaria, Nigeria.

The antimicrobial screening of the extracts:

The antimicrobial activity of the plant extracts were determined using agar well diffusion method as described by Reuben et al 2008. The bacterial and the fungal isolates collected in prepared slants of nutrients agar were sub-cultured into prepared nutrients broth and incubated at 37^oC for 24hours and standardized to 0.5 Mc-Farland scale (10⁸cfu/mL) in a prepared normal saline. The cell suspensions were swabbed onto nutrient agar plates MacConkey agar for Klebsiella pneumoniae. Wells were then bored into the plates of the inoculated organism using sterile cork-borer of 6mm in diameter. 1.0g of each of the methanol and aqueous concentrated extracts were constituted into 10mL of their respective solvents of extraction (methanol and water) to obtain the initial concentration of 100mg/mL. Then 1.0ml of the concentration of the both extracts was introduced to fill the wells created, allowed to stand for 30 minutes at room temperature for proper diffusion and then incubated at 37^oC for 24hours in an incubator. Controls were also set up in parallel, but using the solvents of extractions only. After the incubation 24hours the plates were observed for zones of inhibition and recorded in millimeter (mm).

Minimum inhibitory concentration (MIC):

The MIC of the crude extracts was determined using the method as given by Canales et al 2011. 100mg/ml of each of the extracts were reconstituted into nutrient broth in test tubes and the 100mg/ml was taken as the initial concentration. Four more tubes of 5ml nutrient broth were set up and 5ml of 100mg/ml of the extract was taken and used for two-fold dilution of the four tubes of nutrient broth forming concentrations of 100mg/ml, 50mg/ml, 25mg/ml, 12.5mg/ml and 6.25mg/ml.

Normal saline was used again to prepare turbid suspensions of the microbes; the dilution was done continuously and incubated at 37^oC for 30 minutes. Until the turbidity matched that 0.5 Mcfarland’s standard by visual comparison. At that point the of cells is assumed to be 1.5 x 10⁸cfu/ml. 0.1ml of the cell suspension was inoculated into each of the tubes with the varied concentrations of extracts. All the tubes were incubated at 37^oC for 24 hours. The tube with the lowest concentration which has no growth (turbidity) of the microbes was taken to be the minimum inhibitory concentration (MIC).

RESULTS

Table 1: The phytochemical screening of the extract

Components	Methanolic extract	Aqueous extract
Saponins	+	+
Flavonoids	+	+
Alkaloids	+	+
Sterols (Steroid nucleus)	T	+
Cardiac glycoside	-	-
Tannins	T	+
Glycoside	-	-

KEY: + = Present, - = Absent, T = Trace

Minimum bactericidal concentration (MBC):

The minimum bactericidal concentration (MBC) of the plant extract against the microbes was determined using the method of Ravikumar et al., 2007. The tubes of the MIC that showed no growth of the microbes were sub-cultured by streaking using sterile wire loop on nutrient agar plates, MacConkey agar plate for Klebsiella pneumoniae. The plates were incubated at 37^oC for 24 hours. MBC is the lowest concentration of the extract that showed not any colony growth on the agar plates. It was obtained and recorded.

Figure 1:The antibacterial susceptibility of the extracts showing the zones of inhibitions

Figure 2:The minimum inhibitory concentration (MIC) of the extracts against the bacteria

Figure3:The minimum bactericidal concentration (MBC) of the extracts against the bacteria.

Table 2: The set of controls showing the zones of inhibition in mm

Test Organisms	Negative (solvent) control, methanol	Positive(drug) control, Tetracycline (50 mg/ml
Bacillus subtilis	2	46
Pseudomonas aeruginosa	0	0
Proteus mirabilis	0	39
Escherichia coli	3	41
Klesiella pneumoniae	2	36

DISCUSSION:

The phytochemical screening of the extracts revealed the presence of saponins, alkaloids, phlabatanins, flavonoids. Steroids and tannins were found in trace amounts as presented in Table 1. In the screening for the biological active compounds there was no information about glycoside and cardiac glycoside. These results agreed with the report of Iniaghe et al, 2009. These secondary metabolites exert antimicrobial activity in different mechanism; flavonoids have also exhibited a wide range of biological activities; such as antimicrobial, antioxidant, anti-inflammatory, anti-agionic, analgesic, anti-allergics and cytostatic properties (Hodek et al., 2002). Tannins has been found to react with praline-rich protein to from irreversible complexes (Shimada, 2006), resulting in the inhibition of cell protein synthesis. Herbs that have tannins as their major components are astringent in nature and are used for treating intestinal disorders such as diarrhea and dysentery (Dharmananda , 2003). The presence of saponins lend credence to the use of this plant in managing inflammation (Quinlan et al., 2000) worked on steroidal extracts from medical plants which exhibited antibacterial activities on some bacterial isolates.

The zones of clearance of the pathogens by the extracts as seen in figure 1 indicated that both of the extracts had good activity against the pathogens. This agreed with the report of Verastegui et al., 2008 that ethanol extract is active against range of bacteria including fungi. Pseudomonas aeruginosa in this study was resistant methanol extract this could be as a result of inadequate extraction of the specific bioactive compounds that would have inhibited of the pathogen. The zone of clearance of both the extracts against Escherichia coli was noted to be higher than against other pathogens. This could be as a result of inadequate extraction of the bioactive compounds by the solvent.

The minimum inhibitory concentration (MIC) of the extracts were evaluated as seen in figure 2, and it was discovered that E. coli was inhibited by ethanol extract at concentration of 12.5mg/ml and by aqueous extract it was inhibited at 25mg/ml. Klebsiella pneumonia was another pathogen that was inhibited by methanol extratct at 25mg/ml. This indicate that infection cause by E. coli can be control by aqueous and methanol extracts of this plant specy. Klebsiella infection can be control by methanol extract of the plant. At MIC of 50mg/ml both extracts inhibited B. subtilist and P. aeruginosa.

The minimum bactericidal concentration of the extracts as seen in figure 3 indicate that E. coli was the only pathogen that was killed at lower concentration at 25mg/ml followed by K. pneumoniae which was inhited at 50mg/ml. Other pathogens were killed at concentration of 100ml.

CONCLUSION:

The extracts demonstrated significant biological activity against the test pathogenic organisms and this has introduced the plant as a potential candidate for drug development for the treatment of diseases caused by these pathogens. Of course, there would be the need to ascertain by further studies whether any single or combination of the pure active metabolites would be better, safer and more efficient in treating diseases caused by the selected pathogens than the whole plant (crude extracts).

REFERENCES:

1. Afolayan AJ, Aliero AA. Antimicrobial activity of solanum tomentosum. Afr. J. Biotech, 5(4)., 2006: 369 – 372.

2. Afoloyan AJ. Extacts from the shoots of Aretotis arotoides inhibit the growth of bacteria and fungi. Pharm. Biol 41 (1)., 2003: 22 – 25.

3. Ezekiel CN, Anokwuru CP, Nsofor E, Odusanya OA, Adebanjo O. Antimicrobial Activity of the Methanolic and Crude Alkaloid Extracts of Acalyphawilkesiana cv. macafeeana Copper Leaf. Research Journal of Microbiology, 4., 2009: 269-277.

4. Cowan, M.M. (1999). Plant Products as Antimicrobial Agents clun. micro. Rev., 12(4): 564-582.

5. Dharmananda S.: Gall nuts and the uses of Tannins in Chinese Medicine. In: proceedings of institute for Traditional Medicine, Portland, Oregon, 2003.

6. Gilman EF. Acalyphawilkesiana, Fact sheet FPS-6. University of Florida Extension Service. pp: 1-3., 1999

7. Hammuel C, Abdullahi MS, Mankilik M, Anyim BP, Adesina OB, Inekwe UV, Udiba UU, Batari ML. The phytochemical and antimicrobial activity of oil from the seed of Thevetia peruviana plant. Journal of Applied Environmental and Biological Sciences, 1(12)., 2011: 597-601.

8. Hostettmann K., Marston A. Twenty years of research into medicinal plants: Results and perspectives. Phytochem. Rev., 1., 2002: 275-285.

9. Iniaghe OM, Malomo SO, Adebayo JO. Proximate composition and phytochemical constituents of leaves of some Acalypha species. Pakistan Journal of Nutrition 8., 2009: 256-258.

10. Iwu M. Hand book of African Medical plants, CRE press Boca Raton, FL., pp, 27-44., 1993

11. Kirby GC. Medicinal plants and the control of protozoal disease, with particular reference to malaria. Trans. R. Soc. Trop. Med. Hyg., 90.,1996: 605-609.

12. Maureer-Grimes B, Macbeth DL, Hallihan B, and Delph S. Antimicrobial activity of Medicinal Plants of the Scrophulariaceace and Acanthaceace. International Journal of Pharmacognosy 34 (4)., 1996: 243 – 248.

13. Oladunmoye MK.. Comparative evaluation of antimicrobial activities and phytochemical screening of two varieties of Acalyphawilkesiana. Trends Applied Sci. Res., 1., 2006:538-541.

14. Ravikumar PHS, Makari HK, Gurumurthy H. In vitro Antimicrobial Activity of Ethanol Extract of Thevetia peruviana. Electronic Journal of Environmental, Agriculture and Food Chemistry, 6(9)., 2007:.2318-2322.

15. Stephen UA, Abiodun F, Osahon O, Ewaen E. Phytochemical analysis and antibacterial activity of Khayagrandifoliola stem bark. Journal of Biological Sciences, 9., 2009: 63-67.

16. Verastegui A, Verde J, Garcia S, Heredia N, Oranday A., Rivas C. Species of Agave with antimicrobial activity against selected pathogenic bacteria and fungi. World Journal of Microbial Biotechnology 24., 2008:1249-1252.

17. Yusha’u M, Aliyu BS and Olonitola SO. Preliminary screening of acalypha extracts for in vitro inhibitory activity against extended-spectrum β-lactamase producing enterobacteriaceae. International Journal of Pure and Applied Sciences 2., 2008:1-5

Received on 20.05.2014 Modified on 12.07.2014

Res. J. Pharmacognosy & Phytochem. 6(3): July-Sept.2014; Page 118-121