INTRODUCTION:

Over the past decade herbal medicine has become a topic of global importance, making an impact on both world health and international trade. Medicinal plants continue to play central roles in the healthcare system of large proportion of the world’s population. This is particularly true in the developing countries, where herbal medicine has a long and uninterrupted history of use (Inamul Haq 2004). Recognition and development of medicinal and economic benefits of these plants are on the increase in both developing and industrialized nations (Srinivas et al, 2007).

Continuous usage of herbal medicine by a large proportion of the population in the developing countries is largely due to the high cost of western pharmaceuticals, health care, adverse effects that follow their use (in some case) and the cultural and spiritual point of view of the people of these countries (Srinivas et al, 2007).

In developed countries however, after a downturn in the pace of herbal use inrecent decades, the pace is again quickening as scientists realizethat the effective life span of any antibiotic is limited (Satyejji and Lutfun, 2007). Worldwidespending on finding new anti-infective agents (including vaccines)was expected to increase to 60% from the spending levels in 1993.New sources, especially plant sources, are also being investigated.Secondly, the public is becoming increasingly aware of problemswith the over-prescription and misuse of traditional antibiotics. In addition, many people are interested in having more autonomyover their medical care. All these makes the knowledge of chemical, biological and therapeutic activities of medicinal plants used become necessary. (Fagbohun et al, 2010).

Before the era of Louis Pasteur (1822-1895), world renowned chemist and biologist who proved the germ theory of disease, the notion that tiny organisms could kill vastly larger ones (including human) seemed ridiculous to many people (Karanayil et al 2011; Sheo Singh and John Barrett 2006). Nowadays, it has been accepted that infectious diseases are the number one causes of death worldwide, accounting for approximately one half of all deaths in tropical countries (Iwu et al., 1999). In fact, there are more patients today in hospitals than there are effective drugs due to the development of resistance to available agents.

The use of plant parts as a source of medicine to treat infectious diseases predates history (Sunil Mishra and Singh PN 2011). Nearly all cultures and civilizations from ancient times to the present day have used herbal medicines (Erdemeier et al. 1996; Lino and Deogracious, 2006) to cure infections. The intractable problem of antimicrobial resistance has led to the resurgence of interest in herbal products as sources of novel compounds to fight the ever increasing problems of emergence of newer diseases and preventing the resurgence of older diseases thought to be brought under control (Majorie Murphy Cowan 1999). Herbal medicine practice plays an important role in the primary healthcare delivery system in most developing countries including Nigeria. Even the World Health Organization (WHO, 2002) is actively encouraging national governments of member countries to utilize their traditional systems of medicines with regulations suitable to their national health care systems. The WHO estimates that 80% of the population living in rural areas use or depend on herbal medicine for their health needs (WHO Traditional Medicine Strategy, 2002). Much of the exploration and utilization of natural products as antimicrobials arise from microbial sources (Ajaiyeoba et al 1998). It was the discovery of penicillin that led to later discoveries of antibiotics such as streptomycin, aureomycin and chloromycetin. Though most of the clinically used antibiotics are produced by soil microorganisms or fungi, higher plants have also been a source of antibiotics. Examples of these are the bacteriostatic and antifugicidal properties of Lichens, the antibiotic action of allinine in Allium sativum (garlic), or the antimicrobial action berberines in goldenseal (Hydrastis canadensis). Plant based antimicrobials represent a vast untapped source for medicines. Continued and further exploration of plant antimicrobials needs to occur. Plants based antimicrobials have enormous therapeutic potential (Ajaiyeoba EO. 2000). They are effective in the treatment of infectious diseases while simultaneously mitigating many of the side effects that are often associated with synthetic antimicrobials (Awe S and Omojasola PF 2008). They are effective, yet gentle. Many plants have tropisms to specific organs or systems in the body. Phytomedicines usually have multiple effects on the body.Their actions often act beyond the symptomatic treatment of disease (Ajaiyeoba EO and Okogun JI 1996). An example of this is Hydrastis canadensis. Hydrastis not only has antimicrobial activity, but also increases blood supply to the spleen promoting optimal activity of the spleen to release mediating compounds (Murray 1995; Abdullahi et al 2008).

However, in spite of the obvious and important contribution the herbal medicine makes to primary health care, it continues to be antagonized by majority of allopathic medical practitioners as it is considered to have no scientific basis. This work is therefore a preliminary work to prove that there is scientific evidence to the use of the root of Dichapetalum madascariense in the treatment of diseases.

Taxonomy

Kingdom: Plantae

Phylum: Magnoliophyta

Class: Magnoliopsida

Order: Malpighiales

Family: Dichapetalaceae

Genus: Dichapetalum

Species: Dichapetalum madagascariense

World distribution

Countries:

1) Cameroon,

2) Gabon,

3) Equatorial Guinea,

4) Madagascar,

5) Ghana,

6) Ivory Coast,

7) Benin,

8) Togo,

9) Nigeria,

10) Republic of the Congo,

11) Democratic Republic of the Congo,

12) Guinea,

13) Liberia,

14) Tanzania,

15) Sierra Leone,

16) Central African Republic,

PLANT DESCRIPTION.

Small trees, erect or scandent shrubs.

Leaves are simple, alternate, spirally arranged, often 2-pseudoseriate, entire; stipules 2,deciduous;

petiole are short.

MATERIALS AND METHODS:

MATERIAL:

Ø Standard drug.

§ Pefloxacin and

§ Clotrimazole cream.

Ø Microorganism.

§ Gram positive: Staphylococcus aureus, and Bacillus subtilis.

§ Gram negative: Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi.

BIOLOGICAL MEDIA.

Nutrien agar

Nutrient broth

Saubouraud dextrose agar.

EQUIPMENT.

Electronic weighing balance (Ohaus Model 2610) Laboratory incubator (Sanfa-model No. DNP-9022A). Petri-dishes, syringes, Autoclave (Health team instrument, England) Laboratory oven (Surgienfield instrument, England) Refrigerator (Haier Thermocool). Glass rod.

Digital water bath (Sanfa-model No. DK420). Mortar and pestle, permanent marker, filter paper. Bunsen burner, beakers, test-tubes, muslin cloth.

REAGENT/CHEMICAL.

Methanol, and distilled water were used for extraction.

Dragendorf’s and Hager’s reagent.

Concentrated sulfuric acid,

Fehling solution A and B,

Wagner’s reagents.

Chloroform and ethanol.

PLANT COLLECTION AND PREPARATION

The fresh plant of Dichapetalum madagascariense was collected from Ogidi in Idemili north local Govt. Area in Anambra state, Nigeria in march 2012.

The plant was identified by Mr. Ozioko a taxonomist.

The leaves were air dried in the Pharmacognosy Laboratory and then powdered using locally made corn grinder.

The powder was weighed (320g) and stored in an air tight-container.

EXTRACTION PROCEDURE.

AQUEOUSE EXTRACTION.

Extraction was done with distilled water. 200g of powdered leaves were macerated in 400mls of water for 24hrs with occasional shaking. At the end it was strained using white muslin cloth and then filtered using Whatman No.1 filter paper. The above process was repeated using the marc. The combined filtrates were freeze-dried and the extract was collected.

PHYTOCHEMICAL SCREENING.

Quantitative assay for the presence of secondary plant metabolites were carried out using the standard procedure obtained from (Trease and Evans, 2002).

TEST FOR ALKALOIDS

About 5 g of powdered leave was placed in the test tube and 20ml of methanol added to the tube, the mixture was heated in water bath and allowed to boil for two minutes. It was cooled and filtered and 5ml of the filtrate was tested with two drops Wagner’s reagent (solution of iodine and potassium iodide).

To another 5mL portion of the extract two drops of Hager’s reagent (saturated picric acid solution) was added. The presence of precipitate indicated alkaloid.

TEST FOR STEROIDS:

About 9ml of ethanol was added to 1g of the extracts and refluxed for a few minutes and filtered. The filtrate was concentrated to 2.5ml on a boiling water bath. 5ml of hot distilled water was added to the concentrated solution. The mixture was allowed to stand for 1 hour and the waxy matter was filtered. The filtrate was extracted with 2.5ml of the chloroform using separating funnel. To 0.5ml of the chloroform extract in a test tube, 1ml of concentrated sulfuric acid was added to form a lower layer. A reddish brown interface shows the presence of steroids.

TESTS FOR SAPONINS: About 20ml of water was added to 0.25g of crude extract and boiled gently in a hot water bath for 20 minutes. The mixture was filtered hot and allowed to cool and the filtrate was used for the following tests.

Frothing test: 5ml of filtrate was diluted with 20ml of water and vigorously shaken. The test tube was observed for the presence of stable foam upon standing.

Emulsion test: To the frothing solution, 2 drops of olive oil was added and the content shaken vigorously and observed for the formation of emulsion.

TEST FOR GLYCOSIDES

Fehling’s test: To 5ml of the filtrate was added 5ml of Fehling’s solutions (equal parts of A and B) and the content was heated in a water bath and a reddish precipitate which turns brick red on further heating with sulphuric acid indicates the presence of glycosides.

TANNINS AND PHENOLIC GROUP:

Alcoholic plant extract (0.5ml) was taken in a test tube. Two drops of 1M ferric chloride was added. Appearance of intense greenish-black precipitate indicated the presence of phenolic groups.

SOURCES OF MICROORGANISM

The microorganisms used were both bacteria and fungi obtained from laboratory stock of the Department of Pharmaceutical Microbiology and Biotechnology Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University Awka, Agulu campus.

The organisms include bacteria (Staphylococcus aureus, pseudomonas aeruginosae, Klebsiella Species, Escherichia coli, Bacillus subtilis, Salmonella typhi) and Aspergillus niger, Candida albican were the two fungi used.

ANTIMICROBIAL ACTIVITY SCREENING

PREPARATION OF INOCULUM.

v The test organisms were separately prepared by subculturing the pure isolates in nutrient agar and incubated at 37ºC for 24 hours for bacteria and in sabouraud dextrose agar for fungi.

v Two loopfuls of the microbial culture were collected using sterilized inoculating loop 10ml nutrient broth contained in sterilized bottle and then incubated at 37ºC overnight for subsequent use. 0.2ml of the overnight culture of different organism was then diluted with 20ml nutrient broth to give 1 in 100 diluted equivalents to 1×10E6 CFU/ML which were then used for the study.]

PREPARATION OF THE CULTURE MEDIA

v The nutrient agar was prepared by suspending 28g of the nutrient in one litre of distilled water.

v The suspension was then dissolved completely. It was then sterilized by autoclaving at 121ºC for 15 minutes.

AGAR DILUTION METHOD USING AQUEOUS EXTRACT.

Eight-hundred milligrams (800mg) of the aqueous extract was reconstituted in 2ml of sterile water in test tube. One millilitre,1ml, of the solution was transferred into another tube containing 1ml of sterile water. This step was repeated in four more test tube containing 1ml of sterile water each.

One millilitre(1ml) each of the aqueous extract dilution was poured into six different petri-dishes respectively, and 15ml of molten nutrient agar was aseptically poured into the six different petri-dishes, and rotated appropriately to ensure good mixing and allow the plates to set.

A sterile wire loop was used to streak each of the test organism into the clearly labelled segment of the nutrient agar and incubated at 37ºC for 24 hours the plate was examined for the presence of colony. This was repeated two more times.

Results of Phytochemical Analysis.

Secondary plant metabolites
Alkaliod	++
Glycoside	++
Tannins	++
Saponins	++
Steriods	++

Key: ++ = Presence of the compound.

+ = Trace Amount, = Compound not defected

Agar dilution method using the aqueous extract

	400 mg /ml	200 mg /ml	100 mg /ml	50 mg /ml	25 mg /ml	12.5 mg /ml	PEFLOXACIN 5μg /ml
S.A.	-	-	-	-	-	+	-
E.C.	-	-	-	+	+	+	-
B.S.	-	-	-	-	+	+	-
P.A.	-	-	-	-	+	+	-
S.T.	-	-	-	-	+	+	-

Key: + = Growth, - = No Growth.

Key: S.A = Staphylococcus aureus, P.A = Pseudomonas aeruginosa, Salmonella typhi., E.C = Escherichia coli, B.S = Bacillus subtilis.

Minimum inhibition zone diameter

	AQUEOUS EXTRACT
Staphylococcus aureus	25 mg/ml
E.Coli	100 mg/ml
Bacilus subtilis	50 mg/ml
Pseudomonas aeruginosa	50 mg/ml
Salmonella typhi	50 mg/ml
Candida albicans	--

DISCUSSION AND CONCLUSION:

The study was carried out to evaluate the antimicrobial activity of aqueous extract, the phytochemical constituents of the leaves of Dichapetalum madagascariense.

The phytochemical analysis revealed the presence of alkaloid, tannins, saponins, glycosides and steroids.

These secondary metabolites have been reported to exhibit varied biochemical and pharmacological effect in animals and microorganism when ingested.

The high content of saponins and tannins could be the basis for its antimicrobial activity. Tannins act by coagulating the cell wall protein (Trease and Evans,2002). While saponins causes the lysis of bacteria cell wall his may therefore explain the demonstration of antimicrobial activity of aqueous extract. The observed inhibitory properties of the extract against these clinical bacteria isolate using agar dilution method indicated that Dichapetalum madagascariens possesses a broad spectrum of antimicrobial activity (with reference to the standard antimicrobials used). The emerging antimicrobial resistance to disease have compromised chemotherapy of patient suffering from severe infectious disease especially in Nigeria. These infectious diseases include urinary tract infection and severe diarrhoea caused by Escherichia coli, salmonella typhi and shigella dysentriae. Wound infections, otitis media, genital tract infections, periodontal disease and osteomyelitis in children are mainly caused by streptococcus spp. and B. subtilis (Walker and Whittlessa,2007). The antimicrobial evaluation results of aqueous extract of the leaves of Dichapetalum madagascariense selected clinical isolates: Gram-positive bacteria (staphylococcus aereus, Bacillus subtilis), Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella tphy).and fungus (Candida albicans). Revealed the antimicrobial efficacy of the leaves of this plant. The observed inhibitory properties of the ex- tract against these clinical bacteria and fungi isolates using agar dilution method indicated that Dichapetalum madagascariense possess broad spectrum of antimicrobial activity (with reference to the standard antimicrobials used).

Using plants as the inspiration for new drugs provides an infusion of novel compounds or substances for healing disease.

Evaluating plants from the traditional African system of medicine, provides us with clues as to how these plants can be used in the treatment of disease. Many of the plants presented here show very promising activity in the area of antimicrobial agents, warranting further investigation.

RECOMMENDATION:

In view of the present finding, it seems important to recommend for further bioassay targeted technique studies on the crude extracts.

It would also make a great impact in the health care system and the populace if this work on in vitro studies is extended to in vivo determination of antimicrobial effects using animals.

This is suggested because the extracts which were inactive in vitro may have properties similar to prodrugs which are administered in an inactive form, but their metabolites could be active in vivo (Lino and Deogracios, 2006)

Finally, health foundations should increase their effort on plant research as it is a potential source of broad spectrum antimicrobial agent that could be helpful in reducing the emergence and spread of antimicrobial resistance.

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Received on 21.02.2013 Modified on 05.03.2013

Research J. Pharm. and Tech. 6(4): April 2013; Page 440-444