Anti-microbial agents are substances that kill microorganisms or inhibit their growth. They are widely employed to cure bacterial diseases. Antimicrobial agents that reversibly inhibit the growth of bacteria are called bacteriostatic, whereas those with irreversible lethal action on bacteria are known as bactericidal [1]. Ideally, antimicrobial agents disrupt microbial processes or structures that differ from those of the host. They may damage pathogens by hampering cell wall synthesis, inhibiting microbial protein and nucleic acid synthesis, disrupting microbial membrane structure and function, or blocking metabolic pathways through inhibition of key enzymes [2]. Reported that plant extracts and their products are used in many parts of the world as active principles in herb remedies. They were used locally in the treatment of infections many centuries before scientific studies were discovered. Plant derivatives have made a large contribution to human health as they have been used as a source of preliminary compounds for drugs. Widespread usage of drugs has led to the development of pathogen resistance, hence urging research into new drugs for the treatment of diseases. Active compounds present in medicinal plants provide a bountiful resource of active compounds for the pharmaceutical, cosmetics, and food industries, and more recently in agriculture for pest control.

According to the World Health Organization (WHO, 2002), over 80 percent of the world’s population, especially in the developing world, relies on medicinal plants as sources of medicines for their primary healthcare. The traditional system of medicine, which depends mainly on medicinal plants, is rich in ethnomical knowledge of the uses of medicinal plants in the treatment of infectious conditions [3]. These medicinal plants employed in traditional medicine represent potential sources of cheap and effective standardized herbal medicines (phytomedicine) and lead to the discovery of novel molecules for the development of new chemotherapeutic agents (Farnsworth and Morris, 1976). Several infectious diseases, including malaria, diarrhea, dysentery, gonorrhea, and fungal infections, have been successfully managed in traditional medical practice employing medicinal plants [4].

The Jatropha curcas plant originated in Mexico and was spread to Asia and Africa by Portuguese traders as a hedge plant. It belongs to the family Euphorbiaceae. In many subtropical and semi-arid regions. Traditionally, J. curcas is used for its medicinal properties, and its seeds contain semi-dry oil, which has been found to be useful for medicinal purposes. It has played a major role in the treatment of various diseases, including bacterial and fungal infections. The seeds and leaf extracts of J. curcas have shown molluscidal and insecticidal properties. The extracts of many Jatropha species, including J. curcas, have been shown to display potent cytotoxic, anti-tumor, and antimicrobial activities in different assays. The latex of J. curcas has been shown to possess antibacterial activity against Staphylococcus aureus [5]. However, the antimicrobial activity of the other parts has not been fully investigated.

The plant has thick, glabrous branches, a straight trunk, and grey or reddish bark, masked by large white patches. It has green leaves with short, shallow lobs, which are alternately arranged [6]. J. curcas is widely used in traditional medicine in Africa, Asia, and Latin America to cure various ailments such as skin infections, diarrhea, gonorrhea, fever, and several other diseases caused by microorganisms [7]. J. curcas has also been used as an antidote, remedy, medicine, and potential source of herbal drugs in dental complaints and against constipation [8]. Sap (latex) is applied directly to wounds and cuts as a styptic and astringent to clean teeth and gums and to treat sores on the tongue and in the mouth. It has coagulating effects on blood plasma. The sap is also used as a remedy for alopecia, anasorca, burns, dropsy, eczema, inflammation, paralysis, and yellow fever. The sap is also used for the treatment of dermatomucosal diseases [9].

Profile of the Study Area

The study area is Anyigba town, Dekina Local Government Area, Kogi State. This area falls within the tropical wet and dry climate region. The latitude: 7⁰15-7⁰29N and Longitude: 7⁰11- 7⁰32. The daily temperature range is 25–30° C [10].

Collection and preparation of the plant sap

The Jatropha curcas sap was collected from different locations in Anyigba, Kogi State, in the month of May 2022 and authenticated at the Department of Biological Sciences, Prince Abubakar Audu University, Anyigba, Nigeria. The plant sap from J. curcas was collected directly into microfuge tubes after leaf and stem cutting and kept refrigerated at 4^o C.

Sterilization of glassware and media

All glassware was thoroughly washed with detergent and rinsed with distilled water. They were dried in the hot air oven and then sterilized at a temperature of 121^o C for fifteen minutes using the autoclave. The media was prepared according to the manufacturer's instructions and sterilized by autoclaving at 121^o C for fifteen minutes. Alcohol was used to disinfect the benches before any experiment was carried out to avoid contamination and to ensure aseptic working conditions.

Phytochemical screening of J.curcas sap

The J.curcas sap was qualitatively analyzed for the presence of flavonoids, saponins, carbohydrates, glycosides, steroids, tannins, and alkaloids in accordance with Trease and Evans (2004).

Antimicrobial screening test

Pure clinical isolates of Candida albicans, Trichophyton sp, Staphylococcus aureus, Salmonella sp and Shigella sp were obtained from the Microbiology Department Laboratory Unit of Prince Abubakar Audu University (PAAU). The organisms were grown on nutrient agar in an incubator at 37^o C for 24 hours. The antimicrobial activity of J.curcas was measured by culture media using Mueller-Hinton Agar.

Preparation of Mueller-Hinton Agar

38g of Mueller Hinton agar was weighed using a weighing balance machine. It was dissolved in 1 liter of distilled water. It was then heated to dissolve the media completely. The media was sterilized by autoclaving at 121⁰ C for 15 minutes. It was allowed to cool to 45-50⁰ C. It was mixed and poured into sterile petri dishes.

The organisms used in the culture were as follows: Candida albicans, Trichophyton rubrum, Staphylococcus aureus, Salmonella sp and Shigella sp.

Antimicrobial susceptibility Assay

The agar-well diffusion method was employed to determine the growth inhibition abilities of the test organisms by the sap. The 24-hour-old cultures were transferred into nutrient broth, incubated at 37°C for 5 hours, and standardized to the McFarland standard. A 0.5 mc farland standard was prepared by mixing 0.05 ml of 1.175% barium chloride dehydrate (Bacl2.2H2O) with 9.95 ml of 1% sulfuric acid (H₂SO₄) and was compared visually with a suspension of bacteria and fungi in sterile nutrient broth. The concentrations of 12.5, 25, 50, and 100 ml/ml were used, Mueller-Hinton agar was prepared, and 30 ml of each was poured into sterile petri dishes. The agar was allowed to solidify and dry. The agar was aseptically inoculated uniformly with the test organism by flooding with a 0.2-ml suspension of 106 cfu, which is equivalent to 0.5 McFarland standard. The test culture was preserved undisturbed for 30 minutes. Each of the test organisms from the broth cultures was streaked on 10 different Mueller Hinton agar plates under aseptic conditions and labeled accordingly. With the aid of a sterile 6mm-diameter corkborer, five wells were borne on the agar and sufficiently separated and kept at least 15 mm from the edge of the plate and 25 mm from well to well to prevent overlapping of zones. With the aid of a micropipette, 0.2 ml of a known concentration of the sap at different concentrations was introduced into separate labelled wells (holes). This was done in duplicates, and the inoculated plates were incubated at 37º C for 24 hours. With the aid of a meter rule, the zone diameters of inhibition were measured and recorded to the nearest millimeter. Ciprofloxacin and ketoconazole were used as positive controls, while water was used as a negative control (Okoli et al., 2007).

Minimum inhibitory concentration (MIC)

The MIC of isolates was carried out using the tube dilution technique as described by Doughari et al. (2007). The McFarland standard (10⁶ cfu/ml) was used to standardize the concentration of test organisms. A tube containing 2 ml of 18-hour nutrient broth without extract was seeded with a loopful of the test organism previously diluted to 0.5 McFarland standard to serve as the positive control, while a tube containing 2 ml of 18-hour nutrient broth that was not inoculated served as the negative control. After incubation for 24 hours at 37^o C, the tubes were then examined for microbial growth (turbidity).

Minimum bactericidal concentration (MBC)

The MBC is the lowest concentration of antibacterial substance required to produce a sterile culture. In this technique, the content of the test tube resulting from MIC was streaked using a sterile wire loop on an agar plate and incubated at 37⁰ C for 24 hours. The lowest concentration of the sap, which showed no bacterial growth, was noted and recorded as MBC. (Vollekova and Kostalova, 2001).

Minimum Fungicidal Concentration (MFC)

The MFC is the lowest concentration of antifungal substance required to produce a sterile culture. In this technique, the content of the test tube resulting from MIC was streaked using a sterile wire loop on an agar plate and incubated at 37⁰ C for 24 hours. The lowest concentration of the sap, which showed no fungi growth, was noted and recorded as MFC.

Table 1 shows the result of the phytochemical screening. It was observed that J.curcas sap was positive for alkaloids, flavonoids, carbohydrates, glycosides, saponins and tanins while steroids was absent.

Table 1: Qualitative Phytochemicals Analysis of Jatropha curcas Sap.

Key:

Positive = +

Negative = -

Table 2 shows the result of antibacterial activity of J.curcas sap at different concentrations of 100 ml/ml, 50 ml/ml, 25 ml/ml and 12.5 ml/ml. The inhibitory zone diameter on test bacterial isolates expressed at various concentrations of the sap ranges from 5mm to 25mm.Shigella sp, Staphylococcus aureus and Salmonella sp were all sensitive to the sap.

Table 2: Inhibitory Zone Diameter of Jatropha curcas sap on Test Bacterial Isolates.

Table 3 shows the result of antifungal activity of J.curcas sap at different concentrations of 100 ml/ml, 50 ml/ml, 25ml/ml and 12.5 ml/ml. The zones of inhibition expressed at various concentrations of the sap shows that both Candida albicans and Trichophyton rubrum were all sensitive to the sap but Candida albicans was more sensitive and has the highest zone of inhibition.

Table 3: Inhibitory Zone Diameter of Jatropha curcas sap on Test Fungi Isolates.

Table 4 shows the Minimum Inhibitory Concentration [MIC] and Minimum Bactericidal Concentration [MBC] of J.curcas sap against test organisms.

Table 4: Minimum Inhibitory Concentration and Minimum Bactericidal Concentration of Jatropha curcas Against Test Organisms.

Table 5 shows the Minimum Inhibitory Concentration [MIC] and Minimum Fungicidal Concentration [MFC] of J.curcas sap against test organisms.

Table 5: Minimum Inhibitory Concentration and Minimum Fungicidal Concentration of Jatropha curcas Against Test Organisms.

Zones of inhibition observed

The J. curcas sap was found to have antibacterial and antifungal effects against all the selected organisms used in this project. (Staphylococcus aureus, Shigella spp., Salmonella spp., Candida albicans, and Trichophyton rubrum).

The most susceptible organism to the sap appeared to be Staphylococcus aureus. Thomas also reported that J.curcas curcas has been shown to possess antibacterial activity against Staphylococcus aureus and has played a major role in the treatment of various diseases, including bacteria and fungi.

The susceptibility of Staphylococcus aureus, Salmonella typhimurium, and Shigella spp. to the sap strengthens the suggestion that it could be useful for eczema and the treatment of dermatomucosal diseases (Dada et al., 2019).

The qualitative phytochemical analysis indicated the presence of secondary metablolites. This finding was in agreement with the previous work of Trease et al. (2004), who reported the presence of flavonoids, tannins, carbohydrate, glycosides, and sapponins in J. curcas sap, which support the traditional medical uses of the sap in the treatment of different infections.

The antimicrobial activity of J. curcas sap inhibited Staphylococcus aureus, Salmonella spp., Shigella spp., Candida albicans, and Trichophyton rubrum with a very small zone of inhibition.

The minimum inhibitory concentration (MIC) and Minimum Bactericidal Concentration (MBC) show inhibition at concentrations of 25 mL/mL and 50 mL/mL.

The minimum fungicidal concentration shows inhibition at a concentration of 50 mL/mL. The highest zone of inhibition of J. curcas sap for bacterial isolates was observed on Staphylococcus aureus, and the lowest zone of inhibition was observed on Shigella spp. While the highest zone of inhibition observed for fungi isolate was Candida albicans, the lowest zone was observed on Trichophyton spp., which shows that J. curcas sap can cure both bacterial and fungal infections.

The antimicrobial screening showed that the sap was active against all the test organisms at 100 mL/mL, which was the highest concentration. Staphylococcus aureus, Salmonella spp., Shigella spp., Candida albicans, and Trichophyton rubrum were 25mm, 21mm, 20mm, 26mm, and 23mm, respectively.

At 50 ml/ml, Staphylococcus aureus, Salmonella spp., Shigella spp., Candida albicans, and Trichophyton rubrum were 16mm, 18mm, 17mm, 21mm, and 20 mm, respectively.

At 25 ml/ml, Staphylococcus aureus, Salmonella spp., Shigella spp., Candida albicans, and Trichophyton rubrum were 12mm, 13mm, 14mm, 19mm, and 16 mm, respectively.

At 12.5 ml/ml, Staphylococcus aureus, Salmonella spp., Shigella spp., Candida albicans, and Trichophyton rubrum were 9 mm, 5 mm, 9 mm, 15 mm, and 13 mm respectively.

Based on the result of this study J. curcas is a potential antimicrobial substance active against Gram negative and Gram positive bacteria and some fungi. It also shows that sap can be useful in the treatment of skin diseases cause by Staphylococcus aureus, Salmonella spp., Shigella spp., Candida albicans it is therefore a potential antibiotic.

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