Medicinal & Aromatic Plants
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Phytobiotics Effects of Pawpaw (Carica papaya) Leaves and Fluted Pumpkin (Telferia ocidentalis) Leaves Extracts against Certain Aquatic Pathogens

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Introduction

Fish is a member of a group of organisms that consist of all gill bearing aquatic craniates that lack limbs with digits, like any other animal, fishes suffer from diseases and parasites [1]. The problem of fish diseases may be due to the development of antibiotic resistant bacteria, due to poor water quality and the misuse of antibiotic drugs which lowers the immune system of fishes due to the presence of microbes or pathogen in the water [1]. Recently there is an increasing awareness to determine the efficacy of plants as an alternative to treat microbial diseases of aquatic animal.

A medicinal plant is any plant which in one or more of its organ contains substances that can be used for the synthesis of useful drugs. Medicinal plants contain biologically active chemical substances such as saponins, tannins, essential oils, flavonoids, alkaloids and other chemical compounds which have curative properties. These complex chemical substances of different compositions are found as secondary plant metabolites in one or more of these plants [2]. Such plants include fluted pumpkin leaves and pawpaw leaves, although both plants appear to have broad spectrum of antimicrobial activities [3]. They are various reports on the microbial properties of a number of plant extracts, but their scientific evaluation remains as nascent area for intensive investigations especially with reference to aquatic pathogens.

This study was therefore aimed at screening for the antimicrobial properties of methanolic and ethanolic extracts pawpaw and fluted pumpkin leaves extracts against eight isolated fish pathogenic bacteria, with a possibility of determining the minimum inhibitory concentration by which the plant extracts can be added in fish feeds.

Materials and Methods

Plant collection

Telferia ocidentalis and Carica papaya were obtained in Ayeka, Okitipupa, Nigeria and was identified by Dr. DO Aworinde in the Department of Biological Sciences (Botany Programme), Ondo State University of Science and Technology, Okitipupa, Nigeria.

Preparation and extraction of plants material

Leaves extraction: The extraction of the T. ocidentalis and C. papaya was carried out as described by Ajaiyeoba and Fadare [4]. These plants were air dried for a period of 4 weeks. The air dried T. ocidentalis and C. papaya were grinded into powder, using hammer mill. 170 g of the T. ocidentalis and C. papaya were weighed respectively using a weighing balance. Eight hundred and fifty (850) ml of ethanol and methanol was measured each using a measuring cylinder and was decanted into the cup containing each plant extracts with appropriate labeling. The mixture was then homogenized and kept for 48 hours under room temperature at constant stirring at regular interval. Each labeled mixture was then filtered using a sterile muslin cloth after which the extraction was obtained, the filtrate was kept in the refrigerator at 40°C until required for further use.

Phytochemical screening

Detection of saponins: Froth test: Extracts were diluted with distilled water to 20 ml and this was shaken in a graduated cylinder for 15 minutes. Formation of 1 cm layer of foam indicates the presence of saponins. Foam test: Extract of 0.5 g was shaken with 2 ml of water. If foam produced persists for ten minutes it indicates the presence of saponins.

Detection of phenols ferric chloride test: Extracts were treated with 3-4 drops of ferric chloride solution. Formation of bluish black color indicates the presence of phenol.

Tannin: Extract of 2 ml was taken up in 10 ml distilled water, and filtered. Then few drops of ferric chloride reagent were added to 1 ml of the filtrate. The mixture was observed for the formation of blue, blue black, green or green black coloration or precipitate.

Detection of flavonoids: Alkaline reagent test: Extracts were treated with few drops of sodium hydroxide solution. Formation of intense yellow color indicates the presence of phenol. Glucosinolates: Extracts were treated with few drops of chloroform followed by filtration as described by Adeoye and Oyedapo method [5]. Concentration tetraoxosulphate (IV) acid was carefully layered at the bottom of the tube without disturbing the solution. It was observed for the formation of a sharp brown ring at the chloroform/sulphuric acid interface.

Test for triterpenes and steroids: The Salkowski test: Extract of 3 ml was warmed in 5 ml of chloroform solution was then treated with a small volume of concentrated tetraoxosulphate (IV) acid and shaken. The red colour produced within few minutes indicates a positive reaction.

Detection of protein and amino acids: Xanthoproteic test: The residues were treated with few drops of conc. Nitric acid. Formation of yellow colour indicates the presence of protein.

Media preparation

All media used were prepared according to manufacturer’s instruction. The media were allowed to cool after sterilization to about 400°C before pouring into sterile Petri dishes.

Source of test organisms

The microorganisms isolated from C. gariepinus were A. hydrophila, S. aureus, S. pyogens, B. subtilis, E. coli, S. typhii, and P. aeruginosa. The isolation and characterization of bacteria using a biochemical test was carried out at the Microbiology Laboratory Faculty of Science at University of Ibadan. Aspergillus niger and S. epidermidis were collected from the laboratory stock of Department of Biological Sciences, Ondo State University of Science and Technology. The pure cultures collected were labeled, sub-cultured on nutrient agar slant and nutrient broth(s) and potato dextrose agar (PDA) and were kept inside refrigerator at 40°C until it is required for study.

Antimicrobial assay

A well diffusion assay as described by Bello et al. [6] was used. Pre poured indicator [pathogen (4 mm depth)] was overlaid with a 10 ml soft agar (0.7%) lawn of indicator culture (thus generating a potential mat for the indication of bacteria). Wells of 10 mm diameter were cut into these agar plates using cork borer and 0.1 ml of these plants extract was placed into each well [6]. Distilled water was used as negative control while oxytetracycline (10 mg and 20 mg) was used as positive control. The plates were examined for zone of inhibition which was scored positive, if the width of the clear zone was 10 mm or longer. The diameter of the inhibition zones were taken to be proportional to the logarithm of the antimicrobial of the antimicrobial compounds in T. ocidentalis and C. papaya [7].

Isolation of microorganism/total microbial count

The gills, skin, intestine and liver sample of C. gariepinus were separately macerated and put into sterile capped test tube containing sterilized distilled water and were homogenized [8]. Serial dilution was carried out and 1 ml each from 10^-3 to 10^-5 dilution factor was dispersed into petri dishes that were appropriately labeled and molten sterilized medium was poured aseptically into petri dish. The plates were swirled gently for even distribution of inoculums and allowed to set/gel and then incubated at 37°C for 24 hours. The organism grew into visible different colonies after 24 hours. Total viable counts and Enterobacteriacea counts were determined and the result was expressed in log₁₀ CFU/ml.

Minimum inhibitory concentration of fluted pumpkin and pawpaw leaves

Double dilution of 1700 μg/ml of these plant extract were made into 2 ml volume of broth to 3.37 μg/ml. One row of the test was inoculated with 0.02 ml of 1 in 10 dilution of the broth culture of the organism. The test was incubated at 37°C for 24 hours aerobically. The minimum inhibitory concentration was the lowest concentration that prevented the growth of bacteria after 24 hour incubation [6].

Statistical analysis

The microbial load of fish tissue (skin, gills, intestine and liver) and antimicrobial and antifungal activities (diameter of zone of inhibition, mm) of pawpaw and fluted pumpkin leaves against tested pathogens resulting from the experiment were subjected to one way analysis of variance (ANOVA) using SPSS (Statistical Package for Social Science version 20).

Results

Phytochemical analysis of fluted pumpkin and pawpaw leaves extracts

Preliminary phytochemical screening of fluted pumpkin and pawpaw leaves extracts for primary and secondary metabolites showed the presence of saponins, tannins, alkaloid, amino acid and protein while phenols were not detected in both plants, flavonoids were not detected in fluted pumpkin leaves but the presence of cyanogenic glycoside was not detected in pawpaw leaves but present in fluted pumpkin leaves shown in Table 1.

Table 1: Determination of important phytochemical of ethanolic and methanolic extracts of fluted pumpkin and pawpaw leaves.

Determination of microbial load in Clarias gariepinus

The microbial load of fish tissue (skin, gills, intestine and liver) were determined and the result show that the highest Enterobacteriacea counts was recorded in skin and least in liver while no Enterobacteriacea was recorded on control. Also, the highest total viable count was recorded in skin and least in liver while no total viable counts was recorded in the control as shown in Table 2.

Table 2: Microbial load of skin gills, intestine and liver of C. gariepinus.

Detection of antimicrobial activities of fluted pumpkin and pawpaw leaves extracts

Ethanolic and methanolic extracts of fluted pumpkin and pawpaw leaves respectively shows antibacterial and antifungal properties in the present study. The fluted pumpkin leaves shows the highest activities with all the pathogens investigated. Although both the fluted pumpkin and pawpaw leaves showed no activities against S. pyogens and the methanolic extract of pawpaw leaves shows no activities against B. subtilis while the ethanolic extract of fluted pumpkin leaves shows no activities against S. typhii. Antifungal activity was also recorded against A. niger with the highest inhibition recorded in pawpaw leaves extracts (Table 3).

Table 3: Antibacterial activities (diameter of inhibition zone, mm) of methanolic and ethanolic extracts of fluted pumpkin and pawpaw leaves.

Minimum Inhibitory Concentration (MIC) of pawpaw and fluted pumpkin leaves extracts

The minimum inhibitory concentration of ethanolic and methanolic of fluted pumpkin and pawpaw leaves against eight bacteria isolated from C. gariepinus were examined in the present study and their potency were assessed by minimum inhibitory concentration and it was recorded that 425 μg/ml and 850 μg/ml for methanolic and ethanolic extract of pawpaw and fluted pumpkin leaves respectively against all the tested pathogens (see Table 4A and 4B).

Table 4A: Minimum inhibitory concentration (μg/ml) assay of ethanolic and methanolic extracts of fluted pumpkin leaves on isolated fish pathogen.