Antimicrobial and synergistic potential of Ocimum gratissimum leaves and Petiveria alliacea bark against some selected microorganisms

Background: This study was carried out to investigate the antimicrobial and synergistic potential of the leaves of Ocimum gratissimum and bark of Petiveria alliacea against some tested bacterial and fungal isolates. Fresh and matured leaves of Ocimum gratissimum and bark of Petiveria alliacea were collected from the Institute of Agriculture, Research and Training, Ibadan, Nigeria. The specimens were identi ﬁ ed at the Herbarium unit of the Department of Botany, Obafemi Awolowo University, Ile-Ife, Nigeria. The pathogenic organisms used include bacteria namely, Providencia stuartii, Bacillus cereus, Staphylococcus aureus, Corynebacterium Pyogenes, Streptococcus faecalis, Klebsiella oxytoca, Klebsiella pneumonia, Escherichia coli, Pseudomonas ﬂ uorescence, Serratia rubidae, Proteus mirabilis, Salmonella pullorum; and fungi namely, Trychophyton tonsurans, Candidia albicans, Trychophyton rubrum, Penicillium expansium, Alternaria sp , Fusarium sp , Aspergillus niger, Aspergillus fumigatus, Aspergillus ﬂ avus, and Penicillium camenberti. Methods: Pure isolates of the tested microorganisms were obtained from the department of microbiology, University of Ibadan, Ibadan, Nigeria. The bacterial isolates were maintained on nutrient agar slant and the fungal isolates were on Sabouraud Dextrose Agar (SDA). Antimicrobial sensitivity test (AST) followed by Clinical and Laboratory Standard Institute. Minimal bactericidal and fungicidal concentrations were determined following established protocols. Results: Fungal isolates of Aspergillus ﬂ avus, Penicillium expansiumm, Trychophyton rubrum, and bacterial isolates Klebsiella oxytoca, Klebsiella pneumonia, Escherichia coli, Proteus mirabilis, and Salmonella pullorum were all resistant to the plant extract. Findings from this study opined that ethanolic extract of Ocimum gratissimum leaves is more potent than the methanolic and aqueous extracts of Petiveria alliacea. Conclusion: The plant extracts showed greater antimicrobial activity against bacterial-with respect to fungal isolates suggesting a broader spectrum of activity with ethanolic extract on the gram-positive and the gram-negative bacteria.


Introduction
The various therapeutic roles of plants that fi ght various diseases have led to an increase in the search for alternatives to conventional medicine.Ocimum gratissimum is widely used for medicinal purposes in the treatment of intestinal diseases [1].
It is often used in Nigeria as a condiment in preparing various dishes that gain prominence due to their nutritional power [2].

Medicinal plants are a rich source of anti-bacterial activity.
There are many reports established on the effectiveness of traditional medicine against germs.Therefore, plants are still recognized as a pivot of modern medicine for the treatment of infectious diseases [3].The traditional use of herbs as primary treatment because of their medicinal value is very common in western countries [4].Petiveria alliacea is commonly referred to as "Anamu" or "Yes Aja" in southwestern Nigeria.It is traditionally used to improve memory and treatment of respiratory tract infections.It is also used to strengthen infection, pain, and treatment for a variety of chronic diseases including certain cancers [5].There are many plants with antimicrobial and biological activity [6] currently used in the food industry as antibacterial and antifungal agents [7].
Citation: Oladele IS, Ologundudu FA (2022) Antimicrobial and synergistic potential of Ocimum gratissimum leaves and Petiveria alliacea bark against some selected microorganisms.Ann Environ Sci Toxicol 6(1): 041-046.DOI: https://dx.doi.org/10.17352/aest.000051 Although the main purpose of spices is to provide fl avor and therapeutic properties, antibacterial and antifungal properties have also been used [8].Studies on medicinal plants should include both the phytochemicals and the biological properties of these plants.Numerous studies have been conducted to determine the different antimicrobial and phytochemical components of various medicinal plants that are widely used in the treatment of various microbial infections as alternative therapies for synthetic drugs when many infectious viruses develop resistance [9].Recent scientifi c discoveries in the rational use of medicinal plants and the many therapeutic approaches of local communities can lead to the benefi cial identifi cation of useful techniques, and the conservation and sustainable use of local biodiversity [10].The combination of various antimicrobial agents may result in interaction or opposition in the workplace.Where there is a combination, the antimicrobial effect is achieved at a lower concentration than when applied once [11].fi lter paper under aseptic conditions.Filters evaporated using a rotating evaporator, then stored in the refrigerator at 4 ºC until needed for future use Ladipo, et al. [12]

Sources and preparation of test organism
Pure isolates of bacteria and fungi used in this study were isolated locally from wounds and the environment and

Sensitivity testing
Antibacterial and Antifungal tests are performed according to established criteria by Daoud, et al. [14].One ml of a new culture of bacteria and mold was piped into an empty Petri container.Molten-cooled Muller Hi (PDA) mold was then poured into a Petri container containing the inoculum and blended.After hardening, the springs were drilled using a sterile cork borer (6 mm wide) into agar plates containing inoculum.Thereafter, 100 μl of each extract (20% w/v) was added to the appropriate sources.The plates are transferred to the refrigerator for 30 minutes so that they are evenly distributed.Then, the plates were placed at 37°C for 18 hours.Antimicrobial activity was obtained by measuring the area of the block after the incubation period.

Inoculum suspension
Inocula were prepared from stock cultures stored in nutritious agar at 40C and planted slowly in nutrient solution using a wide wire loop.The density of the suspension inserted in the media to test the trend was determined in relation to the standard 0.5 McFarland Barium sulphate solution Cheesbrough [15].

Minimal bactericidal and fungicidal concentration (MBC and MFC)
Minimal bactericidal concentration (MBC) was determined by incorporating 1ml of direct MIC tubes into the nutrient agar to obtain the bacteriostatic and/or bactericidal effect of the extracts.The MBC plant extraction was determined by a modifi cation of the Spencer and Spencer method [16].

Phytochemical analysis
Phytochemical analysis of samples was performed in accordance with established conventions.
Saponin: Twenty-fi ve grams (25g) of each powdered sample is placed in a bucket and boiled in 25ml of distilled water in a water bath controlled at 100 o C and fi ltered.2.5ml of each fi ltrate is mixed with 5ml of distilled water and stirred vigorously to form a stable foam.The broth was mixed and then stirred vigorously and concentrated to form an emulsion of Obdoni and Ochuko [17].
Steroids: 2 ml (2ml) acetic anhydride added to 0.5g extracted ethanol for each sample and 2ml of tetra oxosulphate (VI) acid added.The color change from violet to blue or green indicated the presence of steroids Kolawole, et al. [15] Flavonoids: Each portion of the powdered plant samples was diluted separately with 10ml of ethyl acetate in a water bath for 30 min.The mixture was fi ltered and 4ml of each fi ltrate was mixed and diluted with 1ml of dilute ammonia solution in a conical fl ask.The formation of a yellow color indicated the presence of fl avonoids Harborne [16].
Tannins: A quantity of powdered sample (0.5g) boiled in 20ml clear water in a test tube and fi ltered in a concrete fl ask.A few (2-3) drops of 0.1% ferric chloride were added and marked with dark brown or blue-black color Trease and Evans [16].
Two grams (2g) of the molten powder were boiled with 50ml of ether from the extract of the phenolic portion for 15min.5ml of the extract was pipette into a 5ml volume conical fl ask and 10ml of distilled water was added 1ml of ammonium hydroxide solution and 5ml concentrated amyl alcohol was also added.Samples were left to react for 30 minutes to improve color.This was measured at 505nm using the UNICO 1100 RS spectrophotometer McDonald, et al. [17] Terpenoids: Five milliliters (5ml) of liquid extract per plant sample mixed with 2ml (2ml) of chloroform in a test tube.
Three milliliters (3ml) of tetraoxosulphate (VI) concentrated tetraoxosulphate (VI) acids are carefully added to the mixture to form a layer.A visible reddish-brown connective tissue is formed when a terpenoids component is present Edeoga, et al. [17].

The biological effect of methanolic (M), Ethanolic (E), and Aqueous (A) extracts of Aspillia Ocimum gratissimum and Petiveria alliacea against some selected bacterial and fungal isolates
The zones of inhibition of the extracts ranged from 1-18mm for bacteria while fungal isolates ranged between 1 and 5mm (Table 1).The highest and lowest zones of inhibition of 18mm  2).E. coli and Proteus mirabilis were resistant to the combined extract of P. alliacea and O. gratissimum among the bacterial isolates.Aspergillus fumgatus and Aspergilus fl avus showed no antimicrobial activity to the combined extract among the fungal isolates.
The susceptibility of the test organisms to antibiotics and the antifungal drug showed that Staphyloccocus aureus and Cornybacterium pyogens primarily sensitive to the plant extracts were found to be resistant to some of the antibiotics used (Table 3).All the plant extracts showed strong antimicrobial activities against Streptococcus faecalis and S. aureus, whereas, these organisms were resistant to synthetic commercial products such as ofl oxacin, sparfl oxacin, chloramphenicol, amoxicillin, ciprofl oxacin, and septrin.However, the antifungal drug (Ketoconazole) was more effective on the test fungi than the plant extracts.Serratia rubidae

Test Fungi
Trychophyton tonsurans       MICs of fungal isolates vary between 150 and 300μg/ml.The easy entry of these fungi into these extracts may explain that most fungi interact with plants in the fi eld and would naturally develop resistance.

Fresh
and mature Occimum gratissimum leaves and Petiveria alliacea bark were obtained from the Institute of Agriculture, Research and Training, Ibadan, Nigeria.Models courtesy of IFE Herbarium.10 g of crushed Ocimum gratissimum leaves and Petiveria alliacea bark are carefully weighed and immersed in 1,000 mL of soluble extract for 72 hours and stirred continuously.The remains were fi ltered using Muslin cloth to fi lter the fi ltrate and were later fi ltered through Whatman No 1 obtained from the Department of Microbiology, OAU, Ile-Ife, Nigeria.Distinguishing bacteria are stored in the agar medium and fungal isolates in the SDA.These alone were placed in a test tube attached to a separate cotton container containing 10ml of Mueller-Hinton broth inoculated at 37 0 C for 24 hours.Bacteria used include Providencia stuartii, Bacillus cereus, Staphylococcus aureus, Corynebacterium Pyogenes, Streptococcus faecalis, Klebsiella oxytoca, Klebsiella pneumonia, Escherichia Poliumsee, Escherichia Salmonsuess, Pseudomonas, Serreptococcus fl our, Pseudomonas, Pseudomonas, Pseudomonas, Candida albicans, Trichophyton rubrum, Penicillium expansium, Alternaria sp, Fusarium sp, Aspergillus niger, Aspergillus and Aspergillus andger, Aspergillus camenberti.Synergistic antimicrobial effects of a variety of fungal and bacterial extracts were determined using a method developed by Oluduro and Omoboye[13].To demonstrate interaction, an equal amount of plant mixture was measured and melted in the right amount of ingredients to give a concentration of 100 mg/ml used for antimicrobial testing Oluduro and Omoboye[13] and 1mm respectively were obtained in Staphyloccocus aureus under the aqueous and methanolic fractions.Bacillus cereus, Cornybacterium pyogenes, Klebsiella pneumonia, and Escherichia coli showed a high degree of resistance to the plant extracts.The fungal isolates; Candida albicans, Tryptophyton rubrum, Penicillium expansium, and Aspergillus fl avus were resistant to Psidium guajava extract since no signifi cant activity was observed Synergistic antimicrobial effects of the plant extracts on the tested bacterial and fungal isolates revealed that the diameter of zones of inhibition ranged from 1mm with combined aqueous extracts and ethanolic extracts to 35mm with ethanolic extracts of P. alliacea and O. gratissimum (Table extract, E = Ethanolic extract, M = Methanolic extract Aqueous extract, E= Ethanolic extract, M= Methanolic extract Citation: Oladele IS, Ologundudu FA (2022) Antimicrobial and synergistic potential of Ocimum gratissimum leaves and Petiveria alliacea bark against some selected microorganisms.Ann Environ Sci Toxicol 6(1): 041-046.DOI: https://dx.doi.org/10.17352/aest.000051rubrum, Penicillum sp. and C. albicans.1969; Begum, et al. 1993; Nwosu and Okafor 1995; Akinyemi, et al. 2004; Janine de Aquino, et al. 2005; Lopez, et al. 2005).(21mm) was obtained by extracting ethanolic P. alliacea against S. aureus.This may be due to the fact that the bioactive compounds in P. alliacea is very soluble in ethanol as a solvent.In addition, ethanol itself has antimicrobial activity when used alone and has the ability to dissolve organic compounds better, thus releasing the active ingredient needed for plant antimicrobial activities (Elegalam, 2005).The results obtained from the minimum inhibitory concentration (MIC) of various extracted plants showed that the plant excretion was strong against bacteria in the MIC of 25μg/ml.The high MIC obtained from the test mold confi rmed their low tendency for almost all tested releases.

Table 1 :
Diameter of zones of inhibition of the plant's extracts on selected bacterial and fungal isolates.

Table 2 :
Synergistic antimicrobial effects of the plant extracts on tested microorganisms.

Table 3 :
Susceptibility of the test organisms to antibiotics and antifungal drug.

Table 4 :
Minimum inhibitory concentration (MIC) of the various extracts on the tested bacterial and fungal isolates (μg/ml).

Table 5 :
Minimum bactericidal and fungicidal concentrations (MBC and MFC) of the various plant extracts on tested bacterial and fungal isolates (μg/ml).