Synergistic Potential of the Leaves of Aspillia Africana (Compositae) and Psidium guajava (Myrtaceae) Against Some Selected Bacterial and Fungal Isolates

Article Info History Received: 20 Nov 2019 Accepted: 8 Jul 2020 Available: 31 Aug 2020 Abstract Background: The plethora of secondary metabolites inherent in plants that could be synthesized and formulated into useful drugs for the treatment of diseases is not fully understood. Objectives: This research was carried out to investigate the antimicrobial potential of the leaves of Aspillia africana and Psidium guajava plants on some selected bacterial and fungal isolates. Methods: Fresh and matured leaves of Aspillia africana and Psidium guajava were collected at the Federal College of Agriculture, Akure and the Botanical Garden, Obafemi Awolowo University (OAU), Ile-Ife, Nigeria. The plants were identified and voucher specimen deposited at IFE Herbarium. The pure isolates of the bacteria and fungi were obtained from Medical Microbiology unit, Department of Microbiology, Obafemi Awolowo University, Ile-Ife, Nigeria. The bacteria isolates were maintained on nutrient agar and the fungal isolates on Sabouraud Dextrose Agar (SDA). Extraction of the plant materials using methanol, ethanol and aqueous fractions were carried out using standard protocols. The isolates were inoculated on a nutrient broth. The Minimum Inhibitory Concentration (MIC) of the extracts was determined by broth dilution method while minimal bactericidal and fungicidal concentrations respectively were determined following established protocols. Results and conclusion: The results showed that Bacillus cereus, Cornybacterium pyogenes, Klebsiella pneumonia and Escherichia coli showed higher degree of resistance to the plant extracts. The fungal isolates; Candida albicans, Tryptophyton rubrum, Penicillium expansium, and Aspergillus flavus were resistant to Psidium guajava extract as no significant biological activity was observed. All the extracts from the plants produced considerable antimicrobial activities with Streptococcus faecalis and Staphyloccocus. aureus. whereas, these organisms were resistant to ofloxacin, sparfloxacin, chloramphenicol, augmentin, ciprofloxacin and septrin. This study concludes that ethanolic extracts (100mg/ml) of the leaf of Aspillia africana is more potent than the methanolic and aqueous extracts of Psidium guajava. This study demonstrated that the therapeutic properties of Aspillia africana and Psidium guajava are better enhanced with their synergistic potential to the tested microorganisms.


INTRODUCTION
Crude extracts of medicinal plants notwithstanding stand out as veritable sources of potential resistance agents and the African biosphere promises to be a potential source of such compounds owing to its rich plant species diversity 1. According to World Health Organization 2 , a larger percentage of the world's population is based mainly on traditional medicine and a significant part of the therapy involves the use of plant materials or their bioactive components. There are many findings on the therapeutic potentials of plant extracts against bacteria 3 , hence, plants are still being recognized as the pivot of modern medicine for the prophylaxis of infectious diseases 4 . Therefore, the contributions of medicinally and pharmaceutically important plants to health care cannot be over-emphasized. There is a growing demand to discourage the usage of drugs with high clinical toxicity. However, the usual practice of oral administration of crude extracts poses threat to human health as the extracts may contain some toxic components 5 . These ambivalent challenges had necessitated the search for antibiotics in institutions, industries and the academic community 6 . Plants have major advantage over synthetic drugs because it's cost effective, readily available, cheaper alternative and pharmacological importance 7 . Several studies have been reported on the antibacterial activities of local plants such as Euphorbia hirta, Kigelia Africana, Hibiscus sabdarifa and Mucuna pruriens 8 .
Aspillia africana (Compositae) is a tropical shrub, semi-woody herb from a perennial root widely grown in Nigeria where it is commonly known as Yunrinyun by the Yorubas, Orangila by Igbos and Tozalin by Hausas. The plant is about 2m high, polymorphic and ubiquitous on western land of Savannah. Its formulations had been used in the treatment of various bacterial infections such as gastro-intestinal disorders, corneal opacity and insect bites. The plant is widely used as haemostatic agent (spontaneous arrest of bleeding from damaged blood vessels). The fresh leaves are effective in wound healing, cuts and sores 9 .
Psidium guajava L. (Mytaceae) is one of such plants in folk medicine that had been extensively used for the treatment of various disease conditions like fever, dysentery, vomiting and inflamed gums 10 . The extract from the leaves were found to display analgesic and antiinflammatory potntial 11 , antimicrobial and antioxidant activities 12 . The alarming increase of resistance to antibiotics by microorganisms cannot be overemphasized. This had necessitated scientist to search for alternative strategies of mitigating this challenge. The objectives of the study are therefore to investigate the antimicrobial potential of the leaves of Aspillia africana and Psidium guajava plants on some selected bacterial and fungal isolates; and to investigate the synergistic antimicrobial effects of the extracts on the test organisms.

Materials and methods Preparation and authentication of plant samples
A vey fresh and matured leaves of Aspillia africana and Psidium guajava were collected at the Federal College of Agriculture, Akure and Botanical Garden, Obafemi Awolowo University (OAU), Ile-Ife, Nigeria. The specimens were identified at IFE Herbarium, Corynebacterium pyogenes ------

Streptococcus faecalis
Performance standards for antimicrobial susceptibility testing. Clinical and laboratory standards institute, 29 th ed, 2019.
Department of Botany, Obafemi Awolowo University, Ile-Ife and voucher deposited. Each plant sample was separately washed and rinsed several times in distilled water. The samples were dried to constant weight in an oven and ground into powdery form using laboratory blender. The powder (100g) obtained were extracted with 500ml of sterile distilled water, 500ml ethanol and 500ml methanol by soaking in water for 5days and then filtered. The filtrates were later concentrated at 45 o C using a motorized rotary evaporator (Resona England). The dried samples were kept in sterile bottles for future use.

Preparation of test organism
The pure isolates of the bacteria and fungi used in this research were obtained from Medical Microbiology unit, Department of Microbiology, Obafemi Awolowo University, Ile-Ife, Nigeria. The bacteria isolates were maintained on nutrient broth and the fungal isolates on SDA. The isolates were constantly sub cultured at fourteen days interval. Before use, the isolates were inoculated into separate test tube that was plugged with adsorbent containing 10ml Mueller-Hinton broth which were incubated for 24h at 37 o C.

Preparation and Extraction of Plant Materials
Freshly harvested sampled leaves of A. africana and P. guajava were thoroughly washed with distilled and rinsed accordingly. Twenty gram each of the leaves was weighed on an electric weighing balance for methanol, ethanol and aqueous extraction. The leaves (20g) were later grounded with mortar and pestle and then transferred into labeled petridishes. Incubation was carried out at 37 o C for 24h. About 90ml each of the solvent were added to the grounded leaves for extraction, vigorously shaken for 30 minutes and allowed to settle.
Sensitivity Test: Antibacterial and Antifungal assay was carried out following established protocols.

Preparation of the medium
40g of Mueller-Hinton agar powder were carefully weighed into a clean conical Flask and 100 ml of sterilized distilled water was dispensed into the conical flask to achieve homogeneity using magnetic stirrer before autoclaving at 121°C for 15 min. The plates were allowed to cool for 2h before a cork borer (No 4) was flamed and used to bore wells (6mm). The plant extracts were filled into the wells and labelled appropriately.

Inoculation of medium
Inoculation of the medium was achieved using spread plate method. The fungal isolates selected were allowed to colonize the Sabouraud Dextrose Agar (SDA) at 25 o C until after sporulation. The fungal spores were harvested by pouring a mixture of sterile glycerol and distilled water (3:100 i.e 3 % glycerol) to the surface of the plate and later the spores were scraped . The harvested fungal spores were standardized before use. The plates were allowed to cool on the laboratory bench for one hour to allow for homogenous diffusion of the extract into the media. Plates were incubated at 25 o C for 96 hours and later observed for zones of inhibition.

Minimal inhibitory concentration (MIC)
The minimum inhibitory concentration (MIC) of the extracts was determined following established protocols. About100mg/ml of the extracts were prepared and introduced into the test tubes containing 8ml of sterile Mueller-Hinton broth, 1ml of the different extract concentration and 1ml broth culture. The mixture was homogenized and then incubated at 37 o C for 24h. The

Proteus mirabilis
Aspergillus niger --- estimation of minimal inhibitory concentration of the plant extracts on the fungi was determined using established protocols. Plant extracts was prepared and 2ml of different concentrations of the solution were added to Sabouraud Dextrose Agar (SDA) at 40 o C to give the appropriate concentration. The medium was then poured to sterile Petri-dishes and allowed to cool. The extracts were then streaked with fungal spores The plates were incubated at 25 o C for 5days and were examined for the presence or absence of fungal growth. The MIC was taking as the lowest concentration that inhibited the growth of the test fungi.

Minimal bactericidal and fungicidal concentration (MBC and MFC)
Minimal bactericidal concentration (MBC) was determined by plating 1ml of the MIC positive tubes on nutrient agar to ascertain the bactericidal effect of the extracts. Samples were taken from plates with no significant growth in the MIC culture and sub-cultured on freshly prepared Mueller-Hinton agar plates for determination of MBC and SDA plates for MFC and later incubated at 25 o C for 5days.

Synergistic antimicrobial effects of the plant extracts against tested bacterial and fungal isolates:
The synergistic antimicrobial effects of the various combinations of the plant extracts for the test microorganisms were determined using the method employed by 14 . To demonstrate synergism, equal quantity of plant combinations were weighed and dissolved in the appropriate quantity of the dilution to give the 100mg/ml concentrations which was used for the antimicrobial test 13 . Antimicrobial susceptibility testing will be compared with clinical and laboratory standard institute (CLSI, 2019) 15 .

Antimicrobial effect of methanolic (M), Ethanolic (E) and Aqueous (A) extracts of Aspillia africana and Psidium guajava on some selected bacterial and fungal isolates
The zones of inhibition of the extracts ranged from 1-15mm for bacteria while fungal isolates ranged between 1 and 7mm respectively. The highest and lowest zones of inhibition of 15mm and 3mm respectively were obtained in Staphyloccocus aureus under the aqueous and methanolic fractions. Bacillus cereus, Cornybacterium pyogenes, Klebsiella pneumonia and Escherichia coli showed high degree of resistance to the plant extracts. The fungal isolates; Candida albicans, Tryptophyton rubrum, Penicillium expansium, and Aspergillus flavus were resistant to Psidium guajava extract as no significant activity was observed. There were no significant effect of the plant extracts against tested fungi isolates (Table 1).

Synergistic antibacterial and antifungal effects of the plant extracts on the tested microorganisms.
The fungal isolates were resistant to the plant extracts as no significant effect was observed. However, the highest zone of inhibition of 21mm was observed in S. aureus under the ethanolic fraction while the lowest (1.2mm) was noticeable in Serratia rubidae under the methanolic extract (Table 2). Escherichia coli and Klebsiella pneumonia were both resistant to the synergistic effect of the plant extracts.

Susceptibility of the test organisms to reference drugs
The susceptibility of the test organisms to antibiotics and antifungal drug is presented in Table 3. Some of the micoorganisms which were sensitive to the plant extracts were found to be resistant to some of the antibiotics used. All the plant extracts produced considerable antimicrobial activities with Streptococcus faecalis and S. aureus, whereas, these organisms were resistant to ofloxacin, sparfloxacin, chloramphenicol, augmentin, ciprofloxacin and septrin. (Table 3). However, the antifungal drug (Ketoconazole) was more effective on the test fungi than the plant extracts.

Minimum inhibitory concentrations (MICs) of the plant extracts against the tested micoorganims
The MIC ranged from 25 to 250µg/ml. The lowest MIC (25µg/ml) was recorded against P. stuartii with ethanolic extract of P. guajava. Also, the aqueous and ethanolic extracts of P. guajava and had a MIC of 25µg/ml on S. aureus. The highest MIC (250µg/ml) was recorded with the aqueous extract of A. africana against P. fluorescence, and methanolic extracts of P. guajava on S. faecalis ( Table 4). The MIC of the plant extracts against selected fungi ranged from 150-300µg/ml. Aqueous and ethanolic extracts of A. africana recorded MIC of 250 µg/ml on T. tonsurans and Fusarium sp. respectively. The highest MIC (300 µg/ml) was obtained with methanolic extract of A. africana on Fusarium sp (Table 4).

Minimum bactericidal concentrations (MBCs) of the plant extracts against the tested organisms
The MBC ranged from 50-400µg/ml. The lowest MBC (50µg/ml) was obtained with ethanolic and aqueous extracts of P. guajava against S. aureus. The highest MBC (400µg/ml) was obtained with the aqueous and ethanolic extracts of A. africana, and methanolic extract of P. guajava on S. faecalis respectively ( Table  5).

Discussion
Nigeria is repleted with reservoir of medicinal plants. The economic burden and health implications of antimicrobial resistance are quite alarming. The unquantifiable extent of the antimicrobial potency of plant extracts orchestrated by characterization of abundant secondary metabolites inherent in these plants necessitated research on traditional medicine. Findings from this study revealed that aqueous and ethanolic extracts of the leaf of Aspillia africana is more potent than the extracts of Psidium guajava. A. Africana posess antibacterial activity against Staphylococcus aureus, Streptococcus feacalis and Pseudomonas fluorescens. 16 reported earlier that the ethanolic extract of the leaf of A. africana posess antibacterial potentials against these microorganisms. However, Psidium guajava exhibited no antimicrobial activity against the tested fungal isolates. Among the bacterial isolates Stapylococcus aureus were the most sensitive to the plant extracts while

CONCLUSION
This study concludes that ethanolic extracts (100mg/ml) of the leaf of Aspillia africana is more potent than the methanolic and aqueous extracts of Psidium guajava. The plant extracts showed greater antimicrobial activity on the bacterial isolates than on the fungal isolates suggesting a broader spectrum of activity with ethanolic extract on the gram positive and the gram negative bacteria. The therapeutic properties of Aspillia africana and Psidium guajava is better enhanced with their synergistic potential to the tested microorganisms.

RECOMMENDATION
A further study on the proper formulation into drugs of the plant extracts for human consumption is highly   recommended. The toxicological studies of the extracts on experimental animals should be determined in order to ascertain its safety threshold on humans.