Evaluation of Antibacterial Activity and GCMS Analysis of Zanthoxylum Ovalifolium Fruit Extracts

Herbal medicines have played a vital role in the evolution of humanity and civilization. The fruits of the plant Zanthoxylum were collected from the study field, washed, dried in the shade and powdered. Soxhlation method was used to make different solvent extract of fruit. The different fruit extracts were examined against P. aeruginosa, P. syringe, S. aureus, S. typhi, K. pneumonia and E. coli by agar well diffusion method and for the detection of biochemical components present in the Zanthoxylum fruit, methanolic fruit extract was analyzed using GC-MS. Results showed that, the methanolic extract of fruit is found to be exhibit activity against P. aeruginosa, P. syringe, S. aureus, S. typhi, K. pneumonia and E. coli when compared to the hexane and ethyl acetate. The discovery of 46 bioactive compounds in methanolic fruit extract was verified by their molecular formula, molecular weight, and area peak percentage. In which 6 bioactive compounds such as 6octadecanoic acid, Pentadecanoic acid, Ethyl oleate, 9-octadecenoic acid (Z)-, methyl ester, Glycerin and 1, 2-dimethoxy-4-(2-methoxyethenyl) benzene compounds showed highest peak area percentage of 26.14, 13.61, 8.56, 6.12 and 6.08% respectively. The compounds found in this research may be responsible for some of the medicinal action of the Z. ovalifolium fruit and may be useful in the development of new medicines. Original Research Article Pavani and Naika; JPRI, 33(30B): 7-17, 2021; Article no.JPRI.69114 8


INTRODUCTION
The value of medicinal herbs was discovered as early as the middle Ages. There were no synthetic drugs available at the moment, so they depended entirely on natural medicines to cure all illnesses. As a result, we can deduce that plants have a wide variety of medicinal properties and are highly helpful to human health and well-being. Biological research is needed to learn more about the healing uses [1].The therapeutic benefit of such noteworthy plants consists in certain chemical compounds that give the human body a definite physiological effect. T he most significant aspect of these bioactive plant components is that, they can be used as chemotherapeutic compounds, in effective curing of different ailments in human b eing and also safer to use without side effects. But in case of synthetic drugs, there may be chances of s ide effects, hypersensitivity and allergic reactions in treated patients [2]. WHO (World health organization) reported that, in developing countries about 80% of the population still rely on traditional system medicine for their medicinal purposes needs mainly plant drug. Unani, Ayurveda, Homeopathy, and Siddha accounted for about 95% of all prescriptions in India. The medicine practitioners used local known medicinal plants in traditional way to tread various ailments [3].
GC-MS is a hyphenated approach that is the most widely used technique for identification and quantification. Through interpreting the spectra and comparing them to reference spectra, the unknown organic compounds in a can be calculated [4].
The plant Zanthoxylum ovalifolium belongs to family Rutaceae (citrus family), many medicinal plants of this Zanthoxylum genus showed pharmacological such as antibacterial, antifungal, analgesic, anti inflammation due to the presence of bioactive compounds in it [5]. Z. ovalifolium fruit have several essential phytochemical compounds such as alkaloids, flavonoids, steroids, tannins, and phenol [6-7], which must be intensely evaluated for the isolation of effective bioactive compound(s) in curing different ailments. The aim of this research is to use GC-MS to identify bioactive chemicals in methanolic leaf extract of Z. ovalifolium and also evaluating the antibacterial activity of different extract.

Collection of Plant Material
From March to August, plant fruits were collected from the study area's plains and mountains. In certain cases, local people's interactions have been used to decide the best site for plants. We tried to use conventional methods. The dried fruits of Z. ovalifolium were used as a source of plant material for the current study. The fruit was harvested from the Sringeri forest in the Chikkamagaluru district of Karnataka. The fruits of Z.ovalifolium were taxonomically classified using normal flora (1, 2, and 3). The herbarium specimen was stored and voucher specimen number KU/AB/RN/PP/002/2017 was placed in the dept. of Botany, Kuvempu University herbarium collection.

Preparation of Plant Sample
The Fresh plant material fruits of Z. ovalifolium were collected from the study area and separated, washed with running water for about 2-3 times and allowed for 20-30 days of shade dried. These dried plant samples were pulverized to the coarse powder of about 1mm in diameter using a mechanical grinder. The powdered materials were kept for 4°C and u sed to for further study [8][9][10][11]

Preparation of Extracts
In this study, different solvents was used to separate the components of Z. ovalifolium fruits based on i ncreasing in the polarity of solvents. Extraction of Z. ovalifolium carried out using a Soxhlet apparatus. E qual amount (250) of powdered fruit sample are extracted using different solvents (Hexane, ethyl acetate a nd methanol) and extraction process continued till the solvent present in the siphon tube turns to colorless then concentrated using rotary evaporator and extracts kept at 4°C until used [12][13][14][15].

Selection of Bacterial Strains
Antibacterial activities of the fruit extracts of Z. ovalifolium were screened against clinical isolated Gram-positive and Gram-negative pathogen bacterial strains were stored in department of Applied Botany, Kuvempu University, shankaraghatta. On nutrient broth at 37°C, pathogenic bacteria were maintained.

Agar Well Radical Diffusion Assay
The antibacterial activity of Z. ovalifolium fruit extracts was evaluated by using standard agar well radial diffusion method against selected pathogenic bacteria. The sterilized nutrient agar medium was poured into sterilized glass petri plates. Liquid broth containing 100 µl of 24 h previous bacterial cultures was spread separately over the solid nutrient agar media plates and it was punched by using sterilized cork borer of size about 6mm diameter. Each well was loaded with 25 µL leaf extract of different solvent (Hexane, Ethyl acetate Methanol) and concentration like, 25, 50, 100 µg/ml and negative control (DMSO) and standard drug 1µg/ml .The culture plates were incubated at 37°C for 24 h. the experiment was triplicate for each extract.

Extract Yield
Due to less extract yield and less confirmation of phytochemicals in hexane and ethyl a cetate extract (1.25 grams and 2.13 grams) we have conducted only antimicrobial activity for these two e xtracts (hexane and ethyl acetate) and only methanolic extract (12.46 grams) is subjected for GCMS as w ell as antimicrobial studies.   Fig. 1)

DISCUSSION
The result revealed that Z. ovalifolium fruit of varies solvent extract (hexane, ethyl acetate and methanol solvent) were showed appreciable antibacterial activity by suppressing the growth of tested human pathogenic as well as plant pathogenic bacteria specimens with the standard drug antibiotic Amoxicillin at varying concentrated doses of 25mg/ml, 50mg/ml and 100mg/ml.
When compared with hexane and ethyl acetate extracts, the methanolic extract showed optimum zone of inhibition and the best activity was observed against Pseudomonas syringae, Staphylococcus aureus, Klebsiella pneumonia, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi (Table1; Fig.1).
Whereas, at a concentration of 10 mg/ml, the standard antibiotic amoxicillin treated against pathogenic bacteria exhibit a higher rate of inhibition zone against the pathogenic bacteria tested. The degree of zone of inhibition was found in the following order i.e., Salmonella typhi > Escherichia coli > Staphylococcus aureus > Pseudomonas aeruginosa> Pseudomonas syringae > Klebsiella pneumonia. (Table1; Fig.  1).
G C-MS analysis of methanolic fruit extract of Z. ovalifolium, confirmed the presence of 46 compounds w ith their molecular formula, molecular weight and area peak percentage. Out of which, 31 compounds w ere known for its biological properties, where, 7 compounds were antimicrobial property, 4 compounds w ere antibacterial compounds, 4 compounds were antifungal activity, 5 compounds were antinflammatory compounds, 9 compounds of antioxidant compounds, 4 compounds were anticancer c ompounds, 7 flavoring compounds, 2 compounds were analgesic, rest of compounds were anti-diabetic, a nti depression, anti-germination, anti-androgenic, plant growth, antihepatotoxic and antidiuretic. R emain 15 compounds biological properties yet to be explored (Table 2; Fig. 2).
T he six major peaks reflecting six major compounds were described and categorized by comparing the m ass spectra of the constituents with compound library such as, 6-octadecanoic acid, Pentadecanoic acid, E thyl oleate, 9octadecenoic acid (Z)-, methyl ester, Glycerin and 1, 2-dimethoxy-4-(2-methoxyethenyl) b enzene compounds were major category of compounds mainly confirmed in the fruit extract (Fig. 3).
The current research contributes to the prediction of the formula and composition of 46 biomolecules of methanolic extract of Z.ovalifolium with antibacterial activity. Further research may result in the isolation of bio-active compounds, as well as their structural characterization and pharmacological activity testing, which will be useful for future clinical trials.

CONSENT
It's not applicable.

ETHICAL APPROVAL
It's not applicable.