A Research Paper On Phytochemical Profiling And In-Vitro Antibacterial Evaluation Of Ethanolic Extract Of GREWIA OPTIVA (BHIMAL TREE)

Medicinal plants remain important sources of structurally diverse natural compounds and continue to be investigated for their potential therapeutic applications. The genus Grewia comprises several species that have traditionally been used as food, fodder and medicinal resources in tropical and subtropical regions. Grewia optiva Drummond ex Burret, commonly known as Bhimal, is a multipurpose tree of the Himalayan region and is particularly important in north-western Himalayan agroforestry systems. Various parts of the plant have been associated with nutritional, traditional and pharmacological applications, while studies on Grewia species have demonstrated the occurrence of numerous bioactive chemical groups, particularly flavonoids and other phenolic constituents. The increasing scientific interest in this genus provides a rationale for further investigation of comparatively less-explored plant parts of G. optiva. [1,2]

The bark of G. optiva represents a promising source for phytochemical investigation because previous chemical studies have demonstrated considerable structural diversity in its constituents. Phytochemical investigations of the stem bark have resulted in the isolation of triterpenes such as betulin, betulinic acid, oleanolic acid and ursolic acid, together with daucosterol. Other investigations have reported the isolation of the compounds grewialin and optivanin along with β-sitosterol, stigmasterol and lupeol. The identification of these compounds indicates that the bark contains chemically diverse secondary metabolites that may contribute to its biological properties. In addition, screening studies of G. optiva bark extracts have reported classes such as alkaloids, flavonoids, tannins, saponins and terpenoids, supporting the need for systematic phytochemical profiling of the bark extract. [3–5]

Bacterial infections remain an important public-health concern, and increasing antimicrobial resistance has intensified the search for new antibacterial substances from natural sources. Plant-derived secondary metabolites, including phenolics, flavonoids, tannins, terpenoids and related compounds, have been investigated for their ability to interfere with microbial growth through different biological mechanisms. Evidence summarized for the genus Grewia indicates antibacterial activity in some species and plant parts, although the available information is not uniform across species, extracts and bacterial strains. For G. optiva, existing research has concentrated more strongly on phytochemistry, antioxidant properties and other biological activities than on a systematic evaluation of ethanolic bark extracts against selected pathogenic bacteria. This creates a useful basis for further antibacterial investigation. [2,6]

Ethanol is widely used in plant extraction because it can solubilize a broad range of phytochemicals and is suitable for preliminary biological screening. Therefore, evaluation of an ethanolic bark extract of G. optiva can provide complementary information regarding its chemical profile and antibacterial potential. The present study is designed to perform preliminary phytochemical profiling of the ethanolic bark extract and assess its in vitro antibacterial activity against selected pathogenic bacterial strains. The findings may help establish a scientific basis for the traditional importance of Bhimal bark and provide preliminary evidence for further isolation and characterization of potentially bioactive constituents. [3,4,6]

1. METHOD AND MATERIALS

Collection and Authentication of Plant Material

Fresh bark of Grewia optiva  (Bhimal tree), belonging to the family Malvaceae, was collected from the Chail Chowk area of Mandi district, Himachal Pradesh, India, during the month of December. Healthy and mature trees were selected for collection of the plant material. The collected bark was carefully separated and washed thoroughly with running tap water followed by distilled water to remove adhering soil, dust and other extraneous materials.

The collected plant material was properly examined and authenticated by experts at the HP CDP JICA ODA, Mandi, Himachal Pradesh. After authentication, the bark material was subjected to further processing. The authenticated bark was subsequently used for the preparation of ethanolic extract and further phytochemical profiling and in-vitro antibacterial evaluation against selected pathogenic bacterial strains.

Preparation of Plant Material

The collected bark will be washed with running tap water followed by distilled water to remove surface contaminants. The material will then be cut into small pieces and dried under shade at room temperature in a well-ventilated area, avoiding direct exposure to sunlight in order to minimize possible degradation of heat- or light-sensitive constituents. Once completely dried, the bark will be pulverized using a clean mechanical grinder to obtain a coarse powder. The powdered material will be passed through a suitable sieve to obtain relatively uniform particle size and stored in a clean, dry, airtight container until extraction.

Preparation of Ethanolic Extract

A measured quantity of powdered Grewia optiva bark will be extracted using ethanol as the extraction solvent. The powdered material will be mixed with an appropriate quantity of ethanol and subjected to extraction by maceration with occasional shaking for adequate contact between the plant material and solvent. After completion of extraction, the mixture will be filtered through suitable filter paper to separate the plant residue. The resulting filtrate will be concentrated using a rotary evaporator or an appropriate low-temperature evaporation procedure until a concentrated extract is obtained. The percentage yield of the extract will be calculated using the weight of the dried extract relative to the initial quantity of powdered plant material. The prepared extract will be transferred into a properly labelled airtight container and stored under suitable conditions until further analysis.

Preliminary Phytochemical Screening

The ethanolic bark extract will be subjected to qualitative phytochemical screening to identify the major groups of secondary metabolites present in the extract. Standard chemical reactions will be performed for the detection of alkaloids, flavonoids, phenolic compounds, tannins, saponins, terpenoids, steroids, glycosides, carbohydrates and proteins. Appropriate reagents will be used for each test, and the formation of characteristic colour changes, precipitates or foam will be recorded as an indication of the corresponding phytochemical group. The results will be documented systematically to establish the preliminary phytochemical profile of the ethanolic bark extract.

Selection and Preparation of Bacterial Strains

Selected pathogenic bacterial strains will be used for evaluating the antibacterial potential of the ethanolic bark extract. Both Gram-positive and Gram-negative bacteria may be included to obtain a broader assessment of antibacterial activity. The bacterial strains will be obtained from an authorized microbiological laboratory or recognized microbial culture collection. Fresh cultures will be prepared before the experiment by subculturing the organisms on suitable microbiological media. A standardized bacterial inoculum will be prepared according to an appropriate laboratory standard to maintain comparable microbial concentrations during antibacterial testing.

In Vitro Antibacterial Evaluation

The antibacterial activity of the ethanolic extract will be investigated using the agar well diffusion method. Mueller–Hinton agar or another suitable antibacterial testing medium will be prepared according to the manufacturer's instructions and sterilized appropriately. The standardized bacterial suspension will be evenly inoculated over the surface of the prepared agar plates. Sterile wells of suitable diameter will then be made in the inoculated agar using a sterile cork borer. Different concentrations of the Grewia optiva bark extract will be introduced into the respective wells. A suitable standard antibacterial drug will be used as a positive control, while the solvent used for preparing the extract will serve as a negative control. Following incubation under appropriate conditions for the selected bacterial organisms, the plates will be examined for clear zones surrounding the wells. The diameter of each zone of inhibition will be measured in millimetres and recorded as an indicator of antibacterial activity.

Determination of Minimum Inhibitory Concentration

Where laboratory facilities permit, the minimum inhibitory concentration (MIC) of the ethanolic bark extract will be determined using a suitable broth dilution or microdilution method. Serial concentrations of the extract will be prepared in an appropriate sterile culture medium and inoculated with standardized bacterial suspensions. After incubation under suitable conditions, bacterial growth will be assessed by visual observation or an appropriate quantitative measurement. The MIC will be recorded as the lowest concentration of the extract that produces no visible bacterial growth compared with the corresponding growth control. This procedure will provide additional quantitative information regarding the antibacterial potency of the extract.

Experimental Controls and Replication

Appropriate positive and negative controls will be incorporated into the antibacterial assay to validate the experimental procedure. The standard antibacterial agent will serve as the positive control, whereas the extraction solvent without plant extract will be used as the negative control. Each experimental condition will be tested in suitable replicates to improve the reliability and reproducibility of the observations. The antibacterial results will be expressed as mean ± standard deviation where applicable.

Statistical Analysis

The experimental observations obtained from the antibacterial assay will be compiled and analyzed using an appropriate statistical method according to the experimental design. Results will be presented as mean ± standard deviation for replicate measurements. Statistical comparisons between the treatment groups and controls will be performed using a suitable statistical test, and a value of p < 0.05 will be considered statistically significant where applicable. The final findings will be presented in tables and figures to facilitate comparison of the antibacterial activity of different concentrations of the ethanolic Grewia optiva bark extract.

2. RESULT

Test materials

In the present study, etahnolic extract of bark of tree Grewia optiva (GO) was prepared using Soxhlet apparatus and was evaluated for its antimicrobial activity against a gram positive and a gram-negative bacteria. A standard concentration of (1000 mg/ml) was prepared from crude plant extract in dimethyl sulfoxide (DMSO).

 Microorganisms are used for antibacterial assay.

Standard strains of Staphylococcus aureus (MTCC-96) and E. coli (MTCC- 443) were used to evaluate the antibacterial potential of ethanolic extract of Grewia optiva (GO).

Antibacterial assay

 Preparation of inoculum

The bacterial strains Staphylococcus aureus (MTCC-96) and E. coli (MTCC- 443) were freshly cultured on nutrient broth media. Inoculum of selected bacterial strains was prepared by transferring 2-3 loop-full of culture growth into 5 mL of sterile normal saline. The suspension was vortexed thoroughly to achieve a homogenous mixture. The turbidity of the suspension was then adjusted to match the 0.5 McFarland standard, corresponding to approximately 1.5×10? CFU/mL. This standardized inoculum was subsequently used for the antibacterial assay.

 Agar well diffusion method

The antibacterial potential of extract FB was investigated using agar well diffusion assay, based on previously reported method (Sharma et al., 2012) with a few modifications. Muller Hinton Agar (MHA) plates were prepared and spread plated with prepared bacterial inoculum using sterile cotton swab. Three equidistant wells (8 mm in diameter) were aseptically punched into each plate using a sterile cork borer. Subsequently, 100 µL of test material (GO- 1000 mg/ml of DMSO) was dispensed into respective wells in two different plates. The second well of each plate was filled with 100 µL of Dimethyl sulfoxide (DMSO), serving as the negative control. The third well received standard antibiotic (AB) Piperacillin/Tazobactam (PIT 100/10 µg), as the positive control. Plates were incubated at 37?°C for 24 hours and observed for the formation of zones of inhibition around the wells. Post-incubation, zones of inhibition were measured in millimetres and recorded comparative analysis.

Results and discussion

In the present study in-vitro evaluation of antibacterial potential of ethanolic extract of Grewia optiva (GO) was evaluated against Staphylococcus aureus and E. coli

Table 1. Details of test material along with controls used for antibacterial assay.

Antibacterial activity of the ethanolic extract of Grewia optiva (GO) against Staphylococcus aureus after 24 hrs of incubation. The formation of distinct zones of inhibition surrounding the wells indicates the antibacterial activity of the extract. Dimethyl sulfoxide (DMSO) served as the negative control, while AB served as the positive control on Mueller–Hinton agar (MHA) plates.

Antibacterial activity of the ethanolic extract of Grewia optiva (GO) against E. coli after 24 hrs of incubation. The formation of distinct zones of inhibition surrounding the wells indicates the antibacterial activity of the extract. Dimethyl sulfoxide (DMSO) served as the negative control, while AB served as the positive control on Mueller–Hinton agar (MHA) plates.

Table 2. Antibacterial assay of ethanolic extract of Grewia optiva (GO).