Article

Introduction

Man uses plants to fulfill his basic food, clothing, and shelter requirements in many ways. Rural and urban cultures use wild plants to make medicines, crafts, and cosmetics. Also, rural areas rely on wild plants as sources of revenue and employment [1]. Significant herbal products include spices, herbal teas, functional food components, medical raw materials, aromatic plants, essential oils, flavouring, fragrant items, and dietary supplements. Substances include terpenoids, flavonoids, sterols, simple phenolic compounds, proteins, enzymes, lipids, oils, carbs, etc. Natural resources still serve as the foundation of the primary healthcare system and are the source of synthetic and conventional herbal medication. Scientists have been researching plants for their potential use in managing chronic wounds and treating certain infectious diseases due to the presence of numerous life-sustaining components in plants [2].

There is considerable research on medicinal plants in India, and India is considered the hub of medicinal plants. Many plants are used as medicine for specific illnesses, and some can also be utilized in the culinary arts. Furthermore, the demand for traditional medicinal plants is growing; in India, the market for medicinal plants is growing at a rate of 20% per year [3]. There is a demand for medicinal plants, and more studies are being conducted worldwide. Murrya koenigii, often known as the curry plant, is a herbal medicine utilized for healing and culinary purposes. The Murraya koenigii or Bergera koenigii is a tropical to sub-tropical tree in the Rutaceae (the rue family), including rue, citrus, and satinwood. It is native to Asia. M. koenigii is additionally known as sweet neem, even though it belongs to a different family than neem, Azadirachta indica, in the Meliaceae family [4]. According to [5] only two Murraya species, M. koenigii and M. paniculate, are found in India. The present study aims to evaluate the phytochemical GC-MS analysis and potential antimicrobial activity of Murraya koeinigii.

Methodology

Plant Collection and Processing
Fresh leaves of Murraya were collected from Tulasi Nursery, Hyderabad, in January. The collected leaves were shade-dried and pulverized into a fine powder using an electric blender. The powdered samples were stored in airtight containers until further use.

Extract Preparation
A total of 500 g of air-dried powdered leaves of Murraya were macerated with 2.5 L of methanol by cold maceration at room temperature for 48 hours with intermittent shaking. The extract was filtered, and the filtrate was concentrated at 40 °C under reduced pressure using a rotary evaporator to obtain the crude dry extract.

Phytochemical Analysis
Qualitative phytochemical screening of the methanolic extract was performed following the method described by [6]. The following tests were conducted:

• Test for Tannins: To 2 ml of extract, 2 ml of distilled water and a few drops of ferric chloride solution were added. A blue-black or greenish colour indicated the presence of tannins.
• Test for Saponins: 3 ml of extract was mixed with 3 ml of distilled water and shaken vigorously. Formation of persistent foam upon heating indicated the presence of saponins.
• Test for Flavonoids: To 1 ml of extract, 1 ml of 10% lead acetate solution was added. No colour change was observed, indicating absence of flavonoids.
• Test for Alkaloids: 3 ml of extract was mixed with 3 ml of 1% HCl in a hot water bath. The solution was divided into two test tubes; 1 ml of Mayer’s reagent was added to one. Formation of a buff-coloured precipitate confirmed the presence of alkaloids.
• Test for Terpenoids: 2 ml of extract was dissolved in 2 ml of chloroform and evaporated to dryness. Concentrated H₂SO₄ (2 ml) was added, and the mixture was heated for 2 minutes. No characteristic colour change was observed, indicating absence of terpenoids.
• Test for Steroids:
  • Salkowski’s Test: 2 ml of extract was dissolved in 2 ml of chloroform, followed by addition of 2 ml concentrated H₂SO₄. Formation of a red colour in the chloroform layer indicated the presence of steroids.
  • Liebermann’s Test: (details can be added if performed).
• Test for Phenols: 1 g of extract was dissolved in 5 ml of distilled water and treated with 5% ferric chloride solution. Formation of a dark green colour confirmed the presence of phenolic compounds.
• Test for Anthraquinones: 2ml of Murraya Koenigii extract was taken in a test tube, and 4ml of concentrated sulphuric acid was added to the test tube, boiled and shaken well, and then 3ml of chloroform was added to the test tube. The chloroform layer was separated, and a pipette containing diluted ammonia was taken out to another test. The appearance of a pink-red/ violet colour at the lower phase indicates the presence of anthraquinones.

II. GC-MS Analysis

The Murray extracts were analyzed using GC-MS (SHIMADZU, Japan) equipped with an Optima 5 ms capillary column (30 m × 0.25 mm, 0.25 μm film thickness), following the method described by Azra & Bhavani (2022).

• Oven program: Initial temperature 60°C, increased at 10°C/min to 160°C, then to 250°C, with a 2-minute hold at each increment.
• Injection parameters: Splitless injection volume of 1.0 μL, split ratio 1:1, injector temperature 200°C.
• MS conditions: Ion source at 230°C, interface temperature 250°C, solvent delay 4.5 min, scan range 50–700 amu. Multiplier voltage 1859, electron ionization (EI) mode at 70 eV.
• Compound identification: Based on retention time, fragmentation patterns, and spectral data compared with the Wiley and NIST libraries (Azra & Bhavani, 2022).

III. Antimicrobial Test

3.1 Preparation of Active Bacterial Cultures

A single colony of pure bacterial culture was inoculated into 50 mL of nutrient broth in a 150 mL conical flask and incubated for 8–12 hours at 37°C.

3.2 Preparation of Sample Concentrations

• Powdered samples: Dissolved in 1 mL of an appropriate solvent (water/methanol/DMSO, etc.), then aliquoted into different concentrations for MIC assay.
• Liquid samples: Used directly or diluted with water/solvent as required.

3.3 Antibacterial Assay

The pour plate method was used.

• 1% of active bacterial culture was mixed into autoclaved agar medium just before solidification and poured into plates.
• Test organisms: Staphylococcus sp. (Gram-positive) and Escherichia coli (Gram-negative).
• Wells were made using a sterile borer, and 100 μL of each sample was loaded. Plates were incubated at 37°C for 18–24 hours.

3.4 Antifungal Assay

• Test organisms: Fusarium sp. and Macrophomina phaseolina.
• Media: Potato Dextrose Agar (PDA) with antibiotics (Streptomycin/Chloramphenicol) to prevent bacterial contamination.
• Procedure: Plates were poured and allowed to solidify. Fungal plugs (5 mm) were placed at the center, and wells (5 mm) were created around them using a sterile cork borer.
• Each well was loaded with 100 μL of samples; one well contained antifungal standard chemicals as control.
• Incubation: Plates were incubated at 25°C for 96 hours, and results were recorded.

IV. Results

4.1 Phytochemical Analysis:  Phytochemical analysis of Murraya’s leaf extract shows saponins, alkaloids, terpenoids, steroids, phenols, and anthraquinones, and compounds like tannins and flavonoids are absent.

4.2 GC-MS Analysis

Based on GC-MS analysis results, it comprises numerous compounds. From the lists of GC-MS analysis, 15 compounds have been selected, which are mentioned in the tablebelow.

4.3 GC-MS -Nature of Compounds

Terpenoids are chemicals present in the essential oils of various plants that have been shown to benefit human and animal health. Sesquiterpenes, one of the most abundant terpenes, are a group of natural compounds with a wide range of beneficial industrial qualities. Copaene (COP) is a sesquiterpene derived from numerous plants [27]. Copaene, or α-Copaene, is the chemical term for an oily liquid hydrocarbon found in many essential oil-producing plants.

The sesquiterpene (E)—caryophyllene is a significant plant volatile oil found in high concentrations in various spice and culinary plants [7]. Caryophyllene is a sesquiterpene found in a variety of plant essential oils. Caryophyllene is thought to have anti-inflammatory, antibacterial, antioxidant, anticarcinogenic, and local anesthetic properties [12]. Campesterol is an anabolic steroid precursor of boldenone. Boldenone undecylenate is a common anabolic steroid used in veterinary medicine to help cattle grow, but it is also one of the most dangerously addictive anabolic steroids in sports.

Antimicrobial Activity

After incubation, bacterial plates were examined for zones of inhibition. Antibacterial activity was evaluated by determining the minimum inhibitory concentration (MIC). For this assay, wells were loaded with 25 µL, 50 µL, 75 µL, and 100 µL of each sample, diluted with water or an appropriate solvent to maintain a final volume of 100 µL per well. The MIC values were expressed as 25, 50, 75, and 100 µL, corresponding to the respective dilutions of the test samples, as sample concentrations varied across different extracts.

Antibacterial Activity and Zone of Inhibition

Minimum Inhibitory concentration (mm)

The fungal plates were observed after incubation time, and the following results were noted.

Antifungal activity and Hypahe intention

Minimum inhibitory concentration (MIC) assay of the given samples

The samples checked for antifungal activity were assayed for their minimum inhibitory concentrations (MIC) by loading 25 µl, 50 µl, 75 µl, and 100 µl of samples diluted with water/solvent to make up the volume to 100 µl in each plate well, respectively. The MIC was designated as 25, 50, 75, and 100, which should be referred to for respective dilutions based on sample concentrations, as different samples have different concentrations.

Minimum inhibitory concentration

From the findings, it can be said that the methanolic extract of curry leaves has antimicrobial activity on various organisms.

Discussion

Murraya koenigii is traditionally used as a stimulant for the management of diabetes, and the leaves are eaten to treat diarrhea and dysentery. Many fatty acids, steroids, and different terpenoid components are seen. According to GC-MS analysis of the leaf extract, highly volatile oil molecules like Copaene, Spathulenol, and Germacrene D are generated from sesquiterpenoid component terpenes. Terpenes are a natural product that includes chemicals with the formula (C₅H₈)n. These unsaturated hydrocarbons, made up of over 30,000 chemicals, are produced mainly by plants, particularly conifers. Monoterpenes (C₁₀), sesquiterpenes (C₁₅), and diterpenes (C₂₀) are the three types of terpenes (C₂₀). Moreover, it also includes steroids, esters, fatty acids and aromatic compounds.

The leaf powder extract was subjected to antimicrobial activity, and the results were investigated. The findings of this study demonstrate that curry leaf methanolic and ethanolic extracts have antibacterial effects on a variety of microorganisms. This discovery paves the way for additional investigation and studies to pinpoint the active ingredients responsible for the biological activity of plants. More studies are required on different strains that have the potential to be used as medicine for other ailments. However, further investigation with purified fractions is required to predict the exact mechanism of action.

Conclusion

This study focused on phytochemical and GC-MS compounds rich in compounds like steroids, fatty acids, and terpenes. I want to proceed with the pharmacological study on plants and work on some compounds obtained through GC-MS analysis based on these studies. More research will be done on the curry leaf’s antibacterial component and how the extracts can be used for clinical and other applications.

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