Exploring the pharmacological significance of Murraya koenigii (L.) Spreng: Insights from North-East India

Abstract Murraya koenigii (L.) Spreng., is a renowned medicinal and aromatic plant having antimicrobial and broad biological activities. In the present study, the essential oil chemical profile of the leaf and floral part were analysed using GC-FID and GC-MS, and the antimicrobial activities were determined by disk diffusion assay. The scientific analysis revealed α-pinene (40.41%) was the major compound of leaf essential oil (MKL) followed by sabinene (29.66%), β-pinene (7.92%), caryophyllene (4.24%), limonene (3.39%), terpinen-4-ol (3.25%), γ-terpinene (2.63%), terpinolene (1.58%), linalool (1.32%), along with other trace components. The floral part (MKF) contains the compounds α-pinene (74.02%) as the major compound followed by β-pinene (7.17%), sabinene (5.72%), limonene (4.19%), β-myrcene (1.98%), γ-terpinene (1.47%) and terpinen-4-ol (1.41%) along with other trace compounds. The MIC result revealed essential oil showed effectiveness against Escherichia coli (40 μg/mL), Micrococcus luteus (80 μg/mL), Aspergillus fumigatus (60 μg/mL) and Aspergillus brasiliensis (80 μg/mL) for MKL while MKF did not exhibit potent antimicrobial activities. The essential oils revealed potent antioxidant potential as determined by DPPH method with IC50 values of 14.02 μL/mL (MKL) and 5.45 μL/mL (MKF) and the ABTS method with IC50 values of 5.07 μL/mL and 8.38 μL/mL in MKL and MKF respectively as compared to that of the standards. The essential oils revealed potent antidiabetic activity with 6.02 μL/mL (MKL) and 8.18 μL/mL (MKF) as that of the standard. However, both MKL and MKF were not much effective in protease inhibitory activity and anti-tyrosinase activity. The study results exhibited a potent bioactivity profile and revealed many new utilization aspects of this essential oil.


Introduction
Murraya koenigii (L.) Spreng. is used in wide therapeutic and industrial purpose, the plant is commonly called curry leaf or "Curry patta" is from the Rutaceae family; the family portrays more than 150 genera and 1600 species as reported 1 .The plant is widely known for its characteristic aroma and medicinal features.The plant is a deciduous shrub that attains height up to 4 m and commonly found across India.The plant is commonly distributed in Assam, Upper and Lower Burma, Chittagong, Andaman Islands, Maharashtra, Tamil Nadu, and Karnataka of India 2 .
The plant is elementary to identify by its general morphological characteristics.It is a short spreading shrub; the primary stem colour is dark green to brownish, possessing dots on it.The leaf structure generally exstipulate, bipinnately compound, about 30 cm in length, with more than 20 leaflets are seen which are lanceolate, with reticulate venation 3 .The flowers are bisexual, white in colour, sweet scented, funnel-shaped, absolute, regular, ebracteate, pentamerous, actinomorphic and hypogynous floral arrangements 1 .
Essential oils are volatile components extracted from natural sources; they are complex compounds according to their structure and can be characterized by an intense aroma; these unique components can be secluded from various parts of the plant like leaves, flowers, buds, bark, fruit, etc. 4 .M. koenigii plant parts also contains essential oil.The significant compounds profile such as flavonoids, saponins, tannins, quinones, carbohydrates, proteins, and essential oils have been isolated and identified as per previous investigations 5 .Carbazole alkaloids, which are abundant in the plant and are widely recognized for their anti-HIV, anti-cancer, anti-bacterial, and anti-fungal properties 6 .Past studies claimed that the selected plant's characteristic aroma is due to p-caryophyllene, p-gurjunene, α-phellandrene and p-elemene 7 .The extracted essential oil from the selected plant also plays a vital role in soap making, skin lotion, aroma therapy, diffusers, and facial steam 8 .
It has been demonstrated in several studies that Murraya koenigii essential oil has antibacterial and antifungal effects.As per a previous report, treatment with essential oil of M. koenigii resulted in biofilm formation reduction in P. aeruginosa PAO1 9 .Essential oil extracted from M. koenigii essential oil exhibited antifungal activity against the pathogenic fungi A. niger, F. moniliforme, P. notatum, M. mucedo and P. funiculosum in a dose-dependent manner 10 .Additionally, M. koenigii bioactive components, including carbazole alkaloids and essential oils, have been linked to the plant's antibacterial activity 11 .Despite these encouraging results, additional study is necessary to completely understand the processes underlying M. koenigii anti-microbial properties and its prospective applications.M. koenigii, have shown substantial anti-oxidant activity and are therefore a topic of study in the field of natural anti-oxidants.M. koenigii essential oil have been shown to have strong free-radical scavenging abilities, however, the number of reports is very few and hence the present study has been proposed.M. koenigii essential oil exhibited superior reducing power and radical scavenging potency which was similar to synthetic counterparts as per previous report 10 .A study on M. koenigii fruit essential oil revealed it was more effective in DPPH scavenging, inhibiting linoleic acid peroxidation, preventing deoxyribose degradation when compared with standard antioxidant butylated hydroxytoluene (BHT), however, it was less effective in scavenging hydroxyl radicals and chelating metal ions 12 .In addition, the presence of bioactive chemicals such flavonoids and phenolic compounds has been linked to M. koenigii antioxidant capability 13 .The antioxidant efficiency of M. koenigii has also been found to be enhanced by endogenous anti-oxidant enzymes 14 .Together, these results highlight M. koenigii potential as a natural source of antioxidants with potential health advantages.Its notable anti-inflammatory effectiveness makes it a possible natural treatment for inflammatory diseases.Studies have confirmed that M. koenigii essential oil has anti-inflammatory effects.The essential oil showed notable anti-inflammatory effects by blocking important pro-inflammatory mediators in research 15 .M. koenigii essential oil has the potential application as an antiinflammatory agent due to the presence of bioactive components such caryophyllene 16 .Additionally, as seen in experimental findings, M. koenigii essential oil revealed the capacity to modify immunological responses and decrease inflammatory pathways 17 .Previous reports revealed that M. koenigii essential oil may be a viable natural source for anti-inflammatory drugs development with potential medical uses.The anti-diabetic benefits of M. koenigii essential oil have been emphasised in investigations.The essential oil showed notable anti-hyperglycemic effects by lowering blood glucose levels in experimental diabetes mice 18 .It has been linked to the presence of bioactive substances including carbazole alkaloids 19 .Additionally, as demonstrated in animal research, M. koenigii essential oil has the capacity to improve insulin sensitivity and modify glucose metabolism 20 .The capacity of M. koenigii essential oil to suppress tyrosinase activity has also been reported.Tyrosinase, a crucial component in the production of melanin, was significantly inhibited by the essential oil 21 .The ability of M. koenigii essential oil to inhibit tyrosinase has been connected to the presence of bioactive chemicals, notably carbazole alkaloids 22 .In addition, M. koenigii essential oil has shown its ability to inhibit melanin synthesis and may be useful in skin hyperpigmentation treatment 23 .
In the present study, the chemical profile of the leaf and floral plant part essential oil from Northeast India has been studied with in-depth biological activities analyzed.The chemical components of the plant may vary as the climate and environmental factors are peculiar to the North Eastern region of India.The study was of great importance and novel because the major components of the leaf and floral part, an integral part of this region again the biological study was new to this region of India.The plant is ubiquitous but extensive study on wide biological activities has not been reported so far for the floral and leaf essential oil which gap is filled by this study.

Collection and identification of plant material
Fresh leaves and flower part of M. koenigii were collected from CSIR-North East Institute of Science and Technology Experimental Farm, Jorhat (CSIR-NEIST), Assam (26°44′10″N; 94°9′30″E) in August 2023 and was identified by plant breeder, Dr. Mohan Lal, Principal Scientist, Agricultural Technology and Rural Development, CSIR-NEIST, Jorhat.The plant name was confirmed through the website of the Plant List.The specimen has been deposited in the herbarium of the department, vide specimen no.RRLJ MKn-0108, and the plants are also maintained in the field gene pool of the institute.

Essential oil isolation and chemical profiling through GC-MS
Fresh floral and leaves part of the plant (each 300 g) were collected, washed, and was subjected to essential oil isolation using a Clevenger apparatus (4 h, 3 L distilled water for floral parts and 6 h, 3 L for leaves) using hydro distillation method with three replications.The essential oil was isolated and the excess water content was removed with anhydrous sodium sulphate treatment.Thereafter, the essential oil was stored at 4°C for further analysis.

GC-FID and GC-MS analysis
The GC-MS analysis was done by using a Gas Chromatography (Agilent Technologies) coupled with MSD 5975 C mass selective detector, a fused silica capillary HP-5MS column (30 mm, 0.25 mm i.d., 0.2 µm film thickness) where the carrier gas was Helium at a rate of 1 mL/min.First, the oven temperature was set at 40°C for 2 min, it was then progressively increased at a rate of 5°C/min till 250°C, and the final temperature was set at 300°C at a rate of 30°C/min and held for 30 minutes.The sample was diluted (sample: acetone, 1:100, v/v) (1 μL) and injected (split syringe, 1:20 for 1 min) maintaining 250°C constant temperature.The GC/MS scan range was 45-650 amu.By comparing the peak mass spectrum data to the NIST/Willey mass spectral library, peaks discovered in the profiles of total ion chromatograms were detected.Representative standard compounds under the same GC conditions were run to generate calibration curves for quantification.Alkanes (C 8 -C 32 ) were used as standards in the determination of retention indices (RI) by the Kovat method.GC-FID (Thermo Scientific TRACE 1110) fused to a TG-WAXMS column (60 m x 0.25 μm) and flame ionization detection was used for further evaluation of the essential oil.The analysis was performed according to the recommended protocol 24 .

Preparation of the inoculums and media
The bacterial strain from the bacterial broth was cultured with MHA and maintained at 37°C for 24 h.The fungal culture in PDB was prepared using the fungal broth and maintained at 28°C for 48 h.In MHA and PDA, the essential oils of MKL and MKF were analyzed for their antibacterial and antifungal properties.For antibacterial and antifungal activities, MKL and MKF with concentrations ranges (50-500 µg/mL) with methanol were made and disk diffusion (6 mm) was carried out.According to the standard technique outlined in literature 25 , the minimum inhibitory concentration (MIC) of MKL and MKF were determined using broth microdilution method for bacterial strain and fungal strain.

Antioxidant activity
The antioxidant activity of MKL and MKF essential oil was determined by the following assays.

DPPH free radical scavenging activity
The procedure described was modified slightly to use ascorbic acid as the standard antioxidant agent for the DPPH assay of MKL and MKF essential oil 26 .A 0.2 mM DPPH solution (1 mL) was added to methanol containing varying amounts of essential oils.After 30 minutes of dark incubation at 37°C, reaction mixture absorbance at 517 nm was measured using a UV-Vis spectrophotometer.The percentage scavenging effect was calculated according to the following formula Where Acontrol= absorbance of DPPH and Asample= absorbance of DPPH + essential oil sample/standard of various concentrations.

ABTS assay
Ascorbic acid was used as a standard in the ABTS (2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) test, which was carried out using a slightly modified version 27 methodology for the MKL and MKF essential oil.

Anti-inflammatory activity
Protein egg albumin denaturation assay Using the protein denaturation experiment previously reported 28 , the anti-inflammatory activity of MKL and MKF essential oil, and sodium diclofenac (standard) were assessed.A reaction mixture of five milliliters was created by adding different amounts of essential oil/drug (2 mL), phosphate buffered saline pH 6.4 (2.8 mL), and egg albumin from fresh hen's egg (0.2 mL).The same volume of double distilled water was used as control.The reaction mixtures were heated in a hot water bath at 70°C for 5 minutes after being incubated for 15 minutes at 37 ± 2°C in a BOD incubator.After letting the mixture cool, the absorbance at 660 nm was measured.The absorption at the same concentrations as the essential oils was measured using the standard, diclofenac sodium.Every experiment was done in triplicates, and the average results were used as the final value.Using the following formula, the inhibition% of protein denaturation was determined.
Where Vc is the absorbance of the control and Vt is the absorbance for the test sample.

Protease inhibitory activity
The protease inhibitory assay of MKL and MKF was carried out with a minor modification utilizing sodium diclofenac as the standard anti-inflammatory medication per protocol 29 .In order to measure the protease inhibitory activity, the essential oils were prepared at different concentrations using 7.5 mL of reaction mixture that contained equal volumes of 1 mol/L trypsin solution (1 mL) and various concentrations of essential oil samples (1 mL).Thereafter, a 2 mL solution containing 0.1% casein was added, thoroughly mixed, and allowed to incubate for 15 minutes.The reaction was stopped by adding 60% per-chloric acid (2.5 mL) to the reaction mixture, and the absorbance was read at 280 nm (Genesis 10 UV spectrophotometer).Protease inhibitory activity was calculated using the following formula: Where Vc was the absorbance of the control and Vt was the absorbance of the essential oil samples.

Antidiabetic activity assay
The antidiabetic activity of MKL and MKF, acarbose (standard) was analysed by a standard method according to the protocol 30 .The reaction mixture was made with 900 µL of 0.01M sodium phosphate buffer pH 6.9 with 0.006 M NaCl containing α-amylase solution 0.5 mg/mL or distilled water (blank) and 100 µL of essential oil/standard solution in the various concentration range.After that, the reaction mixture was incubated for ten minutes at 37ºC.Subsequently, each tube was filled with 500 µL of a 1% starch solution in 0.02 M sodium phosphate buffer pH 6.9 with 0.006 M NaCl.After that, the reaction mixture was incubated for a further 10 minutes at 37ºC.After stopping the reaction with 1 mL of 10% HCl, 300 µL of the working indicator solution, iodine potassium iodide was added.Lastly, 10 mL of distilled water was added to the reaction mixture to dilute it.With the use of a spectrophotometer (Eppendorf Germany), the absorbance was measured and read at 620 nm.The essential oil's ability to inhibit α-amylase activity was assessed using the following formula: Where A control is the absorbance of the control, and A sample is the absorbance of the test sample.

Anti-tyrosinase activity assay
Tyrosinase inhibitory activity of MKL and MKF essential oil was assayed using a modified dopachrome method as per the protocol 31 using standard tyrosinase inhibitory drug, kojic acid.Test tubes containing 125 µL sample solutions were combined with 200 µL of tyrosinase solution and 400 µL of pH 6.8 phosphate buffer, and incubated for 15 minutes at 25°C.Next, 200 µL of L-DOPA was injected to initiate the reaction.Comparably, sample solutions and additional reagents devoid of the enzyme were added to create a blank.At 492 nm, the absorbance of the sample and blank was measured following a 10-minute incubation time at 25°C.The tyrosinase inhibitory activity of the sample and standard kojic acid were compared after the absorbance of the blank was deducted from the absorbance of the sample.The following formula was used to get the% inhibition: Where, Absc was for 100% enzyme activity with only solvent and the enzyme; Abscc was for 0% enzyme activity with solvent in absence of the enzyme; Abss -test sample with tyrosinase; Absb -a blank a test sample without tyrosinase.
The study revealed that the major component in this region was α-pinene, the same compound reported from Pantnagar, Uttarakhand, India.The percentage of the α-pinene is very high in its floral parts, which has not been reported in any previous studies available till date.The above result also reveals that the climatic and environmental conditions can differentiate the essential oil components; however, the concentration of the α-pinene is less as compared to the other regions in the leaf part; the second major component, sabinine is also reported in a study 36,37 .Although similar compounds have been revealed by the analysis, however, there is significant difference in the concentration of the components which may be attributed to the difference of climatic conditions which may affect the biological activities also.

Antimicrobial assay
This study showed that essential oils were less active against the tested microorganisms.The essential oil was active against Escherichia coli in a concentration of 40 µg/mL (MIC).The other organisms on which the essential oil shows its effects were Micrococcus luteus (MIC value of 80 µg/mL) in the case of fungus Aspergillus brasiliensis (MIC value of 80 µg/mL) and Aspergillus fumigatus (MIC value of 60 µg/ mL) (Table S1, S2 and Fig. S1, S2).The study revealed the weak antimicrobial potential of the tested MKL and MKF essential oils.
In  (10  mm) respectively.The study also reported that the major component of the essential oil was effective towards Staphylococcus aureus -210P JTU (12.5±0.5;MIC 50 mg/mL), Pseudomonas aeruginosa -ATCC25619 (16.5±0.5;MIC 25 mg/mL), Klebsiella pneumoniae -SR1-TU (18.5±1.0;MIC 25 mg/mL), Escherichia coli -NI23 JTU (14.5±0.5;MIC 50 mg/mL), Streptococcus pneumoniae -SR16677-PRSP (10.0±1.0;MIC 75 mg/mL) 6 .According to previous studies, the essential oil from the plant leaves can successfully inhibit Listeriainnocua's growth 34 .A previous study revealed that biofilm formation was reduced in P. aeruginosa PAO1 on treatment with the essential oil of M. koenigii 9 .Similar results were reported where essential oil extracted from M. koenigii essential oil exhibited antifungal activity against the pathogenic fungi A. niger, F. moniliforme, P. notatum, M. mucedo and P. funiculosum in a dose dependent manner 10 .According to a report 39 ; the essential oil showed M. koenigii effectively inhibited the pyocyanin production and staphylolytic Las-A activity of P. aeruginosa.Another study reported that in their study they had taken nine microbial strains where five were bacterial strains (  40 .
In the present study, it was seen that the applied concentrations of the essential oil were inactive towards rest of the microbial strains except the reported four strains.The present result differs from earlier reports on the antimicrobial potential of M. koenigii essential oil.The reason behind such results may be due to the compositional difference in the essential oil.

Antioxidant activity DPPH assay
The DPPH assay results revealed that the MKL essential oil exhibited free radical scavenging activity with an IC 50 value of 14.02 µL/mL, which was comparable to that of the standard with an IC 50 value of 13.61 µL/mL.While the highest free radical scavenging activity was exhibited by MKF essential oil with an IC 50 value of 5.45 µL/mL (Table 2).According to a previous study, linalool was reported as the main component of M. koenigii leaves EO, and it inhibited 91.20% of free radicals at a concentration of 0.6 µg/mL.While at a concentration of 20 μg/mL, quercetin's antioxidant activity of 77.8% was significantly less than that of the component extracted from the essential oil 35 .Another literature reported that the leaf essential oil and the stem part essential oil possess antioxidant properties 41 .Another study revealed that the DPPH scavenging activity of the MKLEO was much higher than the used standards, which showed an IC 50 value of 56.83 ± 0.63 μg/mL 36 .Moreover, a finding reported that M. koenigii leaves EO exhibited superior radical scavenging potency and reducing power with IC 50 and RP 50 values close to those of the synthetic antioxidant (standards) 10 .From the study, it was observed that MKL possess antioxidant potential comparable to that of the standard, which is in line with previous reports.Meanwhile, MKF essential oil has been reported for the first time for high antioxidant potential.Thus, it can be judicially used as a green alternative for the synthetic antioxidants.

ABTS assay
The ABTS assay results indicated that MKL essential oil showed best antioxidant potential with an IC 50 value of 5.07 µL/mL, while MKF  3).The values revealed the vigorous activity of MKF and MKL essential oils.There were no previous reports on the ABTS assay of MKL and MKF essential oils.However, earlier reports on the plant extract revealed similar potent antioxidant properties 42 as well as moderate antioxidant activity 43 .The results reveal the active potential of MKF and MKL essential oil as evident from both the antioxidant assays.Therefore, the essential oils could be further evaluated as a green substitute for product formulations.

Anti-tyrosinase activity
The skin whitening ability of the essential oil was analyzed by the tyrosinase inhibitory activity test.The enzyme tyrosinase, which produces melanin in human skin, can cause hyperpigmentation and dark patches if it is overactive.The activity of MKL and MKF exhibited weaker tyrosinase inhibitory property than the standard Kojic acid with an IC 50 value of 14.33 µL/mL (MKL) and 14.25 µL/mL (MKF), than the standard tested Kojic acid with an IC 50 value of 8.78 µL/mL (Table 4).
Earlier in most of the cases, this activity was tested against plant extracts but not in the essential oil.In an earlier study, the essential oil was reported to exhibit significant inhibition of tyrosinase enzyme, a key player in melanin synthesis 21 .The presence of bioactive compounds, including carbazole alkaloids, in Murraya koenigii essential oil has been linked to its anti-tyrosinase potential 22 .Although there have been reports on the tyrosinase inhibitory property of M. koenigii essential oil, however, the present reports differ from that.The difference in the major components might be the key factor behind such variations.Thus, MKL and MKF cannot be considered for tyrosinase inhibitory applications.According to several studies the M. koenigii essential oil has protease inhibitory activity.A study showed that the plant extract possesses high protease inhibitory activity, M. koenigii (IC 50 = 24.93 ± 0.05 µg/ml) 44 .Some other study reported the same for the aqueous extract of the plant 45 .In addition to its protease inhibitory activity, M. koenigii essential oil oil consists of several other biological activities, making it a good natural product for drug development 45 .However, the present results differ from earlier reports for their protease inhibitory activity as the standard diclofenac sodium possess better protease inhibitory activity than MKL and MKF essential oils.The reason behind such deviations may be the difference of agro-climatic conditions and the major components observed.

Antidiabetic activity assay
The anti-diabetic activity of Murraya koenigii essential oil (MKL and MKF) has been extensively studied recently.Numerous research, both in vitro and in vivo, have been carried out to assess M. koenigii EO potential for diabetes management.The antidiabetic assay showed that the α-amylase inhibition percentage increases with increasing concentration; the leaf essential oil showed more potent activity than the floral part essential oil.MKL exhibited an IC 50 value of 6.02 µL/mL, and for MKF it was 8.18 µL/mL, which is better than the values of the standard drug acarbose which is 12.89 µL/mL (Table 6).
According to earlier studies, plant extracts were reported to contain anti-diabetic activity but lack reports in the case of essential oil 45 .In a previous study, M. koenigii ethanolic extracts showed significant blood glucose level reduction at doses of 200 and 400 mg/kg b.w 46 .Similar report has been supported on the anti-diabetic potential of M. koenigii ethanolic extracts on streptozotocin-induced rats 47 .The strong antidiabetic potential of MKF and MKL essential oil suggests the effective utilization as a potential drug with further validations.

Conclusions
The experimental model was able to satisfactorily elucidate that both of the M. koenigii leaf and floral parts essential oil are composed of valuable volatile components with immense potential pharmaceutical applicability.Both MKF and MKL were found to be composed of α-pinene sabinene, β-pinene as the major components.Both essential oils possesses anti-microbial activities in moderate to low levels as well as other potent biological activities such as anti-oxidant and antidiabetic.Both of these essential oils can further be used in food products as it has no adverse effect on gut-friendly microorganisms and may resist fungal invasions in food products which can open doors to industrial researchers.The other biological activities will help to develop and understand new drugs and their interactions with human physiology.The essential oils of M. koenigii leaf and floral part bioactivities are less investigated and may need more research to fill the gap.The floral part of essential oil is not investigated often times and hence can be evaluated by more researchers to fill the gap.On the biological utilization aspect, both MKF and MKL essential oil can be utilized as a potent antioxidant and antidiabetic drug with further clinical trials.

Table 1 .
GC-MS analysis results of Murraya koenigii leaf and floral part essential oil S.

Table 2 .
DPPH free radical scavenging activity results of MKL and MKF essential oil

Table 3 .
ABTS radical scavenging activity results of MKL and MKF essential oil

Table 6 .
Alpha-amylase inhibitory activity result of MKL and MKF essential oil Concentrations (