Research Article - Asian Journal of Biomedical and Pharmaceutical Sciences (2018) Volume 8, Issue 65

Chemical Composition and In vitro Antifungal and Antioxidant Activities of Essential Oil from Murraya koenigii (L.) Spreng. Leaves

YC Tripathi^*, Nishat Anjum and Ashish Rana

Chemistry and Bioprospecting Division, Forest Research Institute, PO New Forest, Dehradun 248006, India

*Corresponding Author:

Y.C. Tripathi
Chemistry Division, Forest Research Institute
PO-New Forest, Dehradun-248006, India
Tel: +91-135- 2752671, 2224207 (O)
Fax: +278615107002
E-mail: tripathiyc@gmail.com

Accepted date: June 07, 2018

Abstract

The chemical composition and the in vitro antifungal and antioxidant activity of essential oil of Murraya koenigii (L.) Spreng leaves have been studied. The yield of M. koenigii leaf essential oil (MKLEO) recorded as 0.52% which is higher than those reported earlier. Altogether 43 compounds were identified by GC-MS analysis representing 99.79% of the total composition of the oil, among which 3-carene, βpinene, α-pinene, linalool, α-eudesmol, p-cymene, γ-terpinene, α-amorphene, allo-ocimene, sabinene, γterpinene, linalyl acetate, myrcene, β- eudesmol, carvone, limonene, β-elemene, α-terpineol were major constituents. Antifungal activity of MKLEO was tested against ten pathogenic fungi and it was found effective in a dose dependent manner. Furthermore, MKLEO was found to exhibit superior radical scavenging potency and reducing power with IC50 and RP50 values close to those of the standards.

Keywords

Murraya koenigii, Leaves, Essential oil, GC-MS, Antifungal, Antioxidant.

Introduction

The essential oils are secondary metabolites produced by plants as a part of their defence mechanism are characterized by a complex mixture of several compounds belonging to different classes viz., hydrocarbons, phenols, terpenes, alcohols, aldehydes, ketones, esters, ethers and others [1-3]. Essential oils derived from aromatic plants apart from their use for flavouring or cosmetic purposes [4], have been widely used in the treatment of various diseases owing to their pharmacological properties such as antimicrobial, antiinflammatory, antioxidant and several other biological activities [5-8]. In view of the interest harmful impacts of synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc. on human health, natural antioxidants are gaining importance in modern therapeutics. Use of such synthetic antioxidants is prohibited as it leads to liver damage [9]. Furthermore, use of essential oils in food processing and packaging represent a valid alternative to prevent autoxidation and prolong shelf life of food products [10].

Murraya koenigii (L.) Spreng commonly known as Curry leaf is a shrub or a small tree under the family Rutaceace, growing up to 6 m height. The plant is native to India, Pakistan, Sri Lanka, Bangladesh and the Andaman Islands. It is also cultivated widely in South-East Asia and some parts of the United States and Australia. In tropical Africa it is planted in many countries, including Nigeria, Kenya, Tanzania and most of the Indian Ocean Islands, where Indian immigrants settled [11]. Leaves of the plant are widely use in Indian cookery for centuries and have a versatile role in traditional medicine [12].

Out of the 14 species globally reported under the Murraya genus, only two i.e. M. koenigii (L.) Spreng and M. paniculata (L.) Jack is found in India; of which, the former is most popular on account of its diverse medicinal properties and its use as a flavouring agent in different curries and foods since the ancient times [13]. In traditional system of medicine, the plant is regarded as analgesic, cooling, alexiteric, antiemetic, anthelmintic, antidiarrhoeal, febrifuge, carminative, purgative, stomachic and stimulant and used to alleviate body temperature, blood disorders, diarrhoea, dysentery, eruption, inflammation, itching, kidney pain, leukoderma, piles, snakebite, thirst, vomiting and blood purification. The leaves of the plant are used traditionally in the Indian Ayurveda system to treat diabetes [14].

Phytochemical investigations have led to the isolation and characterization of several chemical constituents from every part of the plant. The phytoconstituents present in M. koenigii leaves include phenols, steroids, saponins, quinones, alkaloids, flavonoids, tannins, carbohydrates, proteins, and volatile oils [15]. Almost all parts of the plant contain carbazole alkaloids well known for their various pharmacological activities, including anti-HIV, anticancer, antibacterial and antifungal activities [16,17]. Bark contains carbazole alkaloids namely mukoenine-A, B and C and murrastifoline-F, bis-2-hydroxy-3- methyl carbazole, bismahanine, bi koeniquinone-A, bismurrayaquinone-A, murrayacine, murrayazolidine, murrayazoline, mahanimbine, girinimbine, koenioline and xynthyletin [18]. Leaves contains koenimbine, O- methyl murrayamine, O- methyl mahanine, isomahanine, bismahanine, bispyrayafoline, glycozoline, 1-formyl-3 methoxy- 6-methyl carbazole, 6, 7-dimethoxy-1-hydroxy-3-methyl carbazole, koenigine, koenine, koenidine and (-) mahanine, mahanimbine, isomahanimbine, koenimbidine, murrayacine, Isomahanimbicine, euchrestine B, mahanine, mahanimbicine, mahanimbine, bismurrayafoline E, mahanimbicine, bicyclomahanimbicine, cyclomahanimbine, bicyclomahanimbine, mahanimbidine, mukonicine, 1-formyl-3 methoxy-6-methyl carbazole and 6, 7- dimethoxy-1- hydroxy-3 methyl carbazole [19-22]. The leaves of Murraya koenigii also consist of protein, carbohydrates, fibre, minerals, carotene, vitamin C, Nicotinic acid [22]. Bioactive carbazole alkaloids including murrayanol, murrayagetin, marmesin-1”-Orutinoside, mukoline, mukolidine, girinimbine and koenoline from roots; mahanimbine, koenimbine, isomahanine murrayanol, mahanimbine, murrayazolidine, girinimbine, koenimbine and mahanine from fruits and iskurryam, koenimbine, koenine, mahanimbine, girinimbine, koenimbine, mahanine and isomahanine alongwith indicolactone, anisoalctone and 2‟,3‟epoxyindicolactone and minor furocoumarins such as xanthotoxin, isobyaknagelicol, byakangelicol, isogosferol. isoheraclenin, isoimperatonin, oxypeucedanin, isopimpinellin and bergaptanwere have been extracted from the seeds [23]. The most important chemical constituents responsible for its intense characteristic aroma are p-gurjunene, p-caryophyllene, p-elemene and o-phellandrene [24]. Essential oils from M. koenigii serves as an important part in soap making ingredients, lotions, massage oils, diffusers, potpourri, scent, air fresheners, body fragrance, perfume oils, aromatherapy products, bath oils, towel scenting, spa's, incense, facial steams, hair treatments, and more [25,26]. Chemical constituents of M. koenigii leaves reported to exhibit anticancer, hepatoprotective, antipyretic, antioxidant, antiobesity, antimutagenic, antimicrobial, antifungal, insecticidal, antibacterial effect [13]. Previous studies recorded high antioxidant activity in leaves which is mainly attributed to the presence of chemical constituents namely mahanimbine, murrayanol and mahanine [27,28]. These constituents have also been found responsible for antibacterial and antifungal activities of the leaves [19]. The work presented was aimed to investigate the chemical composition of the essential oil from Murraya koenigii leaves collected from Dehradun, Uttarakhand (India) and to evaluate antifungal and antioxidant activities of the essential oil.

Materials and methods

Plant material

Fresh leaves of Murraya koenigii used for the study were collected from suburbs of Dehradun, located in the Doon Valley on the foot hills of the Himalayas and the capital city of the state of Uttarakhand, India during the onset of monsoon and were authenticated by Systematic Botany Section of Botany Division, FRI. A voucher specimen of the collected material is preserved in the Chemistry Division for future reference.

M. koenigii leaves (200 g) were taken into round bottom flask and soaked in distilled water in the ratio of 1:3 and then hydro distilled for 10 h using a Clevenger type apparatus. The distillate was extracted with diethyl ether (3 × 50 ml); the ethereal layer was dried over anhydrous sodium sulphate and the diethyl ether was removed on a gently heated water bath (300°C). The yield of an essential oil was calculated on air dried basis. For accuracy the experiments were repeated three times. The process was further repeated several times to isolate sufficient quantity of the essential oil for the analysis. The percentage yield of M. koenigii leaf essential oil (MKLEO) was calculated with respect to the quantity of leaf material used for essential oil extraction.

GC-MS analysis of MKLEO

Essential oils are a complex mixture mainly of mono- and sesquiterpenes containing hundreds of components and thus it is not possible to isolate pure constituents by column chromatography. However, the chemical components of the essential oil can be qualitatively and quantitatively characterized by sophisticated Gas Chromatography (GC) and Gas Chromatography-Mass Spectrometry (GC-MS) techniques. The GC-MS analysis of MKLEO was carried out with Gas Chromatography-Mass spectrometry (GC-MS) system (Agilent 7697B) fitted with a DB-5 ms capillary column (30 m × 250 μm × 0.25 μm, Agilent 122-S002). The injector, GC-MS interface, ion source, and MS Quadrupole temperature were maintained at maintained at 220°C and the transfer line to be held at 220°C. The oven temperature for volatile oil was programmed at 40°C (1 min), 40 to 220°C (3°C/min), 220 °C (20 min). Oil was diluted to 1% (w/v) in hexane and 1 μL injected. Detection was performed by mass spectrometer in the EI mode (ionization energy of 70 eV, ion source temperature of 180°C, emission current of 220 μA). Acquisition was made in full scanning mode (mass range 50-900 m/z; 3 scans/second). Maximum ionization time was 25 milisecs. Helium flow rate through the column was 1 mL min-1 with a 30:1 split. Identification of the individual oil components was accomplished by comparison of retention times with standard substances and by matching mass spectral data with MS libraries (NIST and Wiley 275.l) using a computer search and literature [29].

Evaluation of Antifungal Activity of MKLEO

Tested fungi

Antifungal activity of MKLEO was evaluated against altogether 10 pathogenic fungi viz., Alternaria alternata (AA), Aspergillus flavus (AF), Aspergillus niger (AN), Aspergillus parasiticus (AP), Fusarium oxysporum (FO), Fusarium moniliforme (FM), Mucor mucedo (MM), Penicillium notatum (PN), Penicillium funiculosum (PF), and Trichoderma viride (TV). These fungi were isolated from the infected saplings and spoiled foods by Standard Blotter Method [30] and identified based on growth characteristic, mycelial morphology, spore morphology and other important characters using standard protocol [31,32]. Pure cultures of each of the selected fungal species were made separately and maintained at on potato dextrose agar (PDA). These pure cultures were used for antifungal assay.

Antifungal activity assay

The antifungal activity was determined using the disc diffusion method [33]. Initially, the medium was prepared by dissolving potato dextrose agar (Hi Media) in distilled water and autoclaving at 121°C for 15 minutes. 20 ml of sterile PDA media was poured in sterilized petridishes (9 cm diameter) and allowed to solidify which were used for antifungal assy. Spore suspension was prepared in 0.9% saline water and adjusted to give a final concentration of 1-5 × 10⁵ cfu/ml. The essential oil was diluted with Tween 40 to obtain the final concentrations of 1000, 750, 500, 250, 100, 50, 25 μg/ml, respectively. A plug of 1-week-old fungal culture (5 mm diameter) was placed on the centre of the sterilised plates containing PDA. About 10 μl of each concentration was injected to the sterile disc papers (6 mm diameter). Then the prepared discs were placed on the culture medium. Carbendazim (2 mg/ml) and Tween 40 were served as positive and negative control respectively. The plates were then incubated at 30°C for 4-5 days, and colony diameter was measured and recorded after 5 days. The growth inhibition of each fungal strain was calculated as the percentage inhibition of a radial growth relative to the control as:

Inhibition (%)=[(1-A/B] × 100

Where A=mean diameter of fungal colony in treatment (mm); B=mean diameter of fungal colony in control (mm). All experiments were performed in triplicate.

Determination of MIC

The minimum inhibitory concentration (MIC) was determined through the broth dilution method [34,35]. Fungi were first grown in the potato dextrose broth for 24 h and then the inoculums were diluted for five times (10-5 dilution) to control its vigorous growth. Then each test tube was added with 1.8 ml of potato dextrose broth and different concentrations MKEO followed by inoculation of 0.2 ml of respective fungi and kept at 28°C for 48 h. The tubes were examined for visual turbidity. Lowest concentrations of the extracts showing no turbidity (without microbial growth) were considered as the minimal inhibitory concentration.

Evaluation of antioxidant activity of MKLEO

The antioxidant activity of MKLEO was evaluated by means of the 2,2-diphenyl-1-picrylhydrazil (DPPH) radical scavenging method [36-38]. Briefly, different amounts of the tested sample (50-250 μg/mL) were added to 5 mL of a 0.004% methanol solution of DPPH. Finally, the absorbance was read against a blank at 515 nm after 30 min of incubation in the dark. All the observations were taken as triplicate. BHT, catechin, gallic acid and ascorbic acid were used as the standard antioxidants. Inhibition of free radicals by DPPH in per cent (IC%) was calculated using the following equation-

IC%=[(Ao-As)/Ao)] × 100,

Where Ao and As are the absorbance values of the control and test sample, respectively. Per cent inhibition was plotted against concentrations and the equation for the line was used to obtain the IC₅₀ value.

Reducing power assay of MKEO

The reducing power of essential oil and various extracts was determined by the method reported earlier [39]. Varying concentrations of tested sample (50-250 μg/ml) were mixed with 2.5 ml of the phosphate buffer (200 mM, pH 6.6) and 2.5 ml of 1% potassium ferricyanide (K₃Fe(CN)₆). The mixtures were incubated at 50°C for 20 min. After incubation, 2.5 ml of 10% trichloroacetic acid was added to the mixtures, followed by centrifugation at 650.g for 10 min. The upper layer (5 ml) was mixed with 5 ml of distilled water and 1 ml of 0.1% ferric chloride (FeCl₃) and absorbance of the resulting solution were measured at 700 nm using spectrophotometer. All the readings were taken in triplicate and BHT, catechin, gallic acid and ascorbic acid were taken as the standard. The reducing power of samples was calculated by the following formula:

RP (%)=[(Ao-As)/Ao)] × 100

Where Ao and As are the absorbance values of the control sample and the test sample, respectively. Percent (%) inhibition was plotted against concentration, and the equation for the line was used to obtain the RP₅₀ value.

Statistical analysis

Experiments were performed in triplicate and results were expressed as means ± standard deviations (SD). Statistical comparisons were made using one-way analysis of variance (ANOVA) by using SPSS 16.

Results and Discussion

Yield and composition of essential oil

The yield of essential oil obtained through hydro distillation of M. koenigii leaves was 0.52% (v/w fresh material) higher than those reported earlier [40,41]. This suggested that M. koenigii plant located in the foothill of Himalayas possesses a higher content of essential oil. Further, the yield of oil also influenced by characteristic pedoclimatic conditions of the area where the source plant is grown, time and mode of harvesting and extraction efficiency [42] which prevent yield retarding factors like peroxidation, isomerization, or rearrangement of products due to temperature, light, and oxygen availability [43].

The GC-MS analysis of the essential oil obtained from M. konegii leaves allowed the identification of altogether 43 compounds representing 99.79% of the total composition of the oil. The essential oil composition, with retention time and percentages are presented in Table 1.

Table 1: Chemical composition of essential oil from M. konegii leaves.

It is evident from the data presented in table 1 that compounds including 3-carene (18.52%), β-pinene (13.57%), α-pinene (9.38%), linalool (5.42%), α-eudesmol (4.55%), p-cymene (3.61%), γ-terpinene (3.48%), α-amorphene (3.38%), alloocimene (2.75%), Sabinene (2.55%), γ-terpinene (2.48%), linalyl acetate (2.46%), myrcene (2.43%), β- eudesmol (2.16%), carvone (1.68%), limonene (1.58%), β-elemene (1.55%), α-terpineol (1.48%), (Z)-β-ocimene (1.38%), (E)-β- ocimene (1.16%), eucalyptol (1.22%) and α-thujene (1.12%) have been identified as the major chemical constituents present in the M. konegii leaf essential oil. Essential oils of plants in the family Rutaceae are often composed of mono and sesquiterpenes. In the present investigation, the oil was dominated by constituents like α-pinene, 3 carene, β-pinene, α- amorphene, allo-ocimene, α-eudesmol, linalool, p-cymene, myrcene, and γ-terpinene while other components are limonene, (Z)-β-ocimene, (E)-β-ocimene, geranyl acetate, α- terpineol, carvone, linalyl acetate, β-eudesmol respectively. It is worth mentioning that composition of essential oils is influenced by several factors such as local climatic, seasonal, and processing or experimental conditions [44,45]. Presence or absence of specific compounds and their contents vary across the locations, species populations, agroclimatic conditions owing to anthropological, climatological, and ecological factors which has obvious impact on flavour characteristics [46]. In India, diverse flavour characteristics of curry leave from different regions has been previously determined and the differences in the chemical composition of essential oil of curry leaf plants of different origins is also evident in preceding reports [47-51].

Antifungal activity

The antifungal activity of M. koenigii leaves essential oil (MKLEO) determined against altogether ten pathogenic fungi by disc diffusion method. The growth inhibitory activities of the essential oil against the tested fungi at different concentrations are summarized in Table 2.

Table 2. Antifungal activity of MKLEO (AA: Alternaria alternata; AF; Aspergillus flavus; AN: Aspergillus niger; AP: Aspergillus parasiticus; FO: Fusarium oxysporum; FM: Fusarium moniliforme; MM: Mucor mucedo; PN: Penicillium notatum; PF: Penicillium funiculosum; TV: Trichoderma viride.).

The results of antifungal activity assay clearly indicate that the essential oil has varying degree of antifungal activity against the all the tested pathogenic fungi in a dose-dependent manner (Table 1). The MIC values ranged from 25.5 to75 μg/mL. From the results, it is also evident that the essential oil exhibit highest growth inhibition in all the tested fungi at test concentration of 2000 μg/ml and it is almost at par with positive control. The growth inhibition in all the tested fungi is moderate with 1000 μg/mL and 750 μg/mL treatment doses of the essential oil. Of the different treatment doses of essential oil, growth inhibitions in all tested fungi are much less as compared to positive control with treatment does of 500, 250 and 100 μg/mL. Among the tested fungi, maximum growth inhibition was recorded in Alternaria alternata with all the treatment concentrations of the essential oil. Growth inhibition in other tested fungi was also noticeable. The percentage growth inhibition in the tested fungi namely Alternaria alternata, Aspergillus flavus, Aspergillus niger, Aspergillus parasiticus, Fusarium oxysporum, Fusarium moniliforme, Mucor mucedo, Penicillium notatum, Penicillium funiculosum, and Trichoderma viride with different treatment doses ( (100-2000 μg/ml) of M. koenigii leaf essential oil ranged between 5.27-96.59, 3.25-95.15, 2.81-94.69, 3.50-95.63, 4.33-96.47, 4.52-95.93, 3.87-93.86, 2.55-93.15, 3.31-94.87, and 3.75-95.37% respectively. In conclusion, M. koenigii leaf essential oil (MKLEO) found to be remarkably effective antifungal in accordance to the inhibition action against all tested pathogenic fungi. The fungi toxicity of MKLEO might be due to the presence of mono and sesquiterpenoids constituents [52]. Although some of these compounds feature low concentrations, they can have an effect on the overall efficiency of the antimicrobial activity of the essential oil through synergistic interaction with the other constituents [53,54].

Antioxidant activity

The antioxidant activity MKLEO were measured in terms of its free radical scavenging ability following DPPH radical scavenging protocol and reducing power assay. The results of radical scavenging potential in the form of their IC₅₀ values and reducing power as RP₅₀ are summarized in Table 3.

Table 3: Antioxidant activity of MKLEO and standards.

With respect to the results of antioxidant activity, it should be noted that IC₅₀ and RP₅₀ values were inversely related to the percentage of DPPH scavenging capacity and reducing power, i.e., the higher the scavenging and reducing power rate, the lower the IC₅₀ and RP₅₀ values respectively. The analysis of the results of antioxidant activity of the MKLEO demonstrated that there were significant differences in these values between the essential oil and the standard antioxidants. However, the difference in IC₅₀ and RP₅₀ values between standards and the essential oil evidently minor which demonstrates that the MKLEO exhibited superior antioxidant activity (Table 3). The free radical scavenging capacity and reducing of the MKLEO can be attributed to the presence of certain terpenoid and phenolic compounds well recognized for their antioxidant activity and synergistic effect of chemical constituents of the essential oil [55,56].

Conclusion

The yield of essential oil from M. koenigii leaves was 0.52% higher than those reported earlier. The GC-MS analysis of the essential oil obtained from M. konegii leaves allowed the identification of altogether 43 compounds representing 99.79% of the total composition of the oil, among which 3-carene, β- pinene, α-pinene, linalool, α-eudesmol, p-cymene, γ-terpinene, α-amorphene, allo-ocimene, sabinene, γ-terpinene, linalyl acetate, myrcene, β- eudesmol, carvone, limonene, β-elemene, α-terpineol, (Z)-β-ocimene), (E)-β-ocimene, eucalyptol and α- thujene have been identified as the major constituents. The essential oil (MKLEO) was found effective against all the ten pathogenic fungi tested in a dose dependent manner. Regarding the antioxidant activity, the MKLEO was found to exhibit superior radical scavenging potency and reducing power with IC₅₀ and RP₅₀ values close to those of the synthetic antioxidant (standards). The essential oil of M. koenigii has the potential to be used in the food, cosmetics, and pharmaceutical industries, since it exhibits antimicrobial and antioxidant properties. It is important to stress the importance of further studies, mainly for determining the mechanism of action of this essential oil as well as the action of its individual constituents.

Acknowledgements

The authors are grateful to the Director, Forest Research Institute, Dehradun for providing necessary facilities for carrying out this work. Authors are thankful to Prof. Lokesh Upadhyay, Department of Biotechnology, Sarmila Institute of Medicinal Products & Research Academy, Thanjavur, Tamil Nadu, India for his help in antifungal assay.

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