Composition and Biological Activities of Murraya paniculata (L.) Jack Essential Oil from Nepal

Murraya paniculata (L.) Jack, a small tropical evergreen shrub growing in Nepal, has numerous uses in traditional medicine for treatment of abdominal pain, diarrhea, stomach ache, headache, edema, thrombosis, and blood stasis. The present study investigated the chemical composition and bioactivities of the leaf essential oil from M. paniculata from Nepal. The essential oil from leaves was obtained by hydrodistillation and a detailed chemical analysis was conducted by gas chromatography-mass spectrometry (GC-MS). The essential oil was screened for antimicrobial activity using the microbroth dilution test, for nematicidal activity against Caenorhabditis elegans, and for lethality against brine shrimp (Artemia salina). A total of 76 volatile components were identified from the essential oil. The major components were methyl palmitate (11.1%), isospathulenol (9.4%), (E,E)-geranyl linalool (5.3%), benzyl benzoate (4.2%), selin-6-en-4-ol (4.0%), β-caryophyllene (4.0%), germacrene B (3.6%), germacrene D (3.4%), and γ-elemene (3.2%). The essential oil showed no antibacterial activity, marginal antifungal activity against Aspergillus niger (MIC = 313 μg/mL), a moderate activity against A. salina (LC50 = 41 μg/mL), and a good nematicidal activity against C. elegans (LC50 = 37 μg/mL).


Introduction
The genus Murraya (Rutaceae) is made up of about 14 species. Murraya paniculata (L.) Jack is a small tropical evergreen shrub, native to the tropical and subtropical parts of the world, including southern China, Taiwan, India, Nepal, Northeastern Pakistan, Sri Lanka, Southeastern Asia (i.e., Cambodia, Laos, Myanmar, Thailand, Vietnam, Indonesia, Malaysia, and the Philippines), and Northern Australia. It is widely naturalized in the southern part of Australia, Southeastern USA and Central America. M. paniculata is also known as Chalcas exotica, Chalcas paniculata, and Camunium exoticum [1]. M. paniculata is commonly known as orange jasmine or mock orange. In Nepal, it is known as bajardante [2]. The average shrub can grow up to 7 m high. Morphologically, the plant can be distinguished by its alternate, glabrous, and glossy leaves that are once-compound, occurring in 3-7 oddly pinnate leaflets. Leaflets are elliptic to cuneate-obovate, 2-9 cm longˆ1.5-6 cm wide [3,4]. M. paniculata blooms throughout the year. Inflorescences are terminal, corymbose, few-flowered, and dense. Flowers are pentamerous, bisexual, and sweetly fragrant. Petals are 12-18 mm long, narrowly elliptic to oblanceolate, curved backwards, and white to fading cream in color. The fruit is a fleshy berry, oblong-ovoid, red to orange, and grows up to 2.5 cm in length [3,4].
The essential oil of M. paniculata was analyzed by GC-MS using an Agilent 6890 GC with Agilent 5973 mass selective detector (Agilent Technologies, Santa Clara, CA, USA), an HP-5ms fused silica capillary column and an Agilent ChemStation data system [MSD, operated in the EI mode (electron energy = 70 eV), scan range = 40-400 amu, and scan rate = 3.99 scans/s], and an Agilent ChemStation data system as previously described [24]. The GC column was an HP-5ms fused silica capillary with a (5% phenyl)-polymethylsiloxane stationary phase, film thickness of 0.25 µm, a length of 30 m, and an internal diameter of 0.25 mm. The carrier gas was helium with a column head pressure of 48.7 kPa and a flow rate of 1.0 mL/min. Inlet temperature was 200˝C and interface temperature was 280˝C. The GC oven temperature program was used as follows: 40˝C initial temperature, hold for 10 min; increased at 3˝C/min to 200˝C; increased 2˝/min to 220˝C. A 1% w/v solution of the sample in chloroform was prepared and 1 µL was injected using a 10:1 split ratio. Identification of the oil components was based on their retention indices (RI) and by comparison of their mass spectral fragmentation patterns with those reported in the literature [25].

Antimicrobial Screening
The essential oil of M. paniculata was screened for antimicrobial activity against Bacillus cereus (ATCC No. 14579), Aspergillus niger (ATCC No. 16888), and Candida albicans (ATCC No. 10231). The minimum inhibitory concentration (MIC) was determined using the microbroth dilution technique as previously reported [26]. For B. cereus, a dilution of the essential oil were prepared in cation-adjusted Mueller Hinton broth (CAMBH) beginning with 50 µL of a 1% w/w solution of the sample in dimethylsulfoxide (DMSO) plus 50 µL CAMBH. The essential oil solution was serially diluted (1:1) in CAMBH in a 96-well plate. Organisms at a concentration of approximately 1.5ˆ10 8 colony forming units (CFU)/mL were added to each well. Plates were incubated at 37˝C for 24 h; the final minimum inhibitory concentration (MIC) was determined as the lowest concentration without turbidity. Gentamicin was used as a positive antibiotic control. Antifungal activity against C. albicans was determined as above using yeast-nitrogen base growth medium with approximately 7.5ˆ10 7 CFU/mL; amphotericin B was the positive control. Antifungal activity against A. niger was determined as above using potato dextrose broth inoculated with A. niger hyphal culture diluted to a McFarland turbidity of 1.0; amphotericin B was the positive control.

Nematicidal Assay
A nematicidal assay using Caenorhabditis elegans was done using a modification of the procedure of Park and co-workers [27]. Briefly, a 1% solution of M. paniculata leaf oil in dimethylsulfoxide (DMSO) was used to make dilutions for the sample solutions. The sample solutions were prepared in sterile water beginning with 50 µL of the 1% essential oil solution mixed in 50 µL sterile water. This sample solution was serially diluted (1:1) with sterile water in a 96-well plate. Into each well, 10-30 C. elegans (mixtures of juvenile and adult nematodes, male:female:juvenile~1:1:2) per 50 µL of sample solution were added. Sterile water and serially diluted DMSO were used as controls. The dead and living nematodes were counted after 24 h using a microscope. Dead nematodes were identified by their immobility and straight body, even after transfer to clean water. Mean lethal concentration (LC 50 ) values were determined using the method of Reed and Muench [28].

Brine Shrimp Lethality Assay
The brine shrimp (Artemia salina) lethality test was done using a modification of the procedure of McLaughlin [29]. A. salina eggs were hatched in a sea salt solution (Instant Ocean ® , Spectrum Brands, Inc. Madison, WI, USA) (38 g/L) with an incandescent light bulb as the heat source. After 48 h, the newly hatched nauplii were counted using a micropipette and transferred to 20 mL vials. A total of nine vials, each containing 10 A. salina nauplii in 10 mL of sea salt solution (the same as the hatching solution) were prepared. Of these vials, three were labeled as controls with one vial containing no DMSO, a second vial containing 10 µL of DMSO, and the third vial containing 100 µL DMSO. A second set of three replicate vials contained 10 µL of 1% essential oil solution in DMSO, and the remaining three vials were prepared by adding 100 µL of 1% essential oil solution in DMSO. After 24 h, surviving A. salina nauplii were counted in each vial and LC 50 values were determined using the Reed-Muench method [28].
M. paniculata oil showed moderate activity in the brine shrimp (Artemia salina) lethality test with LC 50 value of 41 µg/mL. Essential oils showing A. salina toxicity with LC 50 < 10 µg/mL are considered very active [56], 10 µg/mL < LC 50 < 50 µg/mL are moderately active [51,57], and 50 µg/mL < LC 50 < 100 µg/mL, weakly active. In our nematicidal activity screening against C. elegans, we have found LC 50 values ranging from 18 to 1100 µg/mL (unpublished), and we consider nematicidal activities LC 50 < 100 µg/mL to be very active, LC 50 values between 100 and 200 µg/mL to be moderately active [52,58], between 200 and 300 µg/mL to be weakly active, and > 300 µg/mL to be inactive. Thus, M. paniculata oil was highly nematicidal to Caenorhabditis elegans (LC 50 = 37 µg/mL). It is difficult to speculate as to which compound(s) in the essential oil may be responsible for the brine shrimp lethality or nematicidal activity; there are many components in the leaf oil and none are especially dominant.

Conclusion
The leaf essential oil of Murraya paniculata growing in Nepal has been analyzed by GC-MS and revealed this to be a distinct chemotype, rich in methyl palmitate. Biological screening of the leaf oil showed it to have good nematicidal activity, marginal activity against brine shrimp and Aspergillus niger, and inactive against bacteria. The particular chemotype of this plant could have important implication on its biological activity and traditional medicinal uses.
Author Contributions: P.S. conceived and designed the experiments; P.S. and N.S.D. performed the experiments; P.S. and W.N.S. analyzed the data; T.P.S. identified the plant specimen; W.N.S. contributed reagents/materials/analysis tools; N.S.D., P.S. and W.N.S. wrote the paper.

Conflicts of Interest:
The authors declare no conflict of interest.

Abbreviations
The following abbreviations are used in this manuscript:

GC-MS
Gas chromatography-mass spectrometry RI Retention indices ATCC American type culture collection DMSO Dimethylsulfoxide LC 50 Median lethal concentration