Chemical and Antibacterial Activity Evaluation of Alpinia calcarata and Alpinia zerumbet Grown in Foothills Agroclimatic Conditions of Northern India

A total of 31 compounds identified, forming 89.26-94.32% composition of A. calcarata and A. zerumbet. The leaf and rhizome oil of A. calcarata and A. zerumbet were mainly characterized by 1,8-cineole (15.61-43.63%), β-pinene (5.02-23.52%), terpinen-4-ol (1.00-20.87%), camphor (1.94-11.60%), and (Z)-β-ocimene (0.16-11.86%). endo-Fenchyl acetate (13.12-24.39%) was identified as marker constituents of rhizome essential oil of both A. calcarata and A. zerumbet. The antibacterial assay showed that leaf oil of A. calcarata has good activity against S. mutans, whereas its rhizome oil possess good activity against K. pneumoniae, E. coli, S. aureus, and S. epidermidis. However, the rhizome oil of A. zerumbet showed activity against S. mutans, B. subtilisS. aureus-2940 and S. epidermidis.


INTRODUCTION
Plants synthesize numerous secondary metabolites viz. terpenoids, alkaloids, glycosides, anthocyanins, flavonoids, lignans, tannins, phenolics, caretenoids, steroids and saponins, for diverse applications. Different plants parts (leaves, stem, root, rhizome, flowers, and seeds) of medicinal and aromatic plants accumulate these secondary metabolites of medicinal value and the composition was controlled by genetic factors, geographic and climatic conditions, developmental stage of the plant, extraction methods, etc [1 -5]. Zingiberaceae, the largest monocotyledonous family in India, includes 52 genera and 1400 species distributed in the Indo-Malaysian region of Asia. Among them, 22 genera and 178 species were reported in the north eastern and peninsular regions of India. Various members of this family are of economic importance for food-flavour, spice, medicine and source of essential oils and oleoresins of medicinal and export value [6 -9]. The genus Alpinia, consist ca. 230 species worldwide with major distribution in China, India, East Indies, and Polynesia and are used as spices and food additives agent and in the traditional medicine for the treatment of dyspepsia, gastralgia, and sea-sickness and for abdominal colic pains and as digestive, spleen and liver tonic. These are accredited with various pharmacological activities such as antispasmodic, myorelaxant, anti-oxidant, anti-inflammatory, anti-emetic, antiulcer, anti-inflammatory, anti-parasitic, anti-allergic and spasmolytic properties for their use in various indigenous medicinal formulations [10 -14]. Numerous studies on leaf, flower and rhizome essential oil of A. calcarata and A. zerumbet have been carried out on its uses and applications in many common and rare health problems. Alpinia spp. are characterized by a wide range of volatile compounds in numerous phytochemical studies and mainly found to be dominated by monoterpenoids such as 1,8-cineole, camphor, (E)-methyl cinnamate, terpinene-4-ol, and pinenes, ocimenes, and fenchyl acetate as the major constituent distributed in their essential oils [11 -22]. The chemical composition of the rhizome essential oil of A. calcarataand A. zerumbet revealed the presence of oxygenated monoterpene endo-fenchyl acetate as the distinctive marker constituents along with other commonly distributed monoterpenoids [12]. The present experiment focuses on the chemical evaluation & biological activity of two Alpinia species viz. A. calcarata and A. zerumbet grown in tarai regions at CIMAP resource center Pantnagar, Uttarakhand, India.

Plant Material and Extraction of the Essential Oil
Fresh leaves and rhizomes were collected from the crop raised at the experimental field of CSIR-Central Institute of Medicinal and Aromatic Plants, Pantnagar, Uttarakhand. The experimental site is located between coordinates latitude 29.02°N, longitude 79.31°E and an altitude of 237 m above mean sea level The maximum temperature ranges between 35 and 45°C, and minimum between 2 and 5°C. The soil of the experimental site was sandy-loam in texture, with neutral pH and the climate of the region is sub-tropical and humid. The samples were hydrodistilled in a Clevenger-type apparatus for 3 hours. The amount of essential oil was directly measured from the extraction burette and content (%) was calculated as volume (mL) of essential oil per 100 g of fresh plant material. The hydro distilled oil was collected in vials and dehydrated over anhydrous Na 2 SO 4 and stored in a cool dark place for further analysis.

Analysis of the Essential Oil
The essential oils collected were analysed by GC and GC-MS. For the quantitative analysis of the essential oil of leaves and rhizomes of A. calcarata and A. zerumbet, Gas Chromatography-Flame Ionization Detection (GC-FID) was performed on a Nucon GC-5765, equipped with DB-5 capillary column (30m × 0.25mm i.d. and 0.25 µm film thickness). The oven column temperature ranged from 60°C-230°C, programmed at 3°C/minute, using H 2 as carrier gas, split ratio 1:40 and injection volume 0.02 µL. The detector and injector temperatures were 230°C and 220°C, respectively. The relative amounts of the individual components were calculated based on the relative % peak areas (FID response) in the chromatogram without using a correction factor. GC-MS was performed for the identification of the essential oil constituents.GC-MS analysis of the essential oil samples was carried out on a Clarus 680 GC interfaced with a Clarus SQ 8C mass spectrometer (PerkinElmer) fitted with an Elite-5 MS fused silica capillary column (30m×0.25mm i.d.and 0.25µm film thickness). The oven temperature program ranged was from 60°C to 240°C, at 3°C/minute, and to 270°C at 5°C/minute; injector temperature was 250°C; transfer line and source temperatures were 220°C; injection size 0.03 µL neat; split ratio 1:50; carrier gas He at 1.0 mL/minute; ionization energy 70 ev; mass scan ranges 40-450 amu. The essential oil constituents were identified on the basis of retention index, (RI, determined with reference to homologues series of n-alkanes, C 8 -C 30 ), co-injection with standard compounds, MS library search (NIST and WILEY), and by comparing with the MS literature data [23].

Antibacterial Activity Assay
Antibacterial activity of the essential oil was determined by filter paper disc diffusion assay [24]. Inoculums of the test bacteria Standard 0.5. Uniform bacterial lawns were made using 100 μL inoculums on a nutrient agar plate. Filter paper (Whatman) discs (5.0 mm) soaked with test essential oils were placed overseeded plates. The plates were incubated at 37°C for 24 h. The activity was measured in terms of zone of inhibition (ZI,mm). The net zone of inhibition was determined by subtracting the disc diameter (i.e., 5.0 mm) from the total zone of inhibition shown by the test disc in terms of the clear zone around the disc. Norfloxacin was employed as a positive control, while DMSO served as a negative control. The tests were performed in triplicate. The bacterial strains were obtained from the Microbial Type Culture Collection Centre (MTCC), Institute of Microbial Technology (IMT) Chandigarh, India. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) were determined by a Micro dilution broth assay using 96 'U' bottom micro-titer plates as per CLSI guidelines [25]. Samples were serially diluted two folds (in the range of 1000-1.95 μg/mL) in Mueller Hinton Broth (MHB). The broth was inoculated with 10.0 μL of diluted 24 h grown culture of test organisms with a titre equivalent to 0.5 McFarland standards. The inoculated plates were then incubated at 37ºC for 16-24 h and the growth was recorded spectrophotometrically at 600 nm using Spectramax 190-microplate reader (Molecular Devices, CA, and USA). The MIC value was determined from the turbidimetric data as the lowest concentration showing growth inhibition equal to or greater than 80% as compared to control. Bactericidal endpoints were obtained by spread plating known volume (100 µL) from each well on solid media and the endpoint for complete inhibition was defined as the Minimum Bactericidal Concentration (MBC) of test samples in the original tube, which failed to yield discernible growth when sub-cultured.
Experimental observations were performed in triplicate to rule out any error during the procedure.

Alpinia calcarata
Leaf Oil essential oils from leaves and rhizomes of A. calcarata and A. zerumbet possess aroma chemicals viz. 1,8-cineole, ocimenes, terpinen-4-ol, α-pinene, β-pinene and fenchyl acetate for utilization in perfumery and fragrance related formulation. In the southern part of India, these species are in cultivation and used as raw material for phytochemicals and traditional medicine. The antibacterial activity assay also showed that their essential oils possess significant activities against some of the tested pathogenic bacterial strains. The results showed that the future prospect needs to be carried out in these plants for utilizing different plant parts as a prominent source of essential oils and aroma chemicals for product formulation.

ETHICAL STATEMENT
The followed protocols on Plants for this study were carried out in accordance with the CLSI guidelines.

CONSENT FOR PUBLICATION
Not applicable.

AVAILABILITY OF DATA AND MATERIALS
Not applicable.

FUNDING
None.