The antimicrobial activity of volatile oils has received attention in recent years in parallel with advances in customary approaches of protecting the health of mammals and food against the presence of pathogenic microorganisms. Thus, probe on the antimicrobial activity of plant extracts and essential oils against different pathogens have been performed globally. In the last few decades, there has been a rapid surge in the utilization of aromatic therapeutic plants and their essential oils in technical research and industrial applications in many fields, including spices, fragrances, beauty products, foods and therapy for several health conditions[1–3]. The essential oils of the plants are the main aromatic components with multiple therapeutic efficacy.
They are also known as volatile oils due to the fact that they rapidly vaporize when exposed to the atmosphere at room temperature. In general, the essential oils consist of blend of different types of molecules. Essential oils are extracted from seeds, roots, stems, barks, leaves, fruits, fruit rinds, flowers, or resins of different plants. The medicinal nature of certain therapeutic plants are generally related to their content of secondary metabolites such as alkaloids, terpenes, polyphenols, and Phyto steroids. Hedychium spicatum belonging to family
Zingiberaceae, commonly known as Kapoor kachri is useful in the treatment of respiratory disorders, fever, and pain.
It is a perennial rhizomatous plant, growing in Western and Central Himalayas at altitudes of 3500-7500 ft. Rhizomes are 15-20 cm long, externally yellowish-brown, but change to dark brown on storage. As a part of our research we have performed physicochemical analysis of rhizome, GCMS characterisation of essential oil and checked its anti-microbial efficacy against common selected human pathogenic organisms. The study focuses on antimicrobial efficacy of this indigenous oil which can be used as preservative in cosmetic products.
Materials and Methods
The freshly collected rhizomes of Hedychium spicatum (Batch number. NPD/670/2015) were purchased from local market of Bangalore, Karnataka. The sample was identified as well as authenticated by Dr. Kannan, botanist, Department of Pharmacognosy, The Himalaya Drug Company (Bangalore, Karnataka). The rhizomes of Hedychium spicatum were shade dried and kept in sealed LDPE poly bag.
Source of Bacterial culture
The bacterial cultures of Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 8739), and Yeast; Candida albicans(ATCC10231) were procured from ATCC, Virginia, USA. Whereas, Bacillus subtilis (NCTC 10400) was procured from England. The cultures were maintained in the laboratory by frequently sub culturing into Soybean casein digest agar media (Himedia, Mumbai, India).
Physico chemical analysis of raw material
2 g of Hedychium spicatum rhizomes was air dried, powdered and weighed in a previously weighed silica crucible and incinerated, gently at first, and gradually increased the temperature to 675 ± 250C until free from carbon.
Water soluble extractive value
5 g of the rhizome was air-dried, coarsely powdered and macerated with 100 ml of chloroform- water mixture in a closed flask for 24 hours, continuously shaken for 6 hours and allowed to stand for 18 hours. It was rapidly filtered; preventing any loss of solvent, 25 ml of the filtrate
was dried completely in a tared flat bottomed shallow dish at 1050C and weighed. The percentage of water soluble extractive value was calculated.
Alcohol soluble extractive value
5 g of the rhizome was air-dried, coarse powdered and macerated with 100 ml of absolute alcohol in a closed flask for 24 hours. It was continuously shaken for 6 hours and kept still for 18 hours. It was rapidly filtered; preventing any loss of solvent, 25 ml of the filtrate was dried
completely in a tared flat bottomed shallow dish at 1050C and weighed. The percentage of alcohol soluble extractive value was calculated.
Loss on drying
The sample was cut by shredding about 3 mm in thickness and 10 g of sample was kept in a tared evaporating dish. After keeping the drug in the tared evaporating dish, it was dried at 1100C for 5 hours, and weighed. The weighing and drying was continued for about an hour
interval until difference between two successive weights corresponded to not more than 0.25%.
Volatile oil content
10 g of the Hedychium spicatum rhizomes were weighed accurately and grinded using mixer to obtain coarse powder (30-60 mesh). The coarse powder was taken in a 1000 mL flat bottomed flask and about 600 mL of water was added. The plant material was distilled for 8 hours using a Clevenger apparatus. The oil was partitioned to remove water and trace amount of moisture was removed using anhydrous sodium sulphate. The pure oil is decanted to the amber colored glass vial and stored in room temperature.
1 g of sample was weighed in a 250 ml flat bottomed flask. 10 ml of methanol was added and refluxed at 800C ± 20C for about 35 minutes. The extract was passed through Whatman No.1 Filter paper. 15µl of the filtered extract was spotted in pre-coated thin layer silica plate 60 F 254,10x10cm and developed using solvent system of Toluene: Ethyl acetate (75:25). The dried plate was visualised under UV 254 nm and 366 nm using UV cabinet. The plate was derivatized using vanillin sulphuric acid reagent, it was heated at 1050C for about 10 minutes and
visualized under white light.
The essential oil was analysed using Bruker scion 456 GC-MS equipped with ZB 5 MS column (0.25 mm ID x 30 m L, thickness of 0.25 microns). The H. spicatum essential oil was diluted in ethyl acetate and about 2.0 μL of this solution was injected into column. Helium was used as carrier gas at a flow rate of 1.0 mL/ min. The injector temperature was programmed at 280°C. The initial column temperature was maintained at 90°C for 2 minutes, raised to 150˚C at 5˚C/minute maintained for 4 minutes, then to 280˚C at 10˚C/minute maintained for 5 minutes. The quantitative data (percentage composition) was determined by normalization. The mass detector was functionalised in electronic ionization mode (-70 eV). Oven temperature programme and injection procedure were the same as above. Transfer line temperature and filament lamp was maintained at 280°C and 230°C respectively. The components were discovered by comparison of their mass spectra with those from the Wiley Registry of Mass Spectral Data, and linear retention indices (LRI) as well as compared to literature data.
Broth dilution method
Microorganisms were inoculated into a liquid growth media in the presence of varied concentrations of an antimicrobial agent. This method was performed to investigate the minimal inhibitory concentration (MIC) of oil against selected pathogens. Antibiotics; Amphotericin B at 50µg/ml was used as standard for yeast and Ciprofloxacin at 50µg/ml was used for bacteria. The procedure involves preparation and dilution of the antimicrobial agent in a liquid growth medium poured in tubes. 200µl of 100% Hedychium spicatum oil was taken as control. The oil samples were diluted with Muller Hilton Broth (MHB) media (M391,
Himedia, USA) at different concentration in the 96-well microtiter plate. Beginning with 100%, 50%, 25%, 12.5%, 6.25%, 3.12% and 1.56 % respectively. MHB was used as negative control. Saline suspension was used for the colony forming count of microorganisms; microbial suspension with MHB is used as experimental test solution. Microbial suspension with antibiotic was taken as the reference standard. Each well was inoculated with 10µl of microbial inoculums prepared with similar medium after dilution of standardised microbial suspension which was made up to 104 cfu/ml of 24hr culture. After that the mixture was incubated without agitation under suitable conditions at 35˚C for 24 to 48hrs.The UV absorbance was checked at 580 nm for control and test samples. All the experiments were carried out in triplicates.
Results and Discussion
Chemical configuration of essential oil
The chemical configuration of the oils obtained from Hedychium spicatum rhizome is given in Table I. The yield of oil was 0.5% w/w based on dry weight. The total essential oil content obtained from rhizomes was found to be 2.96% v/w. The water extractive value and alcohol extractive values were found to be 11.98 %w/w and 3.61 %w/w respectively. The TLC fingerprint profile of Hedychium spicatum oil was developed in Toluene: Ethyl acetate solvent system in the ratio (75:25) as depicted in Figure I at white light and different UV wavelengths of 254 and 366 nm respectively. Thirty-eight components were identified by GC-MS as described in Table II. The main constituents were found to be Ethyl p-methoxycinnamate (50.1 %), Ethyl cinnamate (26.22 %) and Eucalyptol (5.68 %). Apart from that Eicosane (3.35%), Endo-Borneol (2.44 %), 3-Carene (1.10 %), Camphene (1.04 %), α-Gurjunene (0.63 %), β- Copaene (0.48 %), p-Cymen-8-ol (0.38 %), α-Pinene (0.37 %), Verbenone (0.32 %), Limonene oxide (0.28 %), Isobornyl formate (0.26 %), Eucarvone (0.25 %), Thymol (0.25 %), O-Cymene (0.22 %), Longipinocarvone (0.21 %), -Pinene (0.20%), α-acorenol (0.17 %), Camphor (0.15%), Caryophyllene oxide (0.14 %), D-Limonene (0.14 %), (1R)-(-) Myrcenol (0.14 %), Cubenol (0.12 %), (Z)-Pinocarveol (0.12 %), p-Cymene (0.06 %), Trans-α-Bergamottin (0.06%), α-Ylangene (0.06 %), Linalylanthranilate (0.05 %), Pinocarvone (0.05 %), (-)-Spathulenol (0.05 %), α-Calacorene (0.04 %), Cis-Verbenol (0.04 %), 3-Nitro Propionic acid (0.04 %), Linalool oxide (0.04 %), Rotundene (0.04 %), (R)-Lavandulyl acetate (0.02 %) were identified. As reported by Prakash et al.(2010), Bottini et al. (1987), Nigam et al.(1979), Garg et al.(1977), Dixit et al.(1977) and Sabulal et al.(2007) have reported 1,8-cineole and α-eudesmol (17.0),
β-eudesmol (12.6), (E) Caryophyllene (16.6), Linalool (18.0), Linalool (25.6) respectively(9-14).The oil showed significant antimicrobial activity against the tested organisms of Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Candida
Table 1: Physicochemical analysis of Hedychium spicatum rhizomes
Figure 1: TLC fingerprint of Hedychium spicatum rhizomes
Table II: Percentage composition of the essential oil obtained from rhizomes of Hedychium spicatum.
Most of the essential oils contain complex blend of components and thus have various antimicrobial properties; most of this action appears to have derived from oxygenated terpenoids, Being lipophilic compound, essential oils cross the cytoplasmic membrane and cell wall thereby the cytotoxic activity appears to be linked to disarray of the structures present in different layers of polysaccharides, fatty acids and phospholipids. Due to this mechanism of action it acts on various targets at the same time. Permeability, chemical configuration and
surface charge of the outer structures of the microorganisms mainly determined these differences; the lipophilicity of terpenes is linked with the antimicrobial mechanism. Numerous reports have been reported regarding the mechanisms of antimicrobial property of the oils, and some studies have been partly elucidated. The antimicrobial investigation of the oil was reported using Broth dilution method. According to this method, the oil showed antimicrobial activity against the selected pathogens; Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Candida albicans. Amphotericin B and Ciprofloxacin were kept as standards for Candida albicans and for all the bacterial strains respectively. Percent concentration showing complete inhibition against the selected pathogens; Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Candida albicans were found to be 25%,25%,25%, 50% and 1.56% respectively as mentioned in Table III. Amphotericin B and Ciprofloxacin showed activity against all the organisms at 1.56% (0.31µg/ml).
Table III: Percent concentration showing complete inhibition of tested organisms
The Hedychium spicatum rhizome oil showed significant positive antimicrobial activity against all the tested organisms of Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Candida albicans. The essential oil contains 50.1 % of Ethyl p-methoxycinnamate and 26.22% of Ethyl cinnamate as major components identified through GC-MS which is reported for its UV protection activity (15). This oil can be explored further for cosmetic and pharmaceutical applications.
Authors are highly thankful to Dr. Rangesh P, Chief Scientific Officer, Dr. Venugopal, Group leader, Microbiology department and Dr. Vijayakumar M., Senior Research Scientist, Phytochemistry department, The Himalaya Drug Company for providing necessary inputs and facilities in this research to successfully complete the experiment.