Chemical composition and biological activities of rhizome and fruit rind oils of Alpinia mutica from south India

Abstract Volatile oils from dry rhizomes and fruit rinds of Alpinia mutica were isolated and characterized by GC-FID and GC-MS. A. mutica rhizome oil showed forty-seven components of which forty (92.8%) were characterized, and the major components were β-pinene (20.2%), camphor (13.3%), 1,8-cineole (8.9%), camphene (7.9%) and α-pinene (6.2%). Fruit rind essential oil showed sixty-nine components of which sixty-three (97.8%) were identified. Major constituents in A. mutica fruit rind oil were 1,8-cineole (14.8%), camphor (11.7%), β-pinene (7.6%) and camphene (4.8%). Four major constituents in both A. mutica rhizome and fruit rind oils (camphene, β-pinene, 1,8-cineole, camphor) were estimated by external standardization. Refractive index, specific rotation and specific gravity of both volatile oils were determined. Fruit rind oil showed significant antioxidant, cytotoxic and moderate antimicrobial activities. A. mutica dry fruit rind oil has a very pleasant smell with potential applications in fragrances.


Identification of essential oil constituents
Authentic standards of major terpenoids were co-injected with A. mutica rhizome and fruit rind oils on GC-FID (Table 1). Linear Retention Indices (LRI) of oil constituents were determined on the VF-5 column using standard C5-C30 straight chain hydrocarbons (Aldrich Chemical Company, USA). Individual compounds of A. mutica rhizome and rind oils were identified by Wiley and NIST database matching, comparison with LRIs and comparison of mass spectra with Adam's database and other literature reports (18,19) (Table 1).

Plant samples
Fresh rhizome and fruits of Aipinia mutica Roxb. were collected from the medicinal garden of Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Palode, Kerala during October 2013. The plant specimen was taxonomically confirmed by Dr. Mathew Dan, one of the authors, and a voucher specimen (TBGT 79403) was deposited at the herbarium of JNTBGRI. The rhizomes and fruit rinds of A. mutica were chopped and shade dried (separately).

Isolation of essential oils
Dried rhizomes (500 g) and fruit rinds (150 g) of A. mutica were subjected to hydrodistillation (separately) using Clevenger-type apparatus for 6 hours (each). The collected essential oils were dried over anhydrous Na 2 SO 4 . Rhizome oil was yellow and rind oil was pale yellow in color, and both oils were pleasant smelling. The rhizome and rind oils were stored at 4°C for further studies.

Physical parameters
Physical parameters viz., refractive index (J257 Digital Refractometer, Rudolf Research Analytical, USA), specific rotation (Autopol IV Polarimeter, Rudolf Research Analytical, USA) and specific gravity of A. mutica rhizome and fruit rind oils were measured.

GC-FID analysis
Rhizome and fruit rind oils of A. mutica (50 μL each) were diluted to 3 mL in acetone and their GC-FID analyses were carried out by injecting 1 μL (each) of these diluted oils onto a GC-2010 Plus Gas Chromatograph with AOC-20i autoinjector and FID (Shimadzu, Japan), fitted were visualized surrounding the discs. The determinations were done in duplicates. After 24 hours incubation, the plates were examined for inhibition zones. The diameters of the inhibition zones produced by each (concentrations) of the oil/control solutions were measured in millimeters and interpreted using the CLSI zone diameter interpretative standards (20) (Table 3).
gel MS capillary column and the area response in each injection was recorded. Area versus concentration curve of each standard was prepared. A. mutica rhizome and fruit rind oils were prepared (at 15 mg in 2 mL in acetone) and injected onto GC-FID and their area responses were also recorded. The major constituents in A. mutica rhizome and fruit rind oils were quantified from these area response-concentration (essential oils/standards) data (Table 2).

(ii) Superoxide radical scavenging activity
Superoxide radical scavenging activities of A. mutica rhizome and fruit rind oils were tested by light-induced superoxide generation (23). Phosphate buffer (0.069 M, pH 7.4, 2650 μL), 100 μL of nitro blue tetrazolium (1.5 mM) and 200 μL of potassium cyanide (0.0015% in 10 mM EDTA) each were taken in test tubes. To these solution(s), different concentrations of A. mutica rhizome oil, fruit rind oil, quercetin (0.1, 1, 5, 10, 20 μg/mL in methanol, 10 μL each) or methanol (10 μL, vehicle control) were added separately and mixed well. Then 50 μL of riboflavin (0.12 mM) was added to each mixture, and optical densities were recorded at 560 nm before illumination. These reaction mixtures were illuminated with an incandescent lamp for 15 minutes and final optical densities were recorded again at 560 nm and percentage inhibition(s) were calculated (Table 5).
In in vitro hydroxyl radical scavenging activity, A. mutica rhizome oil, fruit rind oil and quercetin, showed varying levels of activity and the values corresponded to 22.2 ± 3.3, 81.0 ± 2.1 and 46.0 ± 0.5% (20 μg/mL) respectively. IC 50 value of the fruit rind oil is highly significant (1.1 μg/mL), which is better than hydroxyl radical scavenging activities of both rhizome oil and standard compound, quercetin. Even at the highest dose of rhizome oil and quercetin tested (20 μg/mL) showed less than 50% inhibition only (Table 6). A. mutica fruit rind oil showed better DPPH radical scavenging activity (56.5 ± 4.6% at 20

Cytotoxic activity
Cytotoxic activities of A. mutica rhizome and fruit rind oils were tested by the trypan blue exclusion method (26). Dalton's lymphoma ascites (DLA) cells were aspirated with phosphate buffered saline (pH 7.4) and a cell suspension of 1 × 10 6 cell/mL in PBS was prepared. Cytotoxicity was assessed by incubating DLA cells (1 × 10 6 cells/mL) in PBS with 0.1% DMSO (vehicle control), different concentrations of A. mutica rhizome and fruit rind oils in 0.1% DMSO (0.1, 1, 5, 10, 20 μg/mL) for 3 hours at 37°C. After incubation the control and test cells were mixed with trypan blue and observed under compound light microscope and cell deaths were determined (Table 8).