2.1. Collection of Plant Samples

The plant samples of different accessions of C. amada were collected from various geographical locations of Odisha [Table 1 and Figure 1] and were later identified by a taxonomist. The identified samples were planted in the green house for sample maintenance in the Siksha O Anusandhan herbarium for further future use.

Table 1: Geographical coordinates of collected Curcuma amada accessions.

Figure 1: Curcuma amada rhizomes collected from different regions of Odisha. [Click here to view]

2.2. Extraction of Essential Oil

Fresh samples of rhizome (100 g) of C. amada were taken for oil extraction through hydro distillation for about 5–6 h with the help of a Clevenger-type apparatus. To remove the moisture content in the extracted oil, it was treated with anhydrous sodium sulfate and was preserved in the refrigerator (4°C) until further analysis. The oil yield percentage was evaluated on the fresh weight basis (v/w).

2.3. Chemical Analysis of Essential Oil

GC-MS analysis was carried out using Clarus 580 Gas Chromatogram (Perkin Elmer, USA) equipped with a MS detector with Helium gas as a carrier gas with flow rate of 1 mL/min. 0.1 μL of rhizome essential oil was injected and the Elite-5 column (30 cm length × 0.25 mm i.d., film thickness 0.25 μm) was used. The oven temperature was equilibrated at 50°C for 1 min, heated at 5°C/min to 230°C with 5 min hold, and finally raised at 15°C/min to 260°C with 1 min hold. At 250°C and 260°C, the temperature of the injector and both the transfer line, and ion source was set, respectively. The total run time was 45 min. The scanning was done over a mass scan range of 50–600 m/z. The ion chromatogram and mass spectra were acquired using Turbo mass TM software 5.4. The n-alkane series was used for retention index (RI) identification, and compound identification was done through Adams Library [22].

2.4. Antioxidant Activities

The antioxidant activity was evaluated by DPPH radical scavenging assay following the protocol of Sahoo et al. with slight modifications [23]. Different concentrations (1, 5, 10, 20, and 30 μg/mL) of methanolic solution of essential oils were mixed with 1 mL of 0.1 mM DPPH. The reaction mixtures were mixed properly and were kept at room temperature for 30 min in dark. At 517 nm, the absorbance of the sample was measured using ultraviolet-visible spectrophotometer (Thermo Scientific, Waltham, MA). Butylated hydroxytoluene and ascorbic acid were taken as the positive control meanwhile methanol and DPPH solution was taken as control and IC₅₀ value was measured.

2.5. Bacterial Strains

The antimicrobial activity of essential oil of C. amada rhizome was illustrated against two Gram-negative bacteria (Escherichia coli and Acinetobacter baumannii) and two Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus). The bacterial strains were collected from the Department of Microbiology, SOADU, Bhubaneswar.

2.6. Antimicrobial Activity

The antimicrobial activity was measured by checking the minimum inhibitory concentrations (MIC) by broth microdilution method as described by the guidelines of Clinical and Laboratory Standards Institute and following the protocol of Dash et al. [24]. The experiment was accomplished using Mueller–Hinton Broth (MHB) for all the bacterial strains. The rhizome essential oils (100 μL) with a concentration of 100 μg/mL were prepared by mixing them with dimethyl sulfoxide in sterile Eppendorf tubes. The viable bacterial culture (10⁶ CFU/mL of microorganisms) was prepared from overnight suspension. The rhizome volatile oils were analyzed by a 2-fold serial-dilution method with MHB in a 96-microtiter (enzyme-linked immunosorbent assay) plate. Ampicillin was taken as standard. MIC was defined as the concentration that showed no growth visibility or turbidity during the highest dilution of the sample.

3.1. GC-MS Analysis

Rhizomes of C.amada yields a good amount of essential oil (0.12–1.35% v/w) [Table 2] which was a pale yellow liquid having a strong aroma. It was reported that 0.5% (v/w) of rhizome oil yield on fresh weight basis taken from the foothills of Uttarakhand, India [25], while 1.25% (v/w) of rhizome oil yield was reported from Kerala Agriculture University, India [26]. Later, in the present experiment, the RO was subjected to GC-MS analysis which detected 56 peaks. The analysis revealed a total of 84.38–97.37% detectable area percentage comprising all major and minor constituents. Parenthetically, the C. amada ROs of all the accessions were composed mainly of monoterpenoids (89%) with 83% hydrocarbons and 6% of oxygenated counterparts [Figure 2]. Alike the present study findings monoterpenoids (97.22%) with a major 96.75% of hydrocarbon fraction and 0.97% of oxygenated ones were found predominantly in C. amada RO [25]. The essential oils of the mango ginger (Curcuma amada) accessions were found to contain a total of 56 compounds. The major constituents identified were myrcene (67.59–72.97%), (Z)-(Z)-Geranyl linalool (4.3–7.79%), (Z)-(E)-Geranyl linalool (4.05–7.23%), β-ocimene (2.9–6.33%), and β-pinene (1.23–4.82%). Additionally, α-pinene (0.1–0.96%) and (E)-caryophyllene (0.01–1.95%) were present as minor constituents in the essential oils [Table 3 and Figure 3]. A similar study on C. amada RO reported myrcene (40%) and β-pinene (11.78%) as the major constituents [7]. On contrary, a phytochemical screening of RO EOs detected 28 constituents in which ar-curcumene (28.1%), camphor (11.2%), β-cumene (11.2%), curzerenone (7.1%), and eucalyptol (6.0%) were identified as the major constituents which deviate from the present study findings [27]. The analysis result showed a mixture of different compounds in which oxygenated sesquiterpenes (17 compounds) were found in majority followed by oxygenated monoterpene (13 compounds) and monoterpene hydrocarbons (12 compounds). The characteristic aroma of C. amada is contributed by the various combinations of compounds, that is, myrcene, ocimene, cis-, and trans-dihydroocimene [28].

Table 2: Yield percentage of Curcuma amada rhizome oil collected from different regions.

Figure 2: Class distribution of compounds studied by gas chromatography-mass spectrometry in the rhizome essential oil of Curcuma amada. [Click here to view]

Table 3: Qualitative phytochemical analysis of rhizome samples of Curcuma amada.

Figure 3: Gas chromatography-mass spectrometry chromatogram of Curcuma amada rhizome oil detecting various volatile constituents. [Click here to view]

3.2. Antioxidant Activity

DPPH free radical-scavenging activity of the C. amada ROs of different accessions was investigated. The results were expressed against different concentrations and the IC₅₀ values were calculated [Table 4]. As per the calculated IC₅₀ values, it was observed that Ca17 has got considerable antioxidant properties with an IC₅₀ value of 32.05 μg/mL, whereas Ca10 has shown the lowest with IC₅₀ value of 38.2 μg/mL, indicating the influence of phytochemical variation, geographic distribution, and edaphic factors on the measurement of antioxidant properties. The present study results are in agreement with one of the previous reports, which showed IC₅₀ value of RO to be 34.7 mg/mL [21]. A report has shown appreciable antioxidant property of RO of C. amada with IC₅₀ of 25 mg/mL [7]. It can be concluded that with an increase in the concentration of the RO, an increase in the scavenging activity was observed. Various reports were presented on different extracts of leaf and rhizome [4,9,29], but to date, very scanty reports were presented on the antioxidant activity of RO [4,9]. Therefore, the present report can be used for further analysis of the scavenging property of RO which can be used in baking industry as natural antioxidant.

Table 4: DPPH free radical scavenging activity of C. amada rhizome essential oil.

3.3. Antimicrobial Activity

The antimicrobial activity of rhizome essential oils of C. amada accessions was evaluated against Gram-positive and Gram-negative bacterial strains by measuring the MIC values. The ROs of all the accessions demonstrated variable degrees of antibacterial potential against tested microbes. The inhibitory activity of C. amada was compared to that of the commercially available antibiotic Ampicillin which was used as the control. The MIC values of rhizome essential oils ranged from 1.56 to 25 μg/mL [Table 5]. It was observed that the rhizome essential oils showed more activity against A. baumannii (MIC: 1.56 μg/mL), followed by S. aureus (MIC: 3.12 μg/mL), E. coli, and B. subtilis (MIC: 6.25 μg/mL, respectively). Among all the accessions, Ca17 showed the highest antimicrobial potential against all the strains (3.12 μg/mL against B. subtilis and S. aureus, 1.56 μg/mL against A. baumannii and 6.25 μg/mL against E. coli). A similar report was showing potential antimicrobial activity by agar disk-diffusion method against S. aureus, E. coli, and B. Subtilis (18 mm, 16 mm, and 16 mm of inhibition zone, respectively) using ROs [19]. In the current study, it has been shown that the growth of the tested bacteria can be inhibited using C. amada ROs and that the bactericidal activity becomes more potent with an increasing concentration of RO. Although, one report exhibited antibacterial activity using ROs against Ralstonia solanacearum showing inhibition zone ranging from 3 to 7 mm [30], but no clear-cut data are available on the MIC values of rhizome essential oil of C. amada till date. The antimicrobial properties of the essential oil are believed to be attributed to its high levels of monoterpenes, which have been found to exhibit effectiveness against a wide range of susceptible microorganisms [19]. The present study findings from the GC-MS analysis of the rhizome samples constituted a rich amount of monoterpenes; its synergistic effects may contribute to its antimicrobial properties. Moreover, the variation in antimicrobial activity may be possible due to various edaphic factors and different geographical locations. From the above results, it can be concluded that the tested microbes are sensitive toward the ROs of C. amada. Therefore, the data can be utilized for making value-added products in the food industry.

Table 5: Minimum inhibitory concentration (MIC in μg/ml) of C. amada essential oils against different strains.