Variation in Yield, Chemical Composition and Biological Activities of Essential Oil of Three Curcuma Species: A Comparative Evaluation of Hydrodistillation and Solvent-Free Microwave Extraction Methods

The essential oils of three medicinally important Curcuma species (Curcuma alismatifolia, Curcuma aromatica and Curcuma xanthorrhiza) were extracted using conventional hydro-distillation (HD) and solvent free microwave extraction (SFME) methods. The volatile compounds from the rhizome essential oils were subsequently analysed by GC–MS. The isolation of essential oils of each species was carried out following the six principles of green extraction and comparison was made between their chemical composition, antioxidant, anti-tyrosinase and anticancer activities. SFME was found to be more efficient than HD in terms of energy savings, extraction time, oil yield, water consumption and waste production. Though the major compounds of essential oils of both the species were qualitatively similar, there was a significant difference in terms of quantity. The essential oils extracted through HD and SFME methods were dominated by hydrocarbon and oxygenated compounds, respectively. The essential oils of all Curcuma species exhibited strong antioxidant activity, where SFME was significantly better than HD with lower IC50 values. The anti-tyrosinase and anticancer properties of SFME-extracted oils were relatively better than that of HD. Further, among the three Curcuma species, C. alismatifolia essential oil showed the highest rates of inhibition in DPPH and ABTS assay, significantly reduced the tyrosinase activity and exhibited significant selective cytotoxicity against MCF7 and PC3 cells. The current results suggested that the SFME method, being advanced, green and fast, could be a better alternative for production of essential oils with better antioxidant, anti-tyrosinase and anticancer activities for application in food, health and cosmetic industries.


Introduction
Nowadays, there is a growing demand for aromatic plants due to the global resurgence of interest in natural products for health care and therapeutic purposes. The therapeutic properties of herbal and aromatic plants are partially attributed to essential oils. Essential oils, a group of interesting natural products, have gained greater popularity in food, cosmetics, aromatherapy and pharmaceutical industries as an alternative to synthetic

Determination of Extraction Parameters and Essential Oil Yield
Mature and thick rhizomes were selected from the harvested Curcuma species to extract their essential oils using HD and SFME methods. The essential oils had a pleasant aroma and were liquid at room temperature. The extraction time, energy consumption, colour of volatile oil and hydration capacity of the two extraction methods are shown in Table 1. A wattmeter was used to measure the power consumption at the entrance of the microwave generator and the electrical heater power source. Figure 1 represents a comparative analysis of essential oil yield of the studied species, which varied in the range of 0.25% and 0.68% (v/w) on dry weight basis, signifying statistical differences at p < 0.05 (Tukey test). Among the three species, the maximum oil yield was recorded for C. aromatica (SFME-0.68 ± 0.05%, HD-0.43 ± 0.04%) followed by C. xanthorrhiza (SFME-0.52 ± 0.04%, HD-0.35 ± 0.02%) and C. alismatifolia (SFME-0.43 ± 0.03%, HD-0.25 ± 0.02%). In C. alismatifolia, the oil yield (0.25%) obtained in the present study by the hydrodistillation method was relatively higher than the yield (0.12%) reported by Theanphong et al. [32,33]. Similarly, the oil yield of C. aromatica (0.43%) and C. xanthorrrhiza (0.35%) obtained through hydrodistillation was relatively less than the oil yields reported in few previous studies [34][35][36]. In comparison to HD, SFME yielded higher amounts of oil in all the species, which might be due to its efficient and homogenous extraction technique, which provides controlled temperature and microwave energy to the sample matrix, as a result of which cells are ruptured by internal superheating, thus facilitating better diffusion of constituents from the matrix and thereby enhancing the recovery of essential oil [37,38]. Several plant species have been found to produce higher amounts of essential oils using a microwave extraction technique [20,29,37]. In an effort to determine the best extraction method, HD and SFME were compared based on the six green extraction principles proposed by Chemat et al. [39]. The analysis was carried out based on raw materials, oil yield, operating duration, waste generation, water and energy consumption.
In Figure 2, the HD and SFME procedures are graphically represented and categorized in accordance with the six green extraction principles. The percentage equivalents of each principle's values were converted and presented on the graph. The advantages of SFME over HD for the extraction of Curcuma essential oil is that it uses less energy, produces less waste, uses less solvent and gives a higher yield over a shorter extraction period.   In Figure 2, the HD and SFME procedures are graphically represented and categorized in accordance with the six green extraction principles. The percentage equivalents of each principle's values were converted and presented on the graph. The advantages of SFME over HD for the extraction of Curcuma essential oil is that it uses less energy, produces less waste, uses less solvent and gives a higher yield over a shorter extraction period.

Comparison of Chemical Composition Obtained by HD and SFME
The identification of compounds of essential oils obtained from both extraction methods was performed using GC-MS analysis; the compounds were classified according to their order of elution on Elite-5 MS column and are presented in Table 2. A total of 85 compounds were identified constituting 90.76% to 94.97% of the total essential oils in the three Curcuma species. In C. alismatifolia, 50 compounds were identified in essential oils

Comparison of Chemical Composition Obtained by HD and SFME
The identification of compounds of essential oils obtained from both extraction methods was performed using GC-MS analysis; the compounds were classified according to their order of elution on Elite-5 MS column and are presented in Table 2. A total of 85 compounds were identified constituting 90.76% to 94.97% of the total essential oils in the three Curcuma species. In C. alismatifolia, 50 compounds were identified in essential oils obtained through HD and SFME methods accounting for 94.02% and 94.84% of the total oils, respectively. Similarly, in C. aromatica essential oil, 49 compounds could be identified by HD and SFME techniques which constituted 93.12% and 94.97% of the total oil, respectively. The essential oil of C. xanthorrhiza had 48 identified chemical constituents. This constitutes 90.76% of the total essential oils isolated through the HD method and 93.82% via the SFME method.   Overall, the chemical makeup of the essential oils of the three studied Curcuma species was dominated by oxygenated sesquiterpenes (36.09 to 73.42%) followed by oxygenated monoterpenes (4.43 to 31.13%), where both the chemical groups were present in substantially higher amounts in oils extracted through SFME method compared to HD. The amount of sesquiterpene hydrocarbons (5.72 to 15.26%) and monoterpene hydrocarbons (3.01 to 15.64%) in the essential oils varied considerably between the two extraction methods. Table 2 shows that HD is more efficient in isolating hydrocarbon molecules, whereas SFME is more effective in isolating most of the oxygenated compounds. Djouahri et al. [37] made a similar observation while extracting Tetraclinis articulata essential oil using hydrodistillation and microwave-assisted extraction. Significant differences were observed in the quantity of major compounds between the two methods which might be due to a reduction in thermal and hydrolytic reactions compared with hydrodistillation, which utilizes large amounts of water and energy [41]. Similar results were obtained in C. longa essential oil that reported differences in major compounds extracted by a microwave-assisted technique [42]. The variations in the main constituents had no significant effect on the bioactivities of the essential oils. Figure 3 shows the percentage composition of the principal components of essential oils of the three Curcuma species using both the techniques. The major compounds identified in the essential oil of C. alismatifolia are curzerenone (40.6% vs. 26.47% for HD vs. SFME), germacrone (8.63% vs. 12.35% for HD vs. SFME), 1,8 cineole (6.47% vs. 16.6% for HD vs. SFME) and camphor (3.78 % vs. 9.61% for HD vs. SFME). Similarly, essential oil of C. aromatica contained curlone (β-turmerone) (14.19% vs. 12.31% for HD vs. SFME), germacrone (6.69% vs. 16.44% for HD vs. SFME), 1,8 cineole (13.02% vs. 7.6% for HD vs. SFME), camphor (8.45% vs. 14.04% for HD vs. SFME) and xanthorrhizol (6.76% vs. 13.07% for HD vs. SFME), whereas curzerenone (56.34% vs. 62.81% for HD vs. SFME), xanthorrhizol (6.23% vs. 2.84% for HD vs. SFME), germacrone (4.21% vs. 4.44% for HD vs. SFME) and camphor (3.33% vs. 2.71% for HD vs. SFME) were detected in C. xanthorrhiza essential oil. Very few reports are available on the chemical composition of C. alismatifolia rhizome essential oil that revealed β-curcumene and xanthorrhizol as its major constituents [32,33]. The current study identified a new curzerenone-rich essential oil bearing chemotype of C. alismatifolia extracted using both HD and SFME methods. The chemical composition of C. aromatica essential oil now reported is consistent with the earlier reports, where curlone (β-turmerone), 1,8 cineole, germacrone, and xanthorrhizol have been found to be the major constituents. Similarly, curzerenone, xanthorrhizol, germacrone, and camphor were detected as predominant components in the essential oil of C. xanthorrhiza [14,35,[43][44][45][46]. Furthermore, compounds such as ar-curcumene, xanthorrhizol and β-curcumene, reported as major compounds in earlier studies, were present in trace amounts in the present investigation [32,47,48]. No qualitative variations in essential oils were observed between the extraction methods of each species as the compounds detected either by HD or SFME were the same. Fiorini et al. [20] and Araujo et al. [21] also observed no qualitative differences between essential oils of Cannabis sativa and Thymus mastichina extracted from the hydrodistillation and microwave extraction methods, respectively. For the first time, the present study utilized HD and SFME methods to conduct a qualitative and quantitative analyses of the yield and chemical composition of the essential oils of C. alismatifolia, C. aromatica and C. xanthorrhiza.

Antioxidant Activity
The in vitro antioxidative capacity of three Curcuma essential oils extracted via HD and SFME was studied using DPPH and ABTS radical scavenging assays. The DPPH and ABTS radicals are prominent substrates for rapid evaluation of antioxidant activity due to their radical stability and efficiency. Butylated hydroxytoluene and ascorbic acid were taken as the standard positive controls for both the assays. Several concentrations ranging from 1-50 µg/mL were used to analyse the percentage of inhibition as shown in Figures 4 and 5. Their IC 50 values were also calculated. In DPPH assay, the Curcuma essential oils exhibited moderate scavenging activity compared with BHT and ascorbic acid, with an IC 50 value of 15.6 ± 0.14 µg/mL and 4.2 ± 0.08 µg/mL, respectively. C. alismatifolia essential oil had the maximum antioxidant activity with an IC 50 value of 28.2 ± 0.19 µg/mL for HD and 19.1 ± 0.16 µg/mL for SFME followed by C. aromatica (36.3 ± 0.21 µg/mL vs. 23.1 ± 0.18 µg/mL for HD vs. SFME) and C. xanthorrhiza (45.2 ± 0.24 µg/mL vs. 36.8 ± 0.14 µg/mL for HD vs. SFME). Similarly, Curcuma essential oils revealed potent scavenging activity in the ABTS assay compared to BHT and ascorbic acid with an IC 50 value of 13.6 ± 0.12 µg/mL and 3.1 ± 0.06 µg/mL, respectively. In this assay, C. alismatifolia essential oil showed the maximum scavenging capacity with an IC 50 value of 19.3 ± 0.22 µg/mL for HD and 15.24 ± 0.17 µg/mL for SFME followed by C. aromatica (28.3 ± 0.27 µg/mL vs. 17.4 ± 0.18 µg/mL for HD vs. SFME) and C. xanthorrhiza (36.7 ± 0.22 µg/mL vs. 29.4 ± 0.17 µg/mL for HD vs. SFME). The current research revealed that C. alismatifolia essential oil had the highest antioxidant capacity, followed by C. aromatica and C. xanthorrhiza in both assays.
Molecules 2023, 28, x FOR PEER REVIEW 10 of 25 Figure 3. Percentage composition of major compounds in C. alismatifolia, C. aromatica and C. xanthorrhiza essential oils obtained using HD and SFME methods. Different letters denote a significant difference in the mean value of constituents at (p < 0.05).

Antioxidant Activity
The in vitro antioxidative capacity of three Curcuma essential oils extracted via HD and SFME was studied using DPPH and ABTS radical scavenging assays. The DPPH and ABTS radicals are prominent substrates for rapid evaluation of antioxidant activity due to their radical stability and efficiency. Butylated hydroxytoluene and ascorbic acid were taken as the standard positive controls for both the assays. Several concentrations ranging from 1-50 µg/mL were used to analyse the percentage of inhibition as shown in Figures 4 and 5. Their IC50 values were also calculated. In DPPH assay, the Curcuma essential oils exhibited moderate scavenging activity compared with BHT and ascorbic acid, with an IC50 value of 15.6 ± 0.14 µg/mL and 4.2 ± 0.08 µg/mL, respectively. C. alismatifolia essential oil had the maximum antioxidant activity with an IC50 value of 28.2 ± 0.19 µg/mL for HD and 19.1 ± 0.16 µg/mL for SFME followed by C. aromatica (36.3 ± 0.21 µg/mL vs. 23.1 ± 0.18 µg/mL for HD vs. SFME) and C. xanthorrhiza (45.2 ± 0.24 µg/mL vs. 36.8 ± 0.14 µg/mL for HD vs. SFME). Similarly, Curcuma essential oils revealed potent scavenging activity in the . Percentage composition of major compounds in C. alismatifolia, C. aromatica and C. xanthorrhiza essential oils obtained using HD and SFME methods. Different letters denote a significant difference in the mean value of constituents at (p < 0.05).
Our findings are in conformity with those of previously published reports that C. alismatifolia extracts have strong antioxidant capacity and good reducing power in which the standard ascorbic acid had an IC 50 value of 3.76 µg/mL [15,49]. Rhizome essential oils of C. aromatica and C. xanthorrhiza are also reported to possess considerable antioxidant activity in both the radical scavenging assays and β-carotene bleaching tests as well. Trolox C, ascorbic acid and butylated hydroxyanisole were used as the standard references in these studies, and had an IC 50 value of 8.82, 7 and 18 µg/mL, respectively [35,[50][51][52][53]. The high antioxidant effect of rhizome essential oils might be due to the presence of major compounds such as 1,8 cineole, xanthorrhizol, germacrone, curzerenone and minor constituents including terpinolene and myrcene, which are considered potential antioxidant molecules [43,54]. Curcuma species are extensively used as therapeutic agents for disorders caused by free radicals [52] and an attempt has been made in the current study to validate the antioxidant potential of essential oil extracted via HD and SFME methods. The essential oil extracted using SFME was found to be potent scavenger as it inhibited more free radicals than HD at lower concentrations and exhibited low IC 50 value in all Curcuma species. The wide range of IC 50 values observed between both extraction methods concluded that the antioxidant property of SFME-extracted oil is significantly better than oil extracted through HD. The difference in the results of the antioxidant assay could be due to several factors including temperature and extraction time. Prolonged extraction time and high temperature might be responsible for the breakdown of antioxidant molecules [55]. As previously reported, C. longa essential oil possessed higher antioxidant properties in microwave-assisted extraction than the conventional Soxhlet method [29]. Similar findings were also observed in essential oils of several plant species [2,34,50].  Our findings are in conformity with those of previously published reports that C. alismatifolia extracts have strong antioxidant capacity and good reducing power in which the standard ascorbic acid had an IC50 value of 3.76 µg/mL [15,49]. Rhizome essential oils of C. aromatica and C. xanthorrhiza are also reported to possess considerable antioxidant activity in both the radical scavenging assays and β-carotene bleaching tests as well. Trolox C, ascorbic acid and butylated hydroxyanisole were used as the standard references in these studies, and had an IC50 value of 8.82, 7 and 18 µg/mL, respectively [35,[50][51][52][53]. The high antioxidant effect of rhizome essential oils might be due to the presence of major compounds such as 1,8 cineole, xanthorrhizol, germacrone, curzerenone and minor constituents including terpinolene and myrcene, which are considered potential antioxi- properties in microwave-assisted extraction than the conventional Soxhlet method [29]. Similar findings were also observed in essential oils of several plant species [2,34,50]. It seems possible that a change in the concentration of any specific molecule could play a significant role in expressing the antioxidant and biological properties of the substance. The high antioxidant activity of SFME-derived essential oils might be attributed to the presence of a greater concentrations of oxygenated compounds in C. alismatifolia (69.29% vs 76.1% for HD vs. SFME), C. aromatica (66.41% vs 83.37% for HD vs. SFME) and C. xanthorrhiza (76.01% vs 79.04% for HD vs. SFME), respectively. The present finding is in agreement with the observations of Berga-Zougali et al. [56] from France, who employed HD and SFME methods to characterize Myrtus communis essential oil using GC-MS and assessed their bioactivities.

Anti-Tyrosinase Activity
Tyrosinase is a vital enzyme that can accelerate melanin production and enzymic browning caused by bacteria, fungi, plants, insects, and ultraviolet radiation. Free radicals serve a major part in melanin biosynthesis that can cause skin pigmentation, freckles and age spots in humans [57]. The in vitro tyrosinase inhibitory activity of three Curcuma essential oils extracted using HD and SFME methods was studied using a spectrophotometric assay. The results indicate a significant percentage inhibition of mushroom tyrosinase enzyme with treatment of essential oil samples and kojic acid at different concentrations It seems possible that a change in the concentration of any specific molecule could play a significant role in expressing the antioxidant and biological properties of the substance. The high antioxidant activity of SFME-derived essential oils might be attributed to the presence of a greater concentrations of oxygenated compounds in C. alismatifolia (69.29% vs 76.1% for HD vs. SFME), C. aromatica (66.41% vs 83.37% for HD vs. SFME) and C. xanthorrhiza (76.01% vs 79.04% for HD vs. SFME), respectively. The present finding is in agreement with the observations of Berga-Zougali et al. [56] from France, who employed HD and SFME methods to characterize Myrtus communis essential oil using GC-MS and assessed their bioactivities.

Anti-Tyrosinase Activity
Tyrosinase is a vital enzyme that can accelerate melanin production and enzymic browning caused by bacteria, fungi, plants, insects, and ultraviolet radiation. Free radicals serve a major part in melanin biosynthesis that can cause skin pigmentation, freckles and age spots in humans [57]. The in vitro tyrosinase inhibitory activity of three Curcuma essential oils extracted using HD and SFME methods was studied using a spectrophotometric assay. The results indicate a significant percentage inhibition of mushroom tyrosinase enzyme with treatment of essential oil samples and kojic acid at different concentrations ( Figure 6). The highest rate of inhibition was shown by C. alismatifolia essential oil having an IC 50 value of 119.1 ± 1.89 µg/mL for HD and 112.4 ± 1.62 µg/mL for SFME, followed by C. xanthorrhiza (157.5 ± 2.27 µg/mL vs. 151.4 ± 0.18 µg/mL for HD vs. SFME) and C. aromatica (2.71 ± 0.23 mg/mL vs. 2.69 ± 0.22 mg/mL for HD vs. SFME). Kojic acid is a natural tyrosinase inhibitor that inactivates the enzyme by chelating copper and inhibiting tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid [58] and was used as a positive control in the present experiment. Though the efficacy of three Curcuma essential oils was lower than the standard kojic acid with an IC 50 value of 82.84 ± 0.12 µg/mL, they can be considered potential tyrosinase inhibitors. The antityrosinase property of the essential oils might be attributed to the presence of specific compounds such as 1,8-cineole, α-pinene, α-terpineol, xanthorrhizol, camphor, which are known enzyme inhibitors [59][60][61]. Different solvent extracts of the rhizomes of C. aromatica, C. xanthorrhiza, C. longa, C. aeruginosa and C. amada have been investigated in recent times and some important findings have been reported. These studies employed kojic acid as the positive control that showed an IC 50 value of 0.01 mg/mL [62]. To date, there are no reports on the enzyme-inhibitory activity of C. alismatifolia essential oil. Extensive studies on tyrosinase inhibitors in C. longa have been made at in silico, in vitro and in vivo levels [63][64][65][66], whereas the remaining species have only been worked on a basic level. Thus, the present observations about the three Curcuma species might improve our knowledge on the anti-tyrosinase activity of different species of the genus Curcuma and lead to the development of plant-based skin whitening cosmetics.
Molecules 2023, 28, x FOR PEER REVIEW 13 of 25 ( Figure 6). The highest rate of inhibition was shown by C. alismatifolia essential oil having an IC50 value of 119.1 ± 1.89 µg/mL for HD and 112.4 ± 1.62 µg/mL for SFME, followed by C. xanthorrhiza (157.5 ± 2.27 µg/mL vs. 151.4 ± 0.18 µg/mL for HD vs. SFME) and C. aromatica (2.71 ± 0.23 mg/mL vs. 2.69 ± 0.22 mg/mL for HD vs. SFME). Kojic acid is a natural tyrosinase inhibitor that inactivates the enzyme by chelating copper and inhibiting tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid [58] and was used as a positive control in the present experiment. Though the efficacy of three Curcuma essential oils was lower than the standard kojic acid with an IC50 value of 82.84 ± 0.12 µg/mL, they can be considered potential tyrosinase inhibitors. The antityrosinase property of the essential oils might be attributed to the presence of specific compounds such as 1,8-cineole, α-pinene, α-terpineol, xanthorrhizol, camphor, which are known enzyme inhibitors [59][60][61]. Different solvent extracts of the rhizomes of C. aromatica, C. xanthorrhiza, C. longa, C. aeruginosa and C. amada have been investigated in recent times and some important findings have been reported. These studies employed kojic acid as the positive control that showed an IC50 value of 0.01 mg/mL [62]. To date, there are no reports on the enzymeinhibitory activity of C. alismatifolia essential oil. Extensive studies on tyrosinase inhibitors in C. longa have been made at in silico, in vitro and in vivo levels [63][64][65][66], whereas the remaining species have only been worked on a basic level. Thus, the present observations about the three Curcuma species might improve our knowledge on the anti-tyrosinase activity of different species of the genus Curcuma and lead to the development of plant-based skin whitening cosmetics.  In C. alismatifolia and C. xanthorrhiza, SFME-extracted oils had a greater inhibitory effect on the mushroom tyrosinase enzyme than HD-extracted oils, whereas essential oils of C. aromatica obtained from either method exhibited almost similar effects on the enzyme. In terms of enzyme-inhibitory action, SFME-extracted oils are comparatively better than HD-extracted oils, which might be due to the higher efficiency of the microwave extractor in the recovery of the bioactive compounds [67]. Similar findings have been reported by Maaiden et al. [68], who reported that microwave-assisted extraction (MAE) exhibited the highest anti-tyrosinase activity compared to maceration and Soxhlet extraction in some medicinal and aromatic plants.

Anti-Cancer Activity
The standard MTT assay was used to evaluate the anti-proliferative activity of Curcuma essential oils against HepG2, MCF7 and PC3 cancerous cell lines. The 3T3-L1 noncancerous/normal cell line was used to investigate the toxicity of the essential oils. When cells were exposed to increasing doses of essential oil (6.25-200 µg/mL), their viability decreased significantly (Figures 7 and 8). To compare the inhibitory action, doxorubicin was used as the standard reference drug that showed an IC 50 value of 1.43 ± 0.21 µg/mL, 1.16 ± 0.19 µg/mL, 2.02 ± 0.38 µg/mL and 811 ± 1.7 µg/mL in HepG2, MCF7, PC3 and 3T3-L1 cells, respectively. The inhibitory action of doxorubicin in the current study is somewhat similar to that reported in previous studies [69][70][71]. The MTT assay results suggested that the essential oil of C. alismatifolia showed significant selective cytotoxicity against MCF7 and PC3 cells, having an IC 50 value of 55.62 ± 0.89 µg/mL vs 52.86 ± 1.23 µg/mL for HD vs SFME and 102.24 ± 2.56 µg/mL vs 98.61 ± 1.87 for HD vs SFME, respectively, confirming the anti-breast and anti-prostate cancer potency. C. aromatica (71.67 ± 1.89 µg/mL vs. 69.92 ± 1.44 µg/mL for HD vs. SFME) and C. xanthorrhiza (97.28 ± 2.81 µg/mL vs. 94.22 ± 1.98 µg/mL for HD vs. SFME) essential oils exhibited promising anti-prostate cancer efficacy against PC3 cells. Additionally, the three Curcuma essential oils showed good anticancer properties against HepG2 cells exhibiting medium IC 50 values (Table 3). Curcuma essential oils on 3T3-L1 revealed a non-toxic effect with high IC 50 values, leading to the conclusion that the essential oils of Curcuma are safe to use and do not have any adverse side effects.    According to previous studies, Curcuma rhizome and leaf essential oils induce apoptosis or cell death in liver and lung cancer cells [72,73]. By inducing apoptotic cell death in multiple cancer cells, the essential oil of C. aromatica was found to have proven anticancer properties [13,74]. C. xanthorrhiza is reported to have anticancer properties against different cancer cell lines including cervical, breast, lung, colon, liver, oral, oesophageal and skin cancers due to the presence of xanthorrhizol [75]. This is the first in vitro anticancer study of C. alismatifolia essential oil with significant inhibitory ability, thus making it the most effective anticancer agent among the three Curcuma species.
Specific compounds like germacrone [76,77], curzerenone [77], camphor [78], 1,8 cineole [79,80], terpinolene [81] and α-pinene [82] which are previously known to cause Anticancer activity of Curcuma essential oils extracted using HD (A,C) and SFME (B,D) methods showing cell viability on MCF-7 and PC-3 cancer cells using the MTT assay after 24 h. The results of three independent experiments are presented as means ± SD (n = 3). ### p < 0.001 compared with the untreated group; ns p > 0.05, * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with the untreated group as determined by Dunnett's multiple comparison test. According to previous studies, Curcuma rhizome and leaf essential oils induce apoptosis or cell death in liver and lung cancer cells [72,73]. By inducing apoptotic cell death in multiple cancer cells, the essential oil of C. aromatica was found to have proven anticancer properties [13,74]. C. xanthorrhiza is reported to have anticancer properties against different cancer cell lines including cervical, breast, lung, colon, liver, oral, oesophageal and skin cancers due to the presence of xanthorrhizol [75]. This is the first in vitro anticancer study of C. alismatifolia essential oil with significant inhibitory ability, thus making it the most effective anticancer agent among the three Curcuma species.
Specific compounds like germacrone [76,77], curzerenone [77], camphor [78], 1,8 cineole [79,80], terpinolene [81] and α-pinene [82] which are previously known to cause apoptosis and signalling disruption in cancer cells, might be attributed to the cytotoxicity of essential oils. The anticancer properties of essential oils were not greatly influenced by the extraction process, while SFME-extracted oils were slightly better than HD-extracted oils due to their low IC 50 values. Very few reports are available on the effect of anticancer properties of oils isolated using different extraction methods, and here too, no correlation between the methods of extraction and anticancer activity could be established [83,84].

Cell lines and Culture Medium
Human hepatocellular adenocarcinoma (HepG2) cells, human prostate adenocarcinoma (PC3) cells, Human breast adenocarcinoma (MCF7) cells, and Mouse non-malignant embryo fibroblast (3T3-L1) cells were obtained from the Cell Bank of the National Centre for Cell Science (NCCS), Pune, India. The HepG2 cells were maintained in DMEM low-glucose media and PC3, MCF7, 3T3-L1 cells were maintained in DMEM high-glucose media supplemented with 10% FBS (Himedia, Thane, India) along with the 1% antibiotic-antimycotic solution and 1% L-Glutamine (200 mM). All cultures were grown in a humidified 5% CO 2 incubator at 37 • C and the cells were sub-cultured every two days.

Plant Materials
The rhizomes of three Curcuma species were harvested and collected in September 2022 from the net house/greenhouse of the Centre for Biotechnology, Siksha O Anusandhan (Deemed to be University), Bhubaneswar, India (20 • 17 1.348" N, 85 • 46 32.37" E, 75 m above sea level). The rhizomes were mature and healthy with uniform size and vigour, and were grown under similar pedoclimatic conditions. The species were authenticated and identified by Prof. Pratap Chandra Panda, Taxonomist, and voucher specimens of the three Curcuma species were preserved in the Herbarium of the Centre for Biotechnology, Siksha 'O' Anusandhan (Deemed to be University), Bhubaneswar, Odisha, India. The following were the voucher specimen numbers: Curcuma alismatifolia (CBT/2441), Curcuma aromatica (CBT/2442) and Curcuma xanthorrhiza (CBT/2443).

Extraction Methods
The essential oils of the three Curcuma species were extracted using conventional hydrodistillation (HD) and solvent-free microwave extraction (SFME) methods.

Hydrodistillation (HD)
Dried rhizomes of Curcuma species were rehydrated and hydrodistillated for 7 h using a Clevenger apparatus. The volatile oils were then extracted until no more oil was obtained in the apparatus and dried with sodium sulphate (anhydrous) where the yield was calculated as the ratio of essential oil volume to dried rhizome weight (%v/w). The essential oils were stored in the refrigerator at 4 • C for subsequent analysis. Further, the essential oils were subjected to chemical profiling using a targeted GC-MS analysis and bio-activities.

Solvent Free Microwave Extraction (SFME)
Solvent-free microwave-assisted extraction for three Curcuma samples was performed using a Milestone ETHOS X (Sorisole, Italy) advanced microwave extraction device ( Figure 9A).
An infrared sensor to track the temperature and two magnetrons combined to deliver a maximum power of 1800 W (2 × 950 W) are included in this 2.45 GHz multimode microwave reactor. At atmospheric pressure, the extraction was initiated using an integrated glass vessel /reactor with a capacity of 2 L covered by a glass lid ( Figure 9B,C). Dried rhizomes were soaked with water for 30 min within the integrated glass vessel and processed through the system. Power was set at 450 W for the first 30 min, then dropped to 400 W for the rest period. The parameters such as time, pressure, power and temperature were regulated by the instrument's programme/software. The Fragrances setup was used to configure the system, which consists of a glass Clevenger apparatus placed above the oven, continuously condensing the volatile substances and enabling water to re-enter the reactor. The volatile oils were collected from the apparatus ( Figure 9D) and treated with sodium sulphate (anhydrous). The yield was determined as the weight of dried rhizomes divided by the volume of essential oil (%v/w). Further, the samples were preserved in a refrigerator at 4 • C for subsequent analysis.

Solvent Free Microwave Extraction (SFME)
Solvent-free microwave-assisted extraction for three Curcuma samples was pe formed using a Milestone ETHOS X (Sorisole, Italy) advanced microwave extraction d vice ( Figure 9A). An infrared sensor to track the temperature and two magnetrons com bined to deliver a maximum power of 1800 W (2 × 950 W) are included in this 2.45 GH multimode microwave reactor. At atmospheric pressure, the extraction was initiated usin an integrated glass vessel /reactor with a capacity of 2 L covered by a glass lid (Figur 9B,C). Dried rhizomes were soaked with water for 30 min within the integrated glass ve sel and processed through the system. Power was set at 450 W for the first 30 min, the dropped to 400 W for the rest period. The parameters such as time, pressure, power an temperature were regulated by the instrument's programme/software. The Fragrance setup was used to configure the system, which consists of a glass Clevenger apparatu placed above the oven, continuously condensing the volatile substances and enabling wa ter to re-enter the reactor. The volatile oils were collected from the apparatus ( Figure 9D and treated with sodium sulphate (anhydrous). The yield was determined as the weigh of dried rhizomes divided by the volume of essential oil (%v/w). Further, the samples wer preserved in a refrigerator at 4 °C for subsequent analysis.

Compound Identification Using GC-MS Analysis
Essential oils extracted from both extraction methods were analysed and quantifie using a Clarus 580 Gas Chromatograph (Perkin-Elmer, Waltham, MA, USA) associate with an SQ8S mass spectrometric detector. A volume of 0.5 µL was employed for the sam ple injection. Helium was utilized as the carrier gas in the experiment at a flow rate of 1 mL/min and an Elite-5MS capillary column (30 m × 0.25 mm I.D. × 0.25 m thickness) wa used. Initially set at 60 °C, the oven's temperature was increased to 220 °C at a rate of °C/min and then held for 7 min. The ion source temperature was kept at 150 °C, while th injector temperature was kept at 250 °C. The ionization energy was set at 70 eV, and range of 50 to 600 amu was scanned. The mass spectra of each separated component wer compared with the mass spectra of NIST database and retention indices (RI) relative to

Compound Identification Using GC-MS Analysis
Essential oils extracted from both extraction methods were analysed and quantified using a Clarus 580 Gas Chromatograph (Perkin-Elmer, Waltham, MA, USA) associated with an SQ8S mass spectrometric detector. A volume of 0.5 µL was employed for the sample injection. Helium was utilized as the carrier gas in the experiment at a flow rate of 1.0 mL/min and an Elite-5MS capillary column (30 m × 0.25 mm I.D. × 0.25 m thickness) was used. Initially set at 60 • C, the oven's temperature was increased to 220 • C at a rate of 3 • C/min and then held for 7 min. The ion source temperature was kept at 150 • C, while the injector temperature was kept at 250 • C. The ionization energy was set at 70 eV, and a range of 50 to 600 amu was scanned. The mass spectra of each separated component were compared with the mass spectra of NIST database and retention indices (RI) relative to a homologous series of n-alkane (C 8 -C 20 ) on the Elite-5MS capillary column were compared to an existing bibliographic database to identify the constituents [40].

DPPH Assay
The antioxidant potential of oil was evaluated by DPPH free radical scavenging ability by following the methodology of Chang et al. [85] with slight modifications. The mixture of 1 mL of the various sample concentrations (1 to 50 µg/mL) and 1 mL of the methanolic DPPH solution (0.1 mM) was maintained at room temperature for 30 min. The same protocol was followed for ascorbic acid and butylated hydroxytoluene (BHT) that were taken as the standards. The absorbency of the samples was recorded using a UV-Visible spectrophotometer at 517 nm. The mixture of methanol and DPPH without sample was used as a control. More radical scavenging activity is indicated by a lower degree of absorption. The formula below was used to determine the amount of DPPH free radical inhibition by the essential oils:

ABTS Assay
The ABTS method described by Kamila et al. [86] was used to evaluate the oil's antioxidant capacity following some minor modifications. An ABTS reagent was prepared by mixing 2.45 mM ammonium persulfate with 7 mM ABTS solution, then the reagent was incubated in the dark for 16 h at 37 • C. An optical density of 0.70 ± 0.08 at 734 nm should be obtained by diluting the ABTS solution with methanol prior to the experiments. Then, 3 mL of ABTS solution was mixed with 0.3 mL of different sample concentrations (1 to 50 µg/mL). The same methodology was used for the standards, ascorbic acid and butylated hydroxytoluene (BHT). A control solution was prepared using the methanol and ABTS reagent without sample. The absorbance was measured at 734 nm and the following formula was used to determine the % inhibition of the ABTS free radical: Inhibition % = (absorbance of control − absorbance of sample/absorbance of control) × 100 (2)

Anti-Tyrosinase Assay
The tyrosinase inhibitory study was performed with minor modifications to the method reported by Rahmani et al. [87]. L-DOPA (0.5 mg/mL), mushroom tyrosinase (1000 units/mg) and kojic acid were taken as the substrate, enzyme and positive control, respectively. L-DOPA and mushroom tyrosinase were prepared using phosphate buffer (PB) (0.1 M) (pH 6.8) whereas the stock solution for essential oil samples and kojic acid were prepared with 1% DMSO solution since the solubility of oil samples was low in 100% DMSO. The subsequent working concentrations/ dilution (12.5-200 µg/mL) of the oil samples and standard kojic acid were made using the phosphate buffer. In a 96-well plate, each sample consisted of four wells labelled as A, B, C and D, where each well contained a reaction mixture of 180 µL. The following are the contents of each well: A (20 µL enzyme + 140 µL PB), B (20 µL 1% DMSO + 140 µL PB), C (20 µL enzyme + 140 µL PB + 20 µL sample) and D (140 µL PB + 20 µL sample). The reaction mixture was properly mixed and incubated at 25 • C for 10-15 min. After the incubation period, 20 µL of L-DOPA was added to all wells (A, B, C and D) and incubated at 25 • C for 20 min. Then, the absorbency of the samples was noted using EPOCH 2 microplate spectrophotometer (Agilent BioTek) at 475 nm. Using the following formula, the amount of tyrosinase inhibition was calculated; Inhibition % = 100 [(A − B) − (C − D)]/(A − B), where A = The change in absorbance before and after incubation without sample, B = The change in absorbance before and after incubation without sample and enzyme, C = The change in absorbance before and after incubation with oil sample, D = The change in absorbance before and after incubation with oil sample but without enzyme. The experiment was conducted in triplicate.

Anti-Cancer Activity
The anticancer activity of the essential oils was evaluated against MCF7, HepG2, and PC3 cancer cells by measuring the rate of cell proliferation using the colorimetric MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide] assay [88,89]. Cells were seeded at a density of 2 × 10 4 cells/well on a 96-well plate and 20 µL of essential oils in were added in various concentrations (6.25-200 µg/mL) to the microplates and incubated for 24 h at 37 • C in a 5% CO 2 atmosphere. After discarding the used media, MTT reagent was added at a final concentration of 0.5 mg/mL of the overall volume, and the mixture was then incubated in a CO 2 incubator at 37 • C for 3-4 h. The supernatants were then aspirated and 100 µL of solubilization solution (DMSO) was added to dissolve the formed formazan crystals. The absorbance was measured using an ELISA reader at 570 nm. DMSO and doxorubicin were taken as control and standard, respectively. Percentage of cell viability was calculated using the formula, % Cell viability = [(A a − A b )/(A c − A b )] × 100, Where A a = Absorbance value of sample (essential oil), A b = Absorbance value of blank and A c = Absorbance value of the control. The IC 50 value of the MTT experiment was defined as the concentration of essential oil that resulted in 50% cytotoxicity and was determined by using a linear or logarithmic regression equation i.e., Y = Mx + C where, Y = 50, M and C values were derived from the regression equation.

Statistical Analysis
The outcomes of independent experiment were performed in triplicates and were expressed as mean ± SD. To analyse the statistical differences in EO yield (%) and quality across Curcuma species, a one-way ANOVA followed by a post-hoc test was carried out using the statistical software (Minitab 17). The data were also analysed using OriginPro

Conclusions
The standard hydro-distillation (HD) method has been compared with the solvent-free microwave extraction (SFME) technique for the extraction and bioactivity study of essential oils from three different Curcuma species viz. C. alismatifolia, C. aromatica and C. xanthorrhiza. The SFME method proved to be significantly advantageous over the conventional HD method in terms of oil yield, extraction time, energy saving, solvent (water) consumption and waste generation. A similar qualitative profile of essential oils of three studied species was revealed by GC-MS, while quantitative variation between the major compounds was detected in the chemical composition using the two extraction methods. A total of 85 compounds were identified among the three Curcuma species, with hydrocarbons and oxygenated rich compounds predominating the essential oils extracted using HD and SFME, respectively. Essential oils extracted through HD and SFME were shown to have strong antioxidant, anti-tyrosinase and anticancer properties, with C. alismatifolia essential oil having most the potent bioactivity. The essential oil extracted by SFME was found to have greater antioxidant, anti-tyrosinase, and anticancer activities than those isolated employing the HD method. The current results suggest that SFME-generated essential oil might be a valuable source of bioactive compounds and constitute potential antioxidant, anti-tyrosinase and anticancer agents for application in food, health and cosmetic products.