Chemical Composition and Biological Activities of the Leaf Essential Oils of Curcuma longa, Curcuma aromatica and Curcuma angustifolia

Curcuma species are widely used as a food additive and also in various medicinal purposes. The plant is a rich source of essential oil and is predominantly extracted from the rhizomes. On the other hand, the leaves of the plants are usually considered as an agrowaste. The valorization of these Curcuma leaf wastes into essential oils is becoming accepted globally. In the present study, we aim to extract essential oils from the leaves of Curcuma longa (LEO), C. aromatica (REO), and C. anguistifolia (NEO). The chemical composition of these essential oils was analyzed by GC-MS. Free radical scavenging properties were evaluated against the radical sources, including DPPH, ABTS, and hydrogen peroxide. The antibacterial activity was assessed by the disc diffusion method and Minimum inhibitory concentration analysis against Gram positive (Staphylococcus aureus) and Gram negative (Escherichia coli, Pseudomonas aeruginosa and Salmonella enterica) bacteria. Results identified the compounds α-phellandrene, 2-carene, and eucalyptol as predominant in LEO. The REO was predominated by camphor, 2-bornanone, and curdione. The main components detected in NEO were eucalyptol, curzerenone, α-lemenone, longiverbenone, and α-curcumene. Antioxidant properties were higher in the LEO with IC50 values of 8.62 ± 0.18, 9.21 ± 0.29, and 4.35 ± 0.16 μg/mL, against DPPH, ABTS, and hydrogen peroxide radicals. The cytotoxic activity was also evident against breast cancer cell lines MCF-7 and MDA-MB-231 cells; the LEO was found to be the most active against these two cell lines (IC50 values of 40.74 ± 2.19 and 45.17 ± 2.36 μg/mL). Likewise, the results indicated a higher antibacterial activity for Curcuma longa essential oil with respective IC50 values (20.6 ± 0.3, 22.2 ± 0.3, 20.4 ± 0.2, and 17.6 ± 0.2 mm). Hence, the present study confirms the possible utility of leaf agrowastes of different Curcuma spp. as a possible source of essential oils with pharmacological potential.


Introduction
Herbal medicines are important tools in the management of health from the ancient days; traditional medicines and folk medicinal systems utilized these plants and plant products [1]. Among the various plants, the predominant ones include the spices that are the part of the daily diet [2]. Several such spices are widely utilized in Ayurvedic and Chinese traditional medicines as dietary agents in the management of infectious and

Determination of the Yield and Chemical Composition of Leaf Essential Oils by GC-MS
As shown in the Table 1, the yield of leaf essential oils of different Curcuma spp. varied much from the others. The highest yield was noticed in the Curcuma longa (1.62 ± 0.34%). However, the lowest level of yield was noticed in the C. agnustifolia (0.37 ± 0.02%). The gas chromatography-mass spectroscopy analysis revealed the presence of various stress volatiles in the leaf essential oils of different Curcuma spp. The chromatograms of all tested essential oils are shown in Figure 1. The chemical components and percentage composition of Curcuma longa (Supplementary Table S1), C. aromatica (Supplementary  Table S2), and C. angustifolia (Supplementary Table S3) have been listed.

Determination of the Yield and Chemical Composition of Leaf Essential Oils by GC-MS
As shown in the Table 1, the yield of leaf essential oils of different Curcuma spp. varied much from the others. The highest yield was noticed in the Curcuma longa (1.62 ± 0.34%). However, the lowest level of yield was noticed in the C. agnustifolia (0.37 ± 0.02%). The gas chromatography-mass spectroscopy analysis revealed the presence of various stress volatiles in the leaf essential oils of different Curcuma spp. The chromatograms of all tested essential oils are shown in Figure 1. The chemical components and percentage composition of Curcuma longa (Supplementary Table S1), C. aromatica (Supplementary Table S2), and C. angustifolia (Supplementary Table S3) have been listed.  The composition of essential oils is presented in Table 2 and Tables S1-S3 from Table S1). In REO, the predominant compounds were camphor (19.82%), 2-bornanone (12.25%), and curdione (15.31%) (Supplementary Table S2). On the contrary, in NEO, eucalyptol (11.58%), curzerenone (25.32%), α-lemenone (13.59%), longiverbenone (9.37%), boldenone (5.04%), and α-curcumene (5.12%) were the major compounds (Supplementary  Table S3). The antioxidant activities of the different leaf essential oils of LEO, REO, and NEO were estimated in terms of DPPH, ABTS, and hydrogen peroxide radical scavenging activities ( Table 3). The LEO was found to be the most active among the tested essential oils in all the antioxidant assays. The respective IC 50 values of LEO against the three radicals were estimated to be 8.62 ± 0.18, 9.21 ± 0.29, and 4.35 ± 0.16 µg/mL. On contrary, the REO and NEO had significantly higher IC 50 values for the three radical scavenging assays in comparison with LEO (p < 0.05). The Curcuma essential oils demonstrated higher hydrogen peroxide scavenging abilities (p < 0.05). The LEO had similar DPHH and ABTS radical scavenging potential as that of ascorbic acid. The detailed statistical analysis of the antioxidant potential is shown in Supplementary Table S4. The anti-neoplastic properties of the leaf essential oils of different Curcuma spp. (LEO, REO, and NEO) were evaluated on human breast cancer lines. The result indicated strong cytotoxic properties in LEO, followed by REO and NEO; it is reflected in the IC 50 values of the essential oils, with LEO having the lowest IC 50 value and NEO being the highest (Table 4). In addition, the cytotoxicity in these cells was dose-dependent ( Figure 2). Comparing among the two breast cancer cell lines used, all the tested essential oils had higher toxicity in MCF-7 cells than MDA-MB-231. The anti-neoplastic properties of the leaf essential oils of different Curcuma spp. (LEO, REO, and NEO) were evaluated on human breast cancer lines. The result indicated strong cytotoxic properties in LEO, followed by REO and NEO; it is reflected in the IC50 values of the essential oils, with LEO having the lowest IC50 value and NEO being the highest (Table 4). In addition, the cytotoxicity in these cells was dose-dependent ( Figure  2). Comparing among the two breast cancer cell lines used, all the tested essential oils had higher toxicity in MCF-7 cells than MDA-MB-231. The half-maximal inhibition concentration of the leaf essential oils are listed in Table  4. The C. longa demonstrated an IC50 value of 40.74 ± 2.19 and 45.17 ± 2.36 µg/mL against MCF-7 and MDA-MB-231. The lowest cytotoxicity was exhibited by C. angustifolia with IC50 values of 64.17 ± 1.95 and 70.31 ± 1.59 µg/mL, respectively, against MCF-7 and MDA-MB-231 cells. The cyclophosphamide was more toxic to these cells with respective IC50 values of 9.46 ± 0.20 and 8.52 ± 0.22 µg/mL. The detailed statistical analysis among essential oils is shown in Supplementary Table S5.  The half-maximal inhibition concentration of the leaf essential oils are listed in Table 4 The antibacterial activity was estimated by the disc-diffusion method. Curcuma longa essential oil was the most active against all the selected bacterial strains ( Table 5). The inhibition zone of LEO was found to be high against Staphylococcus aureus and Pseudomonas aeruginosa (p < 0.01). The REO was effective against E. coli, being statistically significant over NEO, but was, however, lower than the LEO. Among the three tested essential oils, the weakest one was from C. angustifolia. The standard gentamicin is also found to be highly effective against these bacterial strains with respective inhibition zones of 22.4 ± 0.3, 19.7 ± 0.1, 22.5 ± 0.3, and 19.1 ± 0.5 mm against the different bacteria ( Table 5). The complete statistical analysis is listed in Supplementary Table S6. Statistical analysis of the data is given in Supplementary Table S6; Curcuma longa (LEO), C. aromatica (REO), and C. angustifolia (NEO).
The antibacterial activity was also estimated in terms of the MIC values; among the three essential oils tested, the LEO was found to have significant antibacterial activity. In addition, the LEO and REO were equally effective against Pseudomonas aeruginosa with similar MIC values (Table 6). On comparing with the essential oils, the gentamicin treatment was more effective in terms of the MIC values; more statistical operations and details are listed in the Supplementary Table S7. Statistical analysis of the data is given in Supplementary Table S7; Curcuma longa (LEO), C. aromatica (REO), and C. angustifolia (NEO).

Discussion
Spices are important dietary components with potential biological and pharmacological activities [26]. Spices are highly utilized in food industries and therefore it is an important source of biologically active molecules that are referred to as nutraceuticals [27]. Among the various spices used, the Turmeric (Curcuma longa) is considered to be the most accepted one [28,29]. Apart from the C. longa, there are several other species that exist in the genus. In the present study, we evaluated the chemical composition of the leaf essential oils of different Curcuma spp., which is considered to be the important agrowaste. Apart from these, the antibacterial and cytotoxic activities were also evaluated.
Our results indicated a yield between 0.37 to 1.62% for the different Curcuma spp. essential oils. However, previous studies by Kutti Gounderand and Lingamallu [30] and Hong, et al. [31] indicated a yield of 3.05 to 4.45% from rhizomes. However, considering that the present study used leaves as the source of essential oil, a yield of 1.62% may not be considered low.
Besides the chemical constituent analysis, the results also indicated strong free radical quenching potential. The previous reports by Avanço, et al. [34] and Jena, Ray, Banerjee, Sahoo, Nasim, Sahoo, Kar, Patnaik, Panda, and Nayak [16] indicated the antioxidant properties of the rhizome essential oils of different Curcuma spp. Likewise, the radical quenching properties are also attributed to the Curcuma leaf essential oils [17,35]. Furthermore, the bioactive compounds including eucalyptol, α-lemenone, α-phellandrene, 2-carene and α-curcumene are also reported to act as chain breaking antioxidants [36,37]. Since the role of antioxidants in alleviating chronic diseases and preventing infectious disease are evident [38,39], the leaf essential oils from different Curcuma spp. may also have significant health promoting effects.
The present results also indicated strong cytotoxic properties against breast cancer cells. Cytotoxic activity of the essential oil of C. aromatica has also been evident in multiple cancer cells by stimulating apoptotic cell death [40,41]. Previous studies have also been reported that the essential oils of Curcuma rhizome and leaves induce apoptotic cell death in lung and liver cancer cells [42,43]. The cytotoxicity is attributed to the specific compounds such as α-phellandrene [44,45], camphor [46], curdione [47,48], eucalyptol [49,50], terpinolene [51], and α-pinene [52,53], which are already known to induce apoptosis and signaling interruption in cancer cell. The cytotoxic effect was mediated through cell cycle inhibition at the G 2 /S checkpoint [54].
Results also indicated strong antimicrobial properties to the leaf essential oils of different Curcuma spp. against bacterial strains such as E. coli, S. aureus, and S. enterica. These pathogenic microbes are known to cause various health issues in humans and animals [55,56]. The antibacterial activity was also attributed to the different Curcuma essential oils; previous studies have indicated the antibacterial properties of C. longa [57,58], C. aromatica [59,60], C. angustifolia [61]. The antibacterial activity of C. longa rhizome essential oil is also evident against Bacillus subtilis, Staphylococcus aureus, Salmonella typhimurium, and Escherichia coli [34,57]. Likewise, the rhizome essential oil of C. aromatica is also found to be effective against various microorganisms [60]. In addition, the essential oils were also capable of inhibiting the biofilm forming properties of bacteria, including Streptococcus mutans [62]. A recent study by Septama, et al. [63] has also indicated the antibacterial and anti-biofilm formation activities of the C. xanthorrhiza. To support this information, there are bioactive constituents, such as α-phellandrene [64,65], camphor [66][67][68], eucalyptol [69], terpinolene [70], and α-pinene [52,71].
Hence, the present study indicated significant variation in the chemical composition of the leaf essential oils of different Curcuma spp. Further, these essential oils displayed significant radical quenching potential against DPPH, ABTS, and peroxide radicals. The essential oils, especially LEO, exhibited strong antibacterial properties against Gram positive and Gram-negative strains. The cytotoxic activities of the different Curcuma essential oils were also identified against breast cancer cells.

Materials and Chemicals
The chemicals used for the analysis were of reagent grade and purchased from Sigma Aldrich (St. Louis, MO, USA). The chemicals were DPPH, ABTS, hydrocarbon mixture (C8-C30 n-alkanes), ethanol, and hydrogen peroxide. Cell culture reagents include Dulbecco's Modified Eagle Media, sodium pyruvate, fetal bovine serum, non-essential amino acids, and MTT (Gibco, MA, USA). The microbial growth media included Lysogeny broth and Mueller-Hinton agar (Himedia, Mumbai, India).
The human breast cancer cell lines (MCF-7, and MDA-MB-231) were procured from National Centre for Cell Science, Pune. The bacterial strains were obtained from the Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India. The Grampositive bacteria used was Staphylococcus aureus (MTCC740) and Gram-negative bacteria were Escherichia coli (MTCC1610), Pseudomonas aeruginosa (MTCC 741), and Salmonella enterica (MTCC1252) bacteria.

Collection of Curcuma Leaves and Extraction of Essential Oil
Leaves of different species of Curcuma spp. belonging to the Zingiberaceae family were collected from Kerala Agricultural University, Thrissur, India (10.85053 • N, 76.27106 • E) in February 2022. Hydro-distillation was conducted to extract the essential oils from these different species of Curcuma spp. with the help of the modified Clevenger-type apparatus for 5-6 h (100 • C). Briefly, 20 g of Curcuma leaves alone were taken in a 2000 mL flask along with deionized water. Initially, they were heated to boil as described in Table 1; the essential oil was then collected and dried with anhydrous Na 2 SO 4 [72]. Finally, these essential oils were stored in dark amber-colored glass bottles at 4 • C inside the refrigerator until required for experiments. The essential oil yield was determined on a dry weight basis by using the formula yield (%, v/w).

Percentage Yied =
Volume o f dry essential oil Weight o f shade dry leaves × 100

Chemical Component Analysis by GC-MS Analysis
The chemical composition of the essential oils were determined according to our previously published method [73]. The chromatographic equipment used in the analysis was TSQ 8000 Evo system from the Thermo scientific (Waltham, MA, USA). The analytical system was composed of an autosampler, which was a gas chromatographic column (TG-5MS) of dimensions 30 mm × 0.25 mm × 0.25 µm. The helium gas was used as a carrier with 1.0 mL per minute flow rate. The gas chromatographic oven was maintained at 50 • C with a gradual and steady increase to 120 • C (10 • C per minute) and finally changing the temperature to 270 • C (at a rate of 5 • C per minute). The chemical composition of each essential oil was derived by matching the MS spectra of NIST library. Each run was followed by a blank run without essential oil to omit the carry over contamination. We determined the retention index (RI) values by calibrating the instrument with a homologous series of alkanes (C 7 -C 30 n-alkane mixture) using the same conditions. The calculated retention indices of identified chemical components were compared with library reference retention indices in NIST and Wiley libraries [74][75][76][77]. The in vitro antioxidant activities were estimated for the selected essential oils; the essential oils were initially diluted to appropriate concentrations (0-25 µg/mL) and used for the study.

Anti-DPPH Radical Assay
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging was estimated according to the methods of Baliyan, Mukherjee, Priyadarshini, Vibhuti, Gupta, Pandey, and Chang [26]. Briefly, the varying concentrations of the essential oils were mixed with DPPH (0.12 M) solution in methanol. The mixture was kept in dark at 30 • C for 20 min. The reduction in the optical densities in different essential oil doses was compared to the untreated control, and the percentage inhibition was calculated using the formula;

Curcuma Essential Oils and ABTS Radical Quenching Ability
The 2,2 -azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS) radical scavenging was estimated according to the methods of Munteanu and Apetrei [78]. Initially, the ABTS radicals were generated by incubating 8 mM ABTS and 2.5 mM potassium persulfate for 12 h at 30 • C. These radicals were diluted 1:60 to a yield working ABTS solution; 1 mL of this solution was mixed with different doses of Curcuma essential oils and incubated at 30 • C for 10 min, and the change in absorbance was noted at 734 nm using the same formula given in Section 4.4.1

Hydrogen Peroxide Neutralization Assay
The hydrogen peroxide scavenging was estimated according to the methods described by Al-Amiery, et al. [79]. The essential oil of varying concentration (0.1 mL) was mixed with 50 mM phosphate buffer of pH 7.4 ± 0.1, containing 2 mM hydrogen peroxide solution. The mixture was mixed and incubated in the dark for 10 min at 30 • C, and the change in absorbance was noted at 234 nm.

Cytotoxic Activity of the Leaf Essential Oils of Different Curcuma spp.
Two breast cancer cell lines-MCF7 (ATCC, HTB-22™ Estrogen receptor positive) and the MDA-MB-231 (ATCC, HTB-26™ triple negative breast cancer cell) were procured from National Centre for Cell Science (Pune, Maharashtra, India). These cells were maintained in complete Dulbecco's Modified Eagle Media (Gibco, MA, USA) with sodium pyruvate, sodium carbonate, and non-essential amino acids. The cytotoxicity analysis was conducted according to the MTT assay, as mentioned previously [80]. The percentage of cell death was estimated by the formula; OD means the optical density or absorbance.

Antibacterial Activity of Leaf Essential Oil of Different Curcuma spp. by Disc Diffusion Method
The bacterial strains were initially maintained in Lysogeny broth (Himedia, Mumbai, India); the bacteria were inoculated in an Mueller-Hinton agar (Himedia, Mumbai, India) plate of thickness 5 mm. The Whatman No.1 filter paper disc of 8 mm diameter was immersed with the different Curcuma essential oils (10 µL). These filter paper discs were placed in different parts of the plates at a distance of 50 mm diameter apart in a plate. These plates were incubated at 37 • C for 24 h and the zone of inhibition was estimated for each bacterial strain [81]. The MIC value was estimated by the previously described methods [82][83][84]. Briefly, the bacterial inoculum density was maintained to 5 × 10 5 CFU/mL by the spectrophotometry. Further, the 50 µL of inoculum was placed in a 96-well plate and mixed with different concentrations of different Curcuma essential oils prepared in 0.1% agar. The media was then mixed with 10 µL of 2,3,5-triphenyltetrazolium chloride (TTC), a pink dye, which loses its color in the absence of microbial growth. The lowest concentration without pink color (confirmed with control group using spectrophotometer) was estimated to be the MIC value.

Statistical Analysis
The values of antioxidant activity, cytotoxicity, and antimicrobial assays were expressed as mean ± standard deviation of six individual analyses, which are carried out in triplicate. The IC 50 values were calculated using the Probit analysis method in the GraphPad prism. The statistical analysis was carried out by the analysis of variance using GraphPad prism ver. 7.0 (La Jolla, CA, USA).

Conclusions
The results indicated that the leaf waste materials of the Curcuma spp. can be effectively converted to essential oils. The yield of the oil was also good, considering the source of extraction is a waste product. The predominant compounds in C. longa essential oil were α-phellandrene and 2-carene; whereas camphor and curdione predominated in the C. aromatica essential oil and curzerenone and α-elemenone was high in the Curcuma angustifolia essential oil. The highest antioxidant potential, cytotoxicity and antibacterial activity were exhibited by the C. longa essential oils, followed by C. aromatica. Taking together, it is assumed that the leaf wastes of Curcuma spp., especially C. longa, can be converted to commercially useful essential oils with antioxidant, cytotoxic, and antibacterial properties.