Variable Secondary Metabolite Profiles Across Cultivars of Curcuma longa L. and C. aromatica Salisb.

Background: Curcuma spp. (Zingiberaceae) are used as a spice and coloring agent. Their rhizomes and essential oils are known for medicinal properties, besides their use in the flavoring and cosmetic industry. Most of these biological activities were attributed to volatile and nonvolatile secondary metabolites present in the rhizomes of Curcuma spp. The metabolite variations among the species and even cultivars need to be established for optimized use of Curcuma spp.
Objectives: We compared the phytochemical profiles of rhizomes and their essential oils to establish the variability among seven cultivars: five of Curcuma longa L. (Alleppey Supreme, Duggirala Red, Prathibha, Salem, Suguna) and two of C. aromatica Salisb. (Kasturi Araku, Kasturi Avidi). The GC-MS and LC-MS-based analyses were employed to profile secondary metabolites of these selected cultivars.
Methods: Rhizomes of Curcuma spp. were subjected to hydro-distillation to collect essential oil and analyzed by GC-MS. The methanol extracts of fresh rhizomes were subjected to LC-MS analyses. The compounds were identified by using the relevant MS library databases as many compounds as possible.
Results: The essential oil content of the cultivars was in the range of 0.74-1.62%. Several compounds were detected from the essential oils and rhizome extracts by GC-MS and LC-MS, respectively. Of these, 28 compounds (13 from GCMS and 15 from LCMS) were common in all seven cultivars, e.g., α-thujene, and diarylheptanoids like curcumin. Furthermore, a total of 39 new compounds were identified from C. longa L. and/or C. aromatica Salisb., most of them being cultivar-specific. Of these compounds, 35 were detected by GC-MS analyses of essential oils, 1,2-cyclohexanediol, 1-methyl-4-(1-methylethyl)-, and santolina alcohol, to name a few. The other four compounds were detected by LC-MS of the methanolic extracts of the rhizomes, e.g., kaempferol-3,7-O-dimethyl ether and 5,7,8-trihydroxy-2',5'-dimethoxy-3',4'-methylene dioxyisoflavanone. Conclusions: We identified and recorded the variability in the metabolite profiles of essential oils and whole rhizome extracts from the seven cultivars of Curcuma longa L. and C. aromatica Salisb. As many as 39 new metabolites were detected in these seven Indian cultivars of Curcuma spp. Many of these compounds have health benefits.

Introduction

Turmeric (Curcuma longa L.) is a perennial rhizomatous herb that belongs to the family Zingiberaceae (Prasath et al., 2018). It has been used traditionally in India for its medicinal value and as a spice (Srinivasan et al., 2004; Aggarwal et al., 2007; Esatbeyoglu et al., 2012). In Ayurvedic medicine, turmeric is used internally (as a stomachic, tonic, and blood purifier) or externally (prevention and treatment of skin diseases) (Gounder and Lingamallu, 2012). Turmeric was scientifically validated for several pharmacological benefits, including antioxidant, anti-inflammatory, and chemoprotective properties (Miquel et al., 2002; Krup et al., 2013; Kanase and Khan, 2018; Umar et al., 2020). The rhizomes of turmeric are enriched with several bioactive metabolites, though the attention was mostly on curcuminoids. Besides curcumin (a curcuminoid), the essential oil of C. longa L. showed antimicrobial activity and ability to suppress aflatoxins production (Ferreira et al., 2013).

Out of 110 species of genus Curcuma, only ∼20 species were used so far for phytochemical studies (Nahar and Sarker, 2007). Curcuma longa L. is popularly known as turmeric, while C. aromatica Salisb. and C. caesia Roxb. are known as wild turmeric and black turmeric, respectively. C. longa L. and a few other species, including C. aromatica Salisb., produce curcumin, a yellow colored curcuminoid. So far, at least 235 compounds, primarily phenolics, terpenoids, and alkaloids, were identified from Curcuma spp. (Li et al., 2011). About 70 varieties of C. longa L. are cultivated in India (Sasikumar, 2005; Parthasarathy and Chempakam, 2008), but very few are chemically profiled.

The essential oil of Curcuma spp. is used in traditional medicine for many ailments (Dosoky and Setzer, 2018). The volatile component of C. longa’s rhizome is responsible for its aromatic flavor and odor (Gounder and Lingamallu, 2012). Its essential oil is considered safe for human use (Tisserand and Young, 2013). The oils of C. longa L. and C. aromatica Salisb. have applications in the food and pharmaceutical industries due to their antioxidant, antibacterial, and anti-inflammatory properties (Dosoky and Setzer, 2018). The essential oil also improved the bioavailability of curcumin, thereby its bioactivity (Shishu and Maheshwari, 2010). Preliminarily clinical trials indicated that the essential oil from C. longa L. and C. aromatica Salisb. was helpful against cancer, asthma, and other ailments (Cheng et al., 1999; Joshi et al., 2003; Li Y. et al., 2009). Thus, there is a need to identify high-yielding cultivars containing curcuminoids and essential oil.

Since the pharmacological properties of Curcuma spp. are dependent on their chemical profiles, studies on the chemical constituents of turmeric/wild turmeric and their essential oils gained significance. Thin-layer chromatography (TLC) is one of the methods employed to quantify curcumin (Setyaningsih et al., 2016) and other curcuminoids (Phattanawasin et al., 2009) in Curcuma longa L. A few different techniques used were HPTLC (Pathania et al., 2006; Paramasivam et al., 2009), nuclear magnetic resonance (NMR) spectroscopy (Li W. et al., 2009), and the HPLC method (Kulyal et al., 2016).

Our present study on metabolite profiles would pave the way for metabolomics by providing the identity of several metabolites. Metabolomics is a practical approach for the comprehensive profiling and comparison of metabolites in plant systems (De Vos et al., 2007). It is crucial for quality evaluation and scientific validation of medicinal plants and their products (Mukherjee et al., 2016). Mainly information on secondary metabolites of medicinal plants/spices is of great importance in health, food, and nutrition sectors, due to the antioxidant nature, color, or flavor of these secondary compounds (Beekwilder et al., 2005; Dixon et al., 2006; Hall, 2006). The quality of turmeric and other spices depends on factors, such as cultivation, collection, storage, milling, and processing, apart from genetics and adulteration issues. Therefore, metabolomics provides a practical approach for quality control (Mukherjee et al., 2016; Tetali et al., 2021).

Over the past decade, several methods suitable for large-scale analysis of metabolites in plant extracts were developed (Dixon et al., 2006; Hall, 2006). However, to date, no single analytical method can successfully detect the entire metabolome of higher plants, especially of medicinal and aromatic plants, as they are highly rich in chemically diverse metabolites (Tetali et al., 2021). The GC-MS and LC-MS techniques mutually complement each other in unraveling secondary metabolomes comprising a wide range of volatile and nonvolatile compounds. These compounds belonged to terpenes, phenolic acids, phenylpropanoids, saponins, alkaloids, polyamines, and their derivatives (Huhman and Sumner, 2002; Moco et al., 2006).

Essential oils from different Curcuma species, including C. longa L. and C. aromatica Salisb, were studied for their chemical constituents (Choudhury et al., 1996; Angel et al., 2014; Nampoothiri et al., 2015; Dosoky and Setzer, 2018) to establish their variability. Variation in the volatile compositions of Curcuma spp. such as C. longa L. and C. zedoaria, was done using GC-MS (Dosoky et al., 2019). A combination of GC-MS and LC-MS techniques was used for metabolite analysis of C. domestica L. (C. longa L.) (Herebian et al., 2009). In the present study, the volatile (essential oil) and nonvolatile (total extract) components of the fresh rhizome of the seven cultivars of Curcuma spp. were analyzed by the GC-MS and LC-MS techniques. The present study is the first report revealing such detailed metabolite profiles of the selected cultivars to the best of our knowledge. These cultivars, except Alleppey Supreme, are typically cultivated in Telangana and Andhra Pradesh, and these states are among the largest producers of turmeric in India (Parthasarathy and Chempakam, 2008).

Most of the studies worldwide on Curcuma spp., for their curative properties, were with C. longa L., followed by C. aromatica Salisb, C. aeruginosa Roxb. (Simoh and Zainal, 2015), and C. kwangsiensis S. K. Lee & C. F. Liang (Zeng et al., 2009). Several cultivars exist within these species, which vary in their chemical profiles. The present article is the first attempt to characterize both volatile (essential oil) and nonvolatile (crude extract) components of fresh rhizomes of seven cultivars of Curcuma spp. by the GC-MS and LC-MS techniques. Our results using GC-MS and LC-MS analyses revealed high variability in their metabolite profiles of seven cultivars of genus Curcuma. We emphasize that such an approach could be exploited to distinguish cultivars for a specific application based on their metabolite profile.

Materials and Methods

Materials and Reagents

LC-MS grade methanol, water, and acetonitrile were purchased from Fisher Scientific (Pittsburgh, PA, United States). Ammonium formate, formic acid, 4-fluoro-4′-hydroxy benzophenone (97%), and n-hexane were from Sigma-Aldrich, India. Anhydrous sodium sulfate (99.99%) was from Merck Millipore, India.

Fresh rhizomes of four cultivars of Curcuma longa L. (Duggirala Red, Prathibha, Salem, and Suguna) and two cultivars of C. aromatica Salisb. (Kasturi Araku and Kasturi Avidi) were collected from Turmeric Research Station, Kammarpally, Telangana State, India. Alleppey Supreme cultivar of C. longa L. was from the Indian Institute of Spices Research, Marikunnu (IISR) Kozhikode, Kerala, India. The mature rhizome samples were collected during the postharvest season of turmeric (May–Jun) in 2011 and 2012 and cryopreserved at −80°C until extraction and analysis.

Isolation of Essential Oil by Hydrodistillation for GC-MS Analysis

50 g each of fresh turmeric rhizome of five cultivars of C. longa L. cvs. Alleppey Supreme, Duggirala Red, Prathibha, Salem, Suguna, and two cultivars of C. aromatica Salisb. cvs. Kasturi Araku and Kasturi Avidi were taken out from a −80°C freezer, made into pieces, and ground in a pestle with a mortar to a fine powder under liquid nitrogen. The powder was subjected to hydrodistillation in a Clevenger-type apparatus for 7 h. The essential oil obtained after distillation was dried over anhydrous sodium sulfate and kept at −80°C until GC-MS analysis.

GC-MS Running Conditions and Metabolite Identification

The chemical composition of the Curcuma spp. essential oil was analyzed by the GC-MS technique using Agilent 7890 A gas chromatograph coupled with a Leco Pegasus HT TOF mass spectrometer equipped with a 29.8 m × 320 µm HP-5MS 5% phenyl methyl siloxane capillary column with 0.25 µm film thickness. The oven temperature was programmed at 65°C for 2 min and then increased from 65 to 90°C at 5°C/min (held for 3 min). Then the temperature was increased from 90 to 103°C (held for 3 min) and from 103 to 150°C (held for 15 min) at 20°C/min and 8°C/min, respectively. The temperature was raised finally from 150 to 280°C at 20°C/min. The injector, interphase, and ion source were maintained at 250°C, 280°C, and 250°C, respectively. The detector voltage was 1500 V. A solvent delay of 2 min was selected. One microliter (diluted with n-hexane; 1:10) of essential oil sample was injected into the GC-MS system using split mode (50: 1). Helium was used as a carrier gas at a flow rate of 1 ml/min. GC-MS data were measured at 70 eV; mass scan 40–1000 amu.

The compounds were identified by comparing their mass spectra with the data available in the literature, National Institute of Standards Technology NIST, and Leco-Fiehn Rtx5 libraries. The compounds originated from the GC-MS data file were identified by matching most resembling spectra with the NIST library. Each search produced a hit list of compounds according to match factor or similarity with the library spectra. All the compounds showing similarity more than 70% with the NIST library were selected by the software (Software: Version 4.22 optimized for Pegasus®). Software searches (identifies) compound from their mass spectra and includes MS interpretation programs for analyzing mass spectra based on chemical structure, molecular formula, isotopic pattern, etc. The similarity of 70% or above between the m/z values of the compound detected in the respective cultivar and the MS-libraries’ mass fragmentation pattern was considered as identification. Furthermore, mass spectra of all compounds were also matched with ranges available as per their CAS number. Compounds for which the CAS number was not generated, the PubChem CID was used. Compounds below the similarity level of 70% were not considered and grouped as unknown. The data obtained with the samples collected in 2012 are presented in this article.

Preparation of Rhizome Extracts for LC-MS Analysis

Samples for LC-MS analysis were prepared by grinding the fresh rhizome to a fine powder in a mortar and pestle under liquid nitrogen. 1 g of the rhizome powder was suspended in 2 ml of MeOH (LC-MS grade). The samples were sonicated for 30 min and centrifuged for 25 min at 1500 rpm, and the supernatants were separated by filtering through a 0.45-µm Nylon filter disk. These extracts were freshly prepared for the analysis. A 200 µl aliquot of the extract was diluted quantitatively with internal standard (IS) 200 µl 4-fluoro-4′- hydroxy benzophenone solution. It was prepared freshly for each analysis by dissolving in methanol for a final concentration of 0.58 mg/ml. The samples were subjected to LC-MS analysis for the complete metabolite profile. The data obtained with the samples collected in 2012 were presented in this article.

LC-MS/MS Conditions and Metabolite Identification

LC-MS analyses of the crude extract of fresh rhizome of Curcuma spp. were performed according to Jiang et al. (2006) using Agilent 6520 Accurate Q-TOF (Agilent Santa Clara, CA), and the column used was Zorbax Eclipse XDB-C 18, 4.6 × 50 mm, 1.8 µ; Mobile phase: A) buffer (5 mM ammonium formate, 0.1% formic acid, in deionized and distilled H2O) and B) acetonitrile; gradient (in buffer A): 0–2 min, 5% B; 2–57 min, 5–100% B; 57–60 min, 100% B; 60–65 min, 100–5% B; flow rate: 0.25 ml/min; temperature, 40oC; injection volume 5 µl. For the MS detection, Agilent MSD-Trap-SL was equipped with electrospray ionization (ESI) interface as the ion source. The acquisition parameters for the negative mode were: drying N2 temperature, 350oC, 8 l/min; nebulizer pressure 40 psi; HV capillary 4000 V; skimmer 65.0 V; mass range measured: 110–1700 m/z; Spray voltage: 4 kV; scan rate 1.4. We analyzed the results in both the positive and the negative ion mode acquired by Agilent TOF/Q-TOF mass spectrometry and full MS scan, in the form of total ion current (TIC) chromatogram, and the metabolites were identified based on their MS/MS spectra and fragmentation rules reported previously (Jiang et al., 2006).

Results

Essential Oil Content

The oil was obtained by hydro-distillation, in a Clevenger-type apparatus, of the fresh rhizomes of five cultivars (Alleppey Supreme, Duggirala Red, Prathibha, Salem, and Suguna) of Curcuma longa L. and two cultivars (Kasturi Araku and Kasturi Avidi) of C. aromatica Salisb. The yield of essential oil from the seven cultivars was in the range of 0.74–1.62% on a fresh weight basis, with the highest yield of 1.62% in cv. Kasturi Avidi (C. aromatica Salisb.) followed by cv. Alleppey Supreme (C. longa L.) with an amount of 1.42% and the lowest yield of 0.74% in cv. Duggirala Red (C. longa L.). The essential oil yields from the other five rhizomes were in between these values (Table 1). The oil yields of C. longa L. varieties were higher than those of C. aromatica Salisb.

TABLE 1

Essential oil content and total number of compounds detected by GC-MS in the rhizomes of Curcuma species.

Sl. No.	Cultivar (species)	Essential oil (%)	Identified (Reported in Curcuma spp. or another plant species)	Unidentified
				Unknown	Not reported from any plant species
1	Alleppey Supreme	1.42	31	58	111
	(C. longa L.)
2	Duggirala Red	0.74	36	56	108
	(C. longa L.)
3	Prathibha	1.20	44	60	96
	(C. longa L.)
4	Salem	1.00	30	39	131
	(C. longa L.)
5	Suguna	0.80	35	51	114
	(C. longa L.)
6	Kasturi Araku	0.78	29	64	107
	(C. aromatica Salisb.)
7	Kasturi Avidi	1.62	31	60	109
	(C. aromatica Salisb.)

GC-MS Analysis of Essential Oil

Essential oils of seven cultivars of Curcuma spp. were subjected to GC-MS analysis, and the results from one of such studies for each cultivar are presented in this article. The representative TIC chromatograms of these cultivars are shown in Figure 1. Several compounds were detected in each cultivar’s essential oil (Table 1). Only a few of the identified compounds were confirmed based on their match with the compound profiles found in the NIST databases and Leco-Fiehn Rtx5 library. Up to 44 compounds were identified from the five cvs. of C. longa L. and 31 compounds from two cvs. of C. aromatica Salisb. (Table 1). Altogether 80 compounds were grouped into three categories: cultivar-specific (41), present in more than one cultivar (26), and common in all seven cultivars (13).

FIGURE 1

Representative TIC chromatograms from GCMS of essential oil from cultivars (A) Alleppey Supreme, (B) Duggirala Red, (C) Prathibha, (D) Salem, (E) Suguna of Curcuma longa L. and cvs. (F) Kasturi Araku, (G) Kasturi Avidi of C. aromatica Salisb.

These 41 cultivar-specific compounds were detected in the essential oil of one of the cultivars of C. longa L. or C. aromatica Salisb. (Table 2). The essential oil of C. longa L. cv. Prathibha had the highest number of cultivar-specific compounds, whereas C. aromatica Salisb., cv. Kasturi Araku had the least (Table 2). Of these, 23 cultivar-specific compounds were reported for the first time from the genus Curcuma. The chemical structures of these 23 compounds (Table 2, Sl. Nos. 1–23) are presented in Figure 2 (panels 1–23). In addition to 41 cultivar-specific compounds, 26 compounds were present in more than one cultivar (Table 3). Among these, 12 were detected first time in the genus Curcuma (Table 3, Sl. Nos. 1 to 12; Figure 2, panels: 24–35). The remaining 14 were already known in C. longa L. A total of 13 compounds were common in all seven cultivars of C. longa L. and C. aromatica Salisb. (Table 4) and the representative structures of two of these compounds are given in Figure 3 (panel numbers 14–15, corresponding to serial numbers 10 and 13 respectively of Table 4). Most of these compounds belong to mono, di, and sesquiterpene. A summary of all 80 compounds identified by GC-MS in the essential oils of the seven cultivars is presented in Supplementary Table S1.

TABLE 2

Cultivar-specific compounds identified, in one of the seven cultivars of Curcuma longa L. or C. aromatica Salisb. by GC-MS in the essential oil from rhizomes. The structures of the compounds (serial numbers from 1 to 23) are given in Figure 2 (panel numbers: 1–23), and this is the first report of these compounds from the genus Curcuma L. These compounds, however, were reported from genus other than Curcuma L. The compounds from serial numbers 24 to 41 are already reported in Curcuma species. Structures for few compounds (serial numbers 24–30) are given in Figure 3 (panel numbers: 1–7). Abbreviations used: AS, Alleppey Supreme; DR, Duggirala Red; PR, Prathibha; SA, Salem; SU, Suguna; KAr, Kasturi Araku; KAv, Kasturi Avidi.

Sl. No.	Compound name	Cultivar	RT (Min)	Area/abundance	Formula	Mass	Mass fragmentations	Class of compound	Reported from plant species	References
1	1,2-Cyclohexanediol, 1-methyl-4-(1-methylethyl)-	AS	6.38	958955880	C10H20O2	172.146	43, 71, 111	Monoterpenoid	Citrus medica L.	Bhuiyan et al. (2009)
							154		Leaf and peel essential oil
2	Trans, trans-Octa-2,4-dienyl acetate	AS	8.22	84981	C10H16O2	168.115	43, 77,79	Dienyl acetate	Kaempferia galanga L.	Othman et al. (2006)
									Dried rhizomes
3	Phenol, 2-methoxy-3-(2-propenyl)-	AS	17.05	200016	C10H12O2	164.083	77,131	Phenolic monoterpenoid	Dalberga stevensonii Standl.	Jiang et al. (2018)
							164		Wood extracts
4	3-Isopropyl-4-methyl-1-pentyn-3-ol	DR	13.59	805101	C9H16O	140.120	43,97	Alcohol constituent	Anethum sowa Roxb. ex, Fleming	Saleh-e-in et al. (2010)
							107		Leaf and stem essential oil
5	5,9-Tetradecadiyne	DR	19.45	11283632	C14H22	190.172	41,105, 147	Unsaturated hydrocarbon	Ferula vesceritensis Coss. & Durieu ex Trab.	Zellagui et al. (2012)
									Leaves
6	Naphthalene, 5-butyl-1,2,3,4-tetrahydro-	DR	20.62	1290163	C14H20	188.156	91,145	Tetralin	Meconopsis punicea Maxim. and M. delavayi (Franch.) Franch. Ex Prain, essential oil	Yuan et al. (2003)
							188	Type
7	Santolina alcohol	DR	23.63	547909	C10H18O	154.135	59,81	Tertiary alcohol	Achillea filipendulina Lam., aerial part	Sharopov and Setzer (2010)
							121
8	2-Pentanone, 4-mercapto-4-methyl-	PR	5.32	1330293	C6H12OS	132.060	43,55	Ketone	Camellia sinensis (L.) Kuntze	Kumazawa et al. (2005)
							132
9	8-Methylene-3-oxatricyclo[5.2.0.0(2,4)]nonane-	PR	11.72	25554	C9H12O	136.08	40,79, 92	Hydrocarbon	Schisandra chinensis (Turcz.) Baill., essential oil dried fruit	Wang et al. (2005)
10	7-Tetracyclo[6.2.1.0(3.8)0(3.9)]undecanol, 4,4,11,11 tetramethyl-	PR	19.16	21297856	C15H24O	220.182	77,119	Sesquiterpene alcohol	Cyperus articulatus L., essential oil roots/rhizome	Metuge et al. (2014)
							159
11	Bicyclo(2.2.1)hept-2-ene, 2,3-dimethyl-	PR	19.41	23461594	C9H14	122.109	79,94	Cyclic hydrocarbon	Abies alba Mill	Yang et al. (2009)
							122		leaf and twig
12	1H-3a,7-methanoazulene, 2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-, [3R-(3à,3aá,7á,8aà)]-	PR	19.69	1483176	C15H24	204.187	93,119	Sesquiterpene	Lindera aggregata (Sims) Kosterm., essential oil	Hong (2011)
							161
13	Cholesta-8,24-dien-3-ol, 4-methyl-, (3á,4à)-	PR	21.56	84686714	C28H46O	398.354	69,105	Triterpenoid	Parkia speciosa Hassk.	Salman et al. (2006)
							119		seed
14	4-Ethylphenethylamine	PR	25.88	507899185	C10H15N	149.120	63,120	Amine	Psidium guajava L.	Fasola et al. (2011)
									stem bark essential oil
15	Cyclohexanol, 2-methyl-5-(1-methylethenyl)-	PR	26.57	3965291	C10H17O	154.135	67,107	Monoterpenoid	Mentha spicata L.	Mohammed et al. (2017)
							136		aerial parts
16	Cyclohexane, 1,2-dimethyl-3,5-bis(1-methylethenyl)-	PR	26.59	26170712	C14H24	192.187	107,149	Monoterpenoid	Rhanterium adpressum Coss. & Durieu	Kala et al. (2009)
									Aerial parts
17	5,8,11,14-Eicosatetraenoic acid, phenylmethyl ester, (all-Z)-	SA	5.39	15244294	C27H38O2	394.287	67,91	Mster	Petiveria alliacea L., whole plant	Sathiyabalan et al. (2014)
							205
18	11-Dodecen-2-one	SA	36.83	511982	C12H22O	182.167	43,124	Ketone	Ficus hispida L. f	Song et al. (2001)
							182		Fresh male and female receptive figs, leaves
19	E-11-Tetradecenoic acid	SA	37.15	335669	C14H26O2	226.193	41,55,69	Fatty acid	Coriandrum sativum L., leaf oil	Bhuiyan et al. (2009)
20	2-Nonen-4-yn-1-ol, (Z)-	SU	10.68	287984	C9H14O	154.135	41,67	Alcohol	Alpinia speciosa (J.C. Wendl.) K. Schum	Ho (2010)
							95, 138		Seeds and leaves
21	3-Cyclohexen-1-one, 3,5,5-trimethyl-	SU	21.81	10709364	C9H14O	138.104	96, 138	Cyclohexenone	Crocus sativus L.	D’Auria et al. (2006)
									Dried saffron
22	6,10-Dodecadien-1-yn-3-ol, 3,7,11-trimethyl-	SU	23.27	18320904	C15H24O	220.182	41,67, 95, 138	Sesquiterpenoid	Hiptage benghalensis (L.) Kurz, leaves	Venkataramani and Chinnagounder (2012)
23	3-Octen-5-yne, 2,7-dimethyl-, (Z)-	KAv	7.91	132889991	C10H16	136.125	93, 121, 136	Monoterpene	Litsea glutinosa (Lour.) C.B. Rob	Chowdhury et al. (2008b)
									Fruit oil
24	Aromadendrene	PR	21.68	32839807	C15H24	204.187	41,67,161	Hydrocarbon	Curcuma purpurascens Blume, rhizome, essential oil	Hong et al. (2014)
25	Isoborneol	PR	10.16	931077	C10H18O	154.135	41,67,95	Monoterpenoid	Curcuma aromatica Salisb, rhizome	Sasikumar (2005)
									Essential oil
26	β-Elemene	PR	17.87	8265459	C15H24	204.187	41,67193	Sesquiterpene	Curcuma longa L., rhizome	Ma and Gang (2006)
									Essential oil
27	α-Santalene	PR	19.15	167463111	C15H24	204.187	41,94, 122	Sesquiterpene	Curcuma longa L., rhizome	Chowdhury et al. (2008a)
									Essential oil
28	2-Tridecanone	PR	36.82	145399		198.198	43,57	Ketone	Curcuma albiflora Thwaites, rhizome	Herath et al. (2017)
									Essential oil
29	Nonanoic acid	SU	37.82	1054190	C9H18O2	158.1307	41,60,129	Fatty acid	Curcuma longa L., rhizome	Nieman et al. (2012)
									Essential oil
30	Eucalyptol	KAr	6.34	73923505	C10H18O	154.135	51,71, 139	Monoterpene	Curcuma longa L., rhizome	Chowdhury et al. (2008a)
									Essential oil
31	Carvacrol	AS	15.46	800060	C10H14O	150.104	91, 135	Monoterpene	Curcuma longa L., rhizome, essential oil	Awasthi and Dixit (2009)
32	endo-Borneol	PR	25.95	762477	C10H18O	154.135	95, 140	Monoterpene	Curcuma longa L., rhizome	Chowdhury et al. (2008a)
									Essential oil
33	1,3,5-Cycloheptatriene, 3,7,7-trimethyl-	KAv	22.26	44959671	C10H14	134.109.120	41,91,119	Cyclic hydrocarbon	Curcuma longa L., rhizome	Chowdhury et al. (2008a)
									Essential oil
34	p-Cymen-8-ol	KAr	11.08	28783214	C10H14O	150.104	51,91, 135	Monoterpenoid	Curcuma longa L., rhizome	Chowdhury et al. (2008a)
									Essential oil
35	Camphor	PR	9.68	7985363	C10H16O	152.120	41,95,152	Terpenoid ketone	Curcuma longa L., rhizome	Leela et al. (2002)
									Essential oil
36	α-Bisabolol	PR	22.60	26108003	C15H26O	222.198	41,69, 119	Sesquiterpenoid	Curcuma longa L., rhizome	Chowdhury et al. (2008a)
									Essential oil
37	α-Elemenone	PR	22.99	5795701	C15H22O	218.167	67,119, 216	Sesquiterpene	Curcuma longa L., rhizome	Singh et al. (2010)
									Essential oil
38	Caryophyllene oxide	PR	21.85	84236617	C15H24O	220.182	21,96,138	Sesquiterpenoid oxide	Curcuma longa L., rhizome	Chowdhury et al. (2008a)
									Essential oil
39	Citral	KAr	10.81	4942325	C10H16O	152.120	69,119	Monoterpene	Curcuma longa L., rhizome	Chowdhury et al. (2008a)
									Essential oil
40	Neoisolongifolene, 8,9-dehydro-	KAr	20.92	956644398	C15H24	204.187	44,131, 187	Bicyclic hydrocarbon	Curcuma longa L., rhizome	Chowdhury et al. (2008a)
									Essential oil
41	Sabinene hydrate	KAv	19.81	30005835	C10H18O	154.135	79,93,121	Monoterpene	Zingiber Officinale Roscoe, rhizome essential oil	Koo and Gang (2012)

FIGURE 2

Structures of first-time reported (total 39) from the genus Curcuma, identified from cultivars of Curcuma longa L. and C. aromatica Salisb. detected in essential oil (panel numbers 1–23 and 24–35 corresponding to serial numbers 1–23 and 1–12 of Tables 2, 3 respectively) and rhizome extracts (panel numbers 36 and 37–39 corresponding to serial numbers 1 and 1–3 of Tables 6, 7 respectively) by GC-MS and LC-MS, respectively. Details of all these compounds are given in Supplementary Tables S3, S4. (1) 1,2-Cyclohexanediol, 1-methyl-4-(1-methylethyl)-, (2) trans, trans-octa-2,4-dienyl acetate, (3) phenol, 2-methoxy-3-(2- propenyl)-, (4) 3-isopropyl-4-methyl-1-pentyn-3-ol, (5) 5,9-tetradecadiyne, (6) naphthalene, 5-butyl-1,2,3,4-tetrahydro-, (7) santolina alcohol, (8) 2-pentanone, 4-mercapto-4-methyl-, (9) 8-methylene-3-oxatricyclo[5.2.0.0(2,4)]nonane, (10) 7-tetracyclo[6.2.1.0(3.8)0(3.9)]undecanol, 4,4,11,11 tetramethyl-, (11) bicyclo(2.2.1)hept-2-ene, 2,3-dimethyl-, (12) 1H-3a,7-methanoazulene, 2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-, [3R-(3à,3aá,7á,8aà)]-, (13) cholesta-8,24-dien-3-ol, 4-methyl-, (3á,4à)-, (14) 4-ethylphenethylamine, (15) cyclohexanol, 2-methyl-5-(1-methylethenyl)-, (16) cyclohexane, 1,2-dimethyl-3,5-bis(1-methylethenyl)-, (17) 5,8,11,14-eicosatetraenoic acid, phenylmethyl ester, (all-Z)-, (18) 11-dodecen-2-one, (19) E-11-tetradecenoic acid, (20) 2-nonen-4-yn-1-ol, (Z)-, (21) 3-cyclohexen-1-one, 3,5,5-trimethyl-, (22) 6,10-dodecadien-1-yn-3-ol, 3,7,11-trimethyl-, (23) 3-octen-5-yne, 2,7-dimethyl-, (Z)-, (24) 1,3,5-cycloheptatriene, (25) bicyclo(3.1.0)hexane, 4-methyl-1-(1-methylethyl)-, didehydro deriv., (26) bicyclo(3.2.1)oct-2-ene, 3-methyl-4-methylene-, (27) oxirane, 2-(hexyn-1-yl)-3-methoxymethylene-, (28) bergamotol, Z-α-trans-, (29) (1,3-dimethyl-2-methylene-cyclopentyl)-methanol, (30) 12-oxabicyclo(9.1.0)dodeca-3,7-diene, 1,5,5,8-tetramethyl-, [1R-(1R*,3E,7E,11R*)]-, (31) isolongifolene, 4,5,9,10-dehydro-, (32) Z,Z,Z-4,6,9-nonadecatriene, (33) 6-(p-tolyl)-2-methyl-2-heptenol, (34) 6-tridecen-4-yne, (Z)-, (35) 1,4-cyclohexadiene, 1-methyl-, (36) kaempferol-3,7-O-dimethyl ether, (37) 5,7,8-trihydroxy-2′,5′-dimethoxy-3′,4′-methylene dioxyisoflavanone, (38) chavicol, (39) kaempferol-3-O-rutinoside-7-O-glucoside.

TABLE 3

Compounds detected in more than one cultivar of C. longa L. and C. aromatica Salisb. identified by GCMS in the essential oil from rhizomes. The structures of the compounds from serial numbers 1–12 are given in Figure 2 (panel numbers: 24–35), and the compounds with Sl.No. 13–18 are shown in Figure 3, with corresponding panel numbers: 8–13 respectively. Abbreviations used: AS, Alleppey Supreme; DR, Duggirala Red; PR, Prathibha; SA, Salem; SU, Suguna; KAr, Kasturi Araku; KAv, Kasturi Avidi.

Sl No.	Compound name	Cultivar	RT (Min)	Area/abundance	Formula	Mass	Mass fragment ions	Class of compound	Reported from plant species	References
1	1,3,5-Cycloheptatriene	AS, PR, SA, SU, KAr, KAv	2.16	6243556	C7H8	92.0626	65,91	Closed ring organic compound	Ceropegia woodii Schltr	Meng et al. (2010)
2	Bicyclo(3.1.0)hexane, 4-methyl-1-(1-methylethyl)-, didehydro deriv	DR, PR, SA, SU	5.70	89775296	C10H16	136.1252	41, 77, 93	Monoterpene	Zingiber Officinale Roscoe	Tang et al. (2012)
3	Bicyclo(3.2.1)oct-2-ene, 3-methyl-4-methylene-	DR, SU, KAv	9.35	559996	C10H16	134.1096	91, 105, 134	Monoterpene	Seseli daucifolium C.B. Clarke	Mohiuddin et al. (2012)
4	Oxirane, 2-(hexyn-1-yl)-3-methoxymethylene-	DR, KAr, KAv	9.75	71555	C10H14O2	166.0994	79, 110	Cyclic ether and epoxide	Hyptis spicigera Lam	Ladan et al. (2011)
5	Bergamotol, Z-α-trans-	AS, SA	20.90	39326774	C15H24O	220.182	91, 93,119,187	Sesquiterpene alcohol	Pogostemon deccanensis (Panigrahi) Press	Kumar et al. (2019)
6	(1,3-Dimethyl-2-methylene-cyclopentyl)-methanol	AS, DR, SA, SU, KAv	21.05	25037989	C9H16O	140.1201	67, 77, 94, 109	alcohol H	Elsholtzia argyi H. Lév	Peng and Yang (2005)
7	12-Oxabicyclo[9.1.0]dodeca-3,7-diene, 1,5,5,8-tetramethyl-, [1R-(1R*,3E,7E,11R*)]-	DR, KAr	21.67	21304483	C15H24O	220.1827	67, 96, 109, 138	Epoxide	Eugenia Caryophyllus (Spreng.) Bullock & S.G. Harrison	Mani and Boominathan (2011)
8	Isolongifolene, 4,5,9,10-dehydro-	AS, DR, SA, SU, KAr	22.10	350003	C15H20	200.1565	77, 91, 143, 157, 185	Polycyclic hydrocarbon	Cymbopogon citratus (DC.) Stapf	Tajidin (2012)
9	Z,Z,Z-4,6,9-Nonadecatriene	DR, KAv	22.33	169215573	C34H19	262.2661	79, 93	Hydrocarbon	Papaver somniferum L.	Kumaravel et al. (2019)
10	6-(p-Tolyl)-2-methyl-2-heptenol	AS, SU, KAv	22.98	60406564	C15H22O	218.167	91, 119, 202	Aromatic alcohol	Zingiber officinale Roscoe	Choudhari and Kareppa (2013)
11	6-Tridecen-4-yne, (Z)-	DR, PR, SU	23.14	153999724	C13H22	178.1722	43, 79, 94	Hydrocarbon	Ambrosia trifida L.	Wang et al. (2005)
12	1,4-Cyclohexadiene, 1-methyl-	KAr, KAv	23.15	156905042	C7H10	94.0783	55, 79,94	Aromatic alcohol	Capsicum annuum L.	Eggink et al. (2012)
13	Camphene	AS, DR, PR, SU	4.56	930505	C10H16	136.125	93, 121	Monoterpene	Curcuma longa L.	Chowdhury et al. (2008a)
14	α-Phellandrene	AS, DR, SA, SU, KAr	5.72	2645357306	C10H16	136.125	77, 93	Monoterpene	Curcuma longa L.	Chowdhury et al. (2008a)
15	Limonene	AS, SU, KAr, KAv	6.27	202198637	C10H16	136.125	68, 93	Monoterpene	Curcuma longa L.	Singh et al. (2010)
16	α-Terpineol	AS, PR, SA, SU, KAr, KAv	11.25	35019844	C10H18O	154.135	59	Monoterpenoid	Curcuma longa L.	Gopalan et al. (2000)
							93, 121
17	β-Sesquiphellandren	DR, SA, SU, KAv	20.83	962609070	C15H24	204.187	68, 79	Sesquiterpene	Curcuma longa L.	Chowdhury et al. (2008a)
18	Nerolidol	DR, PR, SA, SU, KAv	21.79	15626126	C15H26O	222.198	69, 93	Sesquiterpinol	Curcuma longa L.	Awasthi and Dixit (2009)
19	Bicyclo(4.1.0)hept-2-ene, 3,7,7-trimethyl-	DR, KAv	5.63	227551	C10H16	136.125	93, 121	Monoterpene	Curcuma longa L.	Chowdhury et al. (2008a)
20	α-Terpinene	AS, DR, SA, KAr, KAv	6.00	28366949	C10H16	136.125	93, 121, 136	Monoterpene	Curcuma longa L.	Chowdhury et al. (2008a)
21	cis-Ocimene	DR, PR, SA, SU, KAr, KAv	6.74	466438	C10H16	136.125	41, 93	Monoterpene	Curcuma longa L.	Usman et al. (2009)
22	γ-Terpinene	AS, DR, PR, SA, SU, KAr	7.01	26650014	C10H16	136.125	93, 119,136	Monoterpene	Curcuma longa L.	Curcuma longa L.
										Usman et al. (2009)
23	Linalool	AS, DR, PR, SU	8.15	500594	C10H18O	154.135	71, 93, 121	Alcohol	Curcuma longa L.	Curcuma longa L.
										Leela et al. (2002)
24	Terpinene-4-ol	AS, DR, PR, SA, SU	10.83	2557284	C10H18O	154.135	73, 94, 154	Monoterpene	Curcuma longa L.	Curcuma longa L.
										Singh et al. (2010)
25	cis-α-Bisabolene	PR, KAr	20.40	11244271	C15H24	204.187	67, 93, 161, 204	Sesquiterpene	Curcuma longa L.	Curcuma longa L.
										Chowdhury et al. (2008a)
26	Ar-Tumerone	DR, SA, KAv	25.87	328137262	C15H20O	216.151	83, 119, 173, 216	Sesquiterpene	Curcuma longa L.	Curcuma longa L.
										Chowdhury et al. (2008a)

TABLE 4

Compounds common in the seven cultivars of Curcuma spp detected by GC-MS in essential oil obtained from rhizomes. The structures of the two compounds with serial numbers 10 and 13 are given in Figure 3 (panel numbers: 14–15 respectively).

Sl No.	Compound name	RT (Min)	Area/abundance	Formula	Mass	Mass fragment ions	Class of compound	Reported from plant species	References
1	α-Thujene	4.13	9407392	C10H16	136.125	93, 136	Monoterpenoid	Curcuma longa L.	Raina et al. (2005)
2	1s-α-Pinene	4.27	122103688	C10H16	136.125	39, 41, 93	Monoterpenoid	Curcuma longa L.	Singh et al. (2002)
3	Sabinene	5.05	3901899	C10H16	136.125	93, 136	Monoterpenoid	Curcuma longa L., leaves	Behura et al. (2002)
4	β or m-Cymene	6.20	338719033	C10H14	134.109	65, 91, 119	Aromatic hydrocarbon	Curcuma longa L.	Singh et al. (2002)
5	Terpinolene	7.82	225186296	C10H16	136.125	93, 121	Monoterpenoid	Curcuma longa L.	Leela et al. (2002)
6	trans-α-Bergamotene	18.86	2365810	C15H24	204.187	69, 93, 119, 161	Sesquiterpene	Curcuma longa L.	Raina et al. (2005)
7	α-Caryophyllene	19.23	6097261	C15H24	204.187	93, 121	Sesquiterpene	Curcuma longa L., leaves	Behura et al. (2002)
8	trans-β-Farnesene	19.37	211225438	C15H24	204.187	69, 93, 133	Sesquiterpene	Curcuma longa L.	Singh et al. (2002)
9	Ar-Curcumene	19.88	482956678	C15H22	202.172	132, 202	Sesquiterpene	Curcuma longa L.	Singh et al. (2002)
10	α-Zingiberene	20.25	1117738917	C15H24	204.187	69, 93, 119, 204	Sesquiterpene	Curcuma longa L.	Chowdhury et al. (2008a)
11	Tumerone	22.16	67277186	C15H22O	218.167	83, 157	Sesquiterpene	Curcuma longa L.	Singh et al., 2002)
12	Curlone	27.34	439832546	C15H22O	218.167	83, 120, 218	Sesquiterpene	Curcuma longa L.	Leela et al. (2002)
13	2-Heptadecanone	39.17	152911	C17H34O	254.261	43, 55, 71, 125	Ketone	Curcuma angustifolia  Roxb	Srivastava et al. (2006)

FIGURE 3

Structure of few selected cultivar-specific (panel numbers 1–7 corresponding to serial numbers 24–30 of Table 2); detected in more than one cultivar (panel numbers 8–13 corresponding to serial numbers 13–18 of Table 3) and common (panel numbers 14–15 corresponding to serial numbers 10 and 13 of Table 4) compounds already reported from the genus Curcuma in essential oils isolated from rhizomes by GCMS analysis of seven cultivars of Curcuma L.: five of Curcuma longa L. (cvs. Alleppey Supreme, Duggirala Red, Prathibha, Salem, and Suguna) and two of C. aromatica Salisb. (cvs. Kasturi Araku and Kasturi Avidi). (1) Aromadendrene, (2) isoborneol, (3) β-elemene, (4) α-santalene, (5) 2-tridecanone, (6) nonanoic acid, (7) eucalyptol, (8) camphene, (9) α-phellandrene, (10) limonene, (11) α-terpineol, (12) β-sesquiphellandren, (13) nerolidol, (14) α-zingiberene, (15) 2-heptadecanone.

TABLE 6

Cultivar-specific compounds identified by LC-MS in the rhizome extracts from one of the seven cultivars of Curcuma longa L. and C. aromatica Salisb. The structure of the compound with the serial number “1” is given in Figure 2 (panel number: 36) and compounds with Sl. Nos. 2, 3–9 are given in Figure 4 with the corresponding panel nos. 3, 5–11, respectively. Abbreviations used: AS, Alleppey Supreme; DR, Duggirala Red; PR, Prathibha; SA, Salem; SU, Suguna; KAr, Kasturi Araku; KAv, Kasturi Avidi.

Sl. No.	Compound name	Cultivar	RT (Min)	Area/abundance	Formula	Mass (m/z)	Mass fragment ions	Class of compound	Reported from plant species	References
1	Kaempferol-3,7-O-dimethyl ether	AS	12.77	25537	C17H14O6	313.0721 M-H-	108; 123; 152; 153	Flavonoid	Lumnitzera racemosa Willd and	Nikolova (2006); DeSouza et al. (2010)
							167		Artemisia vulgaris L.
2	Luteolin-7-O-glucoside	AS	42.2	5324	C21H20O11	447.2733	110; 185; 279; 280	Flavonoid	Curcuma Zedoaria (Christm.) Roscoe	Mass bank ACCESSION:TY00-0145; Rahmatullah et al. (2012)
						M-H
3	1,7-Bis(4-hydroxy-3,5-dimethoxyphenyl)-1,6-heptadiene-3,5-dione	PR	32.9	186331	C23H24O8	428.2	109; 123; 137; 159; 191; 209	Diarylheptanoid	Curcuma longa L.	Nurfina et al. (1997)
						M-H
4	1,7-Bis(3,4-dimethoxy phenyl)-1,6-heptadiene-3,5-dione	PR	38.3	335360	C23H24O6	396.2	105; 107; 119; 129; 137; 145; 155; 195	Diarylheptanoid	Curcuma longa L.	Chen et al. (2006)
						M-H
5	(6S)-2-Methyl-6-[(1R,5S)-(4-methene-5-hydroxyl-2-cyclohexen)-2-hepten-4-one	PR	40.2	229164	C15H24O2	236.1	105; 106; 107; 108; 109; 119; 120; 121; 133; 135	Bisabolane	Curcuma longa L.	Li et al. (2011)
						M-H
6	1,7-Bis(4-hydroxyphenyl)-3,5-heptanediol	KAr	22.8	8120	C19H24O4	316.1	105; 106; 107; 119; 121; 147; 148	Diarylheptanoid	Curcuma longa L.	Ma and Gang (2006)
						M-H
7	1,7-Bis(3,5-diethyl-4-hydroxyphenyl)-1,6-heptadiene-3,5-dione	KAr	60.6	129145	C27H32O4	420.3	106; 107; 119; 120; 121; 122; 123; 144; 145; 146; 147; 171; 172; 175; 187; 197; 201; 209; 211; 213; 223; 237; 239; 321; 323	Aromatic	Curcuma longa L.	Li et al. (2011)
						M-H
8	1-(4-Hydroxy-3-methoxyphenyl)-5-(4-hydroxyphenyl)-1,4-pentadiene-3-one	KAv	28.8	8009	C18H16O4	296.1	105; 109; 117; 119; 133; 145; 159; 161; 171; 173; 181; 185; 1207; 209; 223; 233; 239; 251; 279	Diarylheptanoid	Curcuma longa L.	Park and Kim (2002)
						M-H
9	(-)-(12E,2S,3S,4R, 5R,6R, 9S,11S, 15R)-3,15-Dibenzoyloxy-5,6-epoxylathyr-12-en-14-one	KAv	39.8	5683	C34H38O6	542.2	119; 145; 183; 211; 212; 237	Diterpenoid	Euphorbia micractina Boiss	Tian et al. (2011)
						M-H
10	5’-methoxycurcumin	PR	35.56	388655	C22H22O7	398.1	117; 119; 129; 137; 145; 149; 161; 175; 207	Diarylheptanoid	Curcuma longa L.	Ravindran (2000)
						M-H
11	Methyl-7-methoxycoumarin,4-	SU	34.4	67927	C11H10O3	190.1953	115; 116; 117; 119; 120	Coumarin	none	NIST CAS register No. 2555–28–4
										Mass Bank
										ACCESSION: PR100013
12	Hydroferulic acid	KAr	16.8	13992	C10H12O6	195.10	109; 121; 122	Phenolic acid	Curcuma longa L.	Ma and Gang (2006)
						M-H
13	1,2,3,4-Tetraphenylbutane-2,3-diol	KAr	11.1	3926	C28H26O2	394.1	112; 129; 133; 180; 207; 243; 247; 263; 339	Aliphatic diol	none	Pubchem Compound ID: 344369
						M-H
14	4-Hepten-3-one, 5-hydroxy-1,7-bis(4-hydroxyphenyl)-	PR	25.2	1418	C19H20O4	312.1	118; 119; 120; 146; 161	Diarylheptanoid	Curcuma longa L.	Jiang et al. (2006)
						M-H
15	5,7-Dihydroxy-2-(4-hydroxyphenyl)-chroman-4-one	PR	26.8	12833	C15H12O5	272.0	107; 119; 120	Phenolic acids	none	Chromadex
						M-H
16	Tetradecanoic acid/myristic acid	AS	29.47	2381	C14H28O2	228.0	128; 130; 143; 155; 158; 182; 183; 184; 210	Fatty acid	none	NIST CAS
						M-H				544–63–8
17	1-(4-Hydroxy-3-methoxyphenyl)-7-(4-hydroxy-3,5-dimethoxypheny)-4,6-heptadiene-3-one	PR	32.0	194388	C22H24O6	384.1	150; 151; 158; 165	Diarylheptanoid	Curcuma longa L.	Jiang et al. (2006)
						M-H and M + H
18	Tumerone	AS	35.3	6120	C15H22O	218.0	101; 103; 117; 129; 130; 131; 132; 133; 134; 145; 157; 146; 157; 158; 173; 201	Bisabolane sesquiterpene	Curcuma longa L.	He (2000)
						M-H
19	4-Methylene-5-hydroxybisabola-2,10-diene-9-one	PR	44.6	321246	C15H22O2	234.1	105; 107; 115; 117; 119; 121; 122; 129; 135	Sesquiterpene	Curcuma heyneana Valeton & Zijp	Sirat and Meng (2009)
						M + H
20	1,7-Bis(3,4-dimethoxyphenyl)-4,4-dimethyl-1,6-heptadiene-3,5-dione	PR	54.4	9854	C25H28O6	447.2	286; 298; 316; 317	Diarylheptanoid	Curcuma longa L.	Liang et al. (2009)
						M-H
21	25-Benzylpentacyclo-(22.3.1.0.)-octacosa-1(27),3 (8),4,6,10(15),11,13,17(22), 18,20, 24(28), 25-dodecaen	AS	54.96	42646	C35H30	450.2	105; 107; 119; 121; 122	Cyclic diarylheptanoid	Myrica rubra (Lour.) Siebold & Zucc	Ishida et al. (2002)
						M + H
22	Palmitic acid	SA	59.5	16809	C16H32O2	256.2	120; 166	Fatty acid	Curcuma kwangsiensis S.K. Lee & C.F. Liang	Jiang et al. (1989)
						M-H
23	Stearic acid	SA	63.8	76620	C18H36O2	284.1	197; 199; 200	Fatty acid	Curcuma longa L.	Ma and Gang (2006)
						M-H

TABLE 7

Compounds identified by LC-MS in rhizome extracts of more than one cultivar of Curcuma longa L. and C. aromatica Salisb. The compounds from serial numbers 1–3 are reported first time from the genus Curcuma, the structures of these compounds along with few others are given in Figure 2 (panels: 37–39; panels 1–2, 4, 12 corresponding to serial numbers 4–5, 6, 7). Abbreviations used: AS, Alleppey Supreme; DR, Duggirala Red; PR, Prathibha; SA, Salem; SU, Suguna; KAr, Kasturi Araku; KAv, Kasturi Avidi.

Sl. No.	Compound name	Cultivar	RT (Min)	Area/abundance	Formula	Mass (m/z)	Mass fragment ions	Class of compound	Reported from plant species	References
1	5,7,8-Trihydroxy-2′,5′-dimethoxy-3′,4′-methylene dioxyisoflavanone	AS, DR, KAv	2.2	51978	C18H18O9	377.1059	101; 102; 113; 119; 161; 163; 228; 336	Flavonoid	Terminalia ivorensis A. Chev	Ogundare and Olajuyigbe (2012)
						M-H-
2	Chavicol	AS, SU	38.83	55821	C9H20O	135.0794 M + H+	102; 115	Terpenoid	Piper betle L.	NIST CAS No: 501–92–8
							116			Nagori et al. (2011)
3	Kaempferol-3-O-rutinoside-7-O-glucoside	PR, SA	33.2	3752	C33H40O20	755.2655	135; 161; 175; 176; 191; 439; 579; 755; 756	Flavonoid	Lycopersicon esculentum Mill	Le Gall et al. (2003)
						M- H-
4	Kaempferol-3-rhamnoside	AS, DR	10.76	5367	C21H20O10	431.9911	125; 142; 146; 150	Flavonoid	Curcuma Xanthorrhiza Roxb	Ruslay et al. (2007)
						M-H	176; 184
							190; 293; 304; 308
							311; 316; 343; 345
5	3-Acetyl coumarin	AS, PR, SA, SU, KAv	34.65	289624	C11H8O3	188.0534	115; 116; 117; 118	Coumarin	None	Pub Chem ID
							141; 143			24852845
										NIST 3949–36–8
6	Turmeronol	AS, DR, SU	25.36	87260	C15H20O2	232.1436	103; 104; 105	Bisabolane sesquiterpene	Curcuma longa L.	Ma and Gang (2006)
							107; 115; 117; 118
							119; 120; 121; 128
							129; 131; 141; 142; 143
7	7-(3,4-Dihydroxyphenyl)-5-hydroxy-1-phenyl-(1E)-1-heptene	AS, KAv	35.99	16570	C19H22O3	298.1	119; 183; 184	Diarylheptanoid	Curcuma xanthorrhiza Roxb	Suksamrarn et al. (1994)
						M-H
8	1,7-Diphenyl-1,6-heptadiene-3,5-dione	AS, SA	2.49	9697	C19H16O2	276.0	115	Diarylheptanoid	Synthesized the compound	Sundaryono et al. (2003)
9	1-Hepten-3-one, 5-hydroxy-1,7-bis(3,4-dihydroxyphenyl)-	PR, SA, SU, KAr, KAv	21.2	1660	C19H20O6	344.1	107; 121; 134; 135; 136; 159; 161; 162; 177; 178	Diarylheptanoid	Alnus japonica (Thunb.) Steud	Sati et al. (2011)
10	4-(p-Hydroxyphenyl)-3-buten-2-one	AS, DR, SA, KAv	22.16	18641	C10H10O2	162.0	117; 118	Flavonoid	None	NIST CAS 3160–35–8
11	5-Hydroxy-7-(4-hydroxyphenyl)-1-phenyl-(1E)-1-heptene	AS, DR, PR, SA, KAr, KAv	23.11	6169	C20H24 O4	328.1	107; 119; 133; 134; 135; 136; 159; 161; 162; 177; 178	Diarylheptanoid	Curcuma xanthorrhiza Roxb	Suksamrarn et al. (1994)
12	1-(4-Hydroxy-3-methoxyphenyl)-7-(4-hydroxy-3,5-dimethoxypheny)-4,6-heptadiene-3-one	AS, DR, SA, KAr, KAv	25.8	6408	C22H24O6	384.1	133; 134; 147; 148; 150; 151; 158; 165; 175; 176; 186; 187; 188; 189; 203; 204; 232	Diarylheptanoid	Curcuma longa L.	Jiang et al. (2006)
13	1,5-Bis(3,4-methylenedioxyphenyl)-1,4-pentadien-3-one	SU, KAv	26.0	9161	C19H14O5	322.0	115; 119; 121; 133; 143; 235; 237; 247; 263; 275	Diarylheptanoid	Curcuma longa L.	Jiang et al. (2006)
14	1-Hydroxy-1-(3,4-dihydroxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-6-hepten-3,5-dione	AS, KAv	26.7	45589	C20H20O7	372.1	103; 117; 131; 137; 143; 145; 149; 163; 177	Diarylheptanoid	Curcuma longa L.	Jiang et al. (2006)
15	1,7-Bis(4-hydroxyphenyl)-1-heptene-3,5-dione	AS, DR, PR, SA, SU, KAv	27.4	8495	C19H18O4	310.1	117; 118; 119; 145; 146; 161; 175; 176	Diarylheptanoid	Curcuma longa L.	Jiang et al. (2006)
16	1,7-Bis(4-hydroxyphenyl)-1,4,6-heptatrien-3-one	AS, DR, PR, SA, SU, KAv	30.0	7240	C19H16O3	292.1	115; 117; 119; 120; 143; 145	Diarylheptanoid	Curcuma longa L.	Li et al. (2011)
17	7-(4-Hydroxy-3-methoxyphenyl)-1-(4-hydroxy phenyl)-4,6-heptadien-3-one	AS, KAv	31.8	138675	C20H20O4	324.1	107; 117; 119; 120; 122; 123; 131; 135; 137; 145; 146; 147; 148; 163; 195; 223	Diarylheptanoid	Curcuma longa L.	Jiang et al. (2006)
18	Coumaran	AS, DR, SA, SU	34.36	6563	C8H8O	120.0	116; 117; 118; 119	Coumarin	None	Chromadex
19	5,7-Dihydroxy-4-methylcoumarin	AS, SA, SU, KAv	34.57	92188	C10H8O4	192.17	113; 115; 116; 117; 118; 141	Coumarin	None	PubChem CID
										5354284
20	1,7-Bis(3,4,5-trimethoxyphenyl)-l,6-heptadiene-3,5-dione	PR, SU, KAr, KAv	35.82	4972	C25H28O8	456.3	280; 281; 298; 299	Diarylheptanoid	Synthesised the compound	Hahm et al. (2002)
21	Curlone	AS, DR, PR, SU, KAr, KAv	46.50	19540	C15H22O	218.0	101; 103; 117; 129; 130; 131; 132; 133; 134; 145; 157; 146; 157; 158; 173	Bisabolane sesquiterpene	Curcuma longa L.	He (2000)
22	Hydrocinnamic acid	SU, KAr	35.1	3812	C9H10O2	150.0	103; 105; 133	Phenolic acid	Curcuma longa L.	Ma and Gang (2006)
23	Curcumenol	AS, SU, KAr, KAv	36.67	34556	C15H22O2	234.1	105; 107; 109; 117; 123; 125; 133; 137	Sesquiterpene	Curcuma heyneana Valeton & Zijp	Sirat and Meng (2009)
24	Oleic acid	SA, KAr	60.2	15974	C18H34O2	282.1	168; 257	Fatty acid	Curcuma longa L.	Ma and Gang (2006)

LC-MS Analysis of Methanol Extracts

Methanolic extracts of rhizomes from the seven cultivars of Curcuma spp. were subjected to LC-MS analysis. The results from one of such analyses for each cultivar are presented in this article. The use of “positive” and “negative” modes of LC-MS was quite helpful. TIC chromatograms of all seven cultivars of Curcuma longa L. and C. aromatica Salisb. were shown in Supplementary Figure S1A for negative mode and Supplementary Figure S1B for positive mode.

A typical LC-MS analysis of methanolic extracts from rhizomes of C. longa L. cv. Alleppey Supreme revealed the presence of up to 86 compounds. Out of these, 43 were identified, and the remaining 43 compounds remained unknown. The (-) ESI-LC-MS detected 30 known compounds, and the (+) ESI-LC-MS detected 23 known compounds with an overlap of 10 compounds, detected by both negative and positive ion modes. A similar assessment of data was done with all seven cultivars of Curcuma spp. (Table 5). Altogether 62 compounds were identified, as presented in Supplementary Table S2. These compounds were grouped into three categories: cultivar-specific, detected in more than one cultivar, and common. There were 23 cultivar-specific compounds present in any one cultivar of C. longa L. or C. aromatica Salisb. (Table 6). 24 compounds were present in more than one cultivar of C. longa L. and/or C. aromatica Salisb. (Table 7). The remaining 15 were common in all seven cultivars (Table 8). Of these 15 common compounds found in the LC-MS/MS chromatograms, only one was a “Bisabolane” sesquiterpene (Parthasarathy et al., 2009) and all other 14 were diarylheptanoids. These were identified based on the MS/MS spectra reported by Jiang et al. (2006), including curcumin (CU), demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC). Among the other diarylheptanoids, 1-(4-hydroxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-1,4,6-heptatrien-3-one; 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one, etc., were common in all the cultivars of C. longa L. and C. aromatica Salisb. (Table 8). The structure of the five common compounds was given in Figure 4 (panels 13, 14–15, 16, and 17 corresponding to serial numbers 10, 5–6, 15, and 1, respectively, of Table 8). In addition to diarylheptanoids, several other classes (phenolic acids, flavonoids, ketonic sesquiterpenes, and fatty acid derivatives) were also detected in the turmeric rhizomes.

TABLE 5

Total number of compounds detected by LC-MS from the rhizome extract of C. longa L. and C. aromatica Salisb.

Sl. No.	Cultivar (Species)	Total number of Metabolites detected	Metabolites identified	Unknown Metabolites
1	Alleppey Supreme	86	43	43
	(C. longa L.)
2	Duggirala Red	107	23	84
	(C. longa L.)
3	Prathibha	60	28	32
	(C. longa L.)
4	Salem	91	28	63
	(C. longa L.)
5	Suguna	96	30	66
	(C. longa L.)
6	Kasturi Avidi	90	30	60
	(C. aromatica Salisb.)
7	Kasturi Araku	92	29	63
	(C. aromatica Salisb.)

TABLE 8

Compounds commonly detected by LC-MS analysis of rhizomes extract of all seven cultivars of Curcuma spp.: five of Curcuma longa L. (cvs. Alleppey Supreme, Duggirala Red, Prathibha, Salem, and Suguna) and two of C. aromatica Salisb. (cvs. Kasturi Araku, Kasturi Avidi). The structures for the compounds in the serial numbers 10, 5, 6, 15, and 1 are given in Figure 4 (panels 13, 14, 15, 16, and 17 respectively).

Sl. No.	Compound name	RT (Min)	Area/abundance	Formula	Mass (m/z)	Mass fragment ions	Class of compound	Reported from plant species	References
1	1,5-Bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one	23.5	3535	C19H18O5	325.1247	117; 118; 119; 120	Diarylheptanoid	Curcuma longa L.	Li et al. (2011)
						135; 143; 145; 146
						159; 161; 187
2	Ar-Turmerone	24.7	23545	C15H20O	216.1	103; 104; 105; 106; 107; 108; 115; 116; 117; 118; 119; 120	Bisabolane sesquiterpene	Curcuma longa L.	Parthasarathy and Chempakam (2008)
3	Tetrahydroxybisdemethoxycurcumin	25.4	4773	C19H20O4	311.1446	117; 118; 119; 120	Diarylheptanoid	Curcuma longa L.	Jiang et al. (2006)
						146; 161
4	Tetrahydrodemethoxycurcumin	25.9	7734	C20H22O5	341.1272	101; 113; 119	Diarylheptanoid	Piper nigrum L.	Ravindran (2000)
5	1-(4-Hydroxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-1,4,6-heptatrien-3-one	26.1	76458	C20H18O4	321.0938	115; 117; 119; 121	Diarylheptanoid	Curcuma longa L.	Jiang et al. (2006)
						132; 133; 134; 143
						145; 174; 235; 237
						247; 263’264; 274
						275
6	1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,4,6-heptatrien-3-one	26.3	64172	C21H20O5	351.1123	108; 115; 119; 136	Diarylheptanoid	Curcuma longa L.	Jiang et al. (2006)
						143; 148; 164; 195
						207; 223; 224; 235
						245; 251; 261; 262
						263; 279; 291; 307
7	Tetrahydroxycurcumin	26.5	81260	C20H20O7	371.1686	133; 134; 135; 148	Diarylheptanoid	Curcuma longa L.	Jiang et al. (2006)
						149; 175; 176; 177
8	1-(4-Hydroxy-3-methoxyphenyl)-7-(4-hydroxy-3,5-dimethoxyphenyl)-1,4,6-heptatrien-3-one	26.84	58571	C22H22O6	382.1	149; 159; 173; 197; 209; 211; 221; 233; 237; 239; 249; 261; 267; 277; 289; 293; 295; 305; 309	Diarylheptanoid	Curcuma longa L.	Jiang et al. (2006)
9	1,6-Heptadiene-3,5-dione, 1-(3,4-dihydroxyphenyl)-7-(4-hydroxy phenyl)-	31.6	5753	C19H16O5	324.1	134; 135; 136; 143	Diarylheptanoid	Curcuma longa L.	Jiang et al. (2006)
10	1-(3,4-Dihydroxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-hepta-1,6-diene-3,5-dione	32.3	16375	C20H18O6	353.1212	134; 135; 136; 150	Diarylheptanoid	Curcuma longa L.	Jiang et al. (2006)
11	Bisdemthoxycurcumin	34.5	1675644	C19H16O4	307.1132	117; 119; 120; 143	Diarylheptanoid	Curcuma longa L.	Jiang et al. (2006)
						145
12	Demethoxycurcumin	35.4	1302410	C20H18O5	337.1251	117; 119; 120; 132	Diarylheptanoid	Curcuma longa L.	Jiang et al. (2006)
						134; 143; 145; 158
						160; 175; 201
13	Dihydrocurcumin	35.8	5151	C21H22O6	369.1533	132; 134; 135; 149	Diarylheptanoid	Curcuma longa L.	Jiang et al. (2006)
						158; 160; 175
14	Curcumin	36.2	1503497	C21H20O6	367.1374	132; 133; 134; 135	Diarylheptanoid	Curcuma longa L.	Jiang et al. (2006)
						149; 158; 160; 161
						175
15	1-Heptene-3,5-dione, 1,7-bis-(4-hydroxy-3-methoxyphenyl)-	43.2	5078	C20H20O5	339.1473	117; 119; 120; 134	Diarylheptanoid	Curcuma longa L.	Jiang et al. (2006)
						158

FIGURE 4

Structure of few selected cultivar-specific (panels 3, 5–11 corresponding to serial numbers 2, 3–9 of Table 6); detected in more than one cultivar (panels 1–2, 4, 12 corresponding to serial numbers 4–5, 6, 7 of Table 7) and common (panels 13, 14–15, 16, and 17 corresponding to serial numbers 10, 5–6, 15, and 1, respectively, of Table 8) compounds already reported from genus Curcuma in methanolic extract from rhizomes by LCMS analysis of seven cultivars of Curcuma spp.: five of Curcuma longa L. (cvs. Alleppey Supreme, Duggirala Red, Prathibha, Salem, and Suguna) and two of C. aromatica Salisb. (cvs. Kasturi Araku and Kasturi Avidi). (1) Kaempferol-3-rhamnoside, (2) 3-acetyl coumarin, (3) luteolin-7-O-glucoside, (4) turmeronol, (5) 1,7-bis(4-hydroxy-3,5-dimethoxyphenyl)-1,6-heptadiene-3,5-dione, (6) 1,7-bis(3,4-dimethoxyphenyl)-1,6-heptadiene-3,5-dione, (7) (6S)-2-methyl-6-[(1R,5S)-(4-methene-5-hydroxyl-2-cyclohexen)-2-hepten-4-one, (8) 1,7-bis(4-hydroxyphenyl)-3,5-heptanediol, (9) 1,7-bis(3,5-diethyl-4-hydroxyphenyl)-1,6-heptadiene-3,5-dione, (10) 1-(4-hydroxy-3-methoxyphenyl)-5-(4-hydroxyphenyl)-1,4-pentadiene-3-one, (11) (-)-(12E,2S,3S,4R, 5R,6R, 9S,11S, 15R)-3,15-dibenzoyloxy-5,6-epoxylathyr-12-en-14-one, (12) 7-(3,4-dihydroxyphenyl)-5-hydroxy-1-phenyl-(1E)-1-heptene, (13) 1-(3,4-dihydroxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-hepta-1,6-diene-3,5-dione, (14) 1-(4-hydroxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-1,4,6-heptatrien-3-one, (15) 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,4,6-heptatrien-3-one, (16) 1-heptene-3,5-dione, 1,7-bis-(4-hydroxy-3-methoxyphenyl)-, (17) 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one.

Compounds Reported First Time From the Genus Curcuma Using GC-MS and LC-MS Analysis

A total of 39 compounds were detected (Figure 2) for the first time from the genus Curcuma. Out of these, 35 and 4 compounds were identified respectively in the essential oils and whole rhizome extracts of C. longa L. and C. aromatica Salisb. by the GC-MS and LC-MS techniques. Details of the compounds, including the class of compound, molecular weight, are given in Tables 2, 3, 5, and 6; structures of all these compounds are shown in Figure 2 (panels: 1–39). The MS and MS/MS spectra of these compounds are presented in Supplementary Figure S2 (panels: 1–39). These compounds were reported earlier from plants belonging to any genus other than Curcuma, and this is the first report from genus Curcuma. Out of the total of 62 compounds detected by LC-MS analyses of rhizome extracts, four compounds were reported for the first time from the Curcuma genus. One was cultivar-specific (Table 6; Sl. Nos. 1; Figure 2, panels: 36), and three were present in more than one cultivar (Table 7; Sl. Nos. 1–3). The structures of these first-time reported compounds are given in Figure 2 (panels: 37–39), and their corresponding mass fragmentation spectra are shown in Supplementary Figure S2 (panels: 36–39).

A comprehensive table each for GCMS (Supplementary Table S3) and LCMS (Supplementary Table S4) shows the details of compound identification methods used in the present study for the first-time reported compounds from genus Curcuma and the previous literature. A list of 80 (GC-MS) and 62 (LC-MS) compounds can be seen in Supplementary Tables S5, S6 respectively.

Discussion

In one of our previous studies, we reported that the HPLC method could be a valuable tool to differentiate the cultivars of Curcuma spp. based on their curcuminoids content ratios (Kulyal et al., 2016). Curcuminoids play a significant role in food, cosmetics, and medicinal compounds. But there are several other secondary metabolites such as terpenoids (e.g., mono-, sesqui-, di-, tri-, so on), alkenes, aromatic compounds, flavonoids, coumarins, etc. that are responsible for various biological activities. All these secondary metabolites are present in either the volatile essential oil or the nonvolatile fraction of the Curcuma spp. Employing untargeted metabolomics would be the ideal way to identify as many metabolites as possible. Therefore, in the present study, we analyzed these secondary compounds using GC-MS and LC-MS/MS.

Versatility of GC-MS and LC-MS Techniques to Identify a Large Number of Metabolites

GC-MS analysis is an appropriate technique for analyzing volatile compounds, whereas LC-MS is for detecting polar compounds, and thus, these two techniques are mutually complementary to each other. In the present study, several of the volatile compounds present in the cultivars of C. longa L. and C. aromatica Salisb. belonging to mono- and sesquiterpenoids were detected by GC-MS (Tables 2–4). On the other hand, LC-MS analysis detected phenolic (Tables 6–8) compounds, including several diarylheptanoids in the methanolic extracts of both C. longa L. and C. aromatica Salisb. (Figure 4). Electrospray ionization (ESI), coupled with LC/MS/MS, turned out to be a powerful tool in metabolite profiling and metabolomics research. Studies on chemical derivatization and quantification of several metabolites in turmeric powders and fresh rhizome extracts by LC-MS or LC-MS/MS were made. But the rapid screening within the cultivars of C. longa L. of fresh turmeric rhizome has not yet been reported. To the best of our knowledge, we were able to record the presence of several metabolites, which were not reported so far in the C. longa L. and C. aromatica Salisb. (Tables 2, 3, 6, 7, and Figure 2), using the available literature search, Metlin library, mass bank, and NIST library.

Cultivar Variability Based on Secondary Metabolites

Based on the presence or absence of metabolites identified by GC-MS and LC-MS analyses, there was a need to authenticate cultivar variability. Thus, the metabolite library can be constructed based on the cultivar-specific and compounds found in more than one cultivar. There are very few reports on cultivar-specific secondary metabolite variation. Out of a total of 142 compounds identified by both GC-MS and LC-MS, only 28 compounds (13 from GCMS and 15 from LCMS) were common (Tables 4, 8) present in all the cultivars of C. longa L. and C. aromatica Salisb. Ten of 13 common compounds (GCMS) were reported earlier from C. longa L. rhizome. Two compounds, namely sabinene and α-caryophyllene, were reported from the leaves of C. longa L. The remaining one compound, i.e., 2-heptadecanone, was detected for the first time from these two Curcuma species. This compound was earlier reported in the essential oil of Curcuma angustifolia Roxb. rhizome (Srivastava et al., 2006).

As per our analyses, 64 compounds (Tables 2, 6) out of 142 compounds were cultivar-specific. Of these 64 compounds, 41 were identified in essential oils by GC-MS (e.g., carvacrol, endo-borneol) and 23 (e.g., tumerone, methyl-7-methoxycoumarin,4-) in fresh rhizome extracts (LC-MS) of any one of the cultivars of C. longa L. or C. aromatica Salisb. In addition, 50 compounds (Tables 3, 7) were identified to be present in some of the cultivars, present in more than one cultivar but not common to all the cultivars of C. longa L. and C. aromatica Salisb. Out of these 50 compounds, 26 were identified in essential oils through GC-MS. For example, 1,3,5-cycloheptatriene was detected in all six cultivars except cv. Duggirala Red, whereas 12-oxabicyclo(9.1.0)dodeca-3,7-diene, 1,5,5,8-tetramethyl-, [1R-(1R*,3E,7E,11R*)]-, was detected only in cvs. Duggirala Red and Kasturi Araku. The rest 24 compounds were detected in rhizome extracts by LC-MS (e.g., chavicol detected in cvs. Alleppey Supreme and Suguna). The present extensive analyses of both essential oils and whole rhizome secondary metabolome of seven cultivars of C. longa L. and C. aromatica Salisb. established cultivar variability. Variability of the compounds within or/and in between the cultivars of C. longa L. and C. aromatica Salisb. will give a better understanding of their selection. The current study will help select cultivars for use in pharmacology or the food industry.

Discovery of First-Time Reported Metabolites in C. longa L. and C. aromatica Salisb.

In the present study, as many as 142 compounds were identified in the essential oils and rhizome extracts of C. longa L. and C. aromatica Salisb. Out of these, 39 compounds were identified for the first time in the genus Curcuma. However, these compounds were found in other plant genera. The structures of these compounds are shown in Figure 2, and corresponding details, including the class of compound, molecular weight, are given in Tables 2, 3, 6, and 7. As an example, cv. Alleppey Supreme of C. longa L. showed three cultivar-specific compounds. Among these, 1,2-cyclohexanediol, 1-methyl-4-(1-methylethyl)- (oxygenated alcoholic monoterpenoid) was earlier reported from leaf and peel essential oil of Citrus medica L. (Rutaceae). This compound is used as a flavoring agent (Bhuiyan et al., 2009); trans, trans-octa-2,4-dienyl acetate, present in common Malaysian Kaempferia galanga L. (Zingiberaceae), was used for its food-flavoring property (Othman et al., 2006). Phenol, 2-methoxy-3-(2-propenyl)-, an allyl chain-substituted guaiacol was reported from rosewood extracts (Jiang et al., 2018).

The following four compounds were identified from cv. Duggirala Red (C. longa L.): 3-isopropyl-4-methyl-1-pentyn-3-ol (alcohol constituent) containing leaf and stem of Anethum sowa Roxb. ex, Fleming, used for flavoring of food, beverages and also for many medical preparations (Saleh-e-in et al., 2010); 5,9-tetradecadiyne (unsaturated hydrocarbon) was found to be a major component of Ferula vesceritensis Coss. & Durieu ex Trab. leaf essential oil (Zellagui et al., 2012); naphthalene, 5-butyl-1,2,3,4-tetrahydro- (tetralin type of compounds) found in the essential oil of Meconopsis punicea Maxim. and M.delavayi (Franch.) Franch. Ex Prain (Slavík and Slavíková; Yuan et al., 2003); and santolina alcohol was reported from plant Achillea filipendulina Lam. (Sharopov and Setzer, 2010). A total of nine cultivar-specific compounds were detected in cv. Prathibha (C. longa L.) and the examples of these compounds and their source plants, respectively, are 7-tetracyclo[6.2.1.0(3.8)0(3.9)]undecanol, 4,4,11,11 tetramethyl- in Cyperus articulatus L. (Metuge et al., 2014); bicyclo(2.2.1)hept-2-ene, 2,3-dimethyl- in Abies alba Mill. (Yang et al., 2009). Cultivar-specific compounds of three each were detected in cvs. Salem (C. longa L.) and Suguna (C. longa L.) (Table 2). In C. aromatica Salisb., only one cultivar-specific compound was detected in cv. Kasturi Avidi, i.e., 3-octen-5-yne, 2,7-dimethyl-, (Z)-, and this compound was earlier reported from the medicinally important Litsea glutinosa (Lour.) C.B. Rob. fruit essential oil (Chowdhury et al., 2008a).

Some of the compounds identified in our study were present in more than one cultivar. For example, 6-(p-tolyl)-2-methyl-2-heptenol (Table 3) was detected in three cvs.: Alleppey supreme, Suguna of C. longa L., and Kasturi Avidi of C. aromatica Salisb. This compound was earlier reported from Zingiber officinale Roscoe (Zingiberaceae), used as a spice, food products, and beverages (Choudhari and Kareppa, 2013).

Limitations and Strengths of the Present Study

There is significant variability within and between the cultivars of C. longa L. and C. aromatica Salisb, which can be exploited to differentiate the cultivars of Curcuma spp. The feasibility of studies without using any standard compounds was pointed out by Núñez et al. (2020). Similarly, reference compounds were not used in our study to derive arithmetic indices under the experimental conditions. Despite the dilution made in the essential oil sample before injecting into the GC-MS system, the sample was still too concentrated. The high concentration of oil might have restricted the resolution due to overloading the detector. This could be the reason that we could not identify several compounds. We would ensure the further dilution of the oil sample in our future studies. However, the technology employed, GC-TOFMS and LC-QTOFMS, and MS-spectral database/literature search enabled us to establish the cultivar variability of Curcuma spp. The detailed information on the metabolite variability within or/and between the cultivars of C. longa L. and C. aromatica Salisb. may assist us in selecting the cultivars for a specific purpose, like culinary use, coloring, or pharmacological purpose. The studies such as the present one can help to select cultivars, particularly for use in pharmacology or the food industry. Metabolite variability poses a challenge in the use of turmeric in therapy. The practitioners need to be quite careful and use the identified cultivar and avoid mix-up. The caution applies to commercial/industrial use. Once standardized, the protocol should ensure the use of a specific cultivar. Our GC-MS and LC-MS-based metabolite identification is distinct from chemophenetic studies but is a complementary approach to characterize the Curcuma metabolome.

Importance of Curcuma spp. Metabolites for Human Health

Curcuminoids (CU, DMC, and BDMC) were identified as the main bioactive compounds of genus Curcuma and proved to have a broad spectrum of biological activities based on pharmacological studies. However, rhizomes and their essential oils of Curcuma spp. contained several other bioactive (volatile and nonvolatile) compounds. A summary of the pharmacological studies with the metabolites detected in the present study is given in Table 9. Some of the studies demonstrated therapeutic activity with the isolated metabolites, e.g., carvacrol (Suntres et al., 2015), p-cymene (De Oliveira et al., 2015), which are commonly found in essential oils of Curcuma spp. A few other reports correlated anti-inflammatory and antioxidant properties of C. longa L. essential oil with its chemical components ar-tumerone, α-santalene (Singh et al., 2010) (Table 9). Several compounds detected in the present study in the essential oil or rhizome extracts of C. longa L. or C. aromatica Salisb. were also found in the essential oil of other medicinal plants, traditionally used for their health benefits. The examples of such compounds are 5,9-tetradecadiyne, a cultivar-specific compound of Duggirala Red (C. longa L.), earlier reported in Ferula vesceritensis Coss. & Durieu ex Trab. leaf essential oil, exhibiting antibacterial activity; 3-octen-5-yne, 2,7-dimethyl-, (Z)-, a hydrocarbon monoterpene, identified from cv. Kasturi Avidi (C. aromatica Salisb.) was earlier reported from fruit essential oil of the medicinally important plant, Litsea glutinosa (Lour.) C.B. Rob. (Chowdhury et al., 2008a). We suggest that the medicinal use of the genus Curcuma can be not only species but also cultivar-specific.

TABLE 9

Pharmacological activity of metabolites identified, other than major curcuminoids (curcumin, demethoxycurcumin, and bisdemethoxy curcumin), in C. longa L. and C. aromatica Salisb.

Sl. No.	Compound name	Source plant	Tested Compound/essential oil/extract	Pharmacological activity/health benefit of the compound or compound containing plant product	References
					(Activity assay or compound detection)
1	Carvacrol	Curcuma longa L.	Compound	Antibacterial	Suntres et al. (2015)
2	p-Cymene	Curcuma longa L.	Compound	Antioxidant	(De Oliveira et al. (2015)
				Anti-inflammatory, anticancer, and antimicrobial effects	Marchese et al. (2017)
3	Eucalyptol	Curcuma longa L.	Compound	Antitumor anti-inflammatory relevance to Alzheimer’s disease	Murata et al. (2013); Islam et al. (2014)
4	α-Pinene	Curcuma longa L.	Compound	Anti-inflammatory and chondroprotective	Rufino et al. (2014)
5	α-Terpineol	Curcuma longa L.	Compound	Anti-inflammatory	De Oliveira et al. (2012)
6	Terpinolene	Curcuma longa L.	Compound	Anticancer	Okumura et al. (2012)
7	2-Heptadecanone	Curcuma angustifolia Roxb	Dried rhizome essential oil	As a coolant, demulscent	Srivastava et al. (2006)
8	Santolina alcohol	Achillea filipendulina Lam	Aerial part essential oil	Traditional herbal medicine	Sharopov and Setzer (2010)
9	Cyclohexanol, 2-methyl-5-(1-methylethenyl)-	Mentha spicata L.	Aerial parts essential oil	Antifungal	Mohammed et al. (2017)
10	Cyclohexane, 1,2-dimethyl-3,5-bis(1-methylethenyl)-	Rhanterium adpressum Coss. & Durieu	Aerial parts essential oil	Antifungal	Kala et al. (2009)
11	4-Ethylphenethylamine	Psidium guajava L.	Stem bark essential oil	Antioxidant	Fasola et al., 2011)
12	5,9-Tetradecadiyne	Ferula vesceritensis Coss. & Durieu ex Trab	Leaves essential oil	Antibacterial	Zellagui et al. (2012)
13	E-11-Tetradecenoic acid	Coriandrum sativum L.	Leaf essential oil	Spice, flavoring agent, antimicrobial	Bhuiyan et al. (2009)
14	6,10-Dodecadien-1-yn-3-ol, 3,7,11-trimethyl-	Hiptage benghalensis (L.) Kurz	Leaves essential oil	Treatment of skin diseases, cough, asthma, leprosy	Venkataramani and Chinnagounder (2012)
15	Chavicol	Piper betle L.	Leaf oil	Antifungal, antiseptic, and anthelmintic	Nagori et al. (2011)
16	1,2-Cyclohexanediol, 1-methyl-4-(1-methylethyl)-	Citrus medica L.	Leaf and peel essential oil	Antibiotic	Bhuiyan et al. (2009)
17	3-Isopropyl-4-methyl-1-pentyn-3-ol	Anethum sowa Roxb. ex, Fleming	Leaf and stem essential oil	Flavoring of food and beverages, antimicrobial, antioxidant	Saleh-e-in et al. (2010)
18	Bicyclo(2.2.1)hept-2-ene, 2,3-dimethyl-	Abies alba Mill	Leaf and twig essential oil	Radical scavenging activity	Yang et al. (2009)
19	2-Nonen-4-yn-1-ol, (Z)-	Alpinia speciosa (J.C. Wendl.) K. Schum	Seeds and leaves essential oil	As a food and herbal medicine, mosquito larvicidal activity	Ho (2010)
20	8-Methylene-3-oxatricyclo[5.2.0.0(2,4)]nonane	Schisandra chinensis (Turcz.) Baill	Dried fruit essential oil	Antioxidant	Wang et al. (2005)
21	3-Cyclohexen-1-one, 3,5,5-trimethyl-	Crocus sativus L.	Dried saffron oil	Antitumor	(D’Auria et al. (200
22	Ar-Tumerone	Curcuma longa L.	Rhizome essential oil	Antioxidant	Singh et al. (2010)
	α-Turmerone
	β-Turmerone
	α-Santalene
	Ar-Curcumene
23	7-Tetracyclo[6.2.1.0(3.8)0(3.9)]undecanol, 4,4,11,11 tetramethyl-	Cyperus articulatus L.	Roots/rhizome essential oil	Anti-onchocera activity	Metuge et al. (2014)
24	Cholesta-8,24-dien-3-ol, 4-methyl-, (3á,4à)-	Parkia speciosa Hassk.	Seed essential oil	High nutritional and medicinal value	Salman et al. (2006)
25	3-Octen-5-yne, 2,7-dimethyl-, (Z)-	Litsea glutinosa (Lour.) C.B. Rob	Fruit essential oil	Antirheumatic	Chowdhury et al. (2008b)
26	5,8,11,14-Eicosatetraenoic acid, phenylmethyl ester, (all-Z)-	Petiveria alliacea L.	Whole plant essential oil	Used as folk medicine to enhance memory and in treatment of common cold, flu, other viral, or bacterial infections	Sathiyabalan et al. (2014)
27	Naphthalene, 5-butyl-1,2,3,4-tetrahydro-	Meconopsis punicea Maxim. and M. delavayi (Franch.) Franch. Ex Prain	Whole plant essential oil	As a traditional medicinal plant for anti-inflammatory and analgesic activity	Yuan et al. (2003)
28	1H-3a,7-methanoazulene, 2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-, [3R-(3à,3aá,7á,8aà)]-	Lindera aggregata (Sims) Kosterm	Root/tubers essential oil	Treatment of decubitus ulcer	Hong (2011)
29	trans, trans-Octa-2,4-dienyl acetate	Kaempferia galanga L.	Dried rhizomes	Spice, food-flavoring agent	Othman et al. (2006)
				Vasorelaxant
30	11-Dodecen-2-one	Ficus hispida L. f	Fresh male and female receptive figs	Hepatoprotective, anti-inflammatory, antipyretic	Song et al. (2001)
31	Kaempferol-3,7-O-dimethyl ether	Lumnitzera racemosa Willd and Artemisia vulgaris L.	Fresh twig methanolic extract; leaf methanolic extract	Antibacterial	Nikolova (2006); DeSouza et al. (2010)
32	5,7,8-Trihydroxy-2′,5′-dimethoxy-3′,4′-methylene dioxyisoflavanone	Terminalia ivorensis A. Chev	Fresh sawdust methanolic extract	Antifungal	Ogundare and Olajuyigbe (2012)
33	Kaempferol-3-O-rutinoside-7-O-glucoside	Lycopersicon esculentum Mill	Methanolic extract of fruit	Antioxidant	Le Gall et al. (2002)
34	Phenol, 2-methoxy-3-(2- propenyl)-	Dalberga stevensonii Standl	Wood extracts	Used as raw material for medical industries	Jiang et al. (2018)
35	2-Pentanone, 4-mercapto-4-methyl-	Camellia sinensis (L.) Kuntze	Tea leaves	Antioxidant	Kumazawa et al. (2005)

Concluding Remarks

Essential oils from spices and aromatic plants are enriched with bioactive metabolites, easily isolated and used, unlike the difficulties encountered with synthetic chemical products. The low mammalian toxicity and biodegradable nature of the natural secondary products provide an attractive option to develop them also for crop protection. Metabolomics is a practical and dynamic approach to make a comprehensive study. Both GC-MS and LC-MS techniques should be used to characterize the metabolite profiles of as many cultivars as possible for building a reference library. Preparative LC can be helpful to collect individual metabolite fractions and establish their identity. Several metabolites detected in 7 selected cultivars of Curcuma spp. by GC-MS and LC-MS analyses are reported first time in Curcuma spp. We suggest that the seven Indian cultivars of Curcuma spp. employed in our study can be used as sources of such compounds. High-throughput analysis of cultivar-specific and first-time detected compounds in the present study may lead to new drug candidates. The metabolites validated for their medicinal or other users can be quantified using simple techniques such as HPLC or TLC to ensure their presence in the herbal preparations.