Essential Oil Chemotypes of Four Vietnamese Curcuma Species Cultivated in North Alabama

: Curcuma (turmeric) species are important culinary and medicinal plants, and the essential oils of Curcuma rhizomes have demonstrated promising pharmacological properties. The essential in curzerenone, curdione, and germacrone. These adapted turmeric varieties in North Alabama have potential use for medical purposes and medicinal plant oil market demands in the U.S.


Introduction
There are currently 93 recognized species of Curcuma L. (Zingiberaceae) [1]. These perennial rhizomatous herbs originated in subtropical and tropical areas of Asia, Australia, and South America [2], and a number of Curcuma species are cultivated in large scales in India, Nepal, Pakistan, Bangladesh, Indonesia, Malaysia, and Thailand [3]. Curcuma species are herbaceous perennial herbs with tuberous rhizomes (underground stems). Among them, some important species such as Curcuma amada Roxb. (mango ginger), Curcuma angustifolia Roxb. (wild arrowroot), C. aromatica Salisb. (wild turmeric), Curcuma caesia Roxb. (black turmeric), Curcuma decipiens Dalzell, Curcuma kwangsiensis S. G. Lee & C. F. Liang, Curcuma montana Roxb., Curcuma ochrorhiza Valeton, Curcuma pierreana Gagnep., Curcuma roscoeana Wall., and Curcuma zedoaria (Christm.) Roscoe (zedoary) have economical value as they are used in medicine, cosmetics, and both the floricultural and culinary industries [4]. Turmeric is mainly used for culinary, medicinal, and aromatic purposes. Its rhizomes are the ancient colorful spice source and have a bitter and pungent taste and a pepper-like aroma. Turmeric is also known as the "Golden Spice of India" [5] or "Kitchen Queen" [6]. For example, it has been used in curries in India; in Japan and Korea it is popularly served as a herbal tea; and it is used as a preservative and a coloring agent in mustard sauce, cheese, butter, and chips in the Western world [7]. Curcuminoids and the essential oil of turmeric are associated with a myriad of medicinal, culinary and industrial properties of curcuma species [8], which are derived from the underground plant part, rhizomes (actually the stem), which are tuberous, with a rough and segmented skin. The primary rhizome is known as "mother rhizome" or bulb, and is pear-shaped in the center ( Figure 1). The branches of mother rhizomes are the secondary rhizomes, called lateral or "finger rhizomes" [9]. (zedoary) have economical value as they are used in medicine, cosmetics, and both the floricultural and culinary industries [4]. Turmeric is mainly used for culinary, medicinal, and aromatic purposes. Its rhizomes are the ancient colorful spice source and have a bitter and pungent taste and a pepper-like aroma. Turmeric is also known as the "Golden Spice of India" [5] or "Kitchen Queen" [6]. For example, it has been used in curries in India; in Japan and Korea it is popularly served as a herbal tea; and it is used as a preservative and a coloring agent in mustard sauce, cheese, butter, and chips in the Western world [7]. Curcuminoids and the essential oil of turmeric are associated with a myriad of medicinal, culinary and industrial properties of curcuma species [8], which are derived from the underground plant part, rhizomes (actually the stem), which are tuberous, with a rough and segmented skin. The primary rhizome is known as "mother rhizome" or bulb, and is pear-shaped in the center (Figure 1). The branches of mother rhizomes are the secondary rhizomes, called lateral or "finger rhizomes" [9]. Though turmeric has been known for its multiple uses for over 4000 years in India [10], its use as a medicinal and health supplement in the United States is of recent origin. The interest in turmeric in the U.S. has been increasing over the past two decades mainly due to a large number of scientific publications on its medicinal benefits [8]. To meet the growing demand for turmeric, the U.S. imports 90% of its market demand mainly from India. The U.S. import market was estimated at USD 87.28 million in 2018 [11]. The large market for turmeric in the United States suggests that there is opportunity for cultivation of turmeric in this country provided varieties with high curcumin yield and desirable essential oil composition are available.
Curcuma aromatica Salisb. (wild turmeric) is found naturally in South Asia, including southern China, Bhutan, Myanmar, India, Nepal, Sri Lanka [12], and Vietnam [13], and is widely cultivated in China, India, and Japan [14]. The plant is used in traditional medicines throughout its range for its wound-healing, anti-inflammatory, anti-tumor, immunomodulatory, antimicrobial effects and as an antidote for snake venom [15][16][17]. The rhizome essential oils are generally dominated by camphor, curzerenone, germacrone, curdione, and 1,8-cineole [14]. Though turmeric has been known for its multiple uses for over 4000 years in India [10], its use as a medicinal and health supplement in the United States is of recent origin. The interest in turmeric in the U.S. has been increasing over the past two decades mainly due to a large number of scientific publications on its medicinal benefits [8]. To meet the growing demand for turmeric, the U.S. imports 90% of its market demand mainly from India. The U.S. import market was estimated at USD 87.28 million in 2018 [11]. The large market for turmeric in the United States suggests that there is opportunity for cultivation of turmeric in this country provided varieties with high curcumin yield and desirable essential oil composition are available.
Curcuma longa L. (turmeric) is cultivated worldwide, especially in tropical countries in Asia, Australia, and the Neotropics [9]. It is a well-known medicinal agent and culinary ingredient. In addition to curcumin and other non-volatile curcuminoids, the essential oil of turmeric has been employed in the treatment of various maladies in humans and animals [21]. Turmeric essential oils are made up of hundreds of components, the major components, however, are α-turmerone, β-turmerone, ar-turmerone, β-sesquiphellandrene, α-zingiberene, germacrone, terpinolene, ar-curcumene, and α-phellandrene [3,22].
Curcuma zanthorrhiza Roxb. (Javanese turmeric) is often referred to in the literature as Curcuma xanthorrhiza Roxb., however, that name is not recognized by World Flora Online [23]. The plant is native to Indonesia, although is also cultivated in Malaysia, the Philippines, Thailand, Vietnam, and to a lesser extent in China, India, Japan, and South Korea [24]. Traditional medicinal uses of the plant include treatment for stomach illness, liver ailments, constipation, bloody diarrhea, dysentery, arthritis, rheumatism, fevers, hemorrhoids, vaginal discharge, and skin eruptions [24].
As part of our program investigating potential cultivation of Curcuma in Alabama, Curcuma aromatica (both green-and white-colored rhizomes), C. caesia (black-colored rhizome), C. zanthorrhiza (lime-green rhizome), and C. longa (both yellow-, and red-colored rhizomes), obtained from Vietnam, were cultivated in North Alabama ( Figure 1). The rhizome essential oils were obtained by hydrodistillation and analyzed by gas chromatographic methods. Both the "mother" or main rhizomes as well as the "daughter" or finger rhizomes were obtained and analyzed. The six Curcuma varieties used in this study were selected out of 64 genotypes according to three criteria: high yield but low curcuminoid content (variety, CL56), high yield but no curcumin content (CA22, CA46, CC38, and CZ44), and high yield and high curcumin content (CL63) based on unpublished data by the authors, Lam Duong and S.R. Mentreddy at Alabama A&M University.

Plant Material
The six Curcuma varieties used in this study were selected out of 64 genotypes according to three criteria: high yield but low curcuminoid content (variety, CL56), high yield but no curcumin content (CA22, CA46, CC38, and CZ44), and high yield and high curcumin content (CL63) based on studies at Alabama A&M University. The rhizomes of the six turmeric varieties belonging to Curcuma spp. were collected by Lam Duong from various locations in Vietnam: CA22 (Quang Nam province), CA46 (Gia Lai province), CC38 (Nghe An province), CL56 (Bac Giang province), CL63 (Quang Tri province), and CZ44 (Gia Lai province). The Curcuma rhizomes varieties were initially planted in a glass greenhouse at Alabama A&M University (Normal, AL, USA) and subsequently cultivated at the Alabama A&M Winfred Thomas Agricultural Research Station (Hazel Green, AL, USA, 34 89 N, 86 56 W) as previously described for C. longa cultivation [9]. Each of the fresh Curcuma rhizomes were collected on 18 March 2021, and stored at −20 • C until processed. The mother and daughter rhizomes were, separately, chopped and hydrodistilled using a Likens-Nickerson apparatus for 4 h (see Table 1).
Gas chromatography with flame ionization detection (GC-FID) was carried out as previously reported [29]: Shimadzu GC 2010 (Shimadzu Scientific Instruments, Columbia, MD, USA) equipped with flame ionization detector, ZB-5 capillary column (60 m × 0.25 mm i.d.; film thickness 0.25 µm) (Phenomenex, Torrance, CA, USA); oven temperature programmed as above for GC-MS; injector and detector temperatures 260 • C; He carrier gas, flow rate 1.0 mL/min; 0.3 µL of 5% w/v solution in CH 2 Cl 2 were injected, ratio 1:31. The percent compositions of the essential oils were calculated from peak areas with quantification using the external standard method; calibration curves of representative compounds from each class were used for quantification. Chiral GC-MS was carried out as previously reported [29]: Shimadzu GCMS-QP2010S instrument (Shimadzu Scientific Instruments, Columbia, MD, USA), Restek B-Dex 325 column (Restek Corporation, Bellefonte, PA, USA); oven temperature program 50 • C to 120 • C at 1.5 • C/min, then to 200 • C at 2.0 • C/min; 0.1 µL of 5% w/v solutions in CH 2 Cl 2 were injected, with a split ratio of 1:25. The enantiomers were determined by comparison of retention times with authentic samples obtained from Sigma-Aldrich (Milwaukee, WI, USA) and the relative enantiomer percentages were calculated from peak integration.

Chemical Compositions of Curcuma Rhizome Essential Oils
The fresh rhizome samples were hydrodistilled to give colorless or pale-yellow essential oils in yields ranging from 0.41% to 1.13% (Table 1). The chemical compositions of the Curcuma rhizome essential oils are compiled in Table 2. Gas chromatograms of each Curcuma variety are shown in Supplementary Figure S1.
Interestingly, the volatile phytochemistry of C. caesia and C. zanthorrhiza rhizomes are very similar (about 90% similarity). Likewise, the green-and white-colored rhizome essential oils of C. aromatica are very similar (about 95% similarity). The yellow-and red-colored rhizome essential oils of C. longa showed somewhat lower similarity (about 60% similarity).
There are some significant differences in the concentrations of the major componen in Cluster 1 (Figure 3). The concentration of curzerenone is significantly greater in C. cae than in either C. aromatica or C. zanthorrhiza. The concentrations of curdione in C. aromat are significantly lower than those in either C. caesia or C. zanthorrhiza. Germacrone w significantly lower in C. caesia than in either C. aromatica or C. zanthorrhiza.  The significant differences between the essential oils of yellow-and red-colored C. longa are the concentrations of ar-turmerone (much higher in the red rhizome variety) and β-turmerone (also higher in the red rhizome variety). The concentrations of α-turmerone in the red and yellow varieties are not significantly different ( Figure 4). Nevertheless, although the compositions of yellow-and red-colored rhizomes of C. longa are notably different (60% similarity), they are comparable to the respective chemical profiles of C. longa from tropical Asian collections [3]. The significant differences between the essential oils of yellow-and red-colored C. longa are the concentrations of ar-turmerone (much higher in the red rhizome variety) and β-turmerone (also higher in the red rhizome variety). The concentrations of α-turmerone in the red and yellow varieties are not significantly different (Figure 4). Nevertheless, although the compositions of yellow-and red-colored rhizomes of C. longa are notably different (60% similarity), they are comparable to the respective chemical profiles of C. longa from tropical Asian collections [3].
longa are the concentrations of ar-turmerone (much higher in the red rhizome variety) and β-turmerone (also higher in the red rhizome variety). The concentrations of α-turmerone in the red and yellow varieties are not significantly different ( Figure 4). Nevertheless, although the compositions of yellow-and red-colored rhizomes of C. longa are notably different (60% similarity), they are comparable to the respective chemical profiles of C. longa from tropical Asian collections [3].

Conclusions
The rhizome essential oils of C. aromatica, C. caesia, C. longa, and C. zanthorrhiza that were cultivated in North Alabama show wide variation in composition compared to essential oils from other geographical locations. Nevertheless, the essential oil yields and compositions provide evidence that Curcuma can be successfully cultivated in North Alabama and may provide additional sources of these species for both culinary and herbal medicinal uses. The knowledge of their relative oil yields and compositions could help in value-addition for either fresh rhizomes or dry herbal markets. The four species showed specific essential oil components, which are known to have extensive pharmacological activity separately or in combination with curcuminoids. The species can be used to tailor herbal medicines to combat particular ailments. The cultivation of specific varieties to cater to niche markets could not only benefit the farmers, but also have an impact on the socioeconomic sustainability of rural Alabama in particular and the southeastern U.S. in general. As far as we are aware, this is the first report of the essential oil compositions, including enantiomeric distributions for these Curcuma species cultivated in North America. Among the four species, the C. longa species that combine high yield with high curcumin have been found to be suitable for cultivation. However, the remaining species have economic potential, for example C. zanthorrhiza is well known for its antimicrobial activity against common human pathogens to cater to herbal companies interested in varieties that are high in a certain essential oil component.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/horticulturae8050360/s1, Figure S1: Gas chromatograms of rhizome essential oils of Curcuma varieties cultivated in North Alabama; Figure S2: Mass spectra of unidentified components of Curcuma rhizome essential oils.