*2.5. Hierarchical Cluster Analysis*

Agglomerative hierarchical cluster (AHC) analysis was carried out using the chemical compositions of the *C. longa* rhizome essential oils. The compositions were treated as operational taxonomic units, with the percentages of the 16 most abundant components (α-turmerone, *ar*-turmerone, β-turmerone, α-phellandrene, 1,8-cineole, α-zingiberene, β-sesquiphellandrene, terpinolene, (6*S*,7*R*)-bisabolone, *p*-cymene, zingiberenol, *ar*-curcumene, β-bisabolene, 7-*epi-trans*-sesquisabinene hydrate, limonene, and *ar*-tumerol), using XLSTAT Premium, version 2018.1.1.62926. Euclidean distance was used to determine dissimilarity, and Ward's method was used to define the clusters.

## **3. Results and Discussion**

The fresh mother and lateral rhizomes (Figure 1) were chopped and hydrodistilled to give pale yellow to yellow essential oils in yields ranging from 0.204% to 0.695% (Table 1). Varieties CL5, CL9, and CL11 gave better essential oil yields (0.443–0.659%) than CL3 or CL10 (<0.3%). The total oil content of CL5, CL9, and CL11 were similar to those reported for Indian chemotypes of *C. longa* [21,22]. In CL5, CL9, and CL10, the mother rhizomes had higher oil yields than the lateral rhizomes. A similar trend was reported for curcumin concentration of turmeric varieties grown in south–central AL [12].

The chemical compositions of the *C. longa* rhizome essential oils are compiled in Table 2. The major components in the essential oils were α-phellandrene (3.7–11.8%), 1,8-cineole (2.6–11.7%), α-zingiberene (0.8–12.5%), β-sesquiphellandrene (0.7–8.0%), *ar*turmerone (6.8–32.5%), α-turmerone (13.6–31.5%), and β-turmerone (4.8–18.4%). A hierarchical cluster analysis of the *C. longa* rhizome essential oils in this study was carried out based on the concentrations of the 16 most abundant essential oil components (α-turmerone, *ar*-turmerone, β-turmerone, α-phellandrene, 1,8-cineole, α-zingiberene, βsesquiphellandrene, terpinolene, (6*S*,7*R*)-bisabolone, *p*-cymene, zingiberenol, *ar*-curcumene, β-bisabolene, 7-*epi-trans*-sesquisabinene hydrate, limonene, and *ar*-tumerol) (Figure 2).






**Figure 2.** Dendrogram obtained from agglomerative hierarchical cluster analysis of the rhizome essential oils from varieties of *Curcuma longa* cultivated in north Alabama.

The cluster analysis of the *C. longa* varieties in this work revealed two well-defined groups. One group (varieties CL5, CL9, and CL11) was dominated by turmerones (α-turmerone, *ar*-turmerone, and β-turmerone). The second group demonstrated lower concentrations of turmerones, but higher concentrations of other components (e.g., α-zingiberene and β-phellandrene). Previous examination of *C. longa* rhizome essential oils from India as well as other geographical locations showed there to be four clusters based on the relative concentrations of the turmerones [14]: (1) dominated by turmerones, but with relatively large concentrations of other components; (2) dominated by turmerones, especially *ar*-turmerone; (3) dominated by turmerones, especially α-turmerone; and (4) very large concentrations of *ar*-turmerone. The chemical compositions of varieties CL5, CL9, and CL11 placed them into the cluster dominated by turmerones (i.e., cluster 2 of [14]), while varieties CL3 and CL10 had relatively lower concentrations of turmerones with relatively larger concentrations of components other than turmerones (i.e., cluster 1 of [14]). Thus, the essential oil compositions of these turmeric varieties adaptable to the Alabama summer growing season fit in well with essential oil compositions of turmeric varieties cultivated in Asia. The *ar*-turmerone and α-turmerone levels were similar to (CL3) or greater than (CL5, CL9, and CL11) those reported for turmeric grown in a tropical country, Brazil [23]. The *ar*-turmerone and α-turmerone were substantially greater than those reported for turmeric grown in India [24]. The β-turmerone levels were generally lower than those reported for turmeric grown in tropical countries [22–24], but similar to those reported for turmeric grown in Korea [25]. The cluster analysis also showed very little dissimilarity between the mother rhizome essential oil and the lateral rhizome essential oils for each of the varieties.

Xu and coworkers examined the extracts of 160 samples of *C. longa* from China [26]. Gas chromatographic analysis of the volatiles from the extracts revealed three volatile profile types, while high-performance liquid chromatographic (HPLC) analysis showed three types based on curcuminoid content. Unfortunately, Xu et al. identified only 10 volatile components, whereas 79 components were identified in our essential oil work. Furthermore, percent compositions were not reported and only "representative" chromatograms were presented. Nevertheless, although comparison is tenuous, based on the chromatograms, the volatile profile types identified seem to be analogous to essential oil types in our work. Importantly, volatile profile types "a" and "b" correspond to highcurcuminoid type "B" [26]. Therefore, we conclude that high turmerone concentrations are desirable qualities in turmeric essential oil.

The turmerones are responsible for the turmeric-like odor of *C. longa* [27]. In addition to their pungent scent, turmerones are important, biologically active constituents of *C. longa* rhizome essential oils [28], showing in vitro cytotoxic activities against several human tumor cell lines [29–32], anti-inflammatory activity through attenuated expression of proinflammatory cytokines [33,34], antibacterial activity against Gram-positive organisms [35], antifungal activity against phytopathogenic [36] and dermatopathogenic [37] fungi, mosquito larvicidal activity against *Anopheles gambiae* [38] and *Culex pipiens* [39], and insecticidal activity against the agricultural pests *Sitophilus zeamais* and *Spodoptera frugiperda* [40].

#### **4. Conclusions**

The chemical profiles of varieties CL5, CL9, and CL11 tested in this study in north Alabama were comparable to those growing in tropical regions of the world, suggesting that these varieties are suitable for commercialization in this region. However, CL3 and CL10 gave relatively poor essential oil yields and essential oils with lower concentrations of the turmerones. There is a growing market for *Curcuma longa* essential oils, with several varieties showing promise for development in the southeastern United States.

**Author Contributions:** Conceptualization, S.R.M.; methodology, S.R.M. and W.N.S.; formal analysis, W.N.S.; investigation, W.N.S., L.D. and A.P.; resources, S.R.M.; data curation, W.N.S.; writing original draft preparation, W.N.S. and S.R.M.; writing—review and editing, all authors; supervision, S.R.M.; project administration, S.R.M.; funding acquisition, S.R.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research and the APC were funded by USDA/National Institute of Food and Agriculture (NIFA)-Agriculture and Food Research Initiative (AFRI) Project, grant number 2016-68006-24785.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are available from the corresponding authors.

**Acknowledgments:** W.N.S. and A.P. participated in this work as part of the activities of the Aromatic Plant Research Center (APRC, https://aromaticplant.org/). S.R.M. and L.D. thank Andrea Barr, Mounika Pudota, Jasmine Arnold, and Suresh Kumar for their assistance with field trials.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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