**3. Chemical Analysis of the Essential Oil Obtained from** *C. longa* **Rhizomes**

The chemical composition of the essential oil obtained from *C. longa* rhizomes has been widely determined through gas chromatography-mass spectrometry (GC-MS) (Table 2), which is normally used for a sesquiterpenoid analysis [102] alone or combined with gas chromatography-flame ionisation detector (GC-FID) [103–105] to achieve a quantitative analysis. The determination of the chemical composition is key, because the components of the essential oil and their concentration can be considered a fingerprint conferring specific characteristics and properties [106].

As a general rule, oxygenated sesquiterpenes have been identified as the predominant ones (Table 2) and the principal reason of the biological activity of turmeric essential oil [107]. Concretely, turmerones (α-, β- and ar-) represent the major and the most distinctive individual components [108,109] (Table 2 and Figure 1). They give interesting properties to *C. longa* essential oil, such as anticancer, anti-inflammatory, antioxidant and the prevention of dementia [72,110–113]. Even they enhance the bioavailability and activity of other important turmeric components like curcumin [114–116]. In particular, ar-turmerone (6S-2-methyl-6-(4-methylphenyl) hept-2-en-4-one) has been identified as the leading one, followed by α- and β-, in *C. longa* rhizome oil (Table 2). Many authors have reported about the therapeutic potential of ar-turmerone and its numerous benefits for human health [113]. Lee demonstrated its antibacterial activity against human pathogens like *Clostridium perfringens* and *Escherichia coli* [117]. In the same year, he also reported a higher inhibitory effect than aspirin in platelet aggregation induced by collagen and arachidonic acid [118]. Other researchers have proposed ar-turmerone as a natural anticancer and cancer-preventive agent, being considered the α,β-unsaturated ketone of the molecule, the principal pharmacophore, for this activity [51,119–121]. ar-Turmerone has also been observed as useful in the prevention and attenuation of inflammatory diseases like psoriasis and neuronal ones [122,123].

**Figure 1.** Main compounds found in the rhizomes and leaves of turmeric essential oils.

Oxygenated sesquiterpenes also constitute the predominant group in the essential oils obtained from the rhizome of other species included in the genus *Curcuma* [124]. For instance, curzerenone was the main compound in the rhizome oil of *C. angustifolia* and *C. zedoaria*; curdione was the major one in *C. nankunshanensis*, *C. wenyujin* and *C. kwangsiensis*; germacrone in *C. sichuanensis* and *C. leucorhiza*; β-elemenone in *C. nankunshanensis* var. *nanlingensis*; xanthorrhizol in *C. xanthorrhiza* and velleral in *C. attenuata* [124–128]. Turmerones are normally present, being considered the most representative components in general. Nevertheless, their amount may vary between species, probably due to the intrinsic differences between them [129]. The quantification of oxygenated sesquiterpenes, together with the identification of the secondary components, are key for the distinction and quality control of *Curcuma* spp. [17,130].

The sesquiterpenoids are generally followed by smaller quantities of sesquiterpene hydrocarbons in *C. longa* rhizome oil (Table 2 and Figure 1). This group is characterised by great structural diversity, providing a variety of fragrances and characteristic aromas to the essential oil [131]. Specifically, monocyclic bisabolane derivatives with a C6-ring formed in analogy to the menthane skeleton highlighted in turmeric essential oil obtained from rhizomes. Some examples are bisabolene isomers (β-bisabolene), α-zingiberene and ar-curcumene, characteristic of *Curcuma* spp. and ginger. β-caryophyllene is also common, widely spread in food plants and derived from α-humulene, with a C9-ring fused to a cyclobutane ring [132]. Sesquiterpene hydrocarbons predominate over oxygenated ones in the rhizome oil of other *Curcuma* spp., such as *C. aromatica* (Sesquiterpene Hydrocarbons (SH): 8.30% ± 1.90% and Oxygenated Sesquiterpenes (OS): 7.10% ± 2.14%) and *C. kwangsiensis* var *nanlingensis* (SH: 9.76% ± 1.89% and OS: 6.80% ± 1.27%) [124].

The amount of monoterpene hydrocarbons and oxygenated monoterpenes are usually lower in most samples of rhizome essential oil of *C. longa* (Table 2). Contrarily, they constitute the most abundant group in the rhizome oil of other different *Curcuma* spp., such as *C. amada* [133], as well as in the essential oils obtained from the aerial parts of *C. longa* [17,134–137]. Regarding this, the yield of *C. longa* essential oil varied between the leaves (23%), rhizomes (48%) and rhizoids (27%), and the chemical composition was different between the leaf petiole, lamina and rhizoid oils (myrcene, *p*-cymene, etc.) compared to the stem and rhizome ones in which turmerones predominated [138]. α-Phellandrene, terpinolene and 1,8-cineole (Figure 1) are usually the most abundant compounds detected in the essential oil extracted from the leaves of *C. longa* [36,39,43,44], whereas turmerones are found in minor concentrations (Table 2) [109], being also usually found in the essential oils of the aerial parts of *C. longa p*-cymene, α-terpinene, myrcene and pinenes (Table 2) [134,135,137,139,140]. However, in samples of *C. longa* grown in Nigeria, the leaf essential oil was dominated by turmerones, like in rhizomes (Table 2) [141,142]. In addition, important concentrations of C8-aldehyde (20.58%) were found in the essential oil of *C. longa* leaves in a high-altitude research station in Odisha, India [140]. The concentration of these compounds can be increased by enhancing the leaf biomass production [143].

The aerial parts of *C. longa* normally end as waste products. An interest approach is their recycling to obtain biologically active compounds. In this sense, *C. longa* leaf essential oil and its principal component α-phellandrene have demonstrated remarkable insecticidal activity against *Cochliomya macellaria*, causative agents of myasis in humans and animals, as well as against *Lucilia cuprina* [144,145], being also a *C. longa* leaf essential oil highlight because of its medicinal and food-preservation properties, with a significant inhibition of microbial growth and toxin production [146,147].

On the other hand, several studies corroborate that the qualitative and quantitative chemical compositions of turmeric rhizomes essential oil may fluctuate according to many factors [124,148,149]. Sometimes, different chemical compositions come from the intrinsic characteristics of each genotype. In fact, certain traits of a specific variety of *C. longa* can influence the content of rhizome oil, representing good criteria for the selection of high-yield ones. Regarding this, an interesting study observed a direct relationship between plant height and rhizome oil content, as well as a negative correlation between the amount of

essential oil in the dry leaf with the one contained in the fresh rhizome [150]. A clear example of genotype influence is the dissimilar chemical composition between yellow *C. longa* rhizome oil rich in oxygenated sesquiterpenes (ar-turmerone, turmerone, curlone, etc.) and red one with oxygenated monoterpenes (carvacrol, citral, methyl eugenol, geraniol, etc.) as principal compounds more similar to *Origanum* or *Thymus* spp. [151]. Indeed, the rhizome colour is closely related to the beneficial properties of *C. longa* [152]. The influence of the genotype or cultivars have also been reported by other authors who observed significant variations in the yield and chemical composition of rhizome oils of *C. longa* under similar climatic conditions [153–155].

Together with the genetic and environmental factors, the geographic location contributes to the different yields and quality of *C. longa* rhizome oils, even developing different chemotypes [39,109]. In India, the region of production determines the type of turmeric [156]. Samples from Nepal included α- and β-turmerones (8.19% and 17.74%, respectively) between other compounds like *epi*-α-patshutene (7.19%), β-sesquiphellandrene (4.99%), 1,4-dimethyl-2-isobutylbenzene (4.4%), (±)-dihydro-ar-turmerone (4.27%) and zingiberene (4.03%) [33]. The main components of the essential oil from Nigeria were ar-turmerone, α-turmerone and β-turmerone [141,157], while turmerones (approximately 37%), together with terpinolene (15.8%), zingiberene (11.8%) and β-sesquiphellandrene (8.8%), predominated in the rhizome oil from Reunion Island [134]. Turmerones still are also the predominant compounds in samples from Faisalabad (Pakistan) and Turkey [104,158]. In the South American continent, the essential oil isolated from rhizomes grown in Ecuador was rich in ar-turmerone (45.5%) and α-turmerone (13.4%), similar to Colombian samples, while that from Brazil was dominated by zingiberene (11%), sesquiphellandrene (10%), β-turmerone (10%) and α-curcumene (5%) [105,107,159].

The analysis of each *C. longa* habitat's conditions can help to predict the features of the resulting essential oil and enhance its yield and quality; what results especially important for its optimisation and commercialisation. Altitude, humidity, rainfall, temperature, soil pH, organic carbon, nitrogen, phosphorous and potassium are some of the factors that lead to wide variations in the yield and chemical composition of rhizome essential oil. From the development of predictive models and in vivo tests, the altitude, soil pH, nitrogen and organic carbon have been observed as enhancers of rhizome essential oil production. Amongst them, nitrogen and organic carbon raise the turmerone content concretely and phosphorous and potassium the oil yield [40,160–162]. Land configurations involving furrows and thatches surrounding *C. longa* reduce the loss of these soil nutrients, enhancing the rhizome yield [41].

The stage of maturity of *C. longa* rhizomes can also influence in the yield, chemical composition and properties of the essential oil. In relation to this, Garg et al. demonstrated that the percentage of the essential oil content widely varied between fresh and dried rhizomes of 27 accessions of *C. longa* in North India [163]. Similarly, Sharma et al. also observed certain variations in the qualitative and quantitative chemical compositions between the essential oils extracted from a mix of 5–10 month-old rhizomes and eight ones [139]. Furthermore, Singh et al. confirmed that fresh rhizome essential oil contained a major quantity of the active compound turmerone than dry ones, consequently having stronger activity [164]. A different trend was observed by Gounder et al., who reported the higher activity of cured (fresh rhizome boiled in water, dried in shade and polished) and dried rhizome oils over fresh ones [165], probably due to the lower percentage of arturmerone and β-turmerone. Anyway, the control of the drying conditions constitutes an important parameter in order to obtain the highest content of essential oil in the minimum time possible [166,167]. The sun and mechanical drying coexist as drying methods of *C. longa* rhizomes [156]. In particular, Monton et al. confirmed that one hour of microwave drying without conventional drying represented the optimum conditions to obtain the highest content of turmeric essential oil [167].

**Table 2.** Main components of *C. longa* essential oil according to the part of the plant used, origin, method of extraction and analysis. GC-MS: gas chromatography-mass spectrometry, CG-FID: flame ionisation detector, SFE: supercritical fluid extraction, SWE: supercritical water extraction and: CG-FTIR: gas chromatography-Fourier-transform infrared.



**Table 2.** *Cont.*


**Table 2.** *Cont.*

*C. longa* nutrition also has a significant impact in the yield and composition of rhizome oil. Especially, fertilizer use can enhance the productivity of volatile oil of *C. longa* rhizomes 6% [148]. Furthermore, a prior treatment with minerals during in vitro rhizome development followed by a fertilizer treatment in a greenhouse increases the percentage of volatiles

in *C. longa* rhizomes. Particularly remarkable is the interaction of KNO3 and Ca2+, which favours the accumulation of sesquiterpenes in turmeric rhizome [168]. An interesting research proposed the use of arbuscular mycorrhizal fungi instead of chemical fertilizers in the cultivation of *C. longa* rhizomes. These optimise the absorption of nutrients and water, augment the metabolic activity of the plant, etc. In consequence, the root system becomes more robust, and the chemical composition of the essential oil is improved, increasing the production of certain compounds, including caryophyllene, α-curcumene, β-bisabolene and β-curcumene, using sustainable technologies [169,170]. Finally, the postharvest management of turmeric rhizomes also has a noteworthy influence on the quality of the derived products. Concretely, the boiling conditions, way of slicing, type of mill and speed of crushing and presence of heat and oxygen need to be controlled and standardised to obtain essential oils with certain characteristics [156].

In conclusion, the study of the chemical composition of the essential oil from the rhizome of *C. longa* gives us an idea of the characteristics and possible properties that it possesses. Sesquiterpenes are usually the main compounds in *C. longa* rhizome essential oil, highlighting the oxygenated turmerones followed by sesquiterpene hydrocarbons (Figure 1). However, the qualitative and quantitative chemical compositions of the essential oil can vary depending on the genetic and commented on factors. The knowledge of these can help to achieve a high-yield product with useful composition and properties for the agri-food industry.
