Thai Curcuma Species: Antioxidant and Bioactive Compounds

For the functional food applications, antioxidant properties and the bioactive compounds of the 23 Curcuma species commercially cultivated in Thailand were studied. Total phenolic content and DPPH radical scavenging activity were determined. The concentrations of eight bioactive compounds, including curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), 1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (4), germacrone (5), furanodienone (6), zederone (7), and ar-turmerone (8), were determined from the Curcuma by HPLC. While the total phenolic content of C. longa was highest (22.3 ± 2.4 mg GAE/g, mg of gallic acid equivalents), C. Wan Na-Natong exhibited the highest DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) radical scavenging activity. Twenty-three Curcuma species showed characteristic distributions of the bioactive compounds, which can be utilized for the identification and authentication of the cultivated Curcuma species. C. longa contained the highest content of curcumin (1) (304.9 ± 0.1 mg/g) and C. angustifolia contained the highest content of germacrone (5) (373.9 ± 1.1 mg/g). It was noteworthy that 1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (4) was found only from C. comosa at a very high concentration (300.7 ± 1.4 mg/g). It was concluded that Thai Curcuma species have a great potential for the application of functional foods and ingredients.


Introduction
Turmeric, Curcuma longa, is the only Curcuma species extensively cultivated and traded in the world [1]. Taxonomically, it belongs to the Curcuma genus of the Zingiberaceae family, and is mostly distributed in Asia [2,3]. Various beneficial health-promoting effects of C. longa, such as Alzheimer s disease prevention, anti-inflammatory effects, and HIV-1 protease inhibition, were reported [4,5]. Curcuminoids and sesquiterpenoids were identified as the major bioactive constituents of C. longa [6]. More than a hundred species of Curcuma are reported worldwide, and about 30 species among them are cultivated and consumed in Thailand as food additives, cosmetics, and traditional medicines [7]. In particular, many of them are used for the treatment of various diseases due to the existence of bioactive compounds. However, bioactive compounds in other Curcuma species, other than C. longa, have never been studied extensively.
To expand the application of Curcuma species as functional foods, we have collected and cultivated 23 Thai Curcuma species widely consumed in Thailand. Total phenolic content and antioxidant activity were measured in the ethanol extracts of dried Curcuma rhizomes. For the bioactive compound study, four curcuminoids and four sesquiterpenoids were isolated and used as reference compounds after complete characterizations, because these are popularly studied representative phytochemicals in Curcuma species. In detail, the curcuminoids curcumin (1), demethoxycurcumin (2), and bisdemethoxycurcumin (3) were isolated from C. longa, and the other curcuminoid 1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (4) was isolated from C. comosa. The three germacrane-type sesquiterpenoids germacrone (5), furanodienone (6), and zederone (7) were isolated from C. latifolia, and ar-turmerone (8) was isolated from C. zedoaria (Figure 1). The compositions of these representative bioactive compounds in the Curcuma species were also analyzed by HPLC.

Curcuma Species
The rhizomes of 23 Curcuma species were collected in Kanchanaburi in 2013 and cultivated in Tapong, Meaung Rayong, Thailand in 2014 throughout the year. The plants that formed flowers were processed to prepare depository specimens at Bangkok Herbarium, Plant Variety Protection Division, Department of Agriculture, Bangkok, Thailand.

Plant Materials and Extraction
Fresh rhizomes of the 23 Curcuma species were rinsed several times with tap water to make them dust and debris free. The rhizomes were then cut into small pieces and dried at room temperature in the shade for 2-3 days. The dried samples were ground to a fine powder and kept in a deep freeze (−70 • C) for further experiments. For extraction, each sample (1 g) of Curcuma sp. was macerated in EtOH (20 mL) for 24 h. The ethanol extract was filtered and dried under reduced pressure. The residue was weighed for the extraction yield and dissolved in MeOH for the determination of total phenolic content and DPPH radical scavenging assay.

Determination of Total Phenolic Contents
Total phenolic contents of all plant extracts were determined using Folin-Ciocalteu reagent as described by Singleton and Rossi [8]. The plant extracts were dissolved in MeOH (1 mL, 0.5 mg/mL). Samples were added to microtiter plates and mixed with 100 µL of a 10-fold diluted Folin-Ciocalteu reagent and 80 µL of 7.5% sodium carbonate. The absorbance at 765 nm was measured using microplate reader spectrophotometers (Spectramax190, Molecular Device, CA, USA) after 30 min. Total phenolic content was expressed as mg GAE/g. The calibration curve of standard gallic acid solution was obtained as y = 0.005 x (A 765 ) + 0.081, R 2 = 0.998, where y = mg GAE/g, at the region between 5 and 200 µg/mL.

DPPH Radical Scavenging Assay
The antioxidant activity of the Curcuma extracts was evaluated by DPPH radical scavenging. The extract in MeOH (2 mg/mL) and ascorbic acid in MeOH (200 µg/mL) were prepared. The test solution (50 µL) was mixed with 950 µL of DPPH (0.1 mM in MeOH). After 30 min, the absorbance at 516 nm was measured using spectrophotometer (UV/visible detector) in triplicate. DPPH radical scavenging activity was calculated using the following formula: % scavenging = 100 × ((absorbance of control-absorbance of sample test)/absorbance of control).

Validation of HPLC Analysis
Validation of HPLC analysis was performed using the purified reference compounds. Calibration curves were constructed from the HPLC peak areas of the reference standards, 1-8, versus their concentrations. Linearity was calculated by measuring the eight points of the calibration curve in the range of 0.003125-0.4 mM (1-3) and 0.00625-0.8 mM (4-8) with triplicate measurements. The limits of detection (LOD) for 1-8 were found at the micromolar level. Demethoxycurcumin (2) and furanodienone (6) showed the lowest LOD (2.0 µM) and limits of quantification (LOQ) at 420 nm and 245 nm, respectively (Table 1).

Compositional Analysis of Curcuma Species
Each sample (1 g) of the Curcuma species was macerated in EtOH (20 mL) for 24 h. The extract was filtered and dried under reduced pressure. The dried residue was dissolved in DMF (N,N-dimethylformamide, 1 mg/1 mL) for the analysis with a Finnigan Surveyor Plus HPLC system. The mobile phase comprised 0.1% acetic acid in water (solvent A) and 0.1% acetic acid in MeCN (solvent B). The injection volume for HPLC analysis was 10 µL and the flow was 1.0 mL/min. For the quantitative analysis, UV absorption at 420 nm was adopted for 1-3, and UV absorption at 245 nm was adopted for 4-8.

Statistical Analyses
All measurements were performed in triplicate. Data were processed by Microsoft Excel and reported as means ± standard deviation.

Taxonomy of Curcuma Species
Among the 23 Curcuma species, 11 Curcuma species, such as C. manga, C. aeruginosa, C. comosa, C. aurantiaca, C. aromatic, C. latifolia, C. zedoaria, C. longa, C. parviflora, C. angustifolia, and C. petiolate, were identified by the taxonomist at the Bangkok Herbarium. The other 12 species are new species, and designated by common names in this report (Table 2).
From the results, it was clear that total phenolic contents of Curcuma extracts are not necessarily correlated with the antioxidant activity ( Figure 2). Among the three most antioxidant Curcuma rhizomes, C. Wan Na-Natong and C. Wan Chai-Dam are the new species that have never been extensively studied, regardless of their local popularity. The correlation chart (Figure 2) shows that the total phenolic content and DPPH radical scavenging antioxidant activity among the Curcuma species are not necessarily linearly correlated. This is believed to be due to different phytochemical compositions of Curcuma species, as discussed below.  Table 2, and only notable data are labeled in the chart.

Chemical Composition of 23 Curcuma Species
Curcuminoids 1-3 are considered as the most characteristic bioactive secondary metabolites found in turmeric. Therefore, we analyzed the collected Curcuma species to find out whether the rhizomes contain 1-3. The EtOH extracts (1 mg/1 mL) were analyzed with HPLC at 420 nm [17,18], and only ten species were found to contain curcuminoids (Table 3). Furthermore, only three Curcuma species, C. zedoaria, C. longa, and Curcuma Wan Khamintong, contained all three curcuminoids (Figure 3). Trace amounts of curcuminoids were identified from C. manga, Curcuma Wan Rang-Jud, C. comosa, C. latifolia, C. parviflora, Curcuma Wan Pataba, Curcuma Wan Na-Natong, and C. petiolate under LOQ, only when the higher concentrations of the analytes were analyzed by HPLC. It is noticeable that C. longa was found to contain the highest curcuminoid content (653 mg/g). Even though curcumin (1) is the representative bioactive compound of turmeric, only nine Curcuma species contained curcumin (1). Demethoxycurcumin (2) was the major curcuminoid of four other Curcuma species, C. aeruginosa, C. aurantiaca, C. aromatica, and C. zedoaria.  From Indonesian Curcuma species, C. mangga, C. heyneana, C. aeruginosa, and C. soloensis were reported to contain curcuminoids [19]. Our analysis also confirmed that C. mangga and C. aeruginosa contained curcuminoids. C. aurantiaca contained high contents of 1-3, and this is the first report of the identification of 1-3 in the rhizomes of C. aurantiaca. Diarylheptanoid 1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (4) belongs to the curcuminoids, but it was found only from C. comosa in a high content of 300.7 ± 1.4 mg/g. It is noteworthy that C. comosa does not contain 1-3.
There are more than 100 different sesquiterpenoids reported from Curcuma rhizomes [20], and these secondary bioactive metabolites are also responsible for some of the biological activities observed from the rhizomes of Curcuma species. Therefore, we isolated three germacrane-type sesquiterpenoids, germacrone (5), furanodienone (6), and zederone (7), which is the first report from C. latifolia. In addition, ar-turmerone (8) was isolated from C. zedoaria for the first time. Germacrone (5) is known to exhibit anti-inflammatory [21], antiviral [22], and anti-cancer activities [23], and was found in the various Curcuma species, such as C. aeruginosa, C. amanda, C. aromatica, C. xanthrrhiza, and C. zedoaria.
Among the 23 Curcuma species analyzed, 21 species were found to contain at least one of the four sesquiterpenoids, and only two Curcuma species, C. aurantiaca and C. zedoaria, contained all four sesquiterpenoids (Table 4). Generally, germacrone (5) was the most abundant sesquiterpenoid in the Curcuma species, and C. angustifolia contained only 5, out of the eight reference compounds.

Conclusions
The phytochemical property of 23 Curcuma species was studied by means of total phenolic contents and antioxidant activity assays, as well as compositional analysis. Besides, eight bioactive curcuminoids and sesquiterpenoids were isolated from the rhizomes of C. longa, C. comosa, C. latifolia, and C. zedroaria. In particular, the isolation of germacrone (5), furanodienone (6), and zederone (7) is the first report from C. latifolia. From the compositional analysis, C. longa was distinct in its highest curcumin (1) concentration. C. angustifolia was found to contain the highest content of germacrone (5). Out of 23 Curcuma species, five Curcuma species did not contain any curcuminoids 1-4. None of the analyzed Curcuma species contained all eight bioactive compounds. Therefore, the data of phytochemical profiling can be used for the identification and authentication of cultivated Curcuma species. In addition, there is a tremendous potential for the utilization of less-studied Curcuma species as functional foods and ingredients.