Chemicals and reagents

Deionized water for HPLC; HPLC grade acetonitrile (ACN), methanol (MeOH), n-hexane, ethyl acetate (EtOAc), analytical grade formic acid, dimethyl sulfoxide (DMSO) (Scharlau, Spain);2,2-diphenyl-1-picrylhydrazyl (DPPH) (Sigma–Aldrich, USA); ascorbic acid (Merck, USA); ethanol absolute (Chemsol, Vietnam).

Plant material

sp. rhizomes were collected from Tra Vinh Province, Vietnam. The plant was identified at the Research Institute for Biotechnology and Environment (Nong Lam University Ho Chi Minh City). A voucher specimen (NP0220) is deposited in the Center for Research and Technology Transfer, Vietnam Academy of Science and Technology (VAST).

Sample extraction

sp. rhizomes were rinsed thoroughly to remove the sand and soil, naturally dried overnight, and ground into small pieces. 3.5 (kg) of the ground rhizomes were extracted in approximately 20 L of methanol for 24 hours at room temperature. The macerate was filtered successively on Whatman filter paper. The extract was concentrated using a rotavapor (BÜCHI R-300, Switzerland) at 60°C. After three maceration cycles, 70.0 g of the crude extract (CE) was collected. The crude extract was partitioned with n-hexane and ethyl acetate to yield 8.0 (g) of n-hexane extract (HE), 36.0 (g) of ethyl acetate extract (EE), and 26.0 (g) of methanol rest extract (ME).

Fractionation of ethyl acetate extract

The ethyl acetate extract (EE) of sp. was subjected to fractionation on a PLC 2250 system (Gilson Inc., France). The instrumentation was controlled by Gilson Glider CPC Software V5.1d.01 (Gilson Inc., France). Approximately 1.0 (g) of the EE sample was fractionated with a 50 grams RediSep Rf Gold C18 reversed-phase column (Teledyne ISCO, USA). Water (A) and methanol (B) were used as mobile phase solvents. The gradient was as follows: 0–25 min, 15-50% B; 25-35 min, 50-100% B; 35-50 min, 100% B. The flow rate was 20.0 mL/min, and UV detection was performed at 254 nm. The solution was collected in a 32 mL tube (25 mL for each tube). As a result, the EE sample was separated into 5 fractions (F1-F5).

Antioxidant activity

DPPH radical scavenging activity

The DPPH radical scavenging capacity of sp. extract and fractions were measured based on the method reported by Kwang et al.22 with some specific modifications. Briefly, 250 µM DPPH reagent in absolute ethanol was prepared. The samples were dissolved in DMSO at a concentration of 400 ppm and then diluted to different concentrations of 20, 100, 200, and 300 ppm. To evaluate antioxidant activity, 150 μL of DPPH reagent was mixed with 50 μL of sample in each well of a 96-well plate. The experiment was carried out three times for each sample. The reaction mixtures were incubated for 30 minutes at room temperature. A PowerWave HT Microplate Spectrophotometer was used to detect the absorbance of the incubated products at 520 nm (BioTek, USA).

The positive control was ascorbic acid. The IC value (the concentration of the sample required for 50% DPPH radical inhibition) was determined for the sample based on the regression equation.

The following formula was used to calculate the DPPH scavenging effect:

Radical scavenging (%) =

where

A: the absorbance at 520 nm of DPPH without sample.

A: the absorbance at 520 nm of the mixture of DPPH and sample.

Statistical Analysis

Data were processed using Excel 2016 and Rstudio (version 1.4.1717) software. The results were compared by one-way ANOVA and Tukey's HSD post hoc test. If p≤0.05, a difference was considered statistically significant.

UHPLC‐Q‐TOF analysis

All UHPLC‐Q‐TOF experiments were performed on an instrument consisting of an ExionLC UHPLC system and an X500 QTOF mass spectrometer (AB SCIEX, USA). LC separations were carried out on a Hypersil GOLD C18 column (150 x 2.1 mm, 3 µ) (Thermo Fisher Scientific, USA). The mobile phase solvents were water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The gradient elution profile was as follows: 0–1 min, 2% B; 1-25 min, 2-98% B; 25-29 min, 98% B. The flow rate was 0.4 mL/min, and 2 μL of sample was injected for analysis.

The Q‐TOF analysis was acquired in negative electrospray ionization (ESI) mode. The parameters were as follows: ion source temperature, 500°C; curtain gas, 30 psi; nebulizer gas (GS 1), 45 psi; heater gas (GS 2), 45 psi. For the TOF MS scan, the mass range was set at m/z 70–2000. For the TOF MS/MS scan, the mass range was set at m/z 50–1500. For the negative mode, the ion spray voltage was set at −4.5 kV, the declustering potential (DP) was −70 V, the collision energy (CE) was −20 eV, and the collision energy spread (CES) was 10 eV. All the obtained data were processed by SCIEX OS software version 1.2.0.4122 (AB SCIEX, USA).

In this study, only the fraction with the best antioxidant activity was analyzed by UHPLC‐Q‐TOF to identify the bioactive compounds of sp. rhizomes.

Antioxidant activity from Curcuma sp. extracts and fractions

In this experiment, the antioxidant activity of sp. extract and fractions were evaluated by DPPH assay. Table 1 and Table 2 present the antioxidant results of the extracts and fractions of sp., respectively. The lowest antioxidant activity was observed in methanol rest extract (ME) (7.76 ± 0.65%), while the best DPPH inhibition activity was observed in ethyl acetate extract (EE) (74.48 ± 0.80%) with an IC of 29.40 µg/mL. As shown in Table 2, fraction F1 had the lowest antioxidant result (0.82 ± 0.23%), and fraction F3 had the most effective antioxidant activity (84.27 ± 2.53%) with an IC of 20.91 µg/mL. Ascorbic acid was used as the positive control, of which the IC value was calculated to be 17.43 µg/mL.

The regression equations and IC values of the ethyl acetate extract, fraction F3, and vitamin C are presented in Table 3.

UHPLC‐Q‐TOF analysis of fraction F3

According to the antioxidant activity screening result, fraction F3 showed the best DPPH inhibition, which was then analyzed by UHPLC‐Q‐TOF. MS and fragment data of phytochemicals contained in fraction F3 are presented in Table 4. The total ion chromatogram (TIC) of fraction F3 is shown in Figure 1. Figure 2 shows the chemical structures of the phytochemicals that were discovered. The fragmentation pathway of compound 11 at T = 10.52 is shown in Figure 3.

Identification of curcuminoids by MS/MS

Curcuminoid structures can be divided into two parts: a skeleton of a heptane or pentane system and two aryl groups in the side chain.24 Based on the results of UHPLC‐Q‐TOF analysis, all 13 compounds identified in fraction F3 were curcuminoids with heptane skeletons. The skeletons (A, B, and C) and aryl groups (Ar – Ar) were confirmed to exist in fraction F3 of sp. (Figure 2). Compared with type A curcuminoids, type B curcuminoids contained one keto group at C-3 or C-5, while type C curcuminoids had a double bond at the position of C-6,7.

In the negative mode, the product ions of aryl groups Ar, Ar, and Ar were 123, 109, and 93 (Da), respectively. The product ion of 59 (Da) referred to the acetate group at the C-3 or C-5 position of the skeleton. In addition, the [M-H-CH] ion could be observed via loss of a methyl radical from the aryl group. Compounds 5 and 9 were identified as type B, compound 8 was detected as type C, and the remaining 10 compounds were type A curcuminoids.

Correlation of phytochemicals and antioxidant activity of Curcuma sp.

Ibrahim et al. showed the antioxidant activity of three isolated curcuminoids (curcumin, demethoxycurcumin, and bisdemethoxycurcumin) from and with IC values of 0.15, 0.16, and 0.36 µmol/L, respectively.23 Waras et al. stated that the main bioactive substances in the rhizomes of and that have efficacy as antioxidant and anti-inflammatory activities are curcuminoids.25 Jesmin et al. reported that nine purified compounds isolated from the fresh rhizome of turmeric Ryudai gold had antioxidant activity, of which six compounds were curcuminoids.26

The antioxidant activity and chemical constituents of sp. showed that the presence of curcuminoids in the fraction was responsible for the considerable antioxidant activity. Our findings suggested that sp., a new species of the genus, is a promising source of natural antioxidants. Future studies should aim to isolate compounds in fraction F3 to accurately identify the compound(s) responsible for antioxidant activity from sp.