Antioxidant Activity of Different Species and Varieties of Turmeric (Curcuma spp): Isolation of Active Compounds

Keywords : Turmeric, Ryudai gold, Antioxidant, Phenolic, Flavonoid, Active compound

Abstract

There are over 80 species of turmeric (Curcuma spp.), with some species having multiple varieties; for example, Curcuma longa (C. longa) has 70 varieties. These can differ in chemical properties and biological activities. This study compared antioxidant activity, total phenolic, and flavonoid content of different species and varieties of turmeric: C. longa [varieties Ryudai gold (RD) and Okinawa ukon], C. xanthorrhiza, C. aromatica, C. amada, and C. zedoaria. Antioxidant activity was determined using DPPH free radical scavenging, ORAC, reducing power, and 2-deoxyribose (2-DR) oxidation assays. RD had significantly higher total phenolic (157.4 mg gallic acid equivalent/g extract) and flavonoid (1089.5 mg rutin equivalent/g extract) content, as well as higher DPPH radical-scavenging activity (IC₅₀: 26.4 μg/mL), ORAC (14,090 μmol Trolox equivalent/g extract), reducing power (0.33), and hydroxyl radical scavenging activity (IC₅₀: 7.4 μg/mL). RD was selected for the isolation of antioxidant compounds using chromatographic techniques. Structural identification was performed using ¹H NMR, ¹³C NMR, and LC-MS/MS. The purified antioxidant compounds were bisabolone-9-one (1), 4-methyllene-5-hydroxybisabola-2,10-diene-9-one (2), turmeronol B (3), 5-hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-1-hepten-3-one (4), 3-hydroxy-1,7-bis(4-hydroxyphenyl)-6-hepten-1,5-dione (5), cyclobisdemethoxycurcumin (6), bisdemethoxycurcumin (7), demethoxycurcumin (8), and curcumin (9). The IC₅₀ values for DPPH radical-scavenging activity ranged from 18 μM (curcumin) to 621 μM, and for hydroxyl radical-scavenging activity from 1.5 μM (curcumin) to 25.1 μM. These findings suggest the RD variety of C. longa, developed by the University of the Ryukyus, Okinawa, Japan, is a promising source of natural antioxidants.

Introduction

Antioxidants are crucial dietary components that prevent oxidative cell damage by scavenging free radicals, which are implicated in chronic diseases such as cancer, cardiovascular diseases, diabetes, aging, and neurodegenerative disorders. While endogenous enzymes like superoxide dismutase, catalase, and glutathione peroxidase regulate free radicals, excessive levels lead to oxidative stress. Synthetic antioxidants used in foods can be toxic or carcinogenic, increasing the demand for natural antioxidants from plant sources.

Turmeric (Curcuma spp., Zingiberaceae) is a widely used spice with documented pharmacological activities, including antioxidant, anti-inflammatory, and antimicrobial effects. There are over 80 species of turmeric, with C. longa having about 70 varieties. However, comparative studies on antioxidant activity among these species and varieties are limited, and purification of active antioxidant compounds from C. longa, especially the Ryudai gold (RD) variety, had not been previously performed.

Materials and Methods
Chemicals:

Rutin, AlCl₃, gallic acid, potassium ferricyanide, FeCl₃, AAPH, H₂O₂, Trolox, Folin-Ciocalteu reagent, sodium carbonate, trichloroacetic acid, methanol, ethyl acetate, ascorbic acid, 2-deoxy-D-ribose, n-hexane, silica gel, DPPH, TBA, sodium fluorescein, Toyopearl HW-40F, and EDTA.

Plant Material Preparation:

Six turmeric species/varieties (RD, Okinawa ukon, C. xanthorrhiza, C. aromatica, C. amada, C. zedoaria) were cultivated in Okinawa, Japan. Rhizomes were harvested, washed, sliced, and dried at 50°C for 72 hours.

Extraction:

20 g of each dried turmeric powder was extracted with 200 mL methanol at 25°C for 2 days, repeated three times. Extracts were filtered and concentrated.

Estimation of Total Phenolic Content (TPC):

Measured using Folin-Ciocalteu reagent and expressed as mg gallic acid equivalents (GAE)/g extract.

Estimation of Total Flavonoid Content (TFC):

Measured using AlCl₃ colorimetric assay and expressed as mg rutin equivalents/g extract.

Antioxidant Activity Assays:

DPPH Radical Scavenging: Absorbance at 517 nm; IC₅₀ calculated.

ORAC: Fluorescence-based assay; results as μmol Trolox equivalents/g extract.

Reducing Power (RPA): Absorbance at 700 nm.

2-Deoxyribose (2-DR) Oxidation: Absorbance at 532 nm; IC₅₀ calculated.

Isolation of Bioactive Compounds:

RD extract was fractionated using solvent partitioning, silica gel and Toyopearl HW-40F column chromatography, and HPLC. Compounds were identified by NMR and LC-MS/MS.

Statistical Analysis:

Data are mean ± SEM of three independent experiments in triplicate. ANOVA and Duncan’s multiple range tests were used (P < 0.05).

Results
Extract Yield:

RD had the highest methanol extract yield (15%), followed by C. xanthorrhiza (14%), C. aromatica (10.5%), Okinawa ukon (10%), C. zedoaria (7.5%), and C. amada (6.5%).

Total Phenolic and Flavonoid Content:

RD had the highest TPC (157.4 ± 1.0 mg GAE/g) and TFC (1089.5 ± 0.9 mg rutin/g). C. zedoaria had the lowest.

Antioxidant Activity:

DPPH IC₅₀: RD (26.4 μg/mL), Trolox (23.2 μg/mL), Okinawa ukon (291.3 μg/mL)

ORAC: RD (14,090 μmol TE/g), C. aromatica (1680 μmol TE/g)

Reducing Power: RD (0.33), highest among all.

2-DR IC₅₀: RD (7.4 μg/mL), Okinawa ukon (28.3 μg/mL)

Strong correlations were found between TPC/TFC and antioxidant activity.

Fractionation and Isolation:

The ethyl acetate fraction of RD showed the strongest antioxidant activity. Further fractionation and purification yielded nine compounds with confirmed structures.

Discussion

Significant variation exists in antioxidant activity, TPC, and TFC among turmeric species and varieties. RD exhibited the highest antioxidant activity and phenolic/flavonoid content. The strong correlation between TPC/TFC and antioxidant activity supports the use of RD as a potent natural antioxidant source.

Nine antioxidant compounds were isolated from RD, several of which (curcumin, demethoxycurcumin, bisdemethoxycurcumin, and others) showed high activity. Structure-activity relationships suggest that electron-donating groups (methoxy) enhance antioxidant activity. The findings support the potential of RD-derived compounds for preventing oxidative stress-related diseases and as food additives.

Conclusion

The Ryudai gold variety of Curcuma longa is a rich source of phenolics, flavonoids, and potent natural antioxidants. Nine active compounds were isolated and characterized, several of which surpass the commercial antioxidant Trolox in activity. RD turmeric is a promising candidate for natural antioxidant development in food and health applications.