Phytochemical and quantification studies of secondary metabolites in Curcuma Xanthorrhiza and Curcuma Heyneana

Abstract

Phytochemical and quantification studies had been carried out on the secondary metabolites in Curcuma xanthorrhiza and C. heyneana. Phytochemical screening of the methanol extracts of both species revealed the presence of flavonoids, steroids, saponins, phenolics, tannins, terpenoids, proteins and cardiac glycosides. Extraction of the dried rhizomes of both species was conducted by cold maceration method using n-hexane, dichloromethane and ethyl acetate to yield n-hexane, dichloromethane and ethyl acetate extracts. Isolation of chemical constituents from all crude extracts was performed using silica gel vacuum liquid chromatography and column chromatography. Purification of the n-hexane, dichloromethane and ethyl acetate extracts of C. xanthorrhiza yielded ar-curcumene, germacrone, xanthorrhizol and curcumin while purification of the n-hexane, dichloromethane and ethyl acetate of C. heyneana obtained ar-turmerone, b-sitosterol, xanthorrhizol and curcumin. Structure of all pure compounds were characterised using 1H NMR, 13C NMR, DEPT, COSY, HMQC, HMBC, FTIR and also by comparison with literature data. Antioxidant activity of the methanol extracts of C. xanthorrhiza and C. heyneana together with the pure compounds were conducted using 2,2-diphenyl-1-picrylhydrazyl scavenging, ferric reducing antioxidant potential and 2,2-azino-bis(3-ethylbenzothiazoline-6- sulphonic acid) assays. The methanol extract of C. xanthorrhiza, curcumin and xanthorrhizol displayed strong antioxidant activity in 2,2-diphenyl-1-picrylhydrazyl assay with SC50 values of 66.15, 71.88 and 79.99 µg/mL, respectively. The methanol extract of C. heyneana, ar-turmerone, germacrone and b-sitosterol showed moderate antioxidant activity in 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) assay with SC50 values of 113.71 143.70, 142.95 and 133.13 µg/mL, respectively. Arcurcumene showed weak antioxidant activity in ferric reducing antioxidant potential assay with SC50 value of 160.62 µg/mL. Curcumin, germacrone and xanthorrhizol were selected as the chemical markers for the quantification studies of both Curcuma extracts. HPLC analysis gave good precision and accuracy, as well as sufficient limit of detection and quantitation. Curcumin was found to be the major compound in C. heyneana with concentration of 16.90 µg/mL while in C. xanthorrhiza, the concentration of curcumin was only 0.65 µg/mL. Xanthorrhizol was major compound in methanol extract of C. xanthorrhiza but minor compound in methanol extract of C. heyneana with the concentrations of 44.12 and 0.82 µg/mL. Germacrone was found in concentration of 4.43 and 8.05 µg/mL in methanol extracts of C. xanthorrhiza and C. heyneana. Identification of compounds by validation method proved that curcumin, germacrone and xanthorrhizol were present in the methanol extracts of both species. HPLC chromatogram displayed curcumin absorption band at 380 nm with retention time of 4.24 min, while germacrone and xanthorrhizol absorption bands were observed at 270 nm with retention time of 13.32 min and 16.54 min, respectively.