Separation and Purification of Sulforaphane from Broccoli
Broccoli is a famous vegetable around the world, belonging to the cruciferous family, which is rich in antioxidants such as vitamin C, quercetin and kaempferol . Medicinal studies have shown that increasing consumption of broccoli can lower the risk of breast , skin and prostate cancers. Recent research showed that glucoraphanin (4-methylsulfinybutyl glucosinolate) is another important constituent in broccoli and it can produces sulforaphane when hydrolyzed by myrosinase. Sulforaphane has attracted researchers’ attention as a promising cancer chemopreventive agent . In many studies, sulforaphane can reduce the incidence of a number of forms of tumor . It is important to develop a rapid and simple method for determination and separation of sulforaphane from broccoli. Several analytical methods such as high-performance liquid chromatography (HPLC), GC/mass spectrometry [and evaporative light-scattering have been used to determine sulforaphane in broccoli. Meanwhile, preparative HPLC and high-speed countercurrent chromatography have been used to purify sulforaphane. However, the enzymatic hydrolysis of glucoraphanin generates a variety of compounds (glucose, sulfate, isothiocyanates, thiocyanates, nitriles) which interfere with the separation and determination of sulforaphane. Therefore, it is necessary to establish a simple and convenient method for the selective extraction and separation of sulforaphane from broccoli.
1. Optimization of Chromatographic Conditions
Selection of optimal HPLC conditions is important for the determination and separation of suforaphane. In this study, different wavelengths (205 nm, 235 nm and 254 nm) were investigated, and the results showed sulforaphane had largest absorbance under 205 nm. Therefore, 205 nm was chosen for further analyses.
Different kinds of mobile phases such as methanol, acetonitrile, and different concentrations of methanol/H2O and acetonitrile/H2O were investigated. Under 205 nm, methanol has strong background absorbance, which is difficult to balance the C18 column. The 20% acetonitrile/H2O (v/v) was proved to provide the best separation, because there is no interference of other impurities and the retention time of sulforaphane is short.
2. Optimization of the Hydrolyzation from Glucoraphanin to Sulforaphane
To optimize the hydrolysis of glucoraphanin to sulforaphane in fresh broccoli, preliminary trials were conducted with different pHs of acidic water (3, 4, 5 and 6), and hydrolysis times (2, 4 and 6 h). The incubation temperature was constant at 35 °C. Table 1 shows that the resulting sulforaphane amount was highest with pH 3.0 and hydrolysis time 4 h.
The optimal conditions were found to be use of a silica SPE cartridge, and ethyl acetate and dichloromethane as washing and eluting solvents, respectively, which could eliminate interferences originating from the broccoli matrix. The extracts were sufficiently clean to be directly injected into high-performance liquid chromatography (HPLC) for further chromatographic analysis. Good linearity was obtained from 0.05 to 200 μg/mL (r = 0.998) for sulforaphane with the relative standard deviations less than 3.6%. The mean recoveries of sulforaphane from broccoli were more than 90.8% and the detection limit (S/N = 3:1) was 0.02 μg/mL. The SPE method provides a higher yield of sulforaphane from crude extracts compared to conventional liquid-liquid extraction.
1. Optimization of Chromatographic Conditions
Selection of optimal HPLC conditions is important for the determination and separation of suforaphane. In this study, different wavelengths (205 nm, 235 nm and 254 nm) were investigated, and the results showed sulforaphane had largest absorbance under 205 nm. Therefore, 205 nm was chosen for further analyses.
Different kinds of mobile phases such as methanol, acetonitrile, and different concentrations of methanol/H2O and acetonitrile/H2O were investigated. Under 205 nm, methanol has strong background absorbance, which is difficult to balance the C18 column. The 20% acetonitrile/H2O (v/v) was proved to provide the best separation, because there is no interference of other impurities and the retention time of sulforaphane is short.
2. Optimization of the Hydrolyzation from Glucoraphanin to Sulforaphane
To optimize the hydrolysis of glucoraphanin to sulforaphane in fresh broccoli, preliminary trials were conducted with different pHs of acidic water (3, 4, 5 and 6), and hydrolysis times (2, 4 and 6 h). The incubation temperature was constant at 35 °C. Table 1 shows that the resulting sulforaphane amount was highest with pH 3.0 and hydrolysis time 4 h.
The optimal conditions were found to be use of a silica SPE cartridge, and ethyl acetate and dichloromethane as washing and eluting solvents, respectively, which could eliminate interferences originating from the broccoli matrix. The extracts were sufficiently clean to be directly injected into high-performance liquid chromatography (HPLC) for further chromatographic analysis. Good linearity was obtained from 0.05 to 200 μg/mL (r = 0.998) for sulforaphane with the relative standard deviations less than 3.6%. The mean recoveries of sulforaphane from broccoli were more than 90.8% and the detection limit (S/N = 3:1) was 0.02 μg/mL. The SPE method provides a higher yield of sulforaphane from crude extracts compared to conventional liquid-liquid extraction.