Purification of sulforaphane from Brassica oleracea seed meal using low-pressure column chromatography

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Abstract

Sulforaphane is an isothiocyanate that is present naturally in widely consumed Brassica oleracea vegetables and has been shown to block the formation of tumors. The contents of sulforaphane in five groups of B. oleracea seeds (broccoli, Brussels sprouts, cabbage, cauliflower and kale) were determined by RP-HPLC using linear gradient of acetonitrile in water. A new low-cost method to isolate and purify natural sulforaphane from B. oleracea seed meal was described in this work. Crude sulforaphane was first separated from B. oleracea seed meal by using immiscible solvent extraction with ethyl acetate, 10% ethanol and hexane, and the crude sulforaphane was used as raw materials to prepare high purity sulforaphane by low-pressure column chromatography of silica gel (200–300 mesh) with different eluents and elution modes. Compared with these different elution methods, the gradient elution was preferable to the isocratic elution for reducing the elution time and the eluent consumption and increasing the purity of sulforaphane product. The purity and recovery of sulforaphane were more than 90% in gradient elution.

Introduction

Epidemiological data show that a diet rich in Brassica oleracea vegetables, such as broccoli, cabbage, Brussels sprouts, cauliflower and kale, can reduce the risk from a number of cancers. The underlying mechanism for the reduction of cancer by B. oleracea vegetables is not clear. However, these vegetables are rich in glucosinolates. When vegetables are ground or chopped, myrosinase enzyme (thioglucoside glucohydrolase, EC 3.2.3.1) and glucosinolates come into contact. Myrosinase breaks the β-thioglucoside bond of glucosinolate molecules, producing glucose, sulfate, and a diverse group of aglycone products. The resultant aglycones then undergo non-enzymetic, intramolecular rearrangement to yield isothiocyanates, thiocyanates or nitriles. Sulforaphane (4-methylsulfinybutyl isothiocyanate), derived from glucoraphanin (4-methylsulfinybutyl glucosinolate), is the most potent naturally occurring inducer of phase II enzymes, including quinone reductase and glutathione S-transferase [1]. Moreover, sulforaphane can reduce the incident of a number of forms of tumors and induce cell cycle arrest and apoptosis in various experimental models [2], [3], [4], [5], [6]. Significantly, even at dietary doses, sulforaphane can modulate the xenobiotic-metabolising enzyme systems, shifting the balance of carcinogen metabolism toward deactivation [7].

With the increase in application of sulforaphane in nourishment, the demands for high purity sulforaphane are rapidly increasing. However, the high purity sulforaphane do not meet the needs of various fields because of the limit to its high price at present. Consequently, a new low-cost technology for the purification of sulforaphane is worth of great importance for extending application.

Purified sulforaphane can be prepared by chemical synthesis [8], [9] and purification from plant [10], [11]. Chemical synthesis requires several highly toxic substances, and final products from these reactions still contain toxic residues and require further purification. This disadvantage limits synthesized sulforaphane to be used as food additives. Thus, natural sulforaphane is more favorable for common consumer. Purified sulforaphane from plant are usually prepared by using preparative reverse phase high performance liquid chromatography (RP-HPLC). Separation efficiency of preparative RP-HPLC is the best since small diameter and suitable expensive packing can be used. However, the sample purified by preparative RP-HPLC has to be treated through elaborate pre-separation in order to remove large quantity of contaminants. Moreover, the equipment of preparative RP-HPLC is expensive and the cost of operation and maintenance are also high. Therefore, preparative RP-HPLC was not appropriate for the purification of sulforaphane as industry process with low cost. Compared with preparative RP-HPLC, low-pressure column chromatography (LPCC) exhibits a great potentiality for industrial production owing to its operation simplicity, low cost and high yield. The aim of the present study is to develop an economic LPCC process for purification of sulforaphane from B. oleracea seed meal. Therefore, irregular, large diameter, and cheap silica gel packing material and simple solvents that are easy to be recovered and reutilized were chosen in our procedure. The samples do not need elaborate treatment before purified by LPCC. All of these are worth decreasing separation cost of LPCC.

Crude sulforaphane extracted from B. oleracea seed meal by using immiscible solvent was used as raw materials to prepare high purity sulforaphane by LPCC. The separation conditions of LPCC were optimized by using various eluents and elution modes.

Section snippets

Chemicals

B. oleracea seeds (broccoli, Brussels sprouts, cabbage, cauliflower and Kale) were kindly provided by Vegetables and Flowers Institute of China Academy of Agriculture Science. Sulforaphane standard was purchased from Sigma Chemical Co. (St. Louis, MO.). Acetonitrile was HPLC grade. Methylene chloride, methanol, ethanol, ethyl acetate, hexane and anhydrous sodium sulfate were of analytical grade. Silica gel (200–300 mesh, irregular) was obtained from Haiyang Chemical Group (Qingdao, China).

Seed extraction

Fifty

High performance liquid chromatography (HPLC)

Bertelli et al. [12] used the mixture of water and tetrahydrofuran as mobile phase for analyzing the contents of sulforaphane in the edible tissues of broccoli. In our study, a rapid HPLC method with the linear gradient of acetonitrile–water as mobile phase was established.

Linear regression analysis of the peak area responses (y) versus the theoretical concentration (x) gave the following equation: y = 11949 + 160377.80x, r2 = 0.9998. The correlation coefficient demonstrated linearity of the method

Conclusion

A new low-cost method for separation and purification of sulforaphane from the seeds of B. oleracea was described in our work. In order to remove large quantity of oil contaminant, the immiscible solvent extraction was applied. As a result, ethyl acetate, 10% ethanol and hexane system was the best method to remove oil contaminant and decrease the loss of the derived compound.

Crude sulforaphane extracts were purified by isocratic or gradient elution with hexane and ethanol as eluent. Compared

Acknowledgement

The authors would to acknowledge the support of Beijing Key Laboratory of Bioprocess (SYS100100421).

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