高效逆流色谱法制备苏木中的氧化巴西木素

doi:10.3724/SP.J.1123.2020.03016

色谱 ›› 2020, Vol. 38 ›› Issue (12): 1363-1368.DOI: 10.3724/SP.J.1123.2020.03016

高效逆流色谱法制备苏木中的氧化巴西木素

和文倩¹^,², 范青飞¹^,², 周兰¹^,², 黄凤梅¹^,², 蒋仙³, 纳智¹, 胡华斌¹^,^*(), 宋启示¹^,^*()

1.中国科学院西双版纳热带植物园, 热带植物资源可持续利用重点实验室, 云南昆明 650223
2.中国科学院大学, 北京 100049
3.普洱学院, 云南普洱 665000

收稿日期:2020-03-18 出版日期:2020-12-08 发布日期:2020-12-01
通讯作者: 胡华斌,宋启示
基金资助:
中国国家科技重大专项“重大新药创制”项目(2017ZX09301045);中国科学院CAS “135”项目(XTBG-F02)

Preparation of brazilein from Caesalpinia sappan by high performance countercurrent chromatography

HE Wenqian¹^,², FAN Qingfei¹^,², ZHOU Lan¹^,², HUANG Fengmei¹^,², JIANG Xian³, NA Zhi¹, HU Huabin¹^,^*(), SONG Qishi¹^,^*()

1. CAS Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming 650223, China
2. University of Chinese Academy of Sciences, Beijing 100049, China
3. Puer University, Puer 665000, China

Received:2020-03-18 Online:2020-12-08 Published:2020-12-01
Contact: HU Huabin, SONG Qishi
Supported by:
National Key Project of Science and Technology of China “Important New Drugs Innovation”(2017ZX09301045);Chinese Academy of Sciences CAS “135” Program(XTBG-F02)

摘要/Abstract

摘要：

氧化巴西木素是苏木的主要化学成分之一,具有广泛的药理活性且常作为染色剂应用于各行各业。采用传统柱色谱法进行分离,不仅会造成色谱柱材料的污染,也会造成活性成分的损失。故采用高效逆流色谱(HPCCC)对苏木中的活性化合物氧化巴西木素进行分离纯化。苏木乙醇提取物经乙酸乙酯萃取后直接进行高效逆流色谱分离。首先利用基于薄层色谱的常用溶剂体系估算法和摇瓶法结合高效逆流色谱分析模式进行溶剂体系筛选。结果表明,最佳溶剂体系为氯仿-甲醇-水(4∶3∶2, v/v/v)。高效逆流色谱以反相模式为洗脱模式,主机转速为1600 r/min,流速为10 mL/min,分离温度为25 ℃,检测波长为285 nm,在氯仿-甲醇-水(4∶3∶2, v/v/v)溶剂体系下,从500 mg苏木乙酸乙酯萃取物中一次性分离制备得到15.2 mg纯度为95.6%的氧化巴西木素及一微量成分caesappanin C。采用高效逆流色谱分离制备氧化巴西木素,可避免苏木中的活性成分氧化巴西木素对色谱柱中的固体填充材料染色和难以洗脱等问题,并缩短分离纯化工作时间,提高工作效率。故可将高效逆流色谱应用到苏木中其他色素化合物或其他染料植物的分离制备工艺研究中。

关键词: 高效逆流色谱, 高效液相色谱, 氧化巴西木素, 苏木

Abstract:

Brazilein is among the main chemical constituents of Caesalpinia sappan. It has diverse pharmacological activities. Modern pharmacological studies have shown that the compound has antitumor, anti-inflammatory, antibacterial, antioxidant, immunomodulatory, and other pharmacological activities. Brazilein is often used as a stain in various industries. The separation of brazilein by traditional column chromatography will not only result in contamination of the chromatographic column materials, but also lead to loss of the active ingredient. Countercurrent chromatography is an advanced liquid-liquid chromatographic separation technique. It has been widely used for natural product separation and isolation as it offers several advantages, such as low solvent consumption, a highly selective solvent system, and high recoveries. Typical countercurrent chromatography techniques include centrifugal partition chromatography (CPC), high-speed countercurrent chromatography (HSCCC), and high performance countercurrent chromatography (HPCCC). It is well known that choosing a suitable solvent system is vital in countercurrent separation. Therefore, two methods were introduced for choosing a suitable solvent system. One is the generally useful estimation of solvent systems (GUESS) method, which employs thin-layer chromatography (TLC) to identify a suitable solvent system with minimal labor for the rapid purification of target compounds, and another is the Shake-Flash method. The solvent system could be determined by observing the distribution of the sample in the upper and lower phases. Two kinds of solvent systems were screened using the TLC-GUESS and Shake-Flash methods, and tested through the analysis mode of the HPCCC instrument. The results showed that chloroform-methanol-water (4∶3∶2, v/v/v) was the optimal solvent system for HPCCC separation. A total of 15.2 mg of brazilein and 5.7 mg of caesappanin C were obtained from an ethyl acetate extract with high purities (95.6% and 89.0%, analyzed by HPLC) in one step using the preparation mode of HPCCC, the reversed-phase liquid chromatography mode with the apparatus rotated at 1600 r/min, a flow rate of 10 mL/min, separation temperature of 25 ℃, and detection wavelength of 285 nm. Their structures were determined by spectroscopic and spectrometric analyses. Brazilein stained the solid packing material in the column and was difficult to elute. The results showed that the use of HPCCC for the separation of brazilein can not only prevent the loss of target active ingredients in Caesalpinia sappan, but also shorten the separation and purification times and improve the operating efficiency. Therefore, HPCCC can be used for the separation and preparation of other pigment compounds in Caesalpinia sappan and other dye plants.

Key words: high performance countercurrent chromatography (HPCCC), high performance liquid chromatography (HPLC), brazilein, Caesalpinia sappan

中图分类号:

O658

和文倩, 范青飞, 周兰, 黄凤梅, 蒋仙, 纳智, 胡华斌, 宋启示. 高效逆流色谱法制备苏木中的氧化巴西木素[J]. 色谱, 2020, 38(12): 1363-1368.

HE Wenqian, FAN Qingfei, ZHOU Lan, HUANG Fengmei, JIANG Xian, NA Zhi, HU Huabin, SONG Qishi. Preparation of brazilein from Caesalpinia sappan by high performance countercurrent chromatography[J]. Chinese Journal of Chromatography, 2020, 38(12): 1363-1368.

图/表 5

图1 苏木乙酸乙酯萃取物的HPLC色谱图

Fig. 1 HPLC chromatogram of the ethyl acetate extract of Caesalpinia sappan

图2 两种溶剂体系分离苏木乙酸乙酯萃取物的HPCCC色谱图

Fig. 2 High performance countercurrent chromatography (HPCCC) chromatograms of the ethyl acetate extract of Caesalpinia sappan with two solvent systems a. petroleum ether-ethyl acetate-methanol-water (3∶7∶3∶7, v/v/v/v); b. chloroform-methanol-water (4∶3∶2, v/v/v).

图 3 苏木乙酸乙酯萃取物的高效逆流色谱图

Fig. 3 HPCCC chromatogram of the ethyl acetate extract of Caesalpinia sappan

图 4 HPCCC分离得到的ccc-39和ccc-32的HPLC色谱图

Fig. 4 HPLC chromatograms of ccc-39 and ccc-32 from the ethyl acetate extract of Caesalpinia sappan by HPCCC

图 5 化合物1和2的结构式

Fig. 5 Chemical structures of compounds 1 and 2

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高效逆流色谱法制备苏木中的氧化巴西木素

Preparation of brazilein from Caesalpinia sappan by high performance countercurrent chromatography

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