东北红豆杉枝叶中原花色素的提取分离及其糖尿病抑制活性

doi:10.3969/j.issn.0253-2417.2022.06.015

摘要/Abstract

摘要：

以东北红豆杉枝叶为原料, 采用酶与超声波辅助耦合提取原花色素, 通过单因素试验和响应面法优化提取工艺, 并通过萃取分离得到了一个原花色素化合物, 另外对不同溶剂萃取物及乙酸乙酯萃取物经大孔吸附树脂分离得到的组分进行了抗糖尿病活性实验。结果表明：酶与超声波辅助耦合提取原花色素的最佳工艺条件为60%乙醇溶液为溶剂、酶质量浓度0.15 g/L、液料比14∶1(mL∶g)、酶解时间50 min、酶解温度40 ℃和超声波功率120 W, 在此条件下提取1次的原花色素提取得率为3.84%(提取率为83.48%)。响应面模型显著性检验发现模型的P值为0.000 2, 表明差异极显著, 回归方程有效, 模型具有统计学意义。乙酸乙酯萃取物经AB-8大孔吸附树脂、葡聚糖凝胶、硅胶柱层析分离纯化得到一个原花色素化合物, 通过TOF-MS结构鉴定和紫外全波长扫描, 确定该化合物为儿茶素与刺槐亭醇的二聚体原花色素。不同溶剂萃取物及乙酸乙酯萃取物的大孔吸附树脂分离得到的7种组分(CBFr.1~CBFr.7)抗糖尿病活性结果表明：乙酸乙酯萃取物对α-淀粉酶活性和α-葡萄糖苷酶的抑制率分别为83.60%和63.03%, 都有较好的抑制作用；CBFr.1~CBFr.5对α-淀粉酶的半数抑制质量浓度(IC₅₀)值在0.036~0.043 g/L之间, 对α-淀粉酶都有较好的抑制能力。CBFr.3对α-葡萄糖苷酶抑制活性最好, 其IC₅₀值为0.04 g/L, 低于阿卡波糖IC₅₀值0.063 g/L；分析表明CBFr.1~CBFr.4有较好的α-葡萄糖苷酶抑制活性, 以及一定的α-淀粉酶抑制活性, 可以达到降血糖的效果, 又减少了因过量降低α-淀粉酶活性而产生的副作用, 故CBFr.1~CBFr.4可作为Ⅱ型糖尿病抑制剂。

关键词: 东北红豆杉, 原花色素, 提取工艺, 糖尿病抑制

Abstract:

The proanthocyanidins were extracted by enzymolysis and ultrasonic-assisted coupling extraction method using Taxus cuspidata branches and leaves(TCBL) as the materials. The optimum extraction process was investigated by the single factor experiment and response surface experiment and a proanthocyanidin compound was obtained by extraction and separation. Then, in vitro antidiabetic activities of the fractions isolated from different solvent extract and the ethyl acetate extract by macroporous adsorption resin were studied. The results showed that the optimum process conditions of enzyme and ultrasonic assisted coupling extraction of proanthocyanidins were 60% ethanol aqueous as solvent, enzyme concentration of 0.15 g/L, liquid-solid ratio of 14∶1(mL∶g), enzymolysis time of 50 min, enzymolysis temperature of 40 ℃, and ultrasonic power of 120 W. The extraction yield of proanthocyanidins for one time was 3.84%(extraction rate was 83.48%) under these conditions. Response surface model significance test found that the P value of model was 0.000 2, indicating that the difference was very obvious, the regression equation was effective, and the model had statistical significance. The ethyl acetate extract was separated and purified by AB-8 macroporous adsorption resin, dextran gel, and silica gel column chromatography to obtain a proanthocyanidin compound, which was identified as a dimeric proanthocyanidins of catechin and robinatinol by TOF-MS structure identification and ultraviolet full-wavelength scanning. The anti-diabetes activities of 7 components(CBFr.1-CBFr.7) isolated from different solvent extracts and ethyl acetate extracts by macroporous adsorption resin were studied. The results showed that ethyl acetate extract had a good inhibitory effect on α-amylase activity and α-glucosidase activity, and the inhibition rates were 83.6% and 63.03%, respectively. The median inhibitory concentration(IC₅₀) values of CBFr.1-CBFr.5 for α-amylase were ranging from 0.036 to 0.043 g/L, and they all had a good inhibitory ability to α-amylase. CBFr.3 showed the best inhibitory activity against α-glucosidase, with an IC₅₀ value of 0.04 g/L, which was lower than that of acarbose(0.063 g/L). Analysis showed that CBFr.1-CBFr.4 had better α-glycosidase enzyme inhibition activity and moderate α-amylase inhibitory activity, which could achieve the effect to lower blood sugar and reduce side effects owing to excessive reduction of α-amylase activity. Therefore, CBFr.1-CBFr.4 could be used as type Ⅱ diabetes inhibitors.

Key words: Taxus cuspidata sieb. et Zucc., proanthocyanidins, extraction technology, antidiabetic activity

中图分类号:

TQ35

姜萍, 曲肼靓, 贾雨欣, 孙智超, 刘思盈, 王飞. 东北红豆杉枝叶中原花色素的提取分离及其糖尿病抑制活性[J]. 林产化学与工业, 2022, 42(6): 108-116.

Ping JIANG, Jingliang QU, Yuxin JIA, Zhichao SUN, Siying LIU, Fei WANG. Extraction and Separation of Total Proanthocyanidins from Taxus cuspidata sieb.et Zucc. Branches and Leaves and Their Antidiabetic Activity[J]. Chemistry and Industry of Forest Products, 2022, 42(6): 108-116.

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方差来源 variance source	平方和 sum of squares	自由度 degree of freedom	均方 mean square	F值 F value	P值 P value	显著性 significant
模型model	1.02	9	1.02	19.90	0.000 2	**
X₁	0.33	1	0.33	58.35	0.000 1	**
X₂	0.065	1	0.065	11.38	0.011 9	*
X₃	0.42	1	0.42	73.54	<0.000 1	**
X₁X₂	0.026	1	0.026	4.50	0.071 6
X₁X₃	0.038	1	0.038	6.68	0.036 2	*
X₂X₃	0.000	1	0.000	0.000	1.000 0
X₁²	0.017	1	0.017	3.05	0.124 1
X₂²	0.053	1	0.053	9.24	0.018 9	*
X₃²	0.078	1	0.078	13.63	0.007 1	**
残差residual	0.040	7	5.692×10^-3
失拟项lack of fit	0.017	3	5.708×10^-3	1.00	0.477 2
纯误差pure error	0.023	4	2.83×10^-5
总和correlation total	1.06	16

萃取物 extract	抑制率inhibition rate/%
萃取物 extract	α-淀粉酶 α-amylase	α-葡萄糖苷酶 α-glucosidase
石油醚petroleum ether	16.45	17.69
二氯甲烷dichloromethane	16.83	16.53
乙酸乙酯ethyl acetate	83.60	63.03
正丁醇n-butyl alcohol	63.03	53.97
水water	13.40	16.32
阿卡波糖acarbose	90.90	85.73

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