基于体外酪氨酸酶抑制活性的枇杷花醇提纯化工艺优化及活性成分初步鉴定

doi:10.3969/j.issn.1004-1524.2023.05.18

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (5): 1144-1153.DOI: 10.3969/j.issn.1004-1524.2023.05.18

基于体外酪氨酸酶抑制活性的枇杷花醇提纯化工艺优化及活性成分初步鉴定

陈倩倩¹^,²(), 陶文扬², 郑美瑜², 马子甲¹^,², 王璐², 陆胜民¹^,²^,^*()

1.浙江师范大学生命科学学院,浙江金华 321000
2.浙江省农业科学院食品科学研究所,浙江省果蔬保鲜与加工技术研究重点实验室,浙江杭州 310021

收稿日期:2022-07-01 出版日期:2023-05-25 发布日期:2023-06-01
作者简介:陈倩倩(1996—),女,浙江桐乡人,硕士研究生,研究方向为果品加工。E-mail:996683796@qq.com
通讯作者: *陆胜民,E-mail: 1010508971@qq.com
基金资助:
浙江省农业科学院淳安县科技合作项目

Optimization of ethanol extraction and purification process of loquat flowers based on in vitro tyrosinase inhibitory activity and preliminary identification of active components

CHEN Qianqian¹^,²(), TAO Wenyang², ZHENG Meiyu², MA Zijia¹^,², WANG Lu², LU Shengmin¹^,²^,^*()

1. College of Life Sciences, Zhejiang Normal University, Jinhua 321000, Zhejiang, China
2. Zhejiang Key Laboratory of Fruit and Vegetable Preservation and Processing Technology, Institute of Food Science, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2022-07-01 Online:2023-05-25 Published:2023-06-01

摘要/Abstract

摘要：

为了探究枇杷花的美白功效及其功能因子,以酪氨酸酶活性抑制率为指标,通过单因素和正交实验对枇杷花具有抑制酪氨酸酶活性的物质的醇提工艺进行了优化;通过静态吸附-解析和动态吸附-洗脱实验分别确定D101大孔树脂纯化工艺中吸附-解析平衡时间和最佳纯化工艺;对纯化物进行HPLC-MS分析,初步鉴定出主要化合物。结果表明,最佳的提取工艺为在料液比(m∶V)1∶20、50 ℃和50%乙醇体积分数条件下,进行重复3次,每次1 h,提取枇杷干花具有抑制酪氨酸酶活性的物质,粗提物得率为30.01%;D101树脂对粗提物的最佳吸附平衡时间为4.5 h,解析平衡时间为3.5 h;最佳纯化工艺为:上样浓度40 mg·mL^-1,上样流速3 BV·h^-1,洗脱剂为60%乙醇体积分数,洗脱速度4 BV·h^-1,纯化物得率为4.39%;纯化物中共鉴定出137种成分,分为黄酮类物质、肉桂酸及其衍生物、苯及其衍生物等26个大类。对比中外文献报道,筛选出槲皮素、异鼠李素、对香豆酸等12种具有酪氨酸酶抑制活性的化合物。本研究为枇杷花在美白方面的应用提供理论基础。

关键词: 枇杷花, 酪氨酸酶抑制活性, 醇提物, 大孔树脂纯化, 成分鉴定

Abstract:

In order to explore the whitening efficacy and its functional factors of loquat flowers, the tyrosinase inhibitory activity was used as an index to optimize the ethanol extraction process of loquat flowers with substances inhibiting tyrosinase activity through single factor and orthogonal experiments. The adsorption-desorption equilibrium time and the optimal purification process with macroporous resin D101 were determined by static and dynamic desorption-elution experiments. The main compounds in purified product were preliminarily identified using HPLC-MS analysis. The results showed that the optimal extraction process was extracting dry loquat flowers with substances inhibiting tyrosinase activity for 3 times with 1.0 h each time under the conditions of ratio of material to liquid 1∶20 (m∶V), 50 ℃, and 50% ethanol and the yield of crude extract was 30.01%. The D101 resin had an optimal adsorption equilibrium time of 4.5 h and elution equilibrium time of 3.5 h for the crude extracts. The optimal purification process was 40 mg·mL^-1 of loading concentration and 3 BV·h^-1 of loading flow rate with the eluent and elution speed of 60% ethanol and 4 BV·h^-1, respectively, and the yield of purified product was 4.39%. There were 137 components identified in the purified product, classified into 26 categories such as flavonoids, cinnamic acid and its derivatives, benzene and its derivatives, among which 12 compounds including quercetin, isorhamnetin and p-coumaric acid were screened that had tyrosinase inhibitory activity after compared with literature at home and abroad. This study provides a theoretical basis for the application of loquat flowers in whitening.

Key words: loquat flower, tyrosinase inhibitory activity, ethanol extract, macroporous resin purification, component identification

中图分类号:

TS255

陈倩倩, 陶文扬, 郑美瑜, 马子甲, 王璐, 陆胜民. 基于体外酪氨酸酶抑制活性的枇杷花醇提纯化工艺优化及活性成分初步鉴定[J]. 浙江农业学报, 2023, 35(5): 1144-1153.

CHEN Qianqian, TAO Wenyang, ZHENG Meiyu, MA Zijia, WANG Lu, LU Shengmin. Optimization of ethanol extraction and purification process of loquat flowers based on in vitro tyrosinase inhibitory activity and preliminary identification of active components[J]. Acta Agriculturae Zhejiangensis, 2023, 35(5): 1144-1153.

图/表 9

参考文献 32

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水平 Level	因素Factors
水平 Level	A 温度 Temperature/ ℃	B 提取次数/次 Extraction times	C 乙醇体积分数 Ethanol volume fraction/%	D 时间 Time/h
1	50	2	30	0.5
2	60	3	50	1.0
3	80	4	70	1.5

水平 Level	因素Factors
水平 Level	A 温度 Temperature/ ℃	B 提取次数/次 Extraction times	C 乙醇体积分数 Ethanol volume fraction/%	D 时间 Time/h
1	50	2	30	0.5
2	60	3	50	1.0
3	80	4	70	1.5

实验号 Test number	因素 Factors				酪氨酸酶活性抑制率 Inhibition rate of tyrosinase activity/%
实验号 Test number	A	B	C	D	酪氨酸酶活性抑制率 Inhibition rate of tyrosinase activity/%
1	3	2	3	1	36.58
2	3	3	1	2	31.81
3	2	1	3	2	43.91
4	2	3	2	1	45.13
5	2	2	1	3	34.19
6	1	3	3	3	45.26
7	1	1	1	1	31.17
8	3	1	2	3	31.29
9	1	2	2	2	59.65
k₁	45.36	35.46	32.39	37.63
k₂	41.08	43.48	45.36	45.12
k₃	33.23	40.73	41.92	36.92
R	12.13	8.02	12.97	8.20

实验号 Test number	因素 Factors				酪氨酸酶活性抑制率 Inhibition rate of tyrosinase activity/%
实验号 Test number	A	B	C	D	酪氨酸酶活性抑制率 Inhibition rate of tyrosinase activity/%
1	3	2	3	1	36.58
2	3	3	1	2	31.81
3	2	1	3	2	43.91
4	2	3	2	1	45.13
5	2	2	1	3	34.19
6	1	3	3	3	45.26
7	1	1	1	1	31.17
8	3	1	2	3	31.29
9	1	2	2	2	59.65
k₁	45.36	35.46	32.39	37.63
k₂	41.08	43.48	45.36	45.12
k₃	33.23	40.73	41.92	36.92
R	12.13	8.02	12.97	8.20

编号 Code	物质名称 Substance name	分子式 Molecular formula	分子量 Molecular weight	化学文摘社编号 Chemical Abstracts Service(CAS)	保留时间 Retention time/ min
黄酮类化合物 Flavonoids
1	槲皮素Quercetin^[13]	C₁₅H₁₀O₇	302.042 0	117-39-5	6.643
2	异鼠李素Isorhamnetin^[14]	C₁₆H₁₂O₇	316.057 7	480-19-3	7.108
3	圣草酚Eriodictyol^[15]	C₁₅ H₁₂ O₆	288.063 0	209-016-4	6.894
肉桂酸及其衍生物 Cinnamic acid and its derivatives
4	三香豆酰亚精胺Tricoumaroyl spermidine^[16]	C₃₄H₃₇N₃O₆	583.267 4	NA	9.236
5	双香豆酰亚精胺Dicoumaramide spermidine^[16]	C₂₅H₃₁N₃O₄	437.230 7	NA	9.225
6	咖啡酸乙酯Ethyl caffeate^[17]	C₁₁H₁₂O₄	208.073 2	102-37-4	9.294
7	邻香豆酸2-Hydroxycinnamic acid^[18]	C₉H₈O₃	164.047 2	614-60-8	5.88
8	香豆酸Coumaric acid^[18]	C₉H₈O₃	164.047 2	614-60-8	10.271
9	反式-4-甲氧基肉桂酸trans-4-Methoxycinnamic acid^[19]	C₁₀H₁₀O₃	178.062 9	943-89-5	7.181
10	阿魏酸Ferulic acid^[20]	C₁₀H₁₀O₄	194.057 7	1 135-24-6	6.894
苯及其衍生物 Benzene and its derivatives
11	对甲氧基苯甲醛4-Methoxybenzaldehyde^[32]	C₈H₈O₂	136.052 4	123-11-5	5.952
12	对甲氧基苯甲酸4-Anisic acid^[32]	C₈H₈O₃	152.047 2	100-09-4	10.034

基于体外酪氨酸酶抑制活性的枇杷花醇提纯化工艺优化及活性成分初步鉴定

Optimization of ethanol extraction and purification process of loquat flowers based on in vitro tyrosinase inhibitory activity and preliminary identification of active components

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试剂 Reagent/μL	组别Group
试剂 Reagent/μL	A	B	C	D
磷酸缓冲液Phosphate buffer saline	100	200	100	200
枇杷花提取物	2	2	—	—
Loquat flower extraction/(mg·mL^-1)
DMSO	—	—	2	2
酪氨酸酶溶液	100	—	100	—
Tyrosinase solution/( U·mL^-1)

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上样液浓度 Concentration of loading solution/(mg·mL^-1)	泄漏点 Leakage point/BV	吸附量 Adsorption capacity/mg
25	30	15.0×10³
30	17	10.2×10³
40	15	12.0×10³
50	8	4.0×10³

上样液浓度 Concentration of loading solution/(mg·mL^-1)	泄漏点 Leakage point/BV	吸附量 Adsorption capacity/mg
25	30	15.0×10³
30	17	10.2×10³
40	15	12.0×10³
50	8	4.0×10³