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

doi:10.3969/j.issn.1004-1524.2023.05.18

Acta Agriculturae Zhejiangensis ›› 2023, Vol. 35 ›› Issue (5): 1144-1153.DOI: 10.3969/j.issn.1004-1524.2023.05.18

• Food Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

TS255

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.

Figures/Tables 9

References 32

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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)

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)

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

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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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

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