Optimization of microwave-assisted extraction of anthocyanins from <i>Bletilla striata</i> flowers

WANG Zhen; WANG Jie; XIANG Haiping; FAN Zepeng; SUN Chengman; XING Bingcong; SHAO Qingsong

doi:10.11833/j.issn.2095-0756.20190581

Volume 37 Issue 5

Oct. 2020

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WANG Zhen, WANG Jie, XIANG Haiping, FAN Zepeng, SUN Chengman, XING Bingcong, SHAO Qingsong. Optimization of microwave-assisted extraction of anthocyanins from Bletilla striata flowers[J]. Journal of Zhejiang A&F University, 2020, 37(5): 1020-1026. doi: 10.11833/j.issn.2095-0756.20190581

Citation:

WANG Zhen, WANG Jie, XIANG Haiping, FAN Zepeng, SUN Chengman, XING Bingcong, SHAO Qingsong. Optimization of microwave-assisted extraction of anthocyanins from Bletilla striata flowers[J]. Journal of Zhejiang A&F University, 2020, 37(5): 1020-1026. doi: 10.11833/j.issn.2095-0756.20190581

Optimization of microwave-assisted extraction of anthocyanins from Bletilla striata flowers

doi: 10.11833/j.issn.2095-0756.20190581

WANG Zhen^{1, 2
,},
WANG Jie^{1, 2},
XIANG Haiping²,
FAN Zepeng²,
SUN Chengman²,
XING Bingcong^{1, 2},
SHAO Qingsong^{1, 2
,
,}

1.
State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China
2.
College of Forestry and Biotechnology, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China

Received Date: 2019-10-08
Rev Recd Date: 2020-03-05

Available Online: 2020-05-28

Publish Date: 2020-08-20

Abstract

Objective This study is aimed at the optimization of microwave-assisted extraction of anthocyanins from Bletilla striata flowers. Method With anthocyanins in B. striata flowers determined and extracted via microwave-assisted extraction and pH Differential Method and the optimum parameters of the four single factors of microwave power, methanol concentration, extraction time and solid-liquid ratio obtained, the study has managed to obtain the optimal extraction parameters of anthocyanins with the quadratic regression equation model established using the Response Surface Methodology (RSM). Result (1) The conditions of the optimal model fit well with actual extraction; (2) The pH differential method was adjusted to a measured wavelength of 530 nm and a solution equilibration time of 80 min; (3) The impact of the four factors of microwave power, material to liquid ratio, methanol concentration and microwave time on the amount of extraction decrease in a progressive order; (4) With the microwave power at 640 W, the methanol concentration as 90%, the microwave time set for 22 min, and the ratio of material (g) to liquid (mL) adjusted as 1∶48, the total anthocyanin extraction in B. striata flower was 6.68 mg·g⁻¹, which was basically consistent with the theoretical prediction value of 6.83 mg·g⁻¹. Conclusion The optimized method can be employed for the extraction and preparation of anthocyanins in B. striata flowers, providing a basis for the comprehensive development and utilization of B. striata flowers. [Ch, 8 fig. 2 tab. 22 ref.]
- Bletilla striata flower,
- anthocyanins,
- pH differential method,
- microwave-assisted extraction,
- response surface methodology

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白及Bletilla striata为兰科Orchidaceae白及属Bletilla多年生草本植物，主产于安徽、浙江、江西、贵州等地^[1]，以干燥块茎入药，又名甘根、白给、白芨等^[2]。具有收敛止血，消肿生肌的功效，对于咯血、吐血、疮疡肿毒、皮肤皲裂等有效^[3]。因白及以其干燥块茎入药，故国内外对白及花的研究较少。白及花期为每年4−5月，花序具花3~10朵，花色为紫红色、粉红色^[4]，具有较高的观赏价值，还富含多种花青素等活性成分，其水提物具有较好的抗氧化能力，可作为饮品进行开发利用^[5]。花青素(anthocyanidins)为水溶性天然色素，属于多酚化合物中的类黄酮化合物，是由花色苷水解得到的有颜色的苷元^[6−7]，但在自然界中很少以游离的苷元而多以糖苷的形式存在^[8]。花青素不仅是植物中的主要呈色物质，还具有抗氧化^[9]、降血脂^[10]、抗癌^[11]、抑制神经炎症、改善血管功能^[12]、平衡肠道微生物群落^[13]、减缓与肝功能及结构有关的衰老恶化^[14]与改善轻度至中度痴呆老年人的特定认知结果^[15]等功效。花青素稳定性差，在提取纯化过程中易发生降解^[16]。微波法对天然色素的提取具有色素产率高、色价高、节省溶剂、节省时间等优点^[17]。pH示差法和高效液相色谱法(HPLC)是定量测定花青素的常用方法，两者具有很高的相关性^[18]，其中pH示差法被农业化学家协会(AOAC)正式作为研究实验室和食品工业中各种水果和蔬菜总花青素的基本测量方法^[19−20]。本研究采用微波法进行响应面优化白及花花青素提取，并采用pH示差法测定白及总花青素，以期为白及花中花青素的综合开发提供理论依据。

1. 材料与仪器

白及花采于浙江省湖州市安吉县上墅乡白及基地。采摘盛花期的白及花，去除花茎部分后，于烘箱50 ℃烘干至恒量(±0.005 g)，打粉后过24目筛。于−20 ℃避光储存，备用。氯化钾(西陇化工股份有限公司)、无水乙酸钠(西陇化工股份有限公司)、盐酸(上海凌峰化学试剂有限公司)、无水甲醇(浙江汉诺化工科技有限公司)均为分析纯。pH计FE20[梅特勒-托利多仪器(上海)有限公司]，AK-1000A 500克摇摆式中药粉碎机(温岭市奥力中药机械有限公司)，DHG-9031A电热恒温干燥箱(上海精宏实验设备有限公司)，FA2204电子分析天平(上海力辰仪器科技有限公司)，NJL07-3型实验微波炉(南京杰全微波设备有限公司)，RE-2000A旋转蒸发器(上海亚荣生化仪器厂)，TDL-50C低速离心机(上海安亭科学仪器厂)，Epoch2酶标仪(美国BioTek仪器有限公司)。

2. 方法

2.1. 总花青素的提取

取白及花粉末加入甲醇溶液(以体积分数为1%盐酸作溶剂)进行微波萃取，离心取上清液，残渣进行再次提取，合并2次上清液于35 ℃旋蒸1 h后用提取剂定容至10 mL，−20 ℃避光保存直至测定。

2.2. pH示差法测定花青素条件优化

2.2.1. 吸收波长的确定

因深色花色苷在可见光区的吸收波长范围为465~560 nm^[7]，所以取花青素提取液0.2与0.6 mL的pH 1.0缓冲液混匀，于450~600 nm波长范围内以步长5 nm进行扫描，以确定最大吸收波长。

2.2.2. 平衡时间的确定

分别取花青素提取液0.2与0.6 mL的pH 1.0缓冲液或pH 4.5缓冲液混匀，室温下避光反应0~120 min，每隔10 min测定D(530)、D(700)，以确定最佳平衡时间。

2.3. 提取次数的确定

精确称取样品，在料液比为1∶40(料单位为g，溶液单位为mL)、体积分数为80%的甲醇溶液(以体积分数为1%盐酸稀释)、微波功率400 W的条件下提取10 min，冷却至室温后4 000 r·min⁻¹离心15 min，取上清液于35 ℃旋蒸，用提取剂定容至10 mL，为第1次提取液。将残渣重复上述操作，为第2次提取液。共进行4次提取，按2.4分别进行总花青素质量浓度测定，以确定最佳提取次数。

2.4. 总花青素质量浓度测定

参考李文峰等^[21]总花青素质量浓度测定方法并略作调整。取0.2 mL花青素提取液，分别与0.6 mL的pH 1.0氯化钾缓冲液、pH 4.5醋酸钠缓冲液混匀。室温下避光静置80 min后测定D(530)、D(700)。3次重复，取平均值。总花青素(T_AC)质量分数以矢车菊素-3-O-葡萄糖苷(C3G)的含量为标准进行计算，计算公式^[22]为：T_AC= {[D(530)_pH1.0− D(700)_pH1.0]−[D(530)_pH4.5−D(700)_pH4.5]}M_WD_F/(εL)。其中，T_AC青素质量浓度(g·L⁻¹)，D为各pH值下不同波长对应的吸光度，M_W为C3G的分子量(449.2)，D_F为稀释倍数，ε为C3G的克分子吸光率常数(26 900)，L为比色皿光程(1 cm时，L=1)。再将总花青素质量浓度换算成质量分数进行对比分析。

2.5. 单因素实验

以微波功率、甲醇体积分数、提取时间和料液比为单因素，分别进行参数范围的确定，考察不同因素对总花青素提取量的影响。精确称取样品，在甲醇体积分数为80%、料液比(溶质单位为g，溶剂单位mL)1∶40、提取20 min的条件下，进行微波功率(160、240、320、400、480、560、640 W，误差为±5 W)的范围确定；在微波功率560 W、料液比1∶40、提取20 min的条件下，确定甲醇体积分数(20%、40%、60%、80%、100%)的区间范围；在微波功率560 W、甲醇体积分数80%、料液比1∶40的条件下，考察微波时间(2、5、10、20、30 min)的影响；在微波功率560 W、提取20 min、体积分数为80%甲醇的条件下，进行料液比(1∶10、1∶20、1∶30、1∶40、1∶50、1∶60)的范围确定。按2.4分别进行总花青素提取量的计算。

2.6. 响应面实验

为获得白及花中花青素的最佳提取工艺条件，在单因素结果的基础上，以总花青素提取量为响应值，选取微波功率(A)、甲醇体积分数(B)、微波时间(C)和料液比(D)为自变量，应用Design-Expert.V8.0.6.1软件，按照Box-Behnken中心组合设计原理，设计4因素3水平的响应面实验。设计因素与水平如表1所示。

水平微波功率
A/W 甲醇体积
分数B/% 微波时间
C/min 料液比
D

−1 480 70 10 1∶30
0 560 80 20 1∶40
1 640 90 30 1∶50

Table 1. Coded levels and corresponding actual levels of independent variables used in Box-Behnken design

4. 结论

本研究采用微波萃取法进行白及花中花青素的提取。在单因素实验的基础上，采用响应面分析法进行提取条件的优化，并建立了微波功率、甲醇体积分数、提取时间和料液比对总花青素提取量的二次回归方程模型。得到最佳的提取工艺条件为：微波功率640 W，甲醇体积分数90%，微波时间22.14 min，料液比1∶48.10。结合实际情况调整参数为：微波功率640 W，甲醇体积分数90%，微波时间22 min，料液比1∶48。在此条件下，白及花中总花青素的提取量为(6.68±0.13) mg·g⁻¹，与预测值6.83 mg·g⁻¹基本吻合。

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方差来源	平方和	自由度	均方	F	P	显著性
样本	30.65	14	2.19	19.85	＜0.000 1	***
A	11.98	1	11.98	108.61	＜0.000 1	***
B	2.89	1	2.89	26.18	0.000 2	***
C	1.52	1	1.52	13.83	0.002 3	**
D	4.16	1	4.16	37.70	＜0.000 1	***
AB	1.56	1	1.56	14.16	0.002 1	**
AC	0.09	1	0.09	0.80	0.387 2
AD	1.57	1	1.57	14.19	0.002 1	**
BC	0.23	1	0.23	2.09	0.170 3
BD	0.08	1	0.08	0.76	0.398 2
CD	0.00	1	0.00	0.02	0.892 0
A²	0.97	1	0.97	8.83	0.010 1	*
B²	0.77	1	0.77	6.99	0.019 3	*
C²	2.91	1	2.91	26.42	0.000 2	***
D²	4.61	1	4.61	41.81	＜0.000 1	***
残差	1.54	14	0.11
失拟项	1.19	10	0.12	1.35	0.413 3
纯误差	0.35	4	0.09
总离差	32.19	28
R²=0.95	R²_Adj=0.90
说明：表示显著(P＜0.05)，表示极显著(P＜0.01)，**表示极显著(P＜0.001)