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Box-Behnken响应面法优化女贞子总三萜纯化工艺 Δ

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高 爽 ，王玉真，薄福民，张 丽，孙 越，杨山景，封安杰，李凌军（山东中医药大学药学院，济南 250355）
中图分类号 R282；R284.2 文献标志码 A 文章编号 1001-0408（2020）18-2196-06
DOI 10.6039/j.issn.1001-0408.2020.18.04

摘 要 目的：提高女贞子总三萜中齐墩果酸和熊果酸的转移率和纯度，优化该类成分的纯化工艺。方法：以齐墩果酸和熊果酸
作为女贞子药材中总三萜类代表成分，采用高效液相色谱法测定两者含量。色谱柱为 Thermo BDS Hypersil C18，流动相为甲
醇-0.02％乙酸铵溶液（80∶20，V/V），检测波长为210 nm，柱温为30 ℃，流速为1.0 mL/min，进样量为20 μL。采用单因素试验，以齐
墩果酸、熊果酸的转移率为指标，考察工艺中的水沉温度、水沉时间和复溶乙醇用量对纯化工艺的影响；以两种成分的转移率和纯
度为指标，考察活性炭用量、析晶乙醇体积分数对纯化工艺的影响。在此基础上，以复溶乙醇用量、活性炭用量、析晶乙醇体积分
数为因素，采用 Box-Behnken 响应面法进一步优化该纯化工艺并验证。结果：最佳纯化工艺为女贞子药材提取浓缩液加 4 倍量
（mL/g，下同）水，在0 ℃静置2 h（水沉）；然后加入药材1倍量的乙醇溶解（复溶）；再加入4％的活性炭（除杂）；最后加水调整乙醇
体积分数为80％，在4 ℃下静置12 h（析晶），离心，干燥。3次验证试验结果显示，采用该优选纯化工艺所制得的总三萜中齐墩果
酸、熊果酸的转移率分别为 61.11％、65.78％，纯度分别为 53.44％、19.79％，RSD 均小于 3％。结论：优化所得纯化工艺提取效率
高、操作简便，可用于女贞子总三萜类成分纯化的工业化生产以及相关制剂的开发。
关键词 女贞子；总三萜；齐墩果酸；熊果酸；纯化工艺

Optimization of the Purification Technology of Total Triterpenoids of Ligustrum lucidum by Box-Behnken
Response Surface Methodology
GAO Shuang，WANG Yuzhen，BO Fumin，ZHANG Li，SUN Yue，YANG Shanjing，FENG Anjie，LI Lingjun
（School of Pharmacy，Shandong University of TCM，Jinan 250355，China）

ABSTRACT OBJECTIVE：To improve the transfer rate and purity of oleanolic acid and ursolic acid in total triterpenoids from
Ligustrum lucidum，so as to optimize the purification technology. METHODS：Oleanolic acid and ursolic acid were used as
representative components of total triterpenoids，and their contents were determined by HPLC. The determination was performed on
Thermo BDS Hypersil C18 column with mobile phase consisted of methanol-0.02％ ammonium acetate solution（80 ∶ 20，V/V）at the
flow rate of 1.0 mL/min. The detection wavelength was set at 210 nm，and column temperature was 30 ℃. The sample size was 20
μ L. In single factor tests，using transfer rate of oleanolic acid and ursolic acid as index，the effects of water precipitation
temperature and time，the amount of redissolved ethanol on the purification technology was investigated；using transfer rate and
purity of two components as indexes，the effects of the amount of activated carbon and volume fraction of crystallization ethanol
were investigated. Based on it，using the amount of redissolved ethanol and activated carbon，volume fraction of crystallization
ethanol as factors，Box-Behnken response surface methodology was used to optimize the purification technology，and validation
tests were performed. RESULTS：The optimal purification technology was adding 4-fold （mL/g，the same below） water in L.
lucidum concentrated solution，placing for 2 hours at 0 ℃（water precipitation）；adding 1-fold ethanol to dissolve（redissolution）；
adding 4％ activated carbon（edulcoration）；finally adding water to adjust the volume fraction of ethanol to 80％，placing at 4 ℃
for 12 hours（crystallization），centrifuging and drying. The results of 3 times of validation tests showed that the transfer rates of
oleanolic acid and ursolic acid in total triterpenoids prepared by optimized technology were 61.11％ and 65.78％，the purities of
them were 53.44％ and 19.79％，and RSDs were both lower than 3％. CONCLUSIONS：The optimized purification technology has
high extraction efficiency and simple operation，which can be used for industrial production of purification of total triterpenoids
from L. lucidum and the development of corresponding preparations.
KEYWORDS Ligustrum lucidum；Total triterpenoids；Oleanolic acid；Ursolic acid；Purification technology

围绝经期是女性必经的生理过程，在此阶段出现的 以及骨质疏松等症状称为围绝经综合征（MPS） 。女
[1-2]
如月经紊乱、心血管系统与代谢异常、泌尿生殖综合征 贞子是木犀科植物女贞[Ligustrum lucidum Ait.]的干燥
Δ 基金项目：国家科技重大专项（重大新药创制）（No.2017ZX093- 成熟果实，其药用历史悠久，具有补气疏肝、滋补肝肾的
01064） 功效，可解虚汗、烦热骨蒸等症，近年来在治疗MPS及其
＊硕士研究生。研究方向：中药新制剂、新剂型及药物体内过 [3-5]
并发症等方面的作用受到了广泛关注 。目前从女贞
程。E-mail：gaoshuang0315@163.com
子中分离到的化合物主要有三萜类、环烯醚萜类、苯乙
# 通信作者：副教授，硕士生导师，博士。研究方向：中药新制剂、
[6]
新剂型及药物体内过程。E-mail：sdzyylilingjun@163.com 醇类以及黄酮类等成分 ，其中女贞子总三萜的有效成

·2196 · China Pharmacy 2020 Vol. 31 No. 18 中国药房 2020年第31卷第18期