Page 18 - 《中国药房》2022年22期

·药学研究·

天麻中脑线粒体靶向化合物的获取及其神经保护活性研究
Δ

＊
余星霖 ，杨丽萍，陈 普，段小花（云南中医药大学云南省傣医药与彝医药重点实验室，昆明 650500）
#
中图分类号 R917；R285.5 文献标志码 A 文章编号 1001-0408（2022）22-2700-06
DOI 10.6039/j.issn.1001-0408.2022.22.03

摘 要 目的 从天麻中获取脑线粒体靶向化合物，并对获取的化合物进行神经保护活性研究。方法 提取大鼠脑组织中线粒体，
通过健那绿和中性红染色评估线粒体的纯度，并通过检测线粒体膜电位（MMP）评估纯化线粒体的活性。以纯化线粒体为对照
组、失活的纯化线粒体为实验组，测定并计算对照组和实验组的峰面积之差与对照组峰面积的比值（ΔP），来验证线粒体靶向化合
物获取方法的可行性。通过超滤离心-高效液相色谱（HPLC）法获取天麻中脑线粒体靶向化合物并进行指认，检测其对离体线粒
体活性的影响。进一步针对活性较高的化合物，采用缺氧缺糖/复氧（OGD/R）损伤HT22细胞研究其神经保护活性。结果 提取所
得的脑线粒体纯度高，具有活性。可行性验证结果显示，超滤离心-HPLC法具有识别分离功能，可用于获取具有线粒体靶向功能
的化合物。从天麻中获取了2个脑线粒体靶向化合物（对羟基苯甲醇、对羟基苯甲醛），两者均可提高离体线粒体活性，其中对羟
基苯甲醇活性更高。细胞实验结果显示，对羟基苯甲醇以给药浓度25、50、100 μmol/L预处理，对HT22细胞存活率影响较小。与
模型组相比，对羟基苯甲醇处理可降低OGD/R损伤细胞死亡率，改善模型细胞形态，提高模型细胞ATP、MMP水平，降低模型细
胞中乳酸脱氢酶、活性氧水平与线粒体通透性转换孔开放程度，大部分差异具有统计学意义（P＜0.05）。结论 从天麻中获取2个
脑线粒体靶向化合物（对羟基苯甲醇和对羟基苯甲醛），其中对羟基苯甲醇对OGD/R诱导的神经细胞损伤有明显的保护作用，可
能是通过线粒体靶向治疗发挥神经保护作用。
关键词 天麻；脑线粒体；超滤离心；高效液相色谱；靶向治疗；神经保护

Acquisition of brain mitochondria targeting compounds in Gastrodia elata and study on their
neuroprotective activities
YU Xinglin，YANG Liping，CHEN Pu，DUAN Xiaohua（Yunnan Key Laboratory of Dai and Yi Medicines，
Yunnan University of Chinese Medicine， Kunming 650500， China）

ABSTRACT OBJECTIVE To obtain mitochondria targeting compounds from Gastrodia elata， and to study the neuroprotective
activities of these compounds. METHODS The mitochondria was extracted from cerebral tissue of rats. The purity of mitochondria
was evaluated with Janus green staining and neutral red staining. The activity of purified mitochondria was evaluated. The purified
mitochondria was included in the control group，and the inactivated mitochondria was included in the experimental group. The ratio
of the difference between the peak areas of the control group and the experimental group to the peak areas of control group（ΔP）
was determined and calculated to verify the feasibility of the method for obtaining the mitochondria targeting compounds. The
mitochondria targeting compounds from G. elata were obtained by ultrafiltration centrifugation-high performance liquid
chromatography （HPLC） and identified， and the effects of the compounds on the acitivities of mitochondria were also detected. The
neuroprotective activities of the compounds with better activities were studied with hypoxia glucose deprivation/reoxygenation
（OGD/R） damaged HT22 cells. RESULTS The extracted mitochondria possessed high purity and activity. Results of feasibility
validation showed that ultrafiltration centrifugation-HPLC method had recognition and separation function， and could be used to
obtain compounds with mitochondria targeting function. Two mitochondria targeting compounds （p-hydroxybenzyl alcohol and p-
hydroxybenzaldehyde） were obtained from G. elata； these 2 compounds could improve the activities of mitochondria in vitro， and
p-hydroxybenzyl alcohol had higher activity. Results of cell experiment showed that pretreatment with p-hydroxybenzyl alcohol （25，
50， 100 μmol/L） had little effect on the survival rate of HT22 cells. Compared with the model group， p-hydroxybenzyl alcohol
could reduce cell mortality of OGD/R damaged cells， improve cell morphology， increase the levels of ATP and MMP， and reduced
LDH and ROS levels and MPTP openness；most of the
Δ 基金项目 国家自然科学基金资助项目（No.81960733）；云南省 differences were statistically significant （P＜0.05）.
科技计划项目（No.2019FB120）
CONCLUSIONS Two brain mitochondria targeting
＊ 第一作者 硕 士 研 究 生 。 研 究 方 向 ：民 族 药 学 。 E-mail：
2515840573@qq.com compounds （p-hydroxybenzyl alcohol and p-
# 通信作者 副教授，硕士生导师，博士。研究方向：药理学。 hydroxybenzaldehyde） are obtained from G. elata，among
E-mail：1047896527@qq.com which p-hydroxybenzyl alcohol has a significant protective

· 2700 · China Pharmacy 2022 Vol. 33 No. 22 中国药房 2022年第33卷第22期