中药远志提取物体外杀精子效果与机制研究

Study on the spermicidal mechanism of the crude extract from Polygala tenuifolia willd in vitro

目的:通过精子低渗肿胀(HOS)实验结合改良伊红染色法观察中草药远志粗提物体外杀精活性及机制。方法:HOS实验结合伊红-姬姆萨(EG)染色(HOS-EG)判断精子膜完整性,将精子分为A(HOS+-EG-)、B(HOS-EG-)、C(HOS+-EG+)、D(HOS-EG+)4型。精子染色质扩散实验(SCD)检测精子DNA碎片。制备远志粗提物,并判断其体外杀精子活性。壬苯醇醚-9(N-9)作为标准参照物,生理盐水为对照组。结果:远志粗提物浓度为50.0mg/ml和20.0mg/ml时体外20s内即可完全杀灭精子(与精液1:1混合)。对照组、远志粗提物组及N-9组精子DNA碎片比例各组比较无差异。对照组A、B、C和D型精子百分比分别为(69.0±8.3)%、(3.4±0.5)%、(10.2±1.7)%和(17.4±2.1)%。远志粗提物组及N-9组精子膜完整性全部被破坏,无A型和B型精子,C型精子较对照组明显减少,D型精子比对照组明显增加(P<0.001)。精子复活实验显示远志粗提物组和N-9组均无活动精子恢复。结论:HOS-EG实验可以同时判断一条精子头膜或尾膜损伤。远志粗提物在体外通过破坏精子膜使精子快速失活。

Objective：To observe the spermicidal mechanism of the crude extract from Polygala tenuifolia willd （PTW） in vitro. Methods： The hypoosmotic swelling （HOS） test and eosin-giemsa （EG） staining were used to detect the integrity of sperm membrane. Four types of sperm morphology were divided by the combined HOS-EG test,Type A （HOS＋-EG-）,B （HOS-EG-）,C （HOS＋-EG＋） and D （HOS-EG＋）. The PTW root was extracted.The ability of the crude extract from PTW root to kill sperm was evaluated by the Sander-Cramer test under in vitro conditions. Sperm chromatin dispersion （SCD） test was performed for determining sperm DNA integrity. Nonoxynol-9 （N-9） was used as a reference standard and semen added physiological saline was as the control. Results： The crude extract from PTW root could immobilize and kill 100% spermatozoa within 20 sec in vitro at the concentration of 50.0 mg/ml and 25.0 mg/ml （1：1 mixed with semen,v ： v）. Sperm DNA fragmentation had no changes in exposure to the crude extract from PTW root and N-9 solution. In the control group,the mean percentage of the type A,B,C and D sperm was （69.0±8.3）%,（3.4±0.5）%,（10.2±1.7）% and （17.4±2.1）%,respectively. In the group of the crude extract from PTW and N-9 solution,the type A and B sperm wasn′t observed,and the type C was significantly decreased,and the type D was significantly increased （compared with the control,P all 〈0.001）. Sperm revival test showed none of the spermatozoa recovered their motility in the PTW group and N-9 group. Conclusion： The head or tail membrane damage in the same sperm can be determined by using HOS-EG test. The crude extracts from PTW root have spermicidal function at a short period of time by disrupting sperm membrane integrity in vitro.