桔梗粗提物及桔梗皂苷D体外杀精子活性机制研究

Mechanism for sperm immobilization activity of extract from Platycodon grandiflorum in vitro

目的 探讨中草药桔梗粗提物及桔梗皂苷D体外杀精活性机制.方法 2006年2月至2009年12月,38名身体健康且已生育的成年男性入选为供精者.制备桔梗粗提物及纯化桔梗皂苷D.分组为桔梗粗提物组、桔梗皂苷D组、壬苯醇醚-9（N-9）组（标准参照物）及生理盐水组（对照组）,每组均38份标本.各组添加剂与精液1∶1混合后分别进行体外杀精子试验（Sander-Cramer法）、精子低渗肿胀试验结合改良的伊红-吉姆萨染色（HOS-EG）试验、精子染色质扩散试验（SCD）及精子复活试验.HOS-EG试验判断精子膜完整性,A型精子为头膜-尾膜均完整[HOS（＋）-EG（-）],B型为头膜完整-尾膜损伤[HOS（-）-EG（-）],C型为头膜损伤-尾膜完整[HOS（＋）-EG（＋）],D型为头膜-尾膜均损伤[HOS（-）-EG（＋）].结果 桔梗粗提物浓度在50.0、20.0 g/L时体外20 s内即可完全杀灭精子,桔梗皂苷D浓度在2.0、1.0 g/L时20 s内体外完全杀灭精子.对照组A、B、C和D型精子平均百分比分别为（69.0±8.3）%、（3.4±0.5）%、（10.2±1.7）%和（17.4±2.1）%;桔梗皂苷D组及N-9组精子膜完整性全部被破坏,A型和B型精子为0,桔梗皂苷D组C型精子[（65.3±3.8）%]和D型精子[（34.7±7.1）%]均明显高于对照组（P＜0.01）.SCD试验,对照组、桔梗皂苷D组及N-9组精子DNA碎片比例各组间比较差异无统计学意义.精子复活试验显示药物组均无活动精子恢复.结论 桔梗粗提物及桔梗皂苷D在体外通过破坏精子膜（主要破坏精子头膜）使精子快速失活.

Objective To explore the mechanism of spermicidal effect of crude extract and platycodin-D from Platycodon grandiflorum （PG） root in vitro. Methods Between February 2006 and December 2009, 38 fertile and healthy adult males were selected as donors. PG root was extracted and platycodin-D purified. Grouping was as follows： crude extract from PG root, platycodin-D, nonoxynol-9 （ N-9, as a reference standard） and semen-added physiological saline （ as control ）. Spermicidal experiments were carried out in vitro （Sander-Cramer test）. The hypo-osmotic swelling （HOS） test and modified EosinGiemsa （EG） staining were used to detect the integrity of sperm membrane. Four types of sperm morphology were divided through HOS-EG test： Type A： spermatozoa with swelling in tails and head white staining HOS （ ＋ ）-EG（-） （membrane intact）; Type B： spermatozoa with no swelling in tails （membrane-damaged） and head white staining HOS （ - ）-EG （ - ）; Type C： spermatozoa with tail swelling and head red HOS （ ＋ ）-EG（ ＋ ）; Type D： spermatozoa with no swelling in tails and head red HOS（ - ）-EG（ ＋ ）. Sperm chromatin dispersion （SCD） test was performed to determine the integrity of sperm DNA. Results The crude extract from PG root could immobilize and kill 100% spermatozoa within 20 s in vitro at the concentrations of 50. 0 g/L and 20. 0 g/L （ v： v = 1： 1 in semen）. When the semen sample was exposed to the concentrations of 2. 0 g/L and 1. 0 g/L of platycodin-D, all spermatozoa were immobilized within 20 s.In the control group, the mean percentage of Types A, B, C and D was （69. 0 ± 8. 3 ） %, （3.4 ± 0. 5） %,（ 10. 2 ± 1.7 ） % and （ 17. 4 ± 2. 1 ） % respectively. In the groups of platycodin-D and N-9 solution, the rate of Types A and B was O. The rate of Types C [ （ 65. 3 ± 3. 8 ） % ] and D [ （ 34. 7 ± 7. 1 ） % ] significantly increased versus control in the platycodin-D group （ P ＜ 0. 01 ）. Sperm DNA fragmentation had no change upon an exposure to the extract from PG root, platycodin-D and N-9 solution. And the sperm revival test showed none of the spermatozoa recovered their motility. Conclusion The extract and platycodin-D from PG root have a quick sperm-killing effect in a short time in vitro by disrupting the integrity of sperm membrane （ main head）.