摘要
目的:
在前期研究基础上,基于生物效应和均匀设计法优化月安煎的制备工艺和组方结构;初步探讨月安煎的作用机理,为后续新药研制提供实验依据。
材料与方法:
1.制备PMS肝郁脾虚证大鼠模型:选取处于动情周期非接受期的大鼠,利用束缚法结合皮下注射苯甲酸雌二醇注射液方法制备PMS肝郁脾虚证大鼠模型。
2.月安煎提取物中丹酚酸B含量测定方法的建立:建立HPLC测定月安煎提取物中丹酚酸B含量的方法,以提取物丹酚酸B的含量作为评价月安煎提取工艺及优化组方的化学指标。
3.月安煎干预实验性经前期综合征制备方法研究:利用药效学指标和化学指标为评价方法,比较水提法、双提法和醇提法3种提取方法制备的月安煎供试样品产物,以优选出该小复方的最佳提取方法。
4.均匀设计法优化月安煎组方最佳配伍研究:基于均匀设计法对月安煎组方进行优化,参照2010版药典一部中5种药材的规定用量范围,并结合临床实际确定各药的剂量范围,将5种药材作为5个因素,设计11个水平,实验采用U11(115)均匀设计,以特异性药效学指标和丹酚酸B的提取率作为评价依据,优化月安煎处方的药物组合及剂量配比。
5.月安煎最佳组方的验证及其治疗PMS机理的研究:对月安煎组方结构优化获取的各项理论值进行比较验证,选定最佳结构和剂量配比关系;测定模型血清中E2、P、5-HT、 T、PRL含量、对大鼠子宫进行病理学检查及电镜观察子宫超微结构变化,探讨最终选定的月安煎干预实验性类PMS肝郁脾虚证的可能作用机制。
结果:
1.制备了PMS肝郁脾虚证大鼠模型。本研究利用阴道涂片镜检法确定正常未孕大鼠的动情周期,选取处于动情周期非接受期的大鼠。将大鼠束缚于特制矿泉水瓶内,每日3h,连续21d,造成大鼠肝郁脾虚证后,每日皮下注射苯甲酸雌二醇注射液0.05mL(50μg),连续7d,即可制备PMS肝郁脾虚证大鼠模型。所制备的模型与正常组大鼠相比,精神萎靡,对外界刺激不敏感;旷场实验的水平得分、垂直得分和总分均显著减少;血清中E2显著升高、P显著降低,E2/P显著升高。
2.建立了HPLC测定月安煎提取物中丹酚酸B含量的方法。对照品溶液的制备方法:精密称取丹酚酸B对照品7.2mmg,置于10mL量瓶中,加甲醇溶解并稀释至刻度,摇匀,即得。供试品溶液的制备方法:取提取液适量,置于25mL量瓶中,加水溶解并稀释至刻度,摇匀,过滤,即得。色谱条件:色谱柱:Synergi Polar-Rp80A (250×4.6mm,4μm);流动相:甲醇-乙腈-水-甲酸(30:10:59:1);流速:1.0mL.min-1;柱温;30℃;检测波长:286nm;进样量:5μL。外标一点法计算提取液中丹酚酸B含量。
3.与模型组相比,月安煎双提法工艺组大鼠旷场实验的水平得分和总分均明显增加。3种提取工艺产物均能显著增加模型大鼠P水平,降低E2/P,双提法产物还可显著降低模型大鼠E2水平。3种工艺提取液中丹酚酸B的含量分别为:水提法70.4mg.mL-1、双提法89.2mg.mL-1、醇提法82.8mg.mL-1、双提法提取的丹酚酸B含量高于其他两种提取方法。
4.将旷场实验得分与E2/P水平进行综合评分,经逐步回归分析,得到回归方程:Y=0.7492+0.0180*X1-0.0031*X5-0.0005*X1*:X1-0.0001*X4*X4-0.0001*X1*X3+0.0002*X1*X4+0.0001*X1*X5+0.0003*X2*X5+0.0001*X3*X4,复相关系数为RR=1.0000,S=0.0001,F=1977622.9881,查表:F0.01(9,1)=602,F>F0.01(9,1),P<0.01,检验通过,方程有显著意义。理论最大值:Ymax=1.2261,得出月安煎理论最佳药物组合及剂量配比为:香附(18.18g)、白术(40.00g)、丹参(10.91g)、石菖蒲(30.00g)。利用逐步线性回归方法分析不同方药配伍对丹参中丹酚酸B提取率的影响,得到回归方程为提取率(%)=香附(g)×0.085一白术(g)×0.206+丹参(g)×0.01+石菖蒲(g)×0.068。由回归结果可见:丹参中丹酚酸B的提取率与菟丝子无关。两种评价结果显示均与菟丝子无关,提示药材菟丝子可以去除,在原方基础上精简了一味药材。
5.经药效学验证,与模型组相比月安煎优化方(后简称新月安煎)低(4.5g生药/kg)、中(9.0g生药/kg)、高(18.0g生药/kg)剂量组,旷场实验的水平得分和总分均显著增加(P<0.05),且量效关系明显,高剂量组垂直得分显著性增加(P<0.05);新月安煎中、高剂量组P明显升高(P<0.01),E2/P明显降低(P<0.05)。新月安煎中、高剂量组5-HT含量明显升高(P<0.05),各给药组T含量虽有所增加,但无显著性差异(P>0.05)。高倍电镜观察大鼠子宫超微结构变化显示,新月安煎低、中、高剂量组对模型大鼠的线粒体、粗面内质网、细胞表面微绒毛损伤有明显的保护作用(P<0.01),提示本品可使细胞膜处于功能活跃状态,从而对经前期综合征的症状有所改善。病理学检查,确定各组大鼠子宫在腺体增生程度、腺上皮分泌情况及间质炎细胞浸润等方面的镜下表现无明显区别。
结论:
1.综合药效学和化学实验结果,并考虑减少醇溶剂的使用与残留,确定双提法为月安煎的最佳提取工艺。
2.采用均匀设计法设计实验,经多元回归分析,得出月安煎理论最佳剂量配比为香附:白术:菟丝子:丹参:石菖蒲=18.18:40.00:0.00:10.91:30.00。
3.优化的月安煎组方在原方基础上精简了一味药材。经药效学验证,可明显改善PMS肝郁脾虚症状,调节雌孕激素分泌紊乱状态,组方更加合理,剂量配比更加科学。
4.月安煎干预PMS肝郁脾虚证的可能作用机制为增加孕激素水平,降低E2/P,调整雌孕激素比例失衡状态。同时,可增加5-HT的合成,从而减轻或消除经前期综合征的精神症状。
Purpose:
In this research, the composing and dosage of the prescription were optimized based on pharmacology and uniform design. Yueanjian's mechanism of treating premenstrual syndrome was also studied.
Material and method:
1. The animal models of PMS with the syndrome of liver-depression and spleen-deficiency were established by chronic restraint stress.
2. The content of Salvianolic acid B in Yueanjian was determined by HPLC.
3. Yueanjian was extracted by three methods:water-extraction, steam-distillation and alcohol-extraction. Behavioral performances and the contents of estradiol and progestin in serum were analyzed after the animal models were treated with these three extracts. The contents of Salvianolic acid B in these three extracts were detected by HPLC. All these four indicators were used to optimize the extraction method of Yueanjian.
4. According to U11(115) uniform design table, five drugs in Yueanjian were treated as five factors, which were divided into11levels. Behavioral performances and the contents of estradiol and progestin in serum were used to optimize the prescription of Yueanjian.
5. The theoretical result was tested and verified by animal models of PMS. The levels of E2, P,5-HT, T and PRL in animal models'serum after treated with Yueanjian were analyzed. The ultrastructure and pathology of uterus were also checked to discuss Yueanjian's mechanism of treating premenstrual syndrome.
Results:
1. Vaginal smear cystourethroscopy were used to determine the estrogen cycle of unpregnant rates. Non-estrus rates were chosen and binded in special plastic bottles three times a day.21days later, Estradiol benzoate was hypodermically injected500μg per day, for7days. Compared with normal animals, model animals were listlessness. Model animals'scores in open field experiments were lower, and the levels of progestin declined significantly, while the levels of estradiol and E2/P increased appreciably.
2. The content of Salvianolic acid B in Yueanjian was determined by HPLC. The reference substance solutions preparation method:Precisely sampling the Salvianolic acid B7.2mg into10mL capacity bottle, ultrasonic dissolving in appropriate methanol, added methanol to scale, shook up. The sample preparation method:Taking some samples into25mL capacity bottle, added water to scale, shook up. Chromatographic condition: Synergi Polar-Rp80A (250×4.6mm,4μm) column. Mobile phase:methanol-acetonitrile-water-formic acid30:0:9:). Flow rate1.0mL·min-1.Column temperature:30℃. Detection wavelength:286nm.
3. The levels of progestin in animals treated with these three extracts were higher than model animals, while the levels of E2/P were lower. Steam distillations extract could also make the level of E2reduce significantly. The behavioral performances of animals treated by steam distillations were also higher than model animals. The content of Salvianolic acid B in the extract of steam-distillation was higher than other two extracts.
4. According to the result of multiple regression analysis of behavioral performances and levels of E2/P, the regression equation was:Y=0.7492+0.0180*X1-0.0031*X5-0.0005*X1*X1-0.0001*X4*X4-0.0001*X1*X3+0.0002*X1*X4+0.0001*X1*X5+0.0003*X2*X5+0.0001*X3*X4, multiple correlation coefficient was1.0000, the equation have prominent meanings. The best compatibility proportion of Yueanjian was rhizoma cyperi (18.18g), white atractylodes roots (40.00g), Salvia miltiorrhiza (10.91g) and rhizoma acori graminei (30.00g). According to the result of multiple regression analysis of extraction ratio of Salvia miltiorrhiza, the regression equation was:Y=0.085*X1-0.206*X2+0.01*X4+0.068*X5. These two result show that the seed of Chinese dodder could be removed.
5.The behavioral performances of animals treated by Yuananjian were higher than model animals. Yuananjian could also raise the level of progestin and reduce the level of E2/P. The content of5-HT in serum raised significantly after treated by Yuanjian. Yuanjian showed good protection to the damage of mitochondria, rough endoplasmic reticulum and microvilli on cell surface.
Conclusion:
1. On the basis of comprehensive analysis, steam-distillation was chosen as Yueanjian's best extraction method.
2. The best compatibility proportion of Yueanjian was rhizoma cyperi (18.18g), white atractylodes roots (40.00g),Salvia miltiorrhiza (10.91g) and rhizoma acori graminei (30.00g).
3. Yueanjian's mechanism of treating premenstrual syndrome maybe related with raising the level of progestin, reducing the level of E2/P and increasing the synthesis of5-HT.
引文
[1]吴丽萍,何仲.经前期综合征和经前焦虑性障碍患病率及影响研究进展[J].护理学报,2011,17(22):4-6.
[2]佟庆,赵春霞.经前期综合征的流行病学及相关因素[J].中国临床医生,2010,39(11):15—17.
[3]认识“经前期综合征”[J].中国药店,2002,3(8):89-90.
[4]刘良鸣.“经前平”给全球妇女带来福音[J].中国药业,2002,11(6):58-58.
[5]文星.为了千万个妇女的身体健康—山东中医药大学基础医学院院长乔明琦研发新药侧记[J].前进论坛,2006,46(5):33-33.
[6]濮凌云.经前期综合征的病因学说[J].中国临床医生,2010,39(11):17-19.
[7]黎莉,孙鹏,梁琼麟,等.白香丹胶囊干预经前期综合征肝气逆证大鼠血清代谢组学研究[J].中成药,2011,(5):39-44.
[8]张玉勤.PMS病理生理学研究[J].国外医学情报,1998,19(6):37-38;39-40.
[9]雅德.经前期紧张与β内啡肽有关[J].国外医学情报,1983,4(13):10-11.
[10]杨淑萍.β-内啡肽在女性生殖内分泌中的调节作用[J].国外医学.妇产科学分册,1996,24(3):4-7.
[11]高兴笑,张惠云.经前期综合征相关神经递质及其受体研究进展[J].医学综述,2011,18(23):26-29.
[12]徐明婧,张惠云.经前平颗粒治疗经前期综合征肝气逆证大鼠的作用机制[J].药学与临床研究,2010,18(2):58-61.
[13]张玉勤.经前期综合征——病理生理学研究[J].国外医学情报,1998,19(6):39-40.
[14]马敏芝.女大学生围经期心身健康状况调查[J].中国心理卫生杂志,1996,10(2):65-67.
[15]付磊强,张庆文,范华强.经前期综合征与心理社会因素的研究进展[J].实用预防医学,2009,16(6):317-318.
[16]涂彬彬,乔杰.口服避孕药治疗经前期综合征的研究进展[J].生殖与避孕,2009,30(10):37-41.
[17]陈淑涛.社会-心理-医学模式下的经前期综合征研究进展[J].现代中西医结合杂志, 2003,12(18):97—100.
[18]刘建勋.催乳素与精神病[J].国外医学.内分泌学分册,1982,3(4):30-32.
[19]江英才.经前期综合征和催乳素[J].国外医学.妇产科学分册,1982,10(5):54.
[20]程艽.维生素B6治疗经前期综合征的效果:系统回顾[J].英国医学杂志(中文版),2000,2(2):9.
[21]育巍.维生素B6可改善经前期综合征症状[J].国外医学情报,2000,21(7):30.
[22]徐焰.经前期综合征的病因及治疗[J].山东医药,1986,30(3):39-42.
[23]濮凌云.经前期综合征的病因学说[J].中国临床医生,2010,39(11):17-19.
[24]罗劲梅.经前期综合征治疗进展及展望[J].海南医学,2009,37(S5):133-135.
[25]姚珍薇.经前期紧张综合征的病因与治疗[J].中国实用妇科与产科杂.志,1995,11(1):19-21.
[26]李遒用.经前期综合征的锌和铜的变化[J].国外医学·计划生育分册,1995,14(9):62-63.
[27]乔明琦,张惠云,于艳红,等.经前期综合征肝气逆证患者月经周期血清雌二醇、孕酮含量的动态变化[J].中医杂志,2006,56(4):56-58.
[28]CommitteeforMedicinalProductsforHumanUse(CHMP):.Guidelineonthetreatmentofpre menstrualdysphoricdisorder(PMDD):.EuropeanMedicinesAgency,2011.
[29]罗劲梅.经前期综合征治疗进展及展望[J].海南医学,2009,37(S5):133-135.
[30]黎萍,郭锡永.应激对女性生殖内分泌疾病影响的研究进展[J].中国妇幼健康研究,2006,17(4):74-76.
[31]李燕琴.PMS的治疗[J].国外医学·妇幼保健分册,2000,11(2):70-75.
[32]李元敏.选择性5-羟色胺重摄取抵制剂严重的PMS的疗效[J].国外医学学分册,2001,28(2):122—123.
[33]姚珍薇.经前期紧张综合征的病因与治疗[J].中国实用妇科与产科杂志,1995,1](1):19-21.
[34]吕秋兰.安体舒通治疗经前期综合症:一项双盲、安慰剂对照研究[J].国外医学.妇产科学分册,1996,24(5):57-57.
[35]文宁,雷磊.月经病的中西医结合诊治思路[J].新中医,2010,42(6):12-14.
[36]育巍.维生素B6可改善经前期综合征症状[J].国外医学情报.,2000.21(7):30.
[37]佟庆.赵春霞.经前期综合征的流行病学及相关因素[J].中国临床医生,2010,39(11): 15-17.
[38]孙晓玲,许丽绵.经前期综合征的中医学研究进展[J].中医药信息,2006,24(3):13-15.
[39]胡春雨,王文燕,乔明琦,等.从肝试论经前期综合征的体质类型[J].辽宁中医杂志,2006,49(11):4344.
[40]姜向坤,张丽,,伍雪梅.从肝失疏泄谈经前期综合征[J].贵阳中医学院学报,2007,29(6):10—12.
[41]杜伟静,赵京燕,张英云,等.经行乳房胀痛的中医认识[J].黑龙江中医药,2009,52(2):66-67.
[42]严灿,邓中炎,潘毅.从现代心理应激理论研究中医肝主疏泄功能[J].广州中医药大学学报,2000,17(3):209-211.
[43]金益强.肝脏象本质研究思路与方法的思考[J].山东中医药大学学报,2000,24(4):242-241.
[44]佟庆,,赵春霞.经前期综合征的流行病学及相关因素[J],中国临床医生,2010,39(11): 15—17.
[45]乔明琦,张珍玉,徐旭杰,等.经前期综合征分布规律的流行病学调查研究[J].中国中医基础医学杂志,1997,3(3):31-33.
[46]姜向坤,张丽,伍雪梅.从肝失疏泄谈经前期综合征[J].贵阳中医学院学报,2007,29(6):10-12.
[47]刘建,王小云.经前期综合征的中医辨证论治探讨[J].辽宁中医杂志,2011,54(10):69-70.
[48]钱著.助阳解郁汤治疗经前期紧张综合征32例[J].江苏中医,2000,20(12):1587.
[49]原荣.月经前期紧张症的辨证施治[J].内蒙古电大学刊,2002,16(4):114.
[50]孙晓玲,许丽绵.经前期综合征的中医学研究进展[J].中医药信息,2006,24(3):13-15.
[51]宋丈武.经前紧张综合征辨治体会[J].河南中医,2003,28(3):50-51.
[52]贺稚平.从肝论治经前期紧张综合征[J].北京中医药大学学报,1995,36(6):47-48.
[53]董陆.经前期综合征从肝论治4法[J].浙江中医杂志,2003,38(7):284
[54]霍清平.调肝法在经前期紧张综合征中的应用[J].河南中医药学刊,1995,10(2):38-39.
[55]曹亚芳,王辉蝶,董岷,等.从“肝失疏泄”论治经前期综合征探讨[J].辽宁中医药大学学报,2009,11(12):49-50.
[56]朱必苓,李芳.柴胡疏肝散加减治疗经前期综合征35例疗效观察[J].世界中医药,2010,5(4):24-25.
[57]陈文英.辨证治疗经前期综合征188例观察[J].实用中医药杂志,2001,17(1):4-5.
[58]董陆.经前期综合征从肝论治4法[J].浙江中医杂志,2003,38(7):284
[59]邱明英.加味龙胆泻肝汤治疗经前期头目胀痛78例[J].新中医,2003,35(4):61—62.
[60]李传香,张胜军,朱艳梅.解郁消胀煎治疗经前期综合征400例——附西药对照组200例[J].辽宁中医杂志,2002,45(4):26-27.
[61]胡翠芳,徐红英.从脏论治月经前后诸症[J].中医杂志,2010,51(6):93—94.
[62]李仁杰,夏冰,周洁,等.中医药治疗经行泄泻的研究进展[J].中日友好医院学报,2010,24(6):3941.
[63]陈小玲.经前期综合征治验[J].陕西中医,2000,21(s):27-28.
[64]杜伟静,赵京燕,张英云,等.经行乳房胀痛的中医认识[J].黑龙江中医药,2009,52(2): 66—67.
[65]原荣.月经前期紧张症的辨证施治[J].内蒙古电大学刊,2002,16(4):114.
[66]孙晓玲,许丽绵.经前期综合征的中医学研究进展[J].中医药信息,2006,24(3):13-15.
[67]周征.疏肝补肾法为主治疗经前期综合征[J].河南中医,2003,23(12):53-53.
[68]陈文英.辨证治疗经前期综合征188例观察[J].实用中医药杂志,2001,17(1):4-5.
[69]宗惠,刘瑞芬,张海琴.经前期综合征中医辨治思路[J].中国中医药信息杂志,2009,]6(2):97-98,
[70]陈小玲.经前期综合征治验[J].陕西中医,2000,2 1(s):27-28.
[71]梁茂新,刘杰.影响和提高中药临床疗效的几个关键问题[J].世界科学技术,2002,4(5):23-28.
[72]梁茂新.建立中药小复方精选系统操作技术平台的基本构想[J].世界科学技术,2002,4(6):3642.
[73]梁茂新,李东安.均匀设计在中药小复方精选中的改良应用[J].世界科学技术-中医药现代化,2006,8(2):63-66.
[74]顾晓路,梁茂新.安眠颗粒结构优化的步骤与方法[J].中国实验方剂学杂志,2008,14(6):26-28.
[75]张会宗,马杰,张慧颖,等.治疗慢性胆囊炎中药小复方优化的思路与方法[.J].世界科学技术-中医药现代化,2007,9(5):37-41.
[76]李东安,梁茂新,张慧颖,等.治疗围绝经期综合征中药小复方的筛选步骤与方法[J].世界科学技术,2005,7(6):41-46.
[77]宫俊,董俊龙,梁茂新,等.基于关联规则的广义药对最适合病证的挖掘方法[J].东北大学学报(自然科学版),2011,17(8):1097-1100.
[78]张惠云,乔明琦,朱武成,等.大鼠模拟经前期综合征肝气逆证血清激素与行为变化[J].中成药,2002,24(2):118-119.
[79]梁茂新,黄会生.《普济方》数据库管理系统(计算机软件著作权登记证书.软著登字第0002781号):中华人民共和国国家版权局,1998.
[80]许廷生,梁秀兰.丹参的临床应用综述[J].中国社区医师(综合版),2004,6(9):8-9.
[81]戈升荣.丹酚酸的药理作用研究进展[J].中药材,2002,25(9):683-687.
[82]中国药典[S].一部.2010.
[83]李雪梅,甘雨,张宏等.月安煎干预实验性经前期综合征制备方法研究[J].中国中药杂志,2012,37(7):20-23.
[84]李雪梅,尤献民,梁茂新.月安煎不同配伍对丹酚酸B提取率的影响[J].中草药,2012,43(5):926-928
[85]李雪梅,尤献民,梁茂新.均匀设计法优化月安煎组方最佳配伍研究[J].西北药学杂志,2012,27(3):197-199.
[86]宋洪运,张惠云.经前期综合征肝气郁证大鼠模型血清激素与行为变化[J].辽宁中医杂志,2010,53(11):175—177.
[87]薛刚,张惠云.经前期综合征动物模型的应用及研究进展[J].医学综述,2010,17(20):121-123.
[88]于艳红.山东中医药大学情志病证研究创新团队科研工作概况[J].山东中医药大学学报,2011,35(4):2.
[89]薛刚,张惠云.经前期综合征动物模型的应用及研究进展[J].医学综述,2010,17(20):121-123.
[90]乔明琦,张继忠,马成海,等.猕猴模拟经前期综合征肝气逆证病变的可行性探索[J].中国中医基础医学杂志,1998,4(6):29-32.
[91]李花,蔡光先,刘柏炎.中医“肝脾相关”理论阐微[J].天津中医药,2010,17(3):40-42.
[92]陈锦锋,王爱成,王玉来,等.肝气郁结证诊断的必备症状以及与抑郁状态的关系探讨[J].辽宁中医杂志,2005,48(1):21-24.
[93]岳利峰,丁杰,陈家旭,等.肝郁脾虚证大鼠模型的建立与评价[J].北京中医药大学学报,2008,50(6):40-44.
[94]王竹风,陈家旭,赵歆,等.探讨肝郁的影响[J].时珍国医国药,2005,16(10):12—13.
[95]高丽,隋波,沈东颖,等.雌性大鼠运动性动情周期抑制与卵巢、子宫显微结构及相关激素水平的变化[J].中国临床康复,2006,20(40):117—119.
[96]陈春萍,程大志,罗跃嘉.雌激素对情绪的影响:心理、神经、内分泌研究[J].中国科学:生命科学,2011,16(11):5-18.
[97]周江宁,闫雪波.抑郁症发病的下丘脑中枢驱动调节机制[J].中国科学技术大学学报,2008,44(8):103-113.
[98]罗国安,梁琼麟,张荣利,等.化学物质组学与中药方剂研究—兼析清开灵复方物质基础研究[J].世界科学技术,2006,9(1):16-25.
[99]高兴笑,张惠云.经前期综合征相关神经递质及其受体研究进展[J].医学综述,2011,17(23):26-29.
[100]张玉勤.经前期综合征—病理生理学研究[J].国外医学情报,1998,19(6):39-40.喻东山.5—羟色胺能在抑郁症中的作用[J].临床精神医学杂志,1994,4(3):184-186.