蒙药手参的长期毒性研究.pdf

蒙药手参的长期毒性研究何乌云嘎1，王秀兰2，3，阿润2，荷叶2，莫日根2，永强2（1.内蒙古民族大学 附属医院，内蒙古 通辽 028000；2.内蒙古民族大学 蒙医药学院，内蒙古 通辽 028043；3.内蒙古蒙医药工程技术研究院，内蒙古 通辽 028000）摘要 目的：通过蒙药手参对SD大鼠连续灌胃给药长期毒性试验，观察手参对大鼠长期给药产生的累积毒性反应和严重程度，毒性反应的靶器官及其延迟性毒性反应的恢复和发展情况，为其临床应用提供实验依据。方法：将120只SD大鼠随机分为空白对照组，手参低、中、高剂量组，每组30只，每天采用灌胃和含药饲料喂养法，连续给药3个月，空白组喂养普通饲料及灌胃等体积蒸馏水。连续观察各组大鼠一般情况、每周测定体重变化、给药45 d、90 d和停药15 d后每组各处死10只，测定血常规指标、血液生化指标、测定主要脏器指数、HE染色观察组织病变，综合评估蒙药手参的长期毒性。结果：实验期间各给药组的动物未见死亡，眼、耳、鼻、口未见异常分泌物，尿液及粪便性状未见异常，自主活动及步态正常；体重和摄食量没有发现显著性差异；与空白对照组相比，给药中期，手参中、高剂量组的MPV显著升高；给药期结束时，高剂量组、中剂量组Crea、TP显著降低。恢复期，MPV、Crea、TP等指标恢复正常，中剂量组CHO明显降低；各组大鼠脏器重量及脏器指数均无显著性差异，脏器病理组织学检查也均未见异常。结论：手参以临床用药量的10倍、20倍及30倍量连续3个月灌胃给药，试验大鼠一般状况、体重、摄食量、脏器指数、血常规指标、血生化指标、组织病理等指标未见明显毒性表现及后续毒性反应。可认为手参长期毒性不明显，临床用药安全。其中，恢复期中剂量组CHO降低的原因有待于进一步探讨。关键词 蒙药；手参；SD大鼠；长期毒性中图分类号 R992文献标志码 A文章编号 1671-0185（2023）04-0366-13Study on the Long-term Toxicity of Mongolian MedicineGymnadenia ConopseaHE Wuyunga1，WANG Xiu-lan2，3，Arun2，Heye2，Morigen2，Yongqiang2（1.Affiliated Hospital，Inner Mongolia Minzu University，Tongliao 028000，China；2.College of Mongolian Medicineand Pharmacy，Inner Mongolia Minzu University，Tongliao 028043，China；3.Inner Mongolia Institute ofMongolian Medicine Engineering and Technology，Tongliao 028000，China）Abstract：Objective：Through long-term toxicity test of continuous intragastric administration of Mongolian medicine Gymnadenia conopsea on SD rats，to observe the cumulative toxic reactions and severity，the target organs oftoxic reactions，and the recovery and development situation of delayed toxic reactions of Mongolian medicineGymnadenia conopsea on SD rats，which provides experimental basis for its clinical application.Methods：Onehundred and twenty SD rats were randomly divided into a blank control group，a Gymnadenia conopsea low-dosegroup，a Gymnadenia conopsea medium-dose group and a Gymnadenia conopsea high-dose group，with 30 rats ineach group.The drug administration groups adopted gavage and drug-containing feed feeding method every day，and were continuously administrated for 3 months；the blank group was fed with ordinary feed and administeredintragastrically the same volume of distilled water.The general condition of rats in each group was observed con基金项目：内蒙古自治区科技计划项目（2021GG0003）作者简介：何乌云嘎，内蒙古民族大学附属医院蒙药士，硕士。通讯作者：王秀兰，内蒙古民族大学蒙医药学院教授，博士，博士研究生导师。DOI：10.14045/ki.15-1220.2023.04.013第38卷第4期内蒙古民族大学学报（自然科学版）Vol.38No.42023年07月Journal of Inner Mongolia Minzu UniversityJuly 2023tinuously，their body weight was measured every week，and 10 rats in each group were sacrificed after 45 d and90 d administration，and 15 d after drug withdrawal.Through measuring rats blood routine indexes，blood biochemical indexes and main organ indexes，and observing tissue lesions by HE staining，to comprehensively evaluate the long-term toxicity of Mongolian medicine Gymnadenia conopsea.Results：During the experiment，therewere no death，no abnormal secretions in eyes，ears，nose and mouth，no abnormal urine and feces in each administration group，and their independent activities and gait were normal.There was no significant difference between body weight and food intake.At the middle stage of drug administration，the MPV in the medium dose andhigh dose groups was significantly higher than that in the blank control group.At the end of drug administration，the Crea and TP were significantly decreased in the high-dose and medium-dose groups.In recovery stage，theMPV，Crea，TP and other indexes returned to normal，but the CHO significantly decreased in the medium dosegroup.There were no significant differences in organ weight and organ index among all groups，and no abnormalities were found in organ histopathology.Conclusion：When Gymnadenia conopsea is given by intragastric administration at 10，20 and 30 times of the clinical dosage for consecutive 3 months，the general status，body weight，food intake，organ index，blood index，blood biochemical index，histopathology and other indexes of experimentalrats don t show obvious toxicity or subsequent toxic reaction.It can be considered that the long-term toxicity ofGymnadenia conopsea is not obvious and the safety risk of clinical use is low.The reason of CHO reduction inthe medium dose group during the recovery stage remains to be further discussed.Key words：Mongolian medicine；Gymnadenia conopsea；SD rats；Long-term toxicity手参记载于 内蒙古蒙药材标准，是传统蒙药材，又叫手掌参、旺拉嘎、佛手参，是兰科多年生草本植物手参（Gymnadenia conopsea R.Br）的块茎。产自中国东北、内蒙古、华北、云南、西北、四川、西藏以及蒙古国、俄罗斯、日本、欧洲等地1。在 传统蒙药与方剂 里记载手参具有补精华、固精、滋补的功能；用于缺乏营养、身体虚弱、肾寒、精液耗损、阳痿、陶赖、赫如虎、巴木病2。识药学 中记载“根据形状而命名，入药部位根如人手因此得名”3。蒙药简明手册 中记载“旺拉嘎滋生体力和精华”4。蒙药单味药的介绍 中记载“滋补、增生体力”5。蒙药学 引 读者之喜 称“旺拉嘎生精壮阳、增生体力、去毒”6。中国医学百科全书（蒙医卷）中记载“小剂量时对家兔和狗有利尿作用，大剂量时有反作用；对蛙后肢血管有收缩作用，但没有清楚的记载用药的剂量”7。长期毒性试验是指频繁多次给药的毒性试验统称，叙述受试物重复给予受试药物后的毒性反应，长期毒性试验可为临床安全用药剂量设定、临床毒副反应监测及血液指标检测提供参考8。目前，手参长期毒性方面尚未见研究报道，缺乏其长期用药的安全性评价实验数据，已成为手参产业化过程中亟待解决的瓶颈问题。笔者按照国家有关规定和要求，进行长期毒性试验，系统观察该蒙药长期大量口服时对机体的影响，以评价其安全性。1材料与方法1.1一般材料1.1.1实验动物SPF级SD大鼠120只，雌雄各半，体重180200 g，购自辽宁长生生物技术股份有限公司。动物生产合格证号：SCXK（辽）2020-0001。动物饲养环境：昼夜12 h明暗交替，温度在2126，湿度在41%62%。1.1.2药品与试剂蒙药手参（购自内蒙古蒙医药工程研究院，经包桂花教授鉴定为蒙药材手参，批号20210826）；HE染色剂（南京建成科技有限公司，批号20220104）、抗血凝采血管（江苏宇力医疗器械有限公司，批号20201130），大鼠灌胃器、生化仪试剂盒均购自南京建成科技有限公司，ALT、AST、TBIL、TP、UREAL、Crea、TG、CHO、GLU、ALP、CK、UA试剂盒批号分别为57490301、57488701、54853701、36607001、57733001、54626401、56863901、57937601、5864420、5687101、57540601、58964901）；血细胞分析用血溶剂（迈瑞南京生物技术有限公司，批号2021051708）。第4期何乌云嘎等：蒙药手参的长期毒性研究367内蒙古民族大学学报2023年1.1.3实验仪器电子天平（日本，型号AUY120EXP）；电热恒温鼓风干燥箱（型号DHG-9053A）；生化培养箱（型号SHP-105）；生物组织包埋机（浙江省金华市科迪仪器设备有限公司，型号KD-BM）；切片机（Lica公司，型号RM2235cwEU）；水浴箱（常州荣华仪器制造有限公司，型号HH-8）；离心机（SIGMA公司，型号3-18KS）；全自动生化分析仪（南京罗氏科技有限公司，型号Codas 311）、全自动血液分析仪（深圳迈瑞生物医疗电子股份有限公司，型号UH-07002848）、光学显微镜。1.2实验方法1.2.1分组及给药顺应环境饲养7 d后，将120只SD大鼠随机分为4组，每组30只，雌雄各半，分别为空白对照组，手参低、中、高剂量组，给药量分别为临床用药量的10倍（5.1 gkg-1）、20倍（10.2 gkg-1）、30倍（15.4 gkg-1）。采用灌胃法和喂养法，每日定时给药及饲料1次。灌胃容量每100 g体重2 mL，药品均用蒸馏水配制，对照组给予普通饲料和灌胃蒸馏水，给药6 d/周，持续3个月。每天查看每只大鼠的行为活动和一般状态，每周称重1次，根据其体重调节给药量，试验期间自由饮水。1.2.2观察指标1.2.2.1一般情况观察给药前后每只大鼠的外观形态，行为活动，大小便形状、颜色，有无异常分泌物等；每周称重1次，记录体重及食物摄入量。1.2.2.2血常规及血生化指标在给药45 d、90 d及停药后15 d，每组大鼠各取10只，雌雄各半，麻醉后取腹主动脉采血，进行血常规指标及生化指标的检测；全血用于检查血常规：血小板、血红蛋白、白细胞数、血细胞比容、平均红细胞体积、红细胞平均血红蛋白浓度、红细胞数、平均红细胞血红蛋白量、血小板平均容积等指标。血液置于37 恒温水浴锅中30 min，3 000 rmin-1离心10 min，分离血清，采用全自动生化分析仪测定血清样本中碱性磷酸酶（ALP）、甘油三酯（TG）、丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）、总胆红素（TBIL）、总蛋白（TP）、尿素氮（UREAL）、肌酐（Crea）、胆固醇（CHO）、血糖（GLU）、肌酸激酶（CK）及血清尿酸（UA）等指标。1.2.2.3脏器检查腹主动脉采血后进行尸体解剖，首先，肉眼观察脏器，记录异常情况，准确称取各脏器重量，并计算脏器指数；然后将器官组织用10%福尔马林液固定，脱水、透明、浸蜡、石蜡包埋、切片，HE染色后在显微镜下观察各脏器组织细胞的变化。1.3统计学分析采用 SPSS 22.0版统计软件进行统计，所有数据以样本x s表示。手参各给药组结果分别与空白对照组进行组间两两比较，P0.05为具有显著性差异。2结果2.1一般情况各组大鼠外观特征、生长发育良好，体重在正常范围内增长，无毒副反应，无死亡；摄食量差异性不显著；行为活动正常，无异常兴奋、身体震颤，大小便颗粒状、粪便呈棕褐色、被毛光顺，眼睛、鼻腔、口腔等无异常分泌物。结果见图1、图2。700500300100摄食量/g0123456789101112131415空白对照组低剂量组中剂量组高剂量组时间/周图1雄性大鼠摄食量曲线图Fig.1 Curve of food intake in male rats368420315210105摄食量/g0123456789101112131415空白对照组低剂量组中剂量组高剂量组时间/周图2雌性大鼠摄食量曲线图Fig.2 Curve of food intake in female rats2.2血常规指标结果如表1所示，给药中期，高、中剂量组MPV显著高于空白对照组（P0.01）。结果如表2、表3所示，给药90 d和停药15 d，各组间各指标比较差异均无统计学意义（P0.05）。表1给药中期大鼠血常规指标（x s，n=10）Tab.1 Blood routine indexes of rats at the middle stage of drug administration（x s，n=10）指标RBC/(1012L-1）WBC/(109L-1）HCT/%MCV/fLMPV/fLMCH/pgPLT/(109L-1）MCHC/（gL-1）HGB/（gL-1）空白对照组6.870 90.652 16.505 62.814 138.265 53.551 155.762 31.691 26.764 50.221 221.743 50.721 3813.026 476.181 3389.90 224.044 2149.004 214.444 2手参低剂量组6.770 90.543 25.680 32.533 538.223 02.093 057.354 22.070 56.584 60.282 123.133 22.523 1828.146 129.122 0402.746 30.510 5148.606 87.950 0手参中剂量组7.224 30.285 55.872 51.824 140.571 31.042 157.052 41.441 47.642 60.222 1*21.832 50.590 4934.177 49.510 3382.683 65.440 0157.402 44.012 0手参高剂量组7.022 10.261 35.649 01.277 240.052 51.056 357.120 81.652 57.5824 0.262 6*21.970 90.872 4917.811 415.244 2384.401 59.044 6154.001 15.440 4注：给药组与空白对照组比较，*P0.01。表2给药末期大鼠血常规指标（x s，n=10）Tab.2Blood routine indexes of rats at the end stage of drug administration（x s，n=10）指标RBC/(1012L-1）WBC/(109L-1）HCT/%MCV/fLMPV/fLMCH/pgPLT/(109L-1）MCHC/（gL-1）HGB/（gL-1）空白对照组7.475 60.762 64.442 42.113 640.011 63.354 353.793 52.551 27.154 60.916 120.287 41.993 5722.314 582.645 1377.364 027.571 3150.525 310.955 2手参低剂量组7.867 40.561 04.341 42.011 041.671 42.111 553.001 11.891 46.830 30.194 120.744 60.802 1832.767 095.401 1390.601 04.721 4162.712 07.891 4手参中剂量组6.911 50.351 04.022 52.213 140.725 39.282 053.236 43.211 06.789 00.340 220.193 71.581 2973.040 386.214 2379.853 412.291 4154.115 332.781 4手参高剂量组7.542 10.970 41.521 70.651 040.219 93.961 553.711 43.033 26.950 10.273 323.552 49.155 4979.612 098.843 0383.113 56.510 3154.514 215.730 2注：给药组与空白对照组比较，均为P0.05。第4期何乌云嘎等：蒙药手参的长期毒性研究369内蒙古民族大学学报2023年表3给药恢复期大鼠血常规指标（x s，n=10）Tab.3Blood routine indexes of rats at recovery stage of drug administration（x s，n=10）指标RBC/(1012L-1）WBC/(109L-1）HCT/%MCV/fLMPV/fLMCH/pgPLT/(109L-1）MCHC/（gL-1）HGB/（gL-1）空白对照组6.481 20.444 44.670 03.414 238.560 45.471 154.970 32.116 06.791 00.242 020.641 31.922 0818.311 372.103 3389.011 26.753 0157.103 17.141 3手参低剂量组6.710 31.141 14.800 22.324 140.062 03.021 055.202 13.184 26.821 30.391 322.011 81.771 1810.914 096.760 2394.803 26.610 3164.604 56.971 0手参中剂量组7.025 40.340 53.530 12.920 039.991 04.622 055.691 43.632 67.044 10.425 221.561 71.343 1804.907 095.891 3389.000 17.052 4165.102 67.471 4手参高剂量组7.302 00.621 03.234 12.231 438.891 63.571 155.720 63.692 57.086 00.394 221.541 21.093 3834.900 395.521 3386.430 46.810 1160.302 69.770 3注：给药组与空白对照组比较，均为P0.05。2.3对血液生化指标的影响手参给药中期各组血液生化指标分别与空白对照组相比均无差异（P0.05）。给药90 d后，手参高、中剂量组的Crea、TP指标与空白对照组比较显著性降低（P0.01）。恢复期后，手参中剂量组的CHO指标与空白对照组对比明显降低（P0.05）。结果见表4表6。表4 给药中期大鼠血液生化指标（x s，n=10）Tab.4 Blood biochemical indexes of rats at the middle stage of drug administration（x s，n=10）指标AST/（UL-1）ALT/（UL-1）UREAL/（mmolL-1）TP/（gL-1）Crea/（molL-1）TG/（mmolL-1）CHO/（mmolL-1）GLU/（mmolL-1）CK/（UL-1）ALP/（UL-1）UA/（molL-1）CK/（UL-1）空白对照组112.603 121.183 237.503 18.161 26.391 30.912 556.580 64.032 233.604 04.101 10.433 30.122 51.363 30.242 14.543 00.901 0776.421 220.880 185.310 022.520 07.408 92.040 8776.421 220.880 1手参低剂量组115.201 329.563 235.245 16.196 36.362 51.053 156.200 24.125 333.821 02.554 90.401 20.174 71.251 10.447 04.870 60.743 5853.101 13.221 090.801 319.091 16.173 10.710 2853.101 13.221 0手参中剂量组105.506 422.157 435.636 06.750 35.436 70.790 455.295 32.940 028.208 53.710 60.4212 20.152 11.524 40.361 44.633 90.836 6725.520 860.770 197.600 829.480 06.280 41.203 5725.520 860.770 1手参高剂量组108.801 113.320 435.840 34.010 45.850 21.941 155.454 52.771 033.324 05.240 00.403 40.140 01.401 10.173 14.801 20.763 2823.643 249.990 085.301 339.180 26.263 21.482 1823.643 249.990 0注：给药组与空白对照组比较，均为P0.05。表5给药末期大鼠血液生化指标（x s，n=10）Tab.5 Blood biochemical indexes of rats at the end stage of drug administration（x s，n=10）指标AST/（UL-1）ALT/（UL-1）UREAL/（mmolL-1）TP/（gL-1）Crea/（molL-1）TG/（mmolL-1）CHO/（mmolL-1）GLU/（mmolL-1）CK/（UL-1）ALP/（UL-1）UA/（molL-1）空白对照组114.461 31.341 439.411 56.227 56.843 60.980 959.001 34.250 033.105 64.350 30.911 00.520 01.610 10.327 06.997 41.283 6303.703 6107.334 267.802 120.568 06.106 51.960 9手参低剂量组113.144 56.461 044.161 020.881 17.03081.420 359.45059.140 232.60224.880 00.65130.180 21.41410.392 05.91412.242 0218.425076.380 255.202125.890 05.03411.120 6手参中剂量组76.561 336.350 038.672 110.360 46.342 41.520 443.633 28.000 0*27.331 11.702 0*0.621 40.300 31.031 50.371 05.975 11.132 2223.201 582.240 247.335 114.980 33.961 10.904 0手参高剂量组79.901 29.450 038.003 114.790 46.791 01.050 244.090 95.100 0*25.561 72.450 0*0.571 10.192 11.410 90.890 05.622 11.944 3187.070 058.680 355.220 519.113 24.710 02.203 2注：给药组与空白对照组比较，*P0.01。370表6给药恢复期大鼠血液生化指标（x s，n=10）Tab.6 Blood biochemical indexes of rats at recovery stage of drug administration（x s，n=10）指标AST/（UL-1）ALT/（UL-1）UREAL/（mmolL-1）TP/（gL-1）Crea/（molL-1）TG/（mmolL-1）CHO/（mmolL-1）GLU/（mmolL-1）GLU/（mmolL-1）CK/（UL-1）ALP/（UL-1）UA/（molL-1）空白对照组85.755 718.443 341.824 39.135 65.962 40.706 645.906 75.523 128.402 44.183 40.726 70.326 41.417 60.334 55.716 80.860 85.715 30.860 1251.503 4100.885 355.000 212.421 34.540 01.080 4手参低剂量组80.435 033.446 437.726 413.944 66.122 41.706 441.882 412.064 626.102 45.943 50.494 50.209 71.140 50.480 84.410 40.620 84.410 40.620 5264.500 660.903 551.205 616.923 45.1431 04.640 1手参中剂量组64.675 229.640 728.936 710.748 65.583 50.864 237.982 38.435 325.203 33.434 60.510 40.300 80.813 40.337 5*4.783 61.586 44.780 01.584 6198.704 279.742 444.107 719.698 63.313 50.830 1手参高剂量组93.282 436.881 152.266 532.702 36.243 51.156 445.413 57.920 028.307 54.844 60.576 40.422 41.152 40.375 44.472 20.924 54.478 70.921 2203.806 761.732 361.006 523.164 53.890 00.831 2注：给药组与空白对照组比较，*P0.05。2.4脏器系数大鼠处死后，摘取主要脏器，肉眼观察脏器表面有无异常变化。受试药物靶器官的损害常以这些脏器的脏器指数异常来界定。在计算脏器指数前应首先排除各种可能影响的因素。给药末期，各器官的脏器指数与空白对照组相比较无异常（P0.05）。恢复期结束后，各组脏器指数与空白对照组相比无明显差异（P0.05）。结果见表7表9。表7给药中期大鼠脏器指数指标（x s，n=10）Tab.7Organ indexes of rats at the middle stage of drug administration（x s，n=10）指标/%肝脏心脏肺脏脾脏肾脏肾上腺胸腺睾丸子宫卵巢附睾脑空白对照组2.474 20.102 70.308 60.023 20.430 50.068 60.173 90.025 70.644 20.042 90.029 10.011 60.107 00.038 60.749 10.043 50.296 50.068 20.070 60.009 20.234 50.031 40.581 10.150 6手参低剂量组2.550 80.291 20.329 70.034 80.407 10.120 80.190 10.015 60.623 70.050 00.035 40.020 20.124 10.047 80.841 70.057 30.440 40.108 40.069 40.010 30.291 20.024 00.429 70.299 2手参中剂量组2.502 40.230 30.324 90.022 70.445 00.083 70.185 50.028 50.666 90.046 10.033 20.014 60.119 20.043 70.801 50.014 60.353 50.125 20.074 70.018 10.275 90.014 60.582 40.142 0手参高剂量组2.457 20.131 20.316 70.033 80.404 60.063 90.191 90.030 30.643 90.064 80.032 60.001 30.124 00.046 80.802 20.051 70.301 00.107 80.068 90.105 80.268 00.022 10.574 30.123 5注：给药组与空白对照组比较，均为P0.05。表8 给药末期大鼠脏器指数指标（x s，n=10）Tab.8Organ indexes of rats at the end stage of drug administration（x s，n=10）肝脏心脏肺脏脾脏肾脏2.545 40.178 10.330 60.047 50.393 40.090 80.167 20.022 80.633 10.120 92.848 10.440 10.319 90.042 40.419 40.074 50.185 00.023 30.624 50.056 72.523 00.187 50.335 40.045 40.369 30.147 40.159 40.027 80.599 40.047 82.522 30.241 60.333 20.038 10.403 20.077 40.163 00.018 00.594 90.085 2指标/%空白对照组手参低剂量组手参中剂量组手参高剂量组第4期何乌云嘎等：蒙药手参的长期毒性研究371内蒙古民族大学学报2023年肾上腺胸腺睾丸子宫卵巢附睾脑0.031 20.015 40.101 00.030 40.653 90.092 90.327 70.060 60.085 30.009 70.301 90.060 00.443 70.162 80.026 20.011 90.075 10.023 90.605 80.132 90.337 40.071 10.064 40.012 90.315 40.102 70.371 40.148 70.028 10.012 40.084 60.761 50.768 30.050 40.257 00.052 60.078 80.005 70.400 30.078 50.455 50.172 80.031 10.013 00.077 60.036 00.786 10.080 00.380 80.079 00.085 70.009 30.372 60.065 00.512 90.118 2指标/%空白对照组手参低剂量组手参中剂量组手参高剂量组注：给药组与空白对照组比较，均为P0.05。表9 给药恢复期大鼠脏器指数指标（x s，n=10）Tab.9 Indicators of organ indexes in rats at recovery stage of drug administration（x s，n=10）指标/%肝脏心脏肺脏脾脏肾脏肾上腺胸腺睾丸子宫卵巢附睾脑空白对照组2.703 91.730 00.348 80.367 40.395 51.351 00.152 50.152 50.609 80.609 80.023 10.023 10.086 30.077 70.602 30.333 80.290 50.306 40.080 00.224 40.314 80.342 50.454 20.484 6手参低剂量组2.532 31.570 00.291 80.330 40.392 71.317 00.145 10.130 60.597 30.597 40.020 30.020 30.083 80.083 90.653 50.480 90.294 80.207 00.068 20.203 20.315 00.316 80.446 20.473 5手参中剂量组2.532 31.735 00.313 80.358 20.426 71.431 00.164 00.164 10.606 00.606 10.021 80.021 80.112 90.113 00.713 70.564 20.228 60.162 40.081 60.307 90.411