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苏克雷1 朱方石3 王晓娜2
（1 南京中医药大学, 江苏南京，210028；2.江苏省中医药研究院，江苏南京，210028；3 郑州市第三人民医院消化科, 河南郑州，450000）
摘要 目的：观察胃萎Ⅰ号颗粒对急性胃黏膜损伤大鼠的胃黏膜保护作用。方法：将SD大鼠随机分为正常
组、模型对照组、胃萎Ⅰ号组和替普瑞酮组，采用60%乙醇灌胃制备急性胃黏膜损伤大鼠模型，观察胃萎
Ⅰ号颗粒对大鼠胃黏膜损伤的影响，并测定血清和胃黏膜组织中PGE2、EGF、SS的含量。结果：胃萎Ⅰ号
组和替普瑞酮组均能明显改善黏膜损伤状况，并能显著提高血清和胃组织中PGE2、EGF、SS的含量，较模
型对照组均有统计学意义（P<0.01）；且胃萎Ⅰ号组对EGF、SS含量的提高优于替普瑞酮组（P<0.05）。结
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
•287•
实 验 研 究
Experimental study
论：胃萎Ⅰ号颗粒具有保护胃黏膜的作用，其机理可能与促进EGF、SS、PGE2等胃黏膜保护因子的生长有
关。
关键词 胃萎Ⅰ号颗粒；胃黏膜损伤；表皮生长因子；生长抑素；前列腺素E2
The Study on Protective Effect of Weiwei No.1 Granule on Gastric Mucosa in Acute Gastric Mucosal
Lesion Model Rats
Su Kelei1 Wang Xiaona2 Zhu Fangshi3
(1 First Clinical College of Nanjing University of TCM, Nanjing, Jiangsu, 210028;2 Jiangsu Province Academy
of Traditional Chinese Medicine, Nanjing, Jiangsu, 210028;3 Department of Gastroenterology of Third People's
Hospital of Zhengzhou, Zhengzhou Henan, 450000)
Abstract Objective: To observe the protective effect of Weiwei No.1 Granule on gastric mucosa in acute gastric
mucosal lesion rats. Methods: SD rats were randomly divided into four groups: normal group, model group,
Weiwei No.1 group and teprenone group. Acute gastric mucosal lesion in rat model was prepared with 60%
ethanol by gavage. Effects of gastric mucosa damage and the content of EGF, SS, and PGE2 in serum and gastric
tissues were observed. Results: Compared to the model group, both Weiwei No.1 treatment group and western
medicine treatment group could improve gastric mucosa, and significantly improve the content of EGF, SS, and
PGE2 in serum and gastric tissues, showing significant difference (P<0.01). And Weiwei No.1 group improve the
content of EGF and SS better than Teprenone group (P<0.05). Conclusion: Weiwei No.1 Granule can protect
gastric mucosa, and its mechanism may be related to the influence on levels of EGF, SS, and PGE2.
Key words Weiwei No.1 Granule; Gastric Mucosa Lesion; EGF; SS; PGE2
胃黏膜具有损伤与自我修复的动态平衡机制，维护着胃正常的生理功能。当外源性胃黏膜损伤因素的
侵袭和刺激，损伤胃黏膜屏障机制，易导致使胃黏膜炎症的发生，久之则有可能向胃黏膜腺体萎缩发展过
度[1]。具有健脾益气作用的“胃萎Ⅰ号”复方是我们临床治疗慢性萎缩性胃炎常用的方剂之一，我们在实施
国家“十一五”科技支撑计划“慢性萎缩性临床治疗方案的优化研究”过程中发现，在对慢性萎缩性胃炎获得
治疗作用的同时，尝试用于急性胃黏膜损伤的患者亦取得了较好的疗效。为探究该方对急性胃黏膜损伤的
保护作用及其机制，我们通过复制急性胃损伤大鼠模型，采用胃萎Ⅰ号免煎颗粒进行干预，观察了解干预
前后大鼠胃黏膜组织病理形态学及血清、组织中PGE2、EGF、SS 的含量变化，并阐述其作用机制。现将
结果报道如下。
1 材料与方法
1.1 材料
1.1.1 实验动物 健康成年清洁级SD 大鼠30 只，雌雄各半，体重(200±20)g，购自上海西普尔-必凯实
验动物有限公司。饲养于南京中医药大学附属中西医结
合医院SPF 级动物实验中心，空调下架式笼养，环境温度(23±3)℃，湿度(55±5)%，每天自由饮水（清
洁自来水）及进食标准饲料。
1.1.2 实验药品 1）胃萎Ⅰ号颗粒由江苏天江药业生产，组成：党参15g，炒白术10g，法半夏6g，
茯苓12g，陈皮6g，广木香6g，炙甘草3g。根据人鼠等效计量换算法，用蒸馏水配成为浓度为806.8mg/mL
的溶液装瓶，置 4℃冰箱备用。2）替普瑞酮：50mg/片，卫材（中国）药业有限公司生产，批号：国药准
字H20093656。研成细末后，根据人鼠等效计量换算法，用蒸馏水配成浓度为1.6mg/mL 的混悬液装瓶，
置 4℃冰箱备用。
1.1.3 主要试剂与仪器 大鼠表皮生长因子酶联免疫试剂盒、生长抑素酶联免疫试剂盒、前列腺素酶
联免疫试剂盒，上海益峰生物有限公司；无水乙醇，南京化学试剂有限公司；苏木精染液，江苏省中医药
研究院分子实验室；二甲苯，杭州中国巨化集团公司。CKX31-12PHP 型倒置生物显微镜，日本Olympus 公
司；酶标仪，法国Thermo Labsystems 公司；L600 型离心机，湖南湘仪集团；常规病理切片机和图像分析
系统，德国LEICA 公司；多聚赖氨酸玻片，福州迈新生物技术开发公司。
1.2 方法
1.2.1 造模方法 预试验：随机抽取6只大鼠，雌雄各半，分为3组，每组2只。造模前24h禁食不禁水，
分别以75%乙醇、60%乙醇、50%乙醇给大鼠灌胃，2mL/只，1h后处死，剖腹暴露全胃。结果发现以75%
乙醇造模后大鼠胃黏膜广泛糜烂，大片溃疡形成，并伴局灶出血，损伤较重；60%乙醇造模后黏膜充血水
肿明显，大片糜烂，浅小溃疡形成；50%乙醇黏膜仅有轻度充血水肿，未见糜烂。按预试验结果选用60%
乙醇建立急性胃黏膜损伤模型。
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
•28 8•
实 验 研 究
Experimental study
1.2.2 分组及给药方法 取6只正常SD大鼠设为正常对照组，正常饲养；取模型SD大鼠18只随机分为3
组，每组6只，分别为模型组（生理盐水0.1mL/kg•d灌胃）；胃萎Ⅰ号组（胃萎Ⅰ号颗粒混悬液0.1mL/ kg•d
灌胃）；替普瑞酮组（替普瑞酮混悬液0.1mL/ kg•d灌胃），所用干预药物剂量按人的等效量换算。干预3天
后处死取标本。
1.2.3 标本采集与制作法 1）血清标本：将各组大鼠眼球摘除，取外周血7.5mL，分别装入3支注有标
记的试管中，作为血清PGE2、EGF、SS的检测标本。2）胃组织标本：用颈椎脱臼处死法处死全部大鼠，
剖开腹部，暴漏全胃，在距贲门和幽门1.5cm处离断，取出全胃，沿胃大弯剪开、展平，观察胃黏膜的大
体情况。剪取胃窦部相同大小的组织，立即用10%中性甲醛溶液固定，作为病理学的检测标本。剩余胃组
织用滤纸擦干，用电子天平称取0.2g左右，加9倍0.9%冰生理盐水后移于离心管中，用眼科小手术剪尽快剪
碎组织块，再用匀浆器制备匀浆，分别作为组织PGE2、EGF、SS的检测标本。
1.2.4 血清及组织中PGE2、EGF、SS的含量测定（ELISA法）1）设标准孔10孔，对其进行梯度稀释。
2）外周血2.5mL，离心15min，3000r/min，取10μL血清，分别加入包被好抗原的96孔酶标板的板孔中。3）
匀浆组织2.5mL，离心15min，3000r/min，取匀浆上清液10μL，分别加入另一板包被好抗原的96孔酶标板
的板孔中。4）37℃温育30min。5）弃除混合物，洗涤液洗涤5次，甩尽板内液体，用吸水纸拍干。6）加
入酶标试剂50μL，轻轻晃动混匀，37℃温育30min。7）洗涤5次，加入显色剂A50μL，显色剂B50μL，混
匀，37℃避光显色15min。8）加入50μL终止液，终止反应。9）在酶标仪450nm波长上，测OD值，计算血
清及胃组织中PGE2、EGF、SS的含量。
1.2.5 胃黏膜损伤判定 剖腹取胃，沿胃大弯剪开胃并将胃展平，可见局限于腺胃部黏膜的点状或条
索状出血性病灶，按Guth标准改良评分：其中点状病灶及病灶长度≤1mm为1分，病灶长度≤2mm为2分，病
灶长度≤3mm为3分，病灶长度≤4mm为4分，若病灶长度≤5mm为5分，若病灶长度≤6mm为6分，若病灶长
度＞6mm为7分，若有穿孔为8分。若伴腺胃部充血，则累计加分如下：1）腺胃部充血面积＜1/3，加1分；
2）腺胃部充血面积1/3～2/3，加2分；3）腺胃部充血面积＞2/3，加3分。最后以全胃病灶分数总和作为该
动物胃黏膜损伤指数。
1.2.6 病理学方法 1）将胃标本取出，酒精脱水，二甲苯将组织透明。2）石蜡包埋，每份蜡块制成
厚度5μm的石蜡切片。3）行HE染色，脱蜡，洗蜡，漂洗，染色，脱水，二甲苯透明，中性树胶封片。4）
光镜下观察胃粘膜组织病理学变化。
1.2.6 统计学方法 采用SPSS17.0统计分析软件进行数据处理。
2 结果
2.1 胃黏膜病理学形态的影响 正常对照组：大鼠胃黏膜形态结构完整，腺体排列规则，未见腺体变
形和坏死等病理改变，无明显炎细胞浸润。模型对照组：大鼠胃黏膜上皮细胞受损、脱落，部分黏膜中断，
腺体结构紊乱，期间有大量红细胞聚集，黏膜下层严重水肿，血管充血，形成多个大小深浅不等的糜烂面，
并有较多炎性细胞浸润，黏膜厚度均较空白组明显变薄，部分标本可见腺管发生细胞变性坏死，细胞核缺
失。胃萎Ⅰ号组：大鼠胃黏膜上皮结构已逐渐恢复正常，黏膜水肿明显减轻，腺管排列整齐，腺体细胞形
态正常，少量炎症细胞浸润。替普瑞酮组：较胃萎Ⅰ号颗粒组恢复差。
图1：空白对照组 图2：模型对照组
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
•289•
实 验 研 究
Experimental study
图3：胃萎Ⅰ号颗粒组 图4：替普瑞酮组
2.2 胃黏膜损伤指数比较
表1 各组大鼠胃黏膜损伤指数（UI）计分的比较（ x ±s）
组别 动物数（n） 胃黏膜损伤指数（UI）
正常对照组 6 -
模型对照组 6 7.99±1.15
胃萎Ⅰ号组 6 2.09±0.84*△
替普瑞酮组 6 3.35±0.84*
注：与模型对照组比较*P<0.01，与替普瑞酮组比较△P<0.05
表1 显示，胃萎Ⅰ号组和替普瑞酮组胃黏膜损伤指数较模型组均较模型组明显降低（P<0.01），且胃
萎Ⅰ号组又低于替普瑞酮组（P<0.05）。
2.3 血清PGE2、EGF、SS 含量的比较
表2 各组大鼠血清PGE2、EGF、SS含量的比较（ng/L x ±s）
组别 动物数 PGE2 EGF SS
正常对照组 6 822.27±224.48* 4799.63±380.59** 616.62±84.51**
模型对照组 6 581.44±70.56 3048.52±147.52 345.53±57.94
胃萎Ⅰ号组 6 918.84±101.77**△ 4990.09±753.65**△△ 785.89±51.60**△△
替普瑞酮组 6 1059.36±103.45** 3382.04±753.65 385.41±29.12
注：与模型组比较*P<0.05，** P<0.01；与替普瑞酮组比较△P<0.05，△△P<0.01。
表2 显示，模型组血清PGE2、EGF、SS 较正常组均明显降低（P<0.05 和P<0.01）；胃萎Ⅰ号组治疗
后各指标较模型组显著升高（P<0.01），且其中EGF、SS 含量的升高程度优于替普瑞酮组（P<0.01）；但
PGE2 含量低于替普瑞酮组（P<0.05）。
2.4 组织PGE2、EGF、SS 含量的比较
表3 各组大鼠组织PGE2、EGF、SS含量的比较（ng/L x ±s）
组别 动物数 PGE2 EGF SS
正常对照组 6 418.97±21.67** 2956.85±246.48** 230.13±32.75**
模型对照组 6 349.77±17.39 1472.50±209.92 143.12±15.74
胃萎Ⅰ号组 6 451.46±25.19**△△ 2376.39±263.81*△ 237.91±38.22**△△
替普瑞酮组 6 503.02±21.25** 2016.30±182.85** 155.35±25.66
注：与模型组比较*P<0.05，** P<0.01；与替普瑞酮组比较△P<0.05，△△P<0.01。
表3 显示：模型组血清PGE2、EGF、SS 较正常组均明显降低（P<0.01）；胃萎Ⅰ号组治疗后各指标显
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
•29 0•
实 验 研 究
Experimental study
著高于模型组（P<0.05 和P<0.01）；其中EGF、SS 含量高于替普瑞酮组（P<0.05，P<0.01）。但PGE2 含量
低于替普瑞酮组（P<0.01）。
3 讨论
胃黏膜的损伤常与酸、碱、乙醇、胆盐和药物的刺激及HP 感染相关[2]，而胃黏膜损伤是导致急慢性
胃炎、胃溃疡、甚至胃癌前病变等多种消化系统疾病的重要病理环节[3]。因此，抗胃黏膜损伤和修复损伤
的胃黏膜是防治常见上消化道疾病的重要举措之一。替普瑞酮属萜烯类衍生物，替普瑞酮已被广泛用于治
疗各种急、慢性胃炎和严重胃黏膜改变疾病，替普瑞酮能促进胃黏膜微粒体中糖质中间体合成，提高黏液
磷脂质浓度，增强黏膜防御能力，强化抗溃疡作用；并可以增加胃黏膜的合成和分泌[4]。本研究显示，胃
萎Ⅰ号颗粒对急性胃黏膜的保护作用与替普瑞酮相当，且在降低胃黏膜损伤指数和改善和调节血清及组织
中PGE2、EGF、SS 含量一定程度上优于替普瑞酮组。本研究显示了从中医药领域寻求胃黏膜保护剂的可
能前景。
胃萎Ⅰ号组方由“香砂六君子汤”加减化裁而成，该方作为“十一五”国家科技支撑计划“慢性萎缩性胃炎
临床治疗方案的优化研究”课题中“脾胃虚弱证”的一组治疗方药，对脾胃气虚型患者疗效肯定。有研究表明，
脾虚与胃粘膜防御机制存在着相关，健脾益气有助于胃粘膜损伤的修复[5-6]。此外，有研究表明，本组方中
“四君子”中主要有效成分能通过增加胃肠循环血量，增加黏液，重建胃肠细胞和亚细胞结构，促进胃肠细
胞更新[7]；而半夏水煎醇沉液能具有保护胃黏膜, 促进胃黏膜的修复的作用[8]；另陈皮水煎剂可促进大鼠正
常胃液的分泌[9]。我们认为，胃萎Ⅰ号方对胃黏膜损伤的保护作用在健脾益气传统功效的基础上，与该方
药物组成所含成分的药理机制有关。
前列腺素（PGE2）是胃黏膜极为重要的防御因子，具有防止各种有害物质对消化道上皮细胞损伤和致
死作用[10]；表皮生长因子（EGF）是一种生长因子类蛋白质，通过与胃肠黏膜细胞上的-表皮生长因子受体
（EGFR）结合，能通过增加胃黏膜血流量，促进黏膜细胞DNA、RNA及蛋白质的合成，增加胃黏膜黏液
糖蛋白的合成与分泌，而发挥胃黏膜的保护作用[11]；生长抑素（SS）是一种调节性抑制肽，能帮助黏膜清
除氧自由基及脂质过氧化物，增加细胞内还原型谷耽甘肤含量, 减轻细胞脂质过氧化, 参与胃黏膜的局部
防御机制[12]。本研究显示，胃萎Ⅰ号颗粒可以提高胃黏膜损伤大鼠血清及组织中PGE2、EGF、SS含量，我
们推测，胃萎Ⅰ号颗粒对胃黏膜损伤的保护作用可能与促进PGE2、EGF、SS等胃黏膜保护因子的合成、加
速胃黏膜自身修复的机制有关，但有待于进一步深入研究。
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[3] 徐婷婷, 朱方石. 外源性胃黏膜损伤因素与脾虚相关性的研究进展[J]. 世界华人消化杂志 2010，18(28): 2981-2984.
[4] 沈爱玲，尚游，袁世荧，等. 替普瑞酮对组织器官的保护作用[J]. 国际麻学与复苏杂志. 2010，31（1）：80-82.
[5] 王晓娜,朱方石.脾虚与胃黏膜防御机制相关性的研究进展及评价[J].辽宁中医杂志,2010,37(6):1176-1179.
[6] 杨晓军,吕东勇,朱焕金,等.健脾益气方药对大鼠急性胃粘膜损伤的保护与修复作用[J].河南中医,2010,30(4):351-353.
[7] 封吉化，尚虎虎. 四君子汤对脾虚证大鼠胃肠电活动的影响[J]．武警医学院学报，2010.19（1）：8-9，56.
[8] 刘守义，尤春来. 半夏抗溃疡作用机理的实验研究[J]．辽宁中医杂志，1992，10：42.
[9] 张志海，王彩云，杨天鸣，等. 陈皮的化学成分及药理作用研究进展[J]．西北药学杂志，2005，20（1）：471.
[10] Martin GR，JL. Gastrointestinal inflammation：a central component of mucosal defense and repair [J]. Exp Biol Med，2006，231:130-137.
[11] 姒健敏. 关注生长因子对胃粘膜的保护作用[J]．中华医学杂志，2005，85(39)：2744-2745.
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第九届世界中医药大会
The 9th World Congress of Chinese Medicine
•291•
实 验 研 究
Experimental study
Comparative pharmacokinetics study of three anthraquinones in rat plasma
after oral administration of Radix et Rhei Rhizoma extract and Dahuang Fuzi
Tang by high performance liquid chromatography-mass spectrometry
Xiao Liu1,2 Huan Li1,2,3 Hui Guo1,2,3 Li Wu1,2
Hao Cai1,2, Weike Zhang1 Baochang Cai1,2,3
（1 College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210046, PR China; 2 Engineering Center of State Ministry of Education for
Standardization of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing, 210029, PR China; 3 Nanjing Haichang Chinese Medicine
Group Corporation, Nanjing, 210061, PR China）
Keywords pharmacokinetics; rhein; aloe-emodin; emodin
摘要 目的：研究灌胃给予大鼠大黄附子汤及大黄提取物后血浆中三种蒽醌类成分的药代动力学特征。方
法：采用LC-MS 方法，色谱柱：Kromasil C18(150×4.6 mm, 5 μm，带保护柱)；流动相：甲醇-3 mM 乙酸铵
缓冲液(75:25, v/v)线性梯度洗脱；柱温40 ℃。流速：1.0 mL/min，检测波长:280 nm。结果：血浆中大黄酸
在24-12,000 ng/mL；芦荟大黄素在23.2-11,600 ng/mL；大黄素在0.5-250 ng/mL 浓度范围内呈现良好的线
性关系。灌胃给予大鼠大黄附子汤和大黄提取物后，大黄酸和大黄素的AUC0-t, AUC0-∞；大黄酸的Cmax，
大黄酸、芦荟大黄素、大黄素的Tmax 有显著性差异。
Abstract A specific and sensitive high performance liquid chromatography-mass spectrometric (HPLC-MS)
method has been developed and validated for simultaneous determination of three anthraquinones of rhein (CAS:
478-43-3), aloe-emodin (CAS: 481-72-1) and emodin (CAS: 518-82-1) in rat plasma after oral administration of
Radix et Rhei Rhizoma extract and Dahuang Fuzi Tang. The analytes were separated on a Kromaisl ® C18 column
within a total running time of 12 min with a mobile phase of methanol : ammonium acetate(3 mM) (75:25, v/v).
The calibration curves for all the anthraquinones showed good linearity in the measured range with correlation
coefficient (r) higher than 0.9978. The precision, accuracy, recovery and stability were deemed acceptable. The
method was successfully applied to the comparative pharmacokinetics study of the anthraquinones in rat plasma
after oral administration of Radix et Rhei Rhizoma extract and Dahuang Fuzi Tang
1 Introduction
Dahuang Fuzi Tang (DFT), composed of three herbs including Radix et Rhizoma Rhei (DH), Radix Aconiti
Lateralis Praeparata (FZ) and Radix et Rhizoma Asari (XX), was originally described in Synopsis of Golden
Chamber (Jin Kui Yao Lue), a treatise on febrile and miscellaneous diseases written by the outstanding physician
Zhang Zhongjing in Han Dynasty. It is widely used to treat appendicitis, biliary colic, chronic dysentery, acute
ileus and adhesive ileus in clinic [1]. Numerous studies reported that rhein, aloe-emodin and emodin were the main
effective components of DH (the monarch herb), which exhibited beneficial pharmacological activity [2-5].
Quantitative analyses of the three anthraquinones in vivo for pharmacokinetic application have been documented
using either High performance liquid chromatography (HPLC-UV) or high performance liquid
chromatography-tandem mass spectrometry (HPLC-MS/MS) methods. Analysis time of HPLC-UV method,
however, was tedious (27 min-30 min) and aloe-emodin as well as emodin could not be detected, whilst
determination of rhein was failed after 12 h, and HPLC-MS/MS method only intended for aloe-emodin or emodin
alone [6-10]. Therefore, this present study developed a simple, rapid and reliable HPLC-MS assay for the
simultaneous determination of rhein, aloe-emodin, emodin in rat plasma to compare their pharmacokinetic
profiles after oral administration of DH extract and DFT.
2 Experimental Procedure
2.1 Reagents and materials FZ, XX and DH were obtained from Nanjing Haichang Chinese medicine group
corporation (NO.12 Yongjing Road, Hi-Tech Industrial Development Zone Nanjing, Jiangsu Province, P. R.
China). 1, 8-Dihydroxyanthraquinone (IS), rhein, aloe-emodin and emodin with purity of 99% or higher were
purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China).
The structures of these four compounds are shown in Fig. 1. HPLC-grade methanol was purchased from E. Merck
(Merck, Darmstadt, Germany). Purified water was from a Milli-Q system (Millipore Corporation, Bedford,
Massachusetts, France). Acetate, ascorbic acid, hydrochloric acid (HCl) and ethyl acetate were of analytical grade
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
•29 2•
实 验 研 究
Experimental study
and obtained from Nanjing Chemical Reagent Company (Nanjing, China). .
2.2 Instrumentation and operating conditions The HPLC-MS system consisted of a Shimadzu LC-10AD HPLC
series liquid chromatograph and a Shimadzu HPLC/MS-2010A single quadrupole mass spectrometer equipped
with electrospray ionization (ESI) inte***ce. HPLC separation was carried out on a Kromasil ® C18 (150 × 4.6 mm,
5 μm, Akzo Nobel, Sweden) with a Kromasil guard column maintained at 40 ℃. The mobile phase consisted of
methanol - ammonium acetate (3 mM) (75:25, v/v) with isocratic elution. Mass spectrometer conditions were
optimized to obtain maximal sensitivity as follows: drying gas 8 L/min, CDL temperature 250 ℃, block
temperature 200 ℃ and detector voltage 1.3 kV. Analytes were quantitated in selected ion monitoring (SIM) mode
at negative ion mode. The [M-H]- ions of 282.80 for rhein, 268.85 for aloe-emodin, 268.85 for emodin and 239.95
for IS were selected as detecting ions.
2.3 Sample preparation To prepare DFT, pieces of DH, FZ and XX were mixed together in a ration (3:4:1,
w/w/w) and macerated in deionized water for 30 min, and then decocted twice with boiling water (1:8, w/v) each
for 20 min, then the solution was filtered through a two-layer mesh, and was combined and concentrated to a
density of 0.96 g/mL under vacuum at 60 ℃. DH extract was prepared in the same way but condensed to a density
of 0.36 g/mL.
2.4 Application A total of 12 rats were randomly divided into two groups. Each group contains 6 rats and was
orally administered a different extract by gavage with a syringe: DFT (1.44 g/100g body weight) and DH extract
(0.54 g/100g body weight), respectively. Blood samples (about 0.5 mL) were collected from the orbital vein at
pre-administration (time = 0) and post-administration (time = 0.033, 0.083, 0.167, 0.250, 0.5, 0.75, 1, 1.5, 2, 4, 6,
12, 24 h). The plasma was separated by centrifugation at 12,000 rpm for 3 min, and 0.1 mL was spiked with 10 μL
of IS and then mixed with 50 μL of pH 5 acetate buffer and 50 μL of ascorbic acid (100 mg/mL) by vortex mixing
for 30 s. The mixture was acidified with 50 μL of 0.1 N HCl by vortex mixing for 30 s and partitioned with 1 mL
of ethyl acetate by vortex mixing for 3 min. The aqueous and organic layers were separated by centrifugation at
4,000 rpm for 3 min and the organic layer was transferred to another tube and evaporated to dryness under
nitrogen at 50 ℃. The residue was reconstituted with 100 μL of mobile phase and vortexed for 30 s and
centrifuged at 12, 000 rpm for 3 min. A volume of 20 μL of the supernatant was injected for analysis. The plasma
concentration versus time for rhein, aloe-emodin and emodin were analyzed with a non-compartmental method
using the WinNonlin software (ver. 5.2; Pharsight, Mountain View, CA, USA).
2.5 Method validation The selectivity of the method was evaluated by comparing the chromatograms of six
blank rat plasma samples with that of six rhein, aloe-emodin, emodin and IS spiked rat plasma samples. Stock
standard solution of analytes were prepared in methanol and calibration standard samples were prepared through
spiking blank plasma (80 μL) with this stock solution (20 μL) to obtain final concentrations of 24-2,000 ng/mL for
rhein, 23.2-11,600 ng/mL for aloe-emodin, and 0.5-250 ng/mL for emodin. Standard curves were fitted by least
square regression using logarithm as weighting factor of the peak area ratio of the analytes to IS (log Y) versus
plasma concentrations(log X). The lower limits of quantitation (LLOQ) served as the lowest concentration on the
standard curve.The accuracy and precision of the established method were evaluated by QC samples at low,
medium and high concentrations. Accuracy was defined as the relative deviation in the calculated value of a
standard from that of its true value, expressed as relative error(RE). Precision was evaluated as the relative
standard deviation (RSD). The intra-day accuracy was determined by assaying five replicates at each
concentration level on 1 day, and inter-day accuracy was determined by analyzing QC samples in five duplicates
during three separate and successive days. QC samples were used to evaluate the stability of the analytes in rat
plasma under different storage conditions: long-term stability at -20 ℃ for 14 days, post-preparative stability at
room temperature for 24 h, and after three freeze-thaw cycles.
3 Result and discussion
3.1 Method validation Chromatograms (Fig. 2-4) of blank samples showed no endogenous peaks interfering
with any of the analytes or internal standard. The LLOQ was 24 ng/mL for rhein, 23.2 ng/mL for aloe-emodin and
0.5 ng/mL for emodin. The calibration curves showed good linearity within the range of 24-12,000 ng/mL for
rhein, 23.2-11,600 ng/mL and 0.5-250 ng/mL for aloe-emodin and emodin respectively. The results of precision
and accuracy were all within ±15%. The recoveries of rhein, aloe-emodin and emodin were 87.8-89.6 %,
90.2-96.7% and 87.9-89.7% respectively.The mean extraction recovery of IS was 96.2%. Here, the matrix effect
was within the range of 97.2-100.2%, indicating that no significant matrix effect was observed for rhein,
aloe-emodin and emodin. The mean matrix effect of the IS was 100.1%. The concentration of analytes deviated
less than ±15% from their nominal concentrations after three-thaw cycles, 24 h at room temperature and 14 days
at -20 ℃storage, showing that the samples were stable during preparation and analytical processes.
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
•293•
实 验 研 究
Experimental study
3.4 Pharmacokinetics studies The validated method was successfully applied to the comparative
pharmacokinetic studies of rhein, aloe-emodin and emodin in rat plasma after administration of DH extract and
DFT. Fig.5 showed the concentration-time curves; and the main pharmacokinetic parameters summarized in Table
1 showed that AUC0-t, AUC0-∞, Cmax , MRT, CL and Tmax of DH extract group statistically differed from those of
DFT group remarkably. After oral administration of DFT, AUC0-t, AUC0-∞ of rhein and emodin and the Cmax, of
rhein decreased significantly (P<0.01), while MRT of rhein increased significantly (P<0.01), as compared with
the values of the DH extract group. On the other hand, peak time of the three anthraquinones in DFT group was
significantly delayed compared to the DH extract group (P<0.05). However, there were no significant difference
in the T1/2 values of the three anthraquinones between DH extract and DFT decoction group. Meanwhile, AUC0-t,
AUC0-∞ and Cmax of aloe-emodin in DFT group also showed no difference in comparison with the DH extract
group. But the MRT of aloe-emodin increased significantly(P<0.05) and CL decrease remarkably (P<0.05) in
DFT group. The results indicated that oral administration of DFT decoction could lead to less absorption of rhein
and emodin with peak time detention of rhein, aloe-rhein and emodin. Our previous study has found that the
content of rhein and emodin, the acidic components in DH, decrease half approximately in DFT compared with
that in DH, which may be resulted from the action of alkaloid in FZ, giving a great contribution to the absorption
decrease of rhein and emodin in DFT, in order that the bitter and cold medicinal properties of DH was restricted
by FZ, which is in conformity with the traditional Chinese medicine theory of antagonistic action. On the other
hand, the combination rationality of DFT was not only based on the antagonistic action simply but on the
detention of peak time of rhein, aloe-empdin and emodin, leading to the restraint of strong and quick effect of DH
owing to the herb ingredient interaction of DFT in vivo.
Acknowledgements
This research was supported by the National Natural Science Foundation of China (No. 81073022)
References
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of hormone-dependent and -independent cancer cells under normoxic or hypoxic conditions in vitro, Chem-Biol Interact. 192 (2011) : 220-232.
[3] Q. Guo, Y. Chen, B. Zhang, M. Kang, Q. Xie, Y. Wu, Potentiation of the effect of gemcitabine by emodin in pancreatic cancer is associated with survivin
inhibition, Biochem. Pharmacol. 77 (2009) : 1674-1683.
[4] S. K. Heo, H. J. Yun, E. K. Noh, S. D. Park, Emodin and rhein inhibit LIGHT-induced monocytes migration by blocking of ROS production, Vasc.
Pharmacolo. 53 (2010) : 28-37.
[5] C. Tabolacci, A. Lentini, P. Mattioli, B. Provenzano, S. Oliverio, F. Carlomosti, S. Beninati, Antitumor properties of aloe-emodin and induction of
transglutaminase 2 activity in B16-F10 melanoma cells, Life.Sci. 87 (2010) : 316-324.
[6] J. Y. Jiang, M. W. Yang, W. Qian, H. Lin, Y. Geng, Z.Q. Zhou, D. W. Xiao, Quantitative determination of rhein in human plasma by liquid
chromatography-negative electrospray ionization tandem mass/mass spectrometry and the application in a pharmacokinetic study, J Pharm Biomed Anal. 57
(2012) : 19-25.
[7] Y. Li, J. Duan, T. Guo, W. Xie, S. Yan, B. Li, Y. Zhou, Y. Chen, In vivo pharmacokinetics comparisons of icariin, emodin and psoralen from gan-kang
granules and extracts of herba Epimedii, Nepal dock root, Ficus hirta yahl, J. Ethnopharmaco. 124 (2009) : 522-529.
[8] W. Liu, Z. Zheng, X. Liu, S. Gao, L. Ye, Z. Yang, M. Hu, Z. Liu, Sensitive and robust UPLC–MS/MS method to determine the gender-dependent
pharmacokinetics in rats of emodin and its glucuronide, J Pharm Biomed Anal. 54 (2011) : 1157-1162.
[9] C. S. Shia, S. Y. Tsai, J.C. Lin, M. L. Li, M. H. Ko, P.D. Chao, Y.C. Huang, Y.C. Hou, Steady-state pharmacokinetics and tissue distribution of
anthraquinones of Rhei Rhizoma in rats, J. Ethnopharmaco. 137 (2011) : 1388-1394.
[10] C. S. Shia, S. H. Juang, S. Y. Tsai, P. H. Chang, S.-C. Kuo, Y. C. Hou, P. D. Chao, Metabolism and Pharmacokinetics of anthraquinones in Rheum
palmatumin Rats andex vivo antioxidant activity, Planta Med. 75 (2009): 1386-1392.
Table 1. The pharmacokinetic data for rhein, aloe-emodin and emodin after oral administration of DH extract and
DFT (mean±SD, n=6).
Data unit rhein aloe-emodin emodin DH DFT DH DFT DH DFT
AUC(0-t) ng h/mL 12333± 2709 3284±909** 1240±811 1559±315. 667±188 382±181**
AUC(0-∞) ng h/mL 15328±2823 3709±1126** 1506±984 2091±357 916±325 465±228**
Cmax ng/mL 10576±4388 1410±679** 235±67 183±65 192±52 101±53
T1/2 h 10.4±5.4 6.5±1.3 9.4±2.8 13.9±4.8 10.3±3.1 8.0±4.2
MRT(0-t) h 6.8±1.2 6.4±0.9 8.3±0.8 10.5±1.4* 8.9±1.7 7.8±1.8
CL mL kg/h 1.6±0.3 4.1±1.5** 2.9±1.1 1.5±0.2* 6.4±2.2 7.0±3.1
Tmax h 0.2±0.1 0.5±0.3* 1.0±0.7 3.4±1.3* 0.3±0.2 0.6±0.3*
注：*P<0.05, **P <0.01 compared with the DH extract group
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
•29 4•
实 验 研 究
Experimental study
Fig.1. Chemical structures and mass spectra of (A) rhein, (B) aloe-emodin, (C) emodin and (D) IS.
Fig.2. Selected ion monitoring chromatograms of (A) blank rat plasma sample, (B) blank plasma sample spiked
with rhein, (C) plasma
sample obtained from rats administrated by DH extract of rhein, (D) plasma sample obtained from rats
administrated by DFT of rhein.
Fig.3. Selected ion monitoring chromatograms of (A) blank rat plasma sample, (B) blank plasma sample spiked
with aloe-emodin and emodin, (C) plasma sample obtained from rats administrated by DH extract of aloe-emodin
and emodin, (D) plasma sample obtained from rats administrated by DFT of aloe-emodin and emodin.1:
aloe-emodin; 2: emodin
Fig.4. Selected ion monitoring chromatograms of (A) blank rat plasma sample, (B) blank plasma sample spiked
with IS, (C) plasma sample obtained from rats administrated by DH extract of IS , (D) plasma sample obtained
from rats administrated by DFT of IS.
from rats administrated by DFT of IS.
Fig.5. Plasma concentration-time curves of (A) rhein, (B) aloe-emodin and (C) emodin in rat plasma after oral
administration of DH extract and DFT.
（1 南京中医药大学, 江苏南京，210028；2.江苏省中医药研究院，江苏南京，210028；3 郑州市第三人民医院消化科, 河南郑州，450000）
摘要 目的：观察胃萎Ⅰ号颗粒对急性胃黏膜损伤大鼠的胃黏膜保护作用。方法：将SD大鼠随机分为正常
组、模型对照组、胃萎Ⅰ号组和替普瑞酮组，采用60%乙醇灌胃制备急性胃黏膜损伤大鼠模型，观察胃萎
Ⅰ号颗粒对大鼠胃黏膜损伤的影响，并测定血清和胃黏膜组织中PGE2、EGF、SS的含量。结果：胃萎Ⅰ号
组和替普瑞酮组均能明显改善黏膜损伤状况，并能显著提高血清和胃组织中PGE2、EGF、SS的含量，较模
型对照组均有统计学意义（P<0.01）；且胃萎Ⅰ号组对EGF、SS含量的提高优于替普瑞酮组（P<0.05）。结
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
•287•
实 验 研 究
Experimental study
论：胃萎Ⅰ号颗粒具有保护胃黏膜的作用，其机理可能与促进EGF、SS、PGE2等胃黏膜保护因子的生长有
关。
关键词 胃萎Ⅰ号颗粒；胃黏膜损伤；表皮生长因子；生长抑素；前列腺素E2
The Study on Protective Effect of Weiwei No.1 Granule on Gastric Mucosa in Acute Gastric Mucosal
Lesion Model Rats
Su Kelei1 Wang Xiaona2 Zhu Fangshi3
(1 First Clinical College of Nanjing University of TCM, Nanjing, Jiangsu, 210028;2 Jiangsu Province Academy
of Traditional Chinese Medicine, Nanjing, Jiangsu, 210028;3 Department of Gastroenterology of Third People's
Hospital of Zhengzhou, Zhengzhou Henan, 450000)
Abstract Objective: To observe the protective effect of Weiwei No.1 Granule on gastric mucosa in acute gastric
mucosal lesion rats. Methods: SD rats were randomly divided into four groups: normal group, model group,
Weiwei No.1 group and teprenone group. Acute gastric mucosal lesion in rat model was prepared with 60%
ethanol by gavage. Effects of gastric mucosa damage and the content of EGF, SS, and PGE2 in serum and gastric
tissues were observed. Results: Compared to the model group, both Weiwei No.1 treatment group and western
medicine treatment group could improve gastric mucosa, and significantly improve the content of EGF, SS, and
PGE2 in serum and gastric tissues, showing significant difference (P<0.01). And Weiwei No.1 group improve the
content of EGF and SS better than Teprenone group (P<0.05). Conclusion: Weiwei No.1 Granule can protect
gastric mucosa, and its mechanism may be related to the influence on levels of EGF, SS, and PGE2.
Key words Weiwei No.1 Granule; Gastric Mucosa Lesion; EGF; SS; PGE2
胃黏膜具有损伤与自我修复的动态平衡机制，维护着胃正常的生理功能。当外源性胃黏膜损伤因素的
侵袭和刺激，损伤胃黏膜屏障机制，易导致使胃黏膜炎症的发生，久之则有可能向胃黏膜腺体萎缩发展过
度[1]。具有健脾益气作用的“胃萎Ⅰ号”复方是我们临床治疗慢性萎缩性胃炎常用的方剂之一，我们在实施
国家“十一五”科技支撑计划“慢性萎缩性临床治疗方案的优化研究”过程中发现，在对慢性萎缩性胃炎获得
治疗作用的同时，尝试用于急性胃黏膜损伤的患者亦取得了较好的疗效。为探究该方对急性胃黏膜损伤的
保护作用及其机制，我们通过复制急性胃损伤大鼠模型，采用胃萎Ⅰ号免煎颗粒进行干预，观察了解干预
前后大鼠胃黏膜组织病理形态学及血清、组织中PGE2、EGF、SS 的含量变化，并阐述其作用机制。现将
结果报道如下。
1 材料与方法
1.1 材料
1.1.1 实验动物 健康成年清洁级SD 大鼠30 只，雌雄各半，体重(200±20)g，购自上海西普尔-必凯实
验动物有限公司。饲养于南京中医药大学附属中西医结
合医院SPF 级动物实验中心，空调下架式笼养，环境温度(23±3)℃，湿度(55±5)%，每天自由饮水（清
洁自来水）及进食标准饲料。
1.1.2 实验药品 1）胃萎Ⅰ号颗粒由江苏天江药业生产，组成：党参15g，炒白术10g，法半夏6g，
茯苓12g，陈皮6g，广木香6g，炙甘草3g。根据人鼠等效计量换算法，用蒸馏水配成为浓度为806.8mg/mL
的溶液装瓶，置 4℃冰箱备用。2）替普瑞酮：50mg/片，卫材（中国）药业有限公司生产，批号：国药准
字H20093656。研成细末后，根据人鼠等效计量换算法，用蒸馏水配成浓度为1.6mg/mL 的混悬液装瓶，
置 4℃冰箱备用。
1.1.3 主要试剂与仪器 大鼠表皮生长因子酶联免疫试剂盒、生长抑素酶联免疫试剂盒、前列腺素酶
联免疫试剂盒，上海益峰生物有限公司；无水乙醇，南京化学试剂有限公司；苏木精染液，江苏省中医药
研究院分子实验室；二甲苯，杭州中国巨化集团公司。CKX31-12PHP 型倒置生物显微镜，日本Olympus 公
司；酶标仪，法国Thermo Labsystems 公司；L600 型离心机，湖南湘仪集团；常规病理切片机和图像分析
系统，德国LEICA 公司；多聚赖氨酸玻片，福州迈新生物技术开发公司。
1.2 方法
1.2.1 造模方法 预试验：随机抽取6只大鼠，雌雄各半，分为3组，每组2只。造模前24h禁食不禁水，
分别以75%乙醇、60%乙醇、50%乙醇给大鼠灌胃，2mL/只，1h后处死，剖腹暴露全胃。结果发现以75%
乙醇造模后大鼠胃黏膜广泛糜烂，大片溃疡形成，并伴局灶出血，损伤较重；60%乙醇造模后黏膜充血水
肿明显，大片糜烂，浅小溃疡形成；50%乙醇黏膜仅有轻度充血水肿，未见糜烂。按预试验结果选用60%
乙醇建立急性胃黏膜损伤模型。
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Experimental study
1.2.2 分组及给药方法 取6只正常SD大鼠设为正常对照组，正常饲养；取模型SD大鼠18只随机分为3
组，每组6只，分别为模型组（生理盐水0.1mL/kg•d灌胃）；胃萎Ⅰ号组（胃萎Ⅰ号颗粒混悬液0.1mL/ kg•d
灌胃）；替普瑞酮组（替普瑞酮混悬液0.1mL/ kg•d灌胃），所用干预药物剂量按人的等效量换算。干预3天
后处死取标本。
1.2.3 标本采集与制作法 1）血清标本：将各组大鼠眼球摘除，取外周血7.5mL，分别装入3支注有标
记的试管中，作为血清PGE2、EGF、SS的检测标本。2）胃组织标本：用颈椎脱臼处死法处死全部大鼠，
剖开腹部，暴漏全胃，在距贲门和幽门1.5cm处离断，取出全胃，沿胃大弯剪开、展平，观察胃黏膜的大
体情况。剪取胃窦部相同大小的组织，立即用10%中性甲醛溶液固定，作为病理学的检测标本。剩余胃组
织用滤纸擦干，用电子天平称取0.2g左右，加9倍0.9%冰生理盐水后移于离心管中，用眼科小手术剪尽快剪
碎组织块，再用匀浆器制备匀浆，分别作为组织PGE2、EGF、SS的检测标本。
1.2.4 血清及组织中PGE2、EGF、SS的含量测定（ELISA法）1）设标准孔10孔，对其进行梯度稀释。
2）外周血2.5mL，离心15min，3000r/min，取10μL血清，分别加入包被好抗原的96孔酶标板的板孔中。3）
匀浆组织2.5mL，离心15min，3000r/min，取匀浆上清液10μL，分别加入另一板包被好抗原的96孔酶标板
的板孔中。4）37℃温育30min。5）弃除混合物，洗涤液洗涤5次，甩尽板内液体，用吸水纸拍干。6）加
入酶标试剂50μL，轻轻晃动混匀，37℃温育30min。7）洗涤5次，加入显色剂A50μL，显色剂B50μL，混
匀，37℃避光显色15min。8）加入50μL终止液，终止反应。9）在酶标仪450nm波长上，测OD值，计算血
清及胃组织中PGE2、EGF、SS的含量。
1.2.5 胃黏膜损伤判定 剖腹取胃，沿胃大弯剪开胃并将胃展平，可见局限于腺胃部黏膜的点状或条
索状出血性病灶，按Guth标准改良评分：其中点状病灶及病灶长度≤1mm为1分，病灶长度≤2mm为2分，病
灶长度≤3mm为3分，病灶长度≤4mm为4分，若病灶长度≤5mm为5分，若病灶长度≤6mm为6分，若病灶长
度＞6mm为7分，若有穿孔为8分。若伴腺胃部充血，则累计加分如下：1）腺胃部充血面积＜1/3，加1分；
2）腺胃部充血面积1/3～2/3，加2分；3）腺胃部充血面积＞2/3，加3分。最后以全胃病灶分数总和作为该
动物胃黏膜损伤指数。
1.2.6 病理学方法 1）将胃标本取出，酒精脱水，二甲苯将组织透明。2）石蜡包埋，每份蜡块制成
厚度5μm的石蜡切片。3）行HE染色，脱蜡，洗蜡，漂洗，染色，脱水，二甲苯透明，中性树胶封片。4）
光镜下观察胃粘膜组织病理学变化。
1.2.6 统计学方法 采用SPSS17.0统计分析软件进行数据处理。
2 结果
2.1 胃黏膜病理学形态的影响 正常对照组：大鼠胃黏膜形态结构完整，腺体排列规则，未见腺体变
形和坏死等病理改变，无明显炎细胞浸润。模型对照组：大鼠胃黏膜上皮细胞受损、脱落，部分黏膜中断，
腺体结构紊乱，期间有大量红细胞聚集，黏膜下层严重水肿，血管充血，形成多个大小深浅不等的糜烂面，
并有较多炎性细胞浸润，黏膜厚度均较空白组明显变薄，部分标本可见腺管发生细胞变性坏死，细胞核缺
失。胃萎Ⅰ号组：大鼠胃黏膜上皮结构已逐渐恢复正常，黏膜水肿明显减轻，腺管排列整齐，腺体细胞形
态正常，少量炎症细胞浸润。替普瑞酮组：较胃萎Ⅰ号颗粒组恢复差。
图1：空白对照组 图2：模型对照组
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Experimental study
图3：胃萎Ⅰ号颗粒组 图4：替普瑞酮组
2.2 胃黏膜损伤指数比较
表1 各组大鼠胃黏膜损伤指数（UI）计分的比较（ x ±s）
组别 动物数（n） 胃黏膜损伤指数（UI）
正常对照组 6 -
模型对照组 6 7.99±1.15
胃萎Ⅰ号组 6 2.09±0.84*△
替普瑞酮组 6 3.35±0.84*
注：与模型对照组比较*P<0.01，与替普瑞酮组比较△P<0.05
表1 显示，胃萎Ⅰ号组和替普瑞酮组胃黏膜损伤指数较模型组均较模型组明显降低（P<0.01），且胃
萎Ⅰ号组又低于替普瑞酮组（P<0.05）。
2.3 血清PGE2、EGF、SS 含量的比较
表2 各组大鼠血清PGE2、EGF、SS含量的比较（ng/L x ±s）
组别 动物数 PGE2 EGF SS
正常对照组 6 822.27±224.48* 4799.63±380.59** 616.62±84.51**
模型对照组 6 581.44±70.56 3048.52±147.52 345.53±57.94
胃萎Ⅰ号组 6 918.84±101.77**△ 4990.09±753.65**△△ 785.89±51.60**△△
替普瑞酮组 6 1059.36±103.45** 3382.04±753.65 385.41±29.12
注：与模型组比较*P<0.05，** P<0.01；与替普瑞酮组比较△P<0.05，△△P<0.01。
表2 显示，模型组血清PGE2、EGF、SS 较正常组均明显降低（P<0.05 和P<0.01）；胃萎Ⅰ号组治疗
后各指标较模型组显著升高（P<0.01），且其中EGF、SS 含量的升高程度优于替普瑞酮组（P<0.01）；但
PGE2 含量低于替普瑞酮组（P<0.05）。
2.4 组织PGE2、EGF、SS 含量的比较
表3 各组大鼠组织PGE2、EGF、SS含量的比较（ng/L x ±s）
组别 动物数 PGE2 EGF SS
正常对照组 6 418.97±21.67** 2956.85±246.48** 230.13±32.75**
模型对照组 6 349.77±17.39 1472.50±209.92 143.12±15.74
胃萎Ⅰ号组 6 451.46±25.19**△△ 2376.39±263.81*△ 237.91±38.22**△△
替普瑞酮组 6 503.02±21.25** 2016.30±182.85** 155.35±25.66
注：与模型组比较*P<0.05，** P<0.01；与替普瑞酮组比较△P<0.05，△△P<0.01。
表3 显示：模型组血清PGE2、EGF、SS 较正常组均明显降低（P<0.01）；胃萎Ⅰ号组治疗后各指标显
第九届世界中医药大会
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实 验 研 究
Experimental study
著高于模型组（P<0.05 和P<0.01）；其中EGF、SS 含量高于替普瑞酮组（P<0.05，P<0.01）。但PGE2 含量
低于替普瑞酮组（P<0.01）。
3 讨论
胃黏膜的损伤常与酸、碱、乙醇、胆盐和药物的刺激及HP 感染相关[2]，而胃黏膜损伤是导致急慢性
胃炎、胃溃疡、甚至胃癌前病变等多种消化系统疾病的重要病理环节[3]。因此，抗胃黏膜损伤和修复损伤
的胃黏膜是防治常见上消化道疾病的重要举措之一。替普瑞酮属萜烯类衍生物，替普瑞酮已被广泛用于治
疗各种急、慢性胃炎和严重胃黏膜改变疾病，替普瑞酮能促进胃黏膜微粒体中糖质中间体合成，提高黏液
磷脂质浓度，增强黏膜防御能力，强化抗溃疡作用；并可以增加胃黏膜的合成和分泌[4]。本研究显示，胃
萎Ⅰ号颗粒对急性胃黏膜的保护作用与替普瑞酮相当，且在降低胃黏膜损伤指数和改善和调节血清及组织
中PGE2、EGF、SS 含量一定程度上优于替普瑞酮组。本研究显示了从中医药领域寻求胃黏膜保护剂的可
能前景。
胃萎Ⅰ号组方由“香砂六君子汤”加减化裁而成，该方作为“十一五”国家科技支撑计划“慢性萎缩性胃炎
临床治疗方案的优化研究”课题中“脾胃虚弱证”的一组治疗方药，对脾胃气虚型患者疗效肯定。有研究表明，
脾虚与胃粘膜防御机制存在着相关，健脾益气有助于胃粘膜损伤的修复[5-6]。此外，有研究表明，本组方中
“四君子”中主要有效成分能通过增加胃肠循环血量，增加黏液，重建胃肠细胞和亚细胞结构，促进胃肠细
胞更新[7]；而半夏水煎醇沉液能具有保护胃黏膜, 促进胃黏膜的修复的作用[8]；另陈皮水煎剂可促进大鼠正
常胃液的分泌[9]。我们认为，胃萎Ⅰ号方对胃黏膜损伤的保护作用在健脾益气传统功效的基础上，与该方
药物组成所含成分的药理机制有关。
前列腺素（PGE2）是胃黏膜极为重要的防御因子，具有防止各种有害物质对消化道上皮细胞损伤和致
死作用[10]；表皮生长因子（EGF）是一种生长因子类蛋白质，通过与胃肠黏膜细胞上的-表皮生长因子受体
（EGFR）结合，能通过增加胃黏膜血流量，促进黏膜细胞DNA、RNA及蛋白质的合成，增加胃黏膜黏液
糖蛋白的合成与分泌，而发挥胃黏膜的保护作用[11]；生长抑素（SS）是一种调节性抑制肽，能帮助黏膜清
除氧自由基及脂质过氧化物，增加细胞内还原型谷耽甘肤含量, 减轻细胞脂质过氧化, 参与胃黏膜的局部
防御机制[12]。本研究显示，胃萎Ⅰ号颗粒可以提高胃黏膜损伤大鼠血清及组织中PGE2、EGF、SS含量，我
们推测，胃萎Ⅰ号颗粒对胃黏膜损伤的保护作用可能与促进PGE2、EGF、SS等胃黏膜保护因子的合成、加
速胃黏膜自身修复的机制有关，但有待于进一步深入研究。
参考文献
[1] 陈韶华. 酒精性胃粘膜损伤的研究进展[J].国际消化病杂志, 2006, 26(4):249-250.
[2] 凤良元. 胃病与胃粘膜保护[M].上海:上海中医药大学出版社,2003: 22.
[3] 徐婷婷, 朱方石. 外源性胃黏膜损伤因素与脾虚相关性的研究进展[J]. 世界华人消化杂志 2010，18(28): 2981-2984.
[4] 沈爱玲，尚游，袁世荧，等. 替普瑞酮对组织器官的保护作用[J]. 国际麻学与复苏杂志. 2010，31（1）：80-82.
[5] 王晓娜,朱方石.脾虚与胃黏膜防御机制相关性的研究进展及评价[J].辽宁中医杂志,2010,37(6):1176-1179.
[6] 杨晓军,吕东勇,朱焕金,等.健脾益气方药对大鼠急性胃粘膜损伤的保护与修复作用[J].河南中医,2010,30(4):351-353.
[7] 封吉化，尚虎虎. 四君子汤对脾虚证大鼠胃肠电活动的影响[J]．武警医学院学报，2010.19（1）：8-9，56.
[8] 刘守义，尤春来. 半夏抗溃疡作用机理的实验研究[J]．辽宁中医杂志，1992，10：42.
[9] 张志海，王彩云，杨天鸣，等. 陈皮的化学成分及药理作用研究进展[J]．西北药学杂志，2005，20（1）：471.
[10] Martin GR，JL. Gastrointestinal inflammation：a central component of mucosal defense and repair [J]. Exp Biol Med，2006，231:130-137.
[11] 姒健敏. 关注生长因子对胃粘膜的保护作用[J]．中华医学杂志，2005，85(39)：2744-2745.
[12] 王玮，孙梅．胃肠粘膜抗损伤和修复新进展[J]. 2005, 15（ 19）:2355-2359.
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
•291•
实 验 研 究
Experimental study
Comparative pharmacokinetics study of three anthraquinones in rat plasma
after oral administration of Radix et Rhei Rhizoma extract and Dahuang Fuzi
Tang by high performance liquid chromatography-mass spectrometry
Xiao Liu1,2 Huan Li1,2,3 Hui Guo1,2,3 Li Wu1,2
Hao Cai1,2, Weike Zhang1 Baochang Cai1,2,3
（1 College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210046, PR China; 2 Engineering Center of State Ministry of Education for
Standardization of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing, 210029, PR China; 3 Nanjing Haichang Chinese Medicine
Group Corporation, Nanjing, 210061, PR China）
Keywords pharmacokinetics; rhein; aloe-emodin; emodin
摘要 目的：研究灌胃给予大鼠大黄附子汤及大黄提取物后血浆中三种蒽醌类成分的药代动力学特征。方
法：采用LC-MS 方法，色谱柱：Kromasil C18(150×4.6 mm, 5 μm，带保护柱)；流动相：甲醇-3 mM 乙酸铵
缓冲液(75:25, v/v)线性梯度洗脱；柱温40 ℃。流速：1.0 mL/min，检测波长:280 nm。结果：血浆中大黄酸
在24-12,000 ng/mL；芦荟大黄素在23.2-11,600 ng/mL；大黄素在0.5-250 ng/mL 浓度范围内呈现良好的线
性关系。灌胃给予大鼠大黄附子汤和大黄提取物后，大黄酸和大黄素的AUC0-t, AUC0-∞；大黄酸的Cmax，
大黄酸、芦荟大黄素、大黄素的Tmax 有显著性差异。
Abstract A specific and sensitive high performance liquid chromatography-mass spectrometric (HPLC-MS)
method has been developed and validated for simultaneous determination of three anthraquinones of rhein (CAS:
478-43-3), aloe-emodin (CAS: 481-72-1) and emodin (CAS: 518-82-1) in rat plasma after oral administration of
Radix et Rhei Rhizoma extract and Dahuang Fuzi Tang. The analytes were separated on a Kromaisl ® C18 column
within a total running time of 12 min with a mobile phase of methanol : ammonium acetate(3 mM) (75:25, v/v).
The calibration curves for all the anthraquinones showed good linearity in the measured range with correlation
coefficient (r) higher than 0.9978. The precision, accuracy, recovery and stability were deemed acceptable. The
method was successfully applied to the comparative pharmacokinetics study of the anthraquinones in rat plasma
after oral administration of Radix et Rhei Rhizoma extract and Dahuang Fuzi Tang
1 Introduction
Dahuang Fuzi Tang (DFT), composed of three herbs including Radix et Rhizoma Rhei (DH), Radix Aconiti
Lateralis Praeparata (FZ) and Radix et Rhizoma Asari (XX), was originally described in Synopsis of Golden
Chamber (Jin Kui Yao Lue), a treatise on febrile and miscellaneous diseases written by the outstanding physician
Zhang Zhongjing in Han Dynasty. It is widely used to treat appendicitis, biliary colic, chronic dysentery, acute
ileus and adhesive ileus in clinic [1]. Numerous studies reported that rhein, aloe-emodin and emodin were the main
effective components of DH (the monarch herb), which exhibited beneficial pharmacological activity [2-5].
Quantitative analyses of the three anthraquinones in vivo for pharmacokinetic application have been documented
using either High performance liquid chromatography (HPLC-UV) or high performance liquid
chromatography-tandem mass spectrometry (HPLC-MS/MS) methods. Analysis time of HPLC-UV method,
however, was tedious (27 min-30 min) and aloe-emodin as well as emodin could not be detected, whilst
determination of rhein was failed after 12 h, and HPLC-MS/MS method only intended for aloe-emodin or emodin
alone [6-10]. Therefore, this present study developed a simple, rapid and reliable HPLC-MS assay for the
simultaneous determination of rhein, aloe-emodin, emodin in rat plasma to compare their pharmacokinetic
profiles after oral administration of DH extract and DFT.
2 Experimental Procedure
2.1 Reagents and materials FZ, XX and DH were obtained from Nanjing Haichang Chinese medicine group
corporation (NO.12 Yongjing Road, Hi-Tech Industrial Development Zone Nanjing, Jiangsu Province, P. R.
China). 1, 8-Dihydroxyanthraquinone (IS), rhein, aloe-emodin and emodin with purity of 99% or higher were
purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China).
The structures of these four compounds are shown in Fig. 1. HPLC-grade methanol was purchased from E. Merck
(Merck, Darmstadt, Germany). Purified water was from a Milli-Q system (Millipore Corporation, Bedford,
Massachusetts, France). Acetate, ascorbic acid, hydrochloric acid (HCl) and ethyl acetate were of analytical grade
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
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实 验 研 究
Experimental study
and obtained from Nanjing Chemical Reagent Company (Nanjing, China). .
2.2 Instrumentation and operating conditions The HPLC-MS system consisted of a Shimadzu LC-10AD HPLC
series liquid chromatograph and a Shimadzu HPLC/MS-2010A single quadrupole mass spectrometer equipped
with electrospray ionization (ESI) inte***ce. HPLC separation was carried out on a Kromasil ® C18 (150 × 4.6 mm,
5 μm, Akzo Nobel, Sweden) with a Kromasil guard column maintained at 40 ℃. The mobile phase consisted of
methanol - ammonium acetate (3 mM) (75:25, v/v) with isocratic elution. Mass spectrometer conditions were
optimized to obtain maximal sensitivity as follows: drying gas 8 L/min, CDL temperature 250 ℃, block
temperature 200 ℃ and detector voltage 1.3 kV. Analytes were quantitated in selected ion monitoring (SIM) mode
at negative ion mode. The [M-H]- ions of 282.80 for rhein, 268.85 for aloe-emodin, 268.85 for emodin and 239.95
for IS were selected as detecting ions.
2.3 Sample preparation To prepare DFT, pieces of DH, FZ and XX were mixed together in a ration (3:4:1,
w/w/w) and macerated in deionized water for 30 min, and then decocted twice with boiling water (1:8, w/v) each
for 20 min, then the solution was filtered through a two-layer mesh, and was combined and concentrated to a
density of 0.96 g/mL under vacuum at 60 ℃. DH extract was prepared in the same way but condensed to a density
of 0.36 g/mL.
2.4 Application A total of 12 rats were randomly divided into two groups. Each group contains 6 rats and was
orally administered a different extract by gavage with a syringe: DFT (1.44 g/100g body weight) and DH extract
(0.54 g/100g body weight), respectively. Blood samples (about 0.5 mL) were collected from the orbital vein at
pre-administration (time = 0) and post-administration (time = 0.033, 0.083, 0.167, 0.250, 0.5, 0.75, 1, 1.5, 2, 4, 6,
12, 24 h). The plasma was separated by centrifugation at 12,000 rpm for 3 min, and 0.1 mL was spiked with 10 μL
of IS and then mixed with 50 μL of pH 5 acetate buffer and 50 μL of ascorbic acid (100 mg/mL) by vortex mixing
for 30 s. The mixture was acidified with 50 μL of 0.1 N HCl by vortex mixing for 30 s and partitioned with 1 mL
of ethyl acetate by vortex mixing for 3 min. The aqueous and organic layers were separated by centrifugation at
4,000 rpm for 3 min and the organic layer was transferred to another tube and evaporated to dryness under
nitrogen at 50 ℃. The residue was reconstituted with 100 μL of mobile phase and vortexed for 30 s and
centrifuged at 12, 000 rpm for 3 min. A volume of 20 μL of the supernatant was injected for analysis. The plasma
concentration versus time for rhein, aloe-emodin and emodin were analyzed with a non-compartmental method
using the WinNonlin software (ver. 5.2; Pharsight, Mountain View, CA, USA).
2.5 Method validation The selectivity of the method was evaluated by comparing the chromatograms of six
blank rat plasma samples with that of six rhein, aloe-emodin, emodin and IS spiked rat plasma samples. Stock
standard solution of analytes were prepared in methanol and calibration standard samples were prepared through
spiking blank plasma (80 μL) with this stock solution (20 μL) to obtain final concentrations of 24-2,000 ng/mL for
rhein, 23.2-11,600 ng/mL for aloe-emodin, and 0.5-250 ng/mL for emodin. Standard curves were fitted by least
square regression using logarithm as weighting factor of the peak area ratio of the analytes to IS (log Y) versus
plasma concentrations(log X). The lower limits of quantitation (LLOQ) served as the lowest concentration on the
standard curve.The accuracy and precision of the established method were evaluated by QC samples at low,
medium and high concentrations. Accuracy was defined as the relative deviation in the calculated value of a
standard from that of its true value, expressed as relative error(RE). Precision was evaluated as the relative
standard deviation (RSD). The intra-day accuracy was determined by assaying five replicates at each
concentration level on 1 day, and inter-day accuracy was determined by analyzing QC samples in five duplicates
during three separate and successive days. QC samples were used to evaluate the stability of the analytes in rat
plasma under different storage conditions: long-term stability at -20 ℃ for 14 days, post-preparative stability at
room temperature for 24 h, and after three freeze-thaw cycles.
3 Result and discussion
3.1 Method validation Chromatograms (Fig. 2-4) of blank samples showed no endogenous peaks interfering
with any of the analytes or internal standard. The LLOQ was 24 ng/mL for rhein, 23.2 ng/mL for aloe-emodin and
0.5 ng/mL for emodin. The calibration curves showed good linearity within the range of 24-12,000 ng/mL for
rhein, 23.2-11,600 ng/mL and 0.5-250 ng/mL for aloe-emodin and emodin respectively. The results of precision
and accuracy were all within ±15%. The recoveries of rhein, aloe-emodin and emodin were 87.8-89.6 %,
90.2-96.7% and 87.9-89.7% respectively.The mean extraction recovery of IS was 96.2%. Here, the matrix effect
was within the range of 97.2-100.2%, indicating that no significant matrix effect was observed for rhein,
aloe-emodin and emodin. The mean matrix effect of the IS was 100.1%. The concentration of analytes deviated
less than ±15% from their nominal concentrations after three-thaw cycles, 24 h at room temperature and 14 days
at -20 ℃storage, showing that the samples were stable during preparation and analytical processes.
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
•293•
实 验 研 究
Experimental study
3.4 Pharmacokinetics studies The validated method was successfully applied to the comparative
pharmacokinetic studies of rhein, aloe-emodin and emodin in rat plasma after administration of DH extract and
DFT. Fig.5 showed the concentration-time curves; and the main pharmacokinetic parameters summarized in Table
1 showed that AUC0-t, AUC0-∞, Cmax , MRT, CL and Tmax of DH extract group statistically differed from those of
DFT group remarkably. After oral administration of DFT, AUC0-t, AUC0-∞ of rhein and emodin and the Cmax, of
rhein decreased significantly (P<0.01), while MRT of rhein increased significantly (P<0.01), as compared with
the values of the DH extract group. On the other hand, peak time of the three anthraquinones in DFT group was
significantly delayed compared to the DH extract group (P<0.05). However, there were no significant difference
in the T1/2 values of the three anthraquinones between DH extract and DFT decoction group. Meanwhile, AUC0-t,
AUC0-∞ and Cmax of aloe-emodin in DFT group also showed no difference in comparison with the DH extract
group. But the MRT of aloe-emodin increased significantly(P<0.05) and CL decrease remarkably (P<0.05) in
DFT group. The results indicated that oral administration of DFT decoction could lead to less absorption of rhein
and emodin with peak time detention of rhein, aloe-rhein and emodin. Our previous study has found that the
content of rhein and emodin, the acidic components in DH, decrease half approximately in DFT compared with
that in DH, which may be resulted from the action of alkaloid in FZ, giving a great contribution to the absorption
decrease of rhein and emodin in DFT, in order that the bitter and cold medicinal properties of DH was restricted
by FZ, which is in conformity with the traditional Chinese medicine theory of antagonistic action. On the other
hand, the combination rationality of DFT was not only based on the antagonistic action simply but on the
detention of peak time of rhein, aloe-empdin and emodin, leading to the restraint of strong and quick effect of DH
owing to the herb ingredient interaction of DFT in vivo.
Acknowledgements
This research was supported by the National Natural Science Foundation of China (No. 81073022)
References
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Table 1. The pharmacokinetic data for rhein, aloe-emodin and emodin after oral administration of DH extract and
DFT (mean±SD, n=6).
Data unit rhein aloe-emodin emodin DH DFT DH DFT DH DFT
AUC(0-t) ng h/mL 12333± 2709 3284±909** 1240±811 1559±315. 667±188 382±181**
AUC(0-∞) ng h/mL 15328±2823 3709±1126** 1506±984 2091±357 916±325 465±228**
Cmax ng/mL 10576±4388 1410±679** 235±67 183±65 192±52 101±53
T1/2 h 10.4±5.4 6.5±1.3 9.4±2.8 13.9±4.8 10.3±3.1 8.0±4.2
MRT(0-t) h 6.8±1.2 6.4±0.9 8.3±0.8 10.5±1.4* 8.9±1.7 7.8±1.8
CL mL kg/h 1.6±0.3 4.1±1.5** 2.9±1.1 1.5±0.2* 6.4±2.2 7.0±3.1
Tmax h 0.2±0.1 0.5±0.3* 1.0±0.7 3.4±1.3* 0.3±0.2 0.6±0.3*
注：*P<0.05, **P <0.01 compared with the DH extract group
第九届世界中医药大会
The 9th World Congress of Chinese Medicine
•29 4•
实 验 研 究
Experimental study
Fig.1. Chemical structures and mass spectra of (A) rhein, (B) aloe-emodin, (C) emodin and (D) IS.
Fig.2. Selected ion monitoring chromatograms of (A) blank rat plasma sample, (B) blank plasma sample spiked
with rhein, (C) plasma
sample obtained from rats administrated by DH extract of rhein, (D) plasma sample obtained from rats
administrated by DFT of rhein.
Fig.3. Selected ion monitoring chromatograms of (A) blank rat plasma sample, (B) blank plasma sample spiked
with aloe-emodin and emodin, (C) plasma sample obtained from rats administrated by DH extract of aloe-emodin
and emodin, (D) plasma sample obtained from rats administrated by DFT of aloe-emodin and emodin.1:
aloe-emodin; 2: emodin
Fig.4. Selected ion monitoring chromatograms of (A) blank rat plasma sample, (B) blank plasma sample spiked
with IS, (C) plasma sample obtained from rats administrated by DH extract of IS , (D) plasma sample obtained
from rats administrated by DFT of IS.
from rats administrated by DFT of IS.
Fig.5. Plasma concentration-time curves of (A) rhein, (B) aloe-emodin and (C) emodin in rat plasma after oral
administration of DH extract and DFT.
