APP下载

藏药麻花秦艽不同部位醇提物的抗炎作用研究

2018-10-19包婷雯左明丽王敏王怡姚政林鹏程

中国药房 2018年22期
关键词:泼尼松麻花小鼠

包婷雯 左明丽 王敏 王怡 姚政 林鹏程

摘 要 目的:观察藏药麻花秦艽不同部位醇提物的抗炎作用。方法:72只雄性SPF级昆明种小鼠随机分为模型组[等体积0.5%羧甲基纤维素钠(CMC-Na)溶液]、醋酸泼尼松组(10 mg/kg)及麻花秦艽地上部位醇提物低、高剂量组(10、30 g/kg)与麻花秦艽地下部位醇提物低、高剂量组(10、30 g/kg),每组12只;灌胃给药,每天2次,连续6次;末次给药40 min后于小鼠右耳廓正反两面均匀涂抹二甲苯(0.05 mL)以建立耳肿胀模型,考察各组小鼠的耳肿胀度,并计算耳肿胀抑制率。分组同上;灌胃给药,每天2次,连续5次;末次给药40 min后于小鼠右足皮下注射1%角叉菜胶0.9%氯化钠溶液(0.05 mL)以建立足肿胀模型,考察各组小鼠的足肿胀度,并计算足肿胀抑制率。无菌条件下将灭菌棉球植于小鼠鼠蹊部皮下以建立肉芽肿模型;分组同上,建模24 h后灌胃给药,每天1次,连续10次;末次给药24 h后剥离棉球,考察各组小鼠的肉芽肿胀度,并计算肉芽肿胀抑制率。84只雄性SD大鼠随机分为正常对照组(等体积0.5% CMC-Na溶液)、模型组(等体积0.5% CMC-Na溶液)、醋酸泼尼松组(7 mg/kg)及麻花秦艽地上部位醇提物低、高剂量组(7、21 g/kg)与麻花秦艽地下部位醇提物低、高剂量组(7、21 g/kg),每组12只;灌胃給药,每天1次,连续30次;灌胃给药4次后于大鼠右后足皮下注射完全弗氏佐剂(0.1 mL)以建立关节炎模型;建模18 h后,考察各组大鼠的足肿胀度,并计算足肿胀抑制率,建模20 d后进行大鼠全身关节病变评分,末次给药24 h后称定大鼠胸腺、脾、肾上腺质量和体质量并计算相应脏器指数。结果:与模型组比较,醋酸泼尼松组、麻花秦艽地上部位醇提物高剂量组和麻花秦艽地下部位醇提物低、高剂量组小鼠耳肿胀度均显著降低(P<0.05或P<0.01),耳肿胀抑制率分别为67.12%、27.39%、19.86%、54.79%;麻花秦艽地下部位醇提物低、高剂量组小鼠足肿胀度均显著降低(P<0.01),足肿胀抑制率分别为34.63%、46.07%;醋酸泼尼松组、麻花秦艽地上、地下部位醇提物低、高剂量组小鼠肉芽肿胀度均显著降低(P<0.05或P<0.01),肉芽肿胀抑制率分别为42.52%、14.02%、23.36%、21.50%、35.58%;醋酸泼尼松组和麻花秦艽地上、地下部位醇提物高剂量组大鼠关节肿胀度均显著降低,全身关节病变评分均显著降低(P<0.05或P<0.01)。与正常对照组比较,模型组大鼠胸腺、脾脏、肾上腺指数均显著降低(P<0.01);与模型组比较,麻花素艽地上、地下部位醇提物低、高剂量组各脏器指数差异均无统计学意义(P>0.05)。结论:麻花秦艽地上、地下部位醇提物均具有一定的抗炎作用。

关键词 麻花秦艽;地上部位;地下部位;醇提物;抗炎作用

中图分类号 R965;R285 文献标志码 A 文章编号 1001-0408(2018)22-3114-05

DOI 10.6039/j.issn.1001-0408.2018.22.20

ABSTRACT OBJECTIVE: To observe the anti-inflammatory activity of ethanol extract from different parts of Tibetan medicine Gentiana straminea. METHODS: Totally 72 male SPF Kunming mice were randomly divided into model group (constant volume of 0.5% CMC-Na solution);prednisone acetate group (10 mg/kg), low-dose and high-dose groups of ethanol extract from aerial part of G. straminea (10, 30 g/kg), low-dose and high-dose groups of ethanol extract from underground part of G. straminea (10, 30 g/kg), with 12 mice in each group. They were given relevant medicine intragastrically twice a day for consecutive 6 times. Fourty minites after last medication, ear-swelling model was induced by evenly smeared with xylene (0.05 mL) on both sides of the right auricle. Fourty minites after modeling, the ear swelling degree and inhibitory rate of ear swelling of mice in each group were calculated. Grouping was the same as above, and then they were given relevant medicine intragastrically twice a day for consecutive 5 times. Fourty minites after last medication, mice were given 1% carrageenan 0.9% sodium chloride solution 0.05 mL subcutaneously via right feet to establish feet swelling model; the feet swelling degree and inhibitory rate of feet swelling of mice in each group were calculated. Sterile cotton balls were planted in groin area of mice under the aseptic condition subcutaneously to establish granuloma model. Twenty hours after modeling, grouping was the same as above, and then they were given relevant medicine intragastrically once a day for consecutive 10 times. Twenty hours after last medication, the cotton balls were removed; the granulation swelling degree and inhibitory rate of feet swelling of mice in each group were calculated. Eighty-four male SD rats were randomly divided into normal contrd group (constant volume of 0.5% CMC-Na solution), model group (constant volume of 0.5% CMC-Na solution), prednisone acetate group (7 mg/kg), aerial part ethanol extract of G. straminea low-dose and high-dose groups (7, 21 g/kg), ethanol extract of G. straminea underground part low-dose and high-dose groups (7, 21 g/kg), with 12 rats in each group. They were given relevant medicine intragastrically once a day for consecutive 30 times. After 4 times of intragastric administration, rats were injected with complete Freunds adjuvant (0.1 mL) into the right hind foot to establish arthritis model. Eighteen hours after modeling, the feet swelling degree and inhibitory rate of feet swelling of rats in each group were calculated; 20 d after modeling, systemic lesion scores of rats in each group were conducted; 24 h after last medication, the weight of thymus, spleen, adrenal gland and body weight were weighed and relevant viscera indexes were calculated. RESULTS: Compared with model group, the auricle swelling degree of mice were decreased significantly in prednisone acetate group, high-dose group of ethanol extract from aerial part of G. straminea, low-dose and high-dose groups of ethanol extract from underground part of G. straminea (P<0.05 or P<0.01); inhibitory rates of ear swelling were 67.12%, 27.39%, 19.86%, 54.79%, respectively. Feet swelling degree were decreased significantly in low-dose and high-dose group of ethanol extract from underground part of G. straminea (P<0.01); inhibitory rates of feet swelling were 34.63% and 46.07%. The granulation swelling degree were decreased significantly in prednisone acetate group, low-dose and high-dose groups of ethanol extract from aerial part of G. straminea, low-dose and high-dose groups of ethanol extract from underground part of G. straminea (P<0.05 or P<0.01); inhibitory rates of granulation swelling were 42.52%, 14.02%, 23.36%, 21.50%, 35.58%, respectively. The joint swelling degree of rats were decreased significantly in prednisone acetate group, low-dose and high-dose groups of ethanol extract from aerial part of G. straminea, low-dose and high-dose groups of ethanol extract from underground part of G. straminea (P<0.05 or P<0.01); while systemic lesion score were decreased significantly (P<0.05 or P<0.01). Compared with normal control group, thymus, spleen, adrenal indexes of rats were increased significantly in model group (P<0.01); compared with model group, viscera indexes of rats were not statistical different in low-dose and high-dose groups of ethanol extract from aerial part of G. straminea, low-dose and high-dose groups of ethanol extract from underground part of G. straminea (P>0.05). CONCLUSIONS: The ethanol extract from aerial part and underground part of G. straminea both have anti-inflammatory effects.

KEYWORDS Gentiana straminea; Aerial part; Underground part; Alcohol extract; Anti-inflammatory effects

麻花秦艽(Gentiana straminea Maxim.)为龙胆科龙胆属植物[1],是青海省道地药材[2],以全草或根和花入药。藏医称秦艽类药材为“解吉那保”,临床主要用于关节炎、肺病发烧、黄疸及二便不通等症的治疗[3]。麻花秦艽具有抗炎、镇痛、保肝、降血压、抗病毒、抗肿瘤等药理作用[4-7],主要化学成分有环烯醚萜苷类、黄酮类、三萜类和木脂素类等[7-8],其中,龙胆苦苷和獐牙菜苦苷是其抗炎的主要活性成分[9]。本研究通过二甲苯致小鼠耳肿胀、角叉菜胶致小鼠足肿胀、小鼠棉球肉芽肿实验及完全弗氏佐剂致大鼠关节炎实验考察麻花秦艽地上、地下部位醇提物的抗炎作用[10-12],以为其临床应用提供参考。

1 材料

1.1 仪器

AL244型电子分析天平[梅特勒-托利多仪器(北京)有限公司];R-215型旋转蒸发仪(瑞士Buchi公司);SHB-Ⅲ型循环水式多用真空泵(郑州长城科工贸有限公司);KQ-300型超声波清洗机(昆山市超声仪器有限公司);YLS型打孔器(上海艾研生物科技有限公司)。

1.2 药品与试剂

醋酸泼尼松片(重庆三新制药厂,批号:061202,规格:5 mg);角叉菜胶(批号:9062-07-1)、完全弗氏佐剂(批号:9007-81-2)均购自美国Sigma公司;羧甲基纤维素钠(CMC-Na)、二甲苯、乙醇均为分析纯,水为蒸馏水。

1.3 药材

麻花秦艽(采自青海省互助县)药材样品经青海民族大学药学院林鹏程教授鉴定为真品。

1.4 动物

SPF级昆明种小鼠216只,雄性,8周龄,体质量为20~24 g;清洁级SD大鼠84只,雄性,32~36周龄,体质量为230~250 g,均购自四川省中药研究所实验动物中心[动物生产合格证号:SCXK(川)2005-19]。标准条件饲养1周,自由进食、饮水,环境温度(22±2)℃,相对湿度65%~70%,人工黑暗和光照交替。

2 方法

2.1 麻花秦艽不同部位醇提物的制备

2.1.1 麻花秦艽地上部位醇提物 取5.0 kg麻花秦艽地上部位药材样品,用25 kg乙醇45 ℃回流提取3次,每次回流1 h,得浸膏1.1 kg。临用前以0.5% CMC-Na溶液制成所需的混悬液。

2.1.2 麻花秦艽地下部位醇提物 取5.0 kg麻花秦艽地下部位药材样品,用25 kg乙醇45 ℃回流提取3次,每次回流1 h,得浸膏0.75 kg。臨用前以0.5% CMC-Na溶液制成所需的混悬液。

2.2 二甲苯致小鼠耳肿胀实验

72只小鼠随机分为模型组(等体积0.5% CMC-Na溶液)、醋酸泼尼松组(阳性对照,10 mg/kg)及麻花秦艽地上部位醇提物低、高剂量组(10、30 g/kg)与麻花秦艽地下部位醇提物低、高剂量组(10、30 g/kg)[醋酸泼尼松片成人临床用量为60 mg,按照实验动物与人用剂量的换算公式[12]计算得小鼠用量为10 mg/kg,大鼠用量为7 mg/kg。依据2015年版《中国药典》(一部)中秦艽药材常用量为3~10 g[1],本实验以口服生药量100 g作为成人最大日剂量;按照实验动物与人用药量的换算公式[12]计算得小鼠低、高剂量用量为10、30 g/kg,大鼠低、高剂量用量为7、21 g/kg],每组12只;灌胃给药,每天2次,连续6 次;末次给药40 min后,于小鼠右耳廓正反两面均匀涂抹二甲苯(0.05 mL)以建立耳肿胀模型;建模30 min后,将小鼠颈椎脱臼处死,剪下双耳,用8 mm打孔器取下左右耳廓同一部位的耳片并称定质量,两耳片质量差值即代表耳肿胀度,并计算耳肿胀抑制率:耳肿胀抑制率=(模型组耳肿胀度-给药组耳肿胀度)/ 模型组耳肿胀度×100%。

2.3 角叉菜胶致小鼠足肿胀实验

分组同“2.2”项下操作;灌胃给药,每天2次,连续5 次;末次给药40 min后,于小鼠右后足皮下注射1%角叉菜胶0.9%氯化钠溶液(0.05 mL)以建立足肿胀模型;建模4 h后,将小鼠颈椎脱臼处死,剪下双足并称定质量,两足质量差即代表足肿胀度,并计算足肿胀抑制率:足肿胀抑制率=(模型组足肿胀度-给药组足肿胀度)/ 模型组足肿胀度×100%。

2.4 小鼠棉球肉芽肿实验

乙醚吸入麻醉小鼠后,于无菌条件下将灭菌棉球植于其鼠蹊部皮下以建立肉芽肿模型;分组同“2.2”项下操作,建模24 h后,灌胃给药,每天1次,连续10次;末次给药24 h后,剥离棉球,然后将棉球置于60 ℃烘箱中烘烤12 h,取出称定质量,以棉球干、湿质量差值代表肉芽肿胀度,并计算肉芽肿胀抑制率:肉芽肿胀抑制率=(模型组肉芽肿胀度-给药组肉芽肿胀度)/ 模型组肉芽肿胀度×100%。

2.5 大鼠完全弗氏佐剂关节炎实验

84大鼠随机分为正常对照组(等体积0.5% CMC-Na溶液)、模型组(等体积0.5% CMC-Na溶液)、醋酸泼尼松组(7 mg/kg)及麻花秦艽地上部位醇提物低、高剂量组(7、21 g/kg)与麻花秦艽地下部位醇提物低、高剂量组(7、21 g/kg),每组12只;灌胃给药,每天1次,连续30 次。给药4次后,于大鼠右后足皮下注射完全弗氏佐剂(0.1 mL)以建立关节炎模型。建模18 h后,以大鼠左右足体积差为足肿胀度[8],并计算足肿胀抑制率(公式同“2.3”项下)。建模20 d后,进行大鼠全身关节病变评分(无肿胀为0分,趾关节稍肿为1分,趾关节和足肿胀为2分,踝关节以下的足爪肿胀为3分,包括踝关节在内的全部足爪肿胀为4分)[12];末次给药24 h后处死大鼠,剥离内脏,称定胸腺、脾、肾上腺质量和体质量并计算相应脏器指数:脏器指数=脏器质量(mg)/100 g体质量。

2.6 统计学方法

采用SPSS 19.0软件对数据进行统计分析。计量资料以x±s表示,组间比较采用单因素方差分析。P<0.05为差异有统计学意义。

3 结果

3.1 各组小鼠耳肿胀度比较

与模型组比较,醋酸泼尼松组、麻花秦艽地上部位醇提物高剂量组和麻花秦艽地下部位醇提物低、高剂量组小鼠耳肿胀度均显著降低,差异均有统计学意义(P<0.05或P<0.01),详见表1。

3.2 各组小鼠足肿胀度比较

与模型组比较,麻花秦艽地下部位醇提物低、高剂量组小鼠足肿胀度均显著减少,差异均有统计学意义(P<0.01),详见表2。

3.3 各组小鼠肉芽肿胀度比较

与模型组比较,醋酸泼尼松组和麻花秦艽地上、地下部位醇提物低、高剂量组小鼠肉芽肿胀度均显著降低,差异均有统计学意义(P<0.05或P<0.01),详见表3。

3.4 各组大鼠足肿胀度、全身关节病变评分、脏器指数比较

正常对照组、模型组、醋酸泼尼松组各有1只大鼠死亡。正常对照组大鼠足无肿胀,全身关节病变评分为0分。与模型组比较,醋酸泼尼松组和麻花秦艽地上、地下部位醇提物高剂量组大鼠关节肿胀度均显著降低,全身关节病变评分显著降低,差异均有统计学意义(P<0.05或P<0.01)。与正常对照组比较,模型组大鼠胸腺、脾、肾上腺指数均显著升高,差异均有统计学意义(P<0.05);与模型组比较,醋酸泼尼松组大鼠胸腺、肾上腺指数均显著降低,差异均有统计学意义(P<0.05),而麻花秦艽地上、地下部分醇提物低、高剂量组各脏器指数差异无统计学意义(P>0.05),详见表4~表6。

4 讨论

二甲苯涂抹于小鼠耳廓,可增加耳廓毛细血管通透性,最终导致渗入组织的液体增多,形成肉眼可见的红肿[13]。本研究结果显示,给予30 g/kg麻花秦艽地上部位醇提物和10、30 g/kg麻花秦艽地下部位醇提物均可显著降低模型小鼠耳肿胀度。角叉菜胶注入小鼠右足皮下,可导致局部前列腺素合成增加,并与血管合成物质和激肽类一起诱发水肿[14]。本研究结果显示,给予10、30 g/kg麻花秦艽地下部位醇提物均可显著降低模型小鼠足肿胀度。棉球植入小鼠体内可引起结缔组织增生,该类增生与临床患者炎症病理性改变相似,多用于评定药物抗结缔组织增生作用。本研究结果显示,给予10、30 g/kg麻花秦艽地上、地下部位醇提物均可显著降低模型小鼠肉芽肿胀度。佐剂性关节炎是一种免疫性炎症模型,其以多发性关节炎为特征,发病机制与病理特征与人类风湿性关节炎类似,对抗炎、免疫药物敏感,多用于筛选和研究上述两类药物[15]。本研究结果显示,给予21 g/kg麻花秦艽地上、地下部位醇提物均可显著降低模型大鼠关节肿胀度和全身关节病变评分。

综上所述,麻花秦艽地上、地下部位醇提物均具有一定的抗炎作用。

参考文献

[ 1 ] 国家药典委员会. 中华人民共和国药典:一部[S]. 2015年版.北京:中国医药科技出版社,2010:270-271.

[ 2 ] 安燕,吴启勋,张明锦,等. 青海道地药材秦艽色谱指纹图谱的研究[J]. 青海师范大学学报(自然科学版),2006(1):58-61.

[ 3 ] 帝玛尔·丹增彭措. 晶珠本草[M]. 上海:上海科学技术出版社,2012:203-205.

[ 4 ] 王佳,王钢,王丽琴,等. 秦艽醇提物治疗类风湿关节炎的实验研究[J]. 西部中医药,2015,28(7):11-14.

[ 5 ] 张鹏,刘瑢,张霞,等. 秦艽对小鼠酒精性肝损伤的保护作用[J]. 中国医院药学杂志,2014,34(21):1822-1825.

[ 6 ] 姚佳,曹晓燕,王喆之. 6种秦艽镇痛和抗炎活性的比较[J]. 现代生物医学进展,2010,10(24):4642-4646.

[ 7 ] 聂安政,林志健,王雨,等. 秦艽化学成分及药理作用研究进展[J]. 中草药,2017,48(3):597-608.

[ 8 ] 张莉,党军,梅丽娟,等. 藏药麻花秦艽化学成分研究[J]. 中药材,2016,39(1):103-106.

[ 9 ] CHEN L,WANG HB,SUN XL. Study on the analgesic and anti-inflammatory activities of gentiopicroside[J]. Nat Pro Res Dev,2008,20(5):903-906.

[10] VOGEL HG,VOGELl WH. Drug discovery and evaluation : pharmacological assays[M]. Berlin:Springer International Publishing,2015:676-677.

[11] 卫生部药政局. 新药(西药)临床前研究指导原则汇编[M]. 北京:中华人民共和国卫生部药政局,1993:121-123.

[12] 陈奇. 中藥药理研究方法学[M]. 3版. 北京:人民卫生出版社,2011:33-34、358-359.

[13] JIA N,LI Y,WU Y,et al. Comparison of the anti-inflammatory and analgesic effects of Gentiana macrophylla Pall. and Gentiana straminea Maxim., and identification of their active constituents[J]. J Ethnopharmacol,2012,144(3):638-643.

[14] 缪璐琳,熊秋韵,高继海,等. 不同产地和叶形附子水煎液的抗炎、镇痛作用比较[J]. 中国药房,2017,28(25):3483-3486.

[15] 郭佳佳,刘海,朱燕红,等. 蒙药芯芭提取物的抗炎、镇痛作用研究[J]. 中国药房,2017,28(1):64-67.

(收稿日期:2018-01-15 修回日期:2018-05-09)

(编辑:张 静)

猜你喜欢

泼尼松麻花小鼠
麻花
小鼠大脑中的“冬眠开关”
肠道细菌改变小鼠更难克服恐惧
麻花
低分子肝素联合泼尼松治疗小儿肾病综合征的临床疗效观察
罐头和麻花都让我委屈
联合拉氧头孢、泼尼松治疗急性附睾炎患者的临床疗效研究
泼尼松联合当归芍药散加味治疗原发性肾病综合征临床疗效研究
来氟米特联合泼尼松治疗难治性原发性肾病综合征的随机对照试验
爆笑成语来袭,开心麻花请接招