CN104490969A - 蒙药漏芦花抗炎有效部位的制备及其应用 - Google Patents
蒙药漏芦花抗炎有效部位的制备及其应用 Download PDFInfo
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- CN104490969A CN104490969A CN201410758224.9A CN201410758224A CN104490969A CN 104490969 A CN104490969 A CN 104490969A CN 201410758224 A CN201410758224 A CN 201410758224A CN 104490969 A CN104490969 A CN 104490969A
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Abstract
本发明公开了蒙药漏芦花抗炎有效部位的制备及其应用。其制备方法主要步骤为漏芦花用乙醇回流提取,回收乙醇,醇提物用D101或AB-8型大孔吸附树脂分离,以50~90%乙醇洗脱,洗脱液浓缩干燥即得。本发明漏芦花有效部位对急慢性炎症具有较好的抗炎活性,且工艺简单、适合工业化生产。
Description
技术领域
本发明属于医药技术领域,具体而言,本发明涉及蒙药漏芦花抗炎有效部位的制备方法(提取和分离)及有效部位的应用。
背景技术
炎症是十分常见而重要的基本病理过程,可发生于机体的任何部位和任何组织,人类的大多数疾病常与炎症过程有关。炎症也是机体最重要的保护性反应,人类得以长期生存离不开炎症反应。但在某些情况下,炎症反应对机体也具有不同程度的危害,严重的炎症反应甚至可危及患者的生命。因此,积极研制各种药物,有目的地防止和控制炎症对机体造成的不利影响,将有着重要的意义。
蒙药漏芦花系菊科植物祁州漏芦(Rhaponticum uniflorum(L.)DC)的干燥花,蒙药名:洪古日朱勒,主产于我国黑龙江、吉林、辽宁、内蒙古、河北、山西、陕西、甘肃、山东等地,具有杀“粘”、止刺痛、清热、解毒、止痛、解表等作用,用于肠刺痛、瘟热、结喉、麻疹、毒热、心热、讧热、炽热、血热、新陈热、伤热等【娜贺雅,白玉霞.蒙药漏芦花的研究进展[J].内蒙古大学学报,2011,26(3):330-332.】。漏芦花为蒙医常用药,在蒙医临床中有较长的使用历史,临床上常与胡琏、丹参、龙丹花配伍应用,治疗血热炽盛、伤热、骚热、毒热、希拉热等症状,如肺骚普清散、骚血普清散等;还可配制十二味漏芦花伞、十味大青伞、三花伞、二十三位冰片散用于治疗粘热证、麻疹、白喉、炭疽、肠刺痛、亚玛病、胆溢帘脉等症【罗布桑.蒙药学[M].内蒙古人民出版社,2006:188蒙医学编辑委员会.中国医学百科全书(蒙医药)[M].上海科技出版社,1992;12:211】。朱培忠[朱培忠,蒋素容.漏芦花泡麻油治疗烧、 烫伤[J].四川中医,1985;(8):47]等用新鲜漏芦花30g,泡麻油250mL治疗烫伤背部起水泡的病人,3~4d即愈,且无瘢痕。斯琴[斯琴.蒙药治疗鼻窦炎53例[J].中国民族民间医药,2012;(3):3]等用蒙药十二味漏芦花丸和胡日查-6治疗鼻窦炎,十天为一个疗程,三个疗程后评价疗效,结果显示病例53例,治愈31例,有效12例,总有效率达81%等。尽管漏芦花在蒙医临床中有较长的使用历史和较强的抗炎药理活性,但是对其抗炎活性及药理机制尚不清楚。本发明应用体内抗炎药效实验模型,确定了漏芦花原药材及其醇提物的抗炎效果;并在体外抗炎药效实验的引导下,对其抗炎有效部位进行系统的化学成分研究,进一步阐明漏芦花中抗炎有效成分,为漏芦花的深入研究和新药开发奠定基础。
发明内容
本发明的第一个目的是提供一种具有抗炎作用的蒙药有效部位。
本发明的第二个目的是提供一种蒙药漏芦花抗炎有效部位的制备方法。
本发明的第三个目的是提供一种蒙药漏芦花有效部位在制备抗炎药物中的应用。
本发明可以通过以下技术方案实现:
本发明所述的漏芦花抗炎有效部位的制备方法,按照下列步骤进行:
(1)将漏芦花药材粉碎成粗粉,按照漏芦花粗粉:溶剂=1:(4~20)(重量)的比例,回流提取2~3次,每次1~4小时,滤液回收溶剂,减压干燥,得到漏芦花醇提取物(CEE),其中所述溶剂为乙醇,质量浓度为50~90%;
(2)将步骤(1)所得漏芦花醇提取物加水稀释,漏芦花醇提取物:水=1:(4~10)(重量),稀释液经大孔吸附树脂柱富集,先用去离子水3~8BV(树脂体积)洗脱去杂后,再用5~10BV乙醇洗脱,收集乙醇洗脱液,减压回收乙醇 溶剂,得到流浸膏,将流浸膏干燥,即得到漏芦花抗炎有效部位提取物,其中所述乙醇浓度为50~90%。
上述步骤(1)中所述漏芦花药材为菊科植物祁州漏芦(Rhaponticum uniflorum(L.)DC)的干燥花。
上述步骤(2)中所述大孔吸附树脂为非极性或弱极性大孔吸附树脂,优选为D101或AB-8型大孔吸附树脂。步骤(2)乙醇浓度优选70-80%。
上述所述采用大孔吸附树脂纯化条件优选为漏芦花药材粗粉与树脂质量比为1:(1~5),树脂层析柱径高比1:(3~9),上样流速1~3mL/min/g。
采用本发明方法制备的漏芦花抗炎有效部位,优选包括以下10种化合物,分别为20-羟基蜕皮激素(1)、β-谷甾醇(2)、α-(反-3-甲氧基-4-o-β-D-吡喃葡萄糖基苯亚甲基)-β-(3,4-二甲氧基苄基)-γ-丁内酯(Hemislin B glucoside)(3)、木樨草素(4)、芹菜素(5)、槲皮素(6)、槲皮苷(7)、阿福豆苷(8)、硬脂酸(9)、没食子酸(10)。
本发明的目的在于提供一种含有蒙药漏芦花抗炎有效部位的药物。
本发明所述的含有漏芦花有效部位的药物用于抑制急慢性炎症的医药用途。
本发明所述的漏芦花抗炎有效部位与药学上可接受的辅料按照常规制剂方法制成口服制剂,优选片剂、胶囊剂。
本发明还提供所述方法获得的漏芦花醇提物有效部位用于抑制急慢性炎症的医药用途。
本发明还提供所述方法获得的漏芦花抗炎有效部位用于制备抑制促发炎性分子生成的医药用途;其中,所述促发炎性分子为抑制一氧化氮(NO)。
本发明方法与现有技术相比具有以下有益效果:
1)本发明是从天然植物中分离得到的具有较强抗炎作用的有效部位,并对该有效部位进行了化学成分分离分析和药理活性测试,具有较明确的活性成分和作用机制。
2)本发明方法为现代提取分离技术,分离效果好,操作性强,适合工业化生产。
附图说明
图1显示本发明漏芦花抗炎有效部位A2-2对一氧化氮抑制率。
图2-9显示本发明分离得到的8种具有抑制一氧化氮生成的单体化合物的抑制率。
具体实施方式
下面结合实施例对本发明作进一步说明,但本发明并不限于以下实施例。
实施例1
(1)将漏芦花药材1Kg粉碎成粗粉,加入75%乙醇15Kg,回流提取2次,每次1.5小时,过滤,滤液回收溶剂,减压干燥,得到漏芦花醇提取物(CEE)。
(2)将上述漏芦花醇提取物加水稀释,漏芦花流浸膏:水=1:8(重量),稀释液经AB-8型大孔吸附树脂柱富集,先用去离子水4BV(树脂体积)洗脱,再用70%乙醇8BV洗脱,收集洗脱液,减压回收溶剂,得到流浸膏,将流浸膏干燥,即得到漏芦花抗炎有效部位提取物0.20Kg。
实施例2
(1)将漏芦花药材1Kg粉碎成粗粉,加入60%乙醇12Kg,回流提取2次,每次1小时,过滤,滤液回收溶剂,减压干燥,得到漏芦花醇提取物(CEE)。
(2)将上述漏芦花醇提取物加水稀释,漏芦花流浸膏:水=1:10(重量),稀 释液经D101型大孔吸附树脂柱富集,先用去离子水3BV(树脂体积)洗脱,再用80%乙醇7BV洗脱,收集洗脱液,减压回收溶剂,得到流浸膏,将流浸膏干燥,即得到漏芦花抗炎有效部位提取物0.23Kg。
实施例3
片剂制备:将从漏芦花中提取得到的漏芦花有效部位提取物200g,加入羟丙基甲基纤维素175g,微粉硅胶60g,硬脂酸镁2g,混合均匀,过80目筛,干法制粒,压片。
实施例4
胶囊剂制备:将从漏芦花中提取得到的漏芦花有效部位提取物200g,加入淀粉80g,以95%乙醇作为粘合剂,过20目筛制软材,60℃干燥,整粒,干颗粒加入过100目的滑石粉3g混匀,装胶囊,制得胶囊剂。
下面试验进一步说明本发明:
1、蒙药漏芦花醇提物抗炎有效部位(CEE)体内抗炎活性筛选药效实验
1.1、对二甲苯致小鼠耳廓肿胀的抑制作用
1.1.1实验目的:
了解受试药对动物急性炎症的作用,为研究受试药抗炎作用机理提供依据。1.1.2实验仪器与试剂:
剪刀、电子天平、直径打孔器、注射器;二甲苯、雷公藤片、生理盐水、羧甲基纤维素钠(CMC-Na)
1.1.3受试物与实验动物:
阳性对照药物:雷公藤片,市售。
受试物:实施例1中得到的蒙药漏芦花醇提物(CEE);漏芦花原药材粉末, 粉碎过筛
实验动物:昆明种小鼠,体重20±2克,雌雄兼用,由沈阳医学院实验动物中心提供,实验动物生产许可证号:SCXK(辽)2003-00160。
1.1.4实验步骤:
取小鼠60只,雌雄各半,体重20±2g,随机分为4组,分别为模型组、漏芦花药粉组、漏芦花醇提物组、阳性对照组,每组15只。从实验第一天开始,模型组灌胃等容量的0.5%CMC-Na溶液,其它各给药组灌胃按照表1-1所示剂量,给予含相应药的0.5%CMC-Na混悬液,给药体积均为0.2m1/10g体重,每天1次,连续7天。末次给药30分钟后,分别取0.lml二甲苯涂于各鼠右耳前后两面致炎。1h后,将小鼠颈椎脱臼处,沿耳廓基线剪下两耳,用直径8mm的打孔器分别在两耳同一部位打下圆耳片,迅速用电子天平称重,每鼠右耳片减去左耳片重量即为耳片炎症肿胀度。计算耳廓肿胀度:
E%=[(WR-WL)/WL]×100,
其中,WR为小鼠右耳片重量,WL为小鼠左耳片重量。
1.1.5实验结果:
与模型组相比,各给药组均明显抑制耳廓肿胀,差异显著(分别P<0.05,P<0.01)。提示,各组药对二甲苯致小鼠耳廓肿胀程度具有不同程度的降低作用。
与雷公藤片组相比,漏芦花醇提物组、漏芦花原药组无明显差异(P>0.05)。提示,漏芦花醇提物、漏芦花原药组抗二甲苯致炎症效果基本相似,结果见表1。
表1漏芦花及漏芦花醇提物组对二甲苯致小鼠耳廓肿胀的影响(n=15)
注:均值±标准差,与模型组比较*p<0.05,**P<0.01
1.2、对角叉菜致大鼠足肿胀的抑制作用
1.2.1实验目的:
了解受试药对动物急性炎症的作用,为研究受试药抗炎作用机理提供依据。1.2.2实验仪器与试剂:
器官充满度测量器、注射器;角叉菜胶溶液、雷公藤片、生理盐水、羧甲基纤维素钠(CMC-Na)
1.2.3受试物与实验动物:
阳性对照药物:雷公藤片,市售
受试物:实施例1中得到的蒙药漏芦花醇提物(CEE);漏芦花原药材粉末,自制粉碎过筛。
实验动物:Wistar大鼠,体重180±10克,雌雄兼用,由沈阳医学院实验动物中心提供,实验动物生产许可证号:SCXK(辽)2003-00160。
1.2.4实验步骤:
取大鼠60只,雌雄兼用,按体重随机分为4组:分别为模型组、漏芦花药粉组、漏芦花醇提物组、阳性对照组,每组15只。从实验第一天开始,各给药组大鼠分别按照表2-1所示剂量灌胃含相应药的0.5%CMC-Na混悬液。模型组灌胃等容量的0.5%CMC-Na溶液,每天1次,连续7天。建立模型前用容积法测量各组大鼠左右足容积。于末次给药30min后,将各组大鼠右后足键膜下注射1%角叉菜胶溶液0.1ml,之后于2h,4h,6h,8h和10h分别测量大鼠右后足趾容积,并计算其足肿胀度,见下式:
足肿胀度(率)=(致炎后足跖体积-致炎前足跖体积)/致炎前足跖体积。1.2.5实验结果:
与模型组相比,各给药组对致炎后8h、10h均明显抑制其足肿胀,差异显著(分别P<0.05,P<0.01)。提示,各组药对角叉菜致足肿胀具有不同程度的治疗作用。
与漏芦花醇提物组相比,雷公藤组、漏芦花组肿胀率明显升高,差异显著(P<0.05)。提示,漏芦花醇提物组抗角叉菜致炎症效果强于雷公藤片、漏芦花组。结果见表2-1。
表2漏芦花及漏芦花醇提物组对角叉菜致大鼠足肿胀的影响(n=15)
注:均值±标准差,与模型组比较*p<0.05,**P<0.01,与漏芦花醇提物组比较◆p<0.05
1.3、对大鼠棉球致肉芽肿的抑制作用
1.3.1实验目的:
了解受试药对动物慢性炎症的作用,为研究受试药抗炎作用机理提供依据。1.3.2实验仪器与试剂:
器官充满度测量器、注射器;戊巴比妥钠、灭菌棉球、青、链霉素、雷公藤片、生理盐水、羧甲基纤维素钠(CMC-Na)
1.3.3受试物与实验动物:
阳性对照药物:雷公藤片,市售
受试物:实施例1中得到的蒙药漏芦花醇提物(CEE);漏芦花原药材粉末,自制粉碎过筛。
实验动物:Wistar大鼠,体重180±10克,雌雄兼用,由沈阳医学院实验动物中心提供,实验动物生产许可证号:SCXK(辽)2003-00160。。
1.3.4实验步骤:
取大鼠60只,雌雄兼用,按体重随机分为4组:模型组、漏芦花药粉组、漏芦花醇提物组、阳性对照组,每组15只。给每只小鼠腹腔注射戊巴比妥钠40mg/kg麻醉,分别与两侧腹股沟皮下各植入灭菌棉球1个(30mg),刀口缝合后以青、链霉素防止感染。次日起给药各组大鼠分别灌胃相应药的0.5%CMC-Na混悬液,模型组灌胃等容量0.5%CMC-Na溶液。每日1次,连续7日。于末次给药1h后,处死大鼠。打开腹腔,取出棉球,剥取肉芽组织,剔尽脂肪组织,60℃干燥至恒重,减去棉球重量即为肉芽肿重量。
1.3.5实验结果:
与模型组相比,各给药组均明显减轻肉芽肿,差异显著(分别为P<0.05,P<0.01,P<0.001)。说明各组药对棉球致肉芽肿具有不同程度的治疗作用。
与漏芦花醇提物组相比,雷公藤组、漏芦花组肿胀率明显升高,差异显著(P<0.05)。提示,漏芦花醇提物组抗棉球致炎症效果强于雷公藤片、漏芦花原药粉,结果见表3-1。
表3漏芦花及漏芦花醇提物组对大鼠棉球致肉芽肿的影响(n=15)
注:均值±标准差,与模型组比较*p<0.05,**P<0.01,***P<0.01,与漏芦花醇提物组比较◆p<0.05
通过上述三项抗炎试验,可得出结论:漏芦花及漏芦花醇提物对急性和慢性炎症均有较好的抑制作用,漏芦花醇提物抗炎有效部位的抑制作用更显著, 对三个体内抗炎模型的抗炎效果均强于阳性对照药物雷公藤片。表明蒙药漏芦花醇提物抗炎有效部位有望制备成新的具有抗炎功效的生化药物。
2、应用RAW264.7体外抗炎模型对漏芦花各提取部位的活性测试
2.1实验材料:
细胞株:小鼠单核巨噬细胞RAW264.7购自中科院典型培养物保藏委员会细胞库;脂多糖(LPS)购自Sigma Chemical(St.Louis,MO),DMEM培养基购自Hyclone(USA)、青霉素、链霉素购自Sinopharm Chemical Reagent Co.Ltd.(Beijing,China)、胰蛋白酶(Trysin)、生理盐水(PBS)胎牛血清购自Gibco BRL(NY,USA);CCK-8购自Dojindo China Co.Ltd(Shanghai,China);Griess检测试剂盒,购自碧云天生物技术研究所,其他常用生化试剂均为国产分析纯。
受试物:与实施例1相同,得到的蒙药漏芦花醇提物(CEE)上大孔吸附树脂,采用水洗脱和水洗脱后再分别采用70%乙醇、90%乙醇洗脱,得到水洗脱物(A1)、70%乙醇洗脱物(A2)、90%乙醇洗脱物(A3);70%乙醇洗脱物回收溶剂后再次上大孔吸附树脂,采用水洗脱和水洗脱后再分别采用30%-90%乙醇洗脱得到水洗脱物(A2-1)、30%乙醇洗脱物(A2-2)、50%乙醇洗脱物(A2-3)、70%乙醇洗脱物(A2-4)、90%;乙醇洗脱物(A2-5)。
2.2细胞培养
本发明选用小鼠单核巨噬细胞株RAW264.7以进行一氧化氮产生量的探讨。RAW264.7细胞在37℃、5%CO2条件下,用含10%胎牛血清、青霉素(1×105U/L)及链霉素(100mg/L)的DMEM培养基培养。待RAW264.7细胞生长至对数期后备用
2.3细胞毒性试验
将对数生长期的RAW264.7细胞接种于96孔培养板上,并调整细胞密度为 1×105个/mL,每孔体积100μl,每组8孔。37℃、5%CO2条件下培养箱培养24h后,加入2.1中不同受试物漏芦花的不同浓度的各提取有效部位,每个浓度设3个复孔。培养24h后,弃去培养基,各孔加100μlCCK-8溶液(cck-8:无血清DMEM=1:9),继续培养1h,以酶标仪于波长450nm处测定OD值。并按公式计算各浓度下漏芦花各提取有效部位对正常RAW264.7细胞的抑制率,绘制标准曲线,找到最大无毒剂量。
抑制率(%)=(细胞对照组光吸收值-药物组光吸收值)/(细胞对照组光吸收值)×100%
2.4NO含量检测
将生长于对数期的单核巨噬细胞用胰蛋白酶消化下来,并用细胞完全培养液将其调整到适宜的细胞密度(大约5×105个/ml)。然后接种到96孔板,每孔体积100μL,置于细胞培养箱中培养,24h后,用不同浓度的漏芦花各提取有效部位(样品)100μl,终浓度(根据毒性试验确定)进行加样,培养2h后,向各孔中加LPS(终浓度1μg/ml)100μl,同时设溶剂空白对照孔(细胞+含与样品中等量溶剂的完全培养液),最大释放组(细胞+LPS+含等量溶剂的完全培养液),每个浓度设4个平行。接种24h后,收集上清液对亚硝酸根离子进行检测。取收集好的上清液50μl接种于96孔板中,再加入等体积的Griess试剂,震荡,室温反应10min,用酶标仪在540nm处测定吸光值。用浓度分别为1.25、2.5、5、10μmol/l的亚硝酸钠绘制标准曲线,根据标准曲线计算细胞培养上清液中亚硝酸根离子的浓度进而计算样品对一氧化碳释放的抑制率,如下式:
抑制率%=(A-B)/(A-C)×100
A-C:NO2-浓度(μM)[A:LPS(+),样品(-);B:LPS(+),样品(+);C:LPS(-),样品(-)].。
2.5结果与结论
结果见表4
表4漏芦花各提取有效部位抑制脂多糖刺激RAW264.7细胞产生NO
注:均值±标准差,与空白对照组比较*p<0.05,**P<0.01,***P<0.01
实验表中结果表明,实施例1中制备得到的漏芦花乙醇总提取物(CEE)、70%乙醇粗洗脱物(A2)、30%乙醇洗脱物(A2-2)、50%乙醇洗脱物(A2-3)均表现出较强的抑制一氧化氮产生的活性,其中A2-2、A2-3为活性较强的有效部位,即为蒙药漏芦花抗炎有效部位。
3、漏芦花抗炎有效部位化学成分的分离
根据体外药理活性实验,对漏芦花抗炎活性有效部位A2-2进行分离,从中得到10种单体化合物。
A2-2经常压硅胶柱色谱分离,以二氯甲烷-甲醇(100∶0→0∶100)梯度洗脱,经TLC检测合并,得到15个组分Fr.1~15,然后Fr.2用石油醚-乙酸乙酯(体积比10:1,5:1,1:1,1:4,1:8),乙酸乙酯,乙酸乙酯-甲醇(体积比1:1,1:5,1:10)梯度洗脱,再用Sephadex LH-20凝胶柱色谱(甲醇洗 脱)反复分离得到化合物2,10;Fr.3经反复重结晶和Sephadex LH-20凝胶柱色谱分离(甲醇洗脱),分别得到化合物3,4,5;Fr.4经反复重结晶和Sephadex LH-20凝胶柱色谱分离(甲醇洗脱),分别得到化合物3,5,6,9;Fr.8经常压硅胶柱色谱(氯仿-甲醇8:1,5:1,2:1,1:1)梯度洗脱,再用凝胶Sephadex LH-20柱色谱(甲醇洗脱)分离纯化,得到化合物8;Fr.10经常压硅胶柱色谱(氯仿-甲醇9:1,5:1,7:3,1:1)梯度洗脱,再用凝胶Sephadex LH-20柱色谱(甲醇洗脱)分离纯化,得到化合物1,7。
以上化合物通过核磁共振氢谱、碳谱、质谱等仪器分析,结合参考文献,确定这10种化合物分别为20-羟基蜕皮激素(1)、β-谷甾醇(2)、α-(反-3-甲氧基-4-o-β-D-吡喃葡萄糖基苯亚甲基)-β-(3,4-二甲氧基苄基)-γ-丁内酯(Hemislin B glucoside)(3)、木樨草素(4)、芹菜素(5)、槲皮素(6)、槲皮苷(7)、阿福豆苷(8)、硬脂酸(9)、没食子酸(10)。
4、应用RAW264.7体外抗炎模型对漏芦花有效部位单体化合物的活性测试
4.1实验材料:
细胞株:小鼠单核巨噬细胞RAW264.7购自中科院典型培养物保藏委员会细胞库;脂多糖(LPS)购自Sigma Chemical(St.Louis,MO),DMEM培养基购自Hyclone(USA)、青霉素、链霉素购自Sinopharm Chemical Reagent Co.Ltd.(Beijing,China)、胰蛋白酶(Trysin)、生理盐水(PBS)胎牛血清购自Gibco BRL(NY,USA);CCK-8购自Dojindo China Co.Ltd(Shanghai,China);Griess检测试剂盒,购自碧云天生物技术研究所,其他常用生化试剂均为国产分析纯。
受试物:从漏芦花有效部位分离得到的10种单体化合物,化学结构见3、漏芦花抗炎有效部位化学成分的分离。
4.2细胞培养
本发明选用小鼠单核巨噬细胞株RAW264.7以进行一氧化氮产生量的探讨。RAW264.7细胞在37℃、5%CO2条件下,用含10%胎牛血清、青霉素(1×105U/L)及链霉素(100mg/L)的DMEM培养基培养。待RAW264.7细胞生长至对数期 后备用
4.3细胞毒性试验
将对数生长期的RAW264.7细胞接种于96孔培养板上,并调整细胞密度为1×105个/mL,每孔体积100μl,每组8孔。37℃、5%CO2条件下培养箱培养24h后,加入不同浓度的各化合物溶液,每个浓度设3个复孔。培养24h后,弃去培养基,各孔加100μlCCK-8溶液(cck-8:无血清DMEM=1:9),继续培养1h,以酶标仪于波长450nm处测定OD值。并按公式计算各浓度下漏芦花各提取有效部位对正常RAW264.7细胞的抑制率,绘制标准曲线,找到最大无毒剂量。抑制率(%)=(细胞对照组光吸收值-药物组光吸收值)/(细胞对照组光吸收值)×100%
4.4NO含量检测
将生长于对数期的单核巨噬细胞用胰蛋白酶消化下来,并用细胞完全培养液将其调整到适宜的细胞密度(大约5×105个/ml)。然后接种到96孔板,每孔体积100μL,置于细胞培养箱中培养,24h后,将不同浓度(浓度范围为0.39,1.5625,6.25,25,100and 400μg/mL,终浓度根据毒性试验确定)的化合物溶液加入,培养2h后,向各孔中加LPS(终浓度1μg/ml)100μl,同时设溶剂空白对照孔(细胞+含与样品中等量溶剂的完全培养液),最大释放组(细胞+LPS+含等量溶剂的完全培养液),每个浓度设4个平行。接种24h后,收集上清液对亚硝酸根离子进行检测。取收集好的上清液50μl接种于96孔板中,再加入等体积的Griess试剂,震荡,室温反应10min,用酶标仪在540nm处测定吸光值。用浓度分别为1.25、2.5、5、10μmol/l的亚硝酸钠绘制标准曲线,根据标准曲线计算细胞培养上清液中亚硝酸根离子的浓度进而计算样品对一氧化碳释放的抑制率,如下式:
抑制率%=(A-B)/(A-C)×100
A-C:NO2-浓度(μM)[A:LPS(+),样品(-);B:LPS(+),样品(+);C:LPS(-),样品(-)].。
4.5结果与结论
结果见附图,由本发明附图可以看出,除了β-谷甾醇、硬脂酸,其他8种单体化合物均表现出一定的抑制一氧化氮生成的活性。相比漏芦花抗炎有效部位A2-2,木樨草素、芹菜素、槲皮素、槲皮苷、阿福豆苷、没食子酸展现出较强的抑制一氧化氮生成的活性;而20-羟基蜕皮激素、α-(反-3-甲氧基-4-o-β-D-吡喃葡萄糖基苯亚甲基)-β-(3,4-二甲氧基苄基)-γ-丁内酯抑制活性相对较弱。
Claims (10)
1.一种蒙药漏芦花抗炎有效部位的制备方法,其特征在于,包括以下步骤:
(1)将漏芦花药材粉碎成粗粉,按照漏芦花粗粉:溶剂=1:(4~20)(重量)的比例,回流提取2~3次,每次1~4小时,滤液回收溶剂,减压干燥,得到漏芦花醇提取物(CEE),其中所述溶剂为乙醇,质量浓度为50~90%;
(2)将步骤(1)所得漏芦花醇提取物加水稀释,漏芦花醇提取物:水=1:(4~10)(重量),稀释液经大孔吸附树脂柱富集,先用去离子水3~8BV(树脂体积)洗脱去杂后,再用5~10BV乙醇洗脱,收集乙醇洗脱液,减压回收乙醇溶剂,得到流浸膏,将流浸膏干燥,即得到漏芦花抗炎有效部位提取物,其中所述乙醇浓度为50~90%。
2.根据权利要求1的方法,其特征在于,步骤(1)中所述漏芦花药材为菊科植物祁州漏芦(Rhaponticum uniflorum(L.)DC)的干燥花。
3.根据权利要求1的方法,其特征在于,步骤(2)中所述大孔吸附树脂为非极性或弱极性大孔吸附树脂。
4.根据权利要求3的方法,其特征在于,所述大孔吸附树脂型号为D101或AB-8型大孔吸附树脂。
5.据权利要求1的方法,其特征在于,步骤(2)乙醇浓度优选70-80%。
6.根据权利要求1的方法,其特征在于,采用大孔吸附树脂纯化条件优选为漏芦花药材粗粉与树脂质量比为1:(1~5),树脂层析柱径高比1:(3~9),上样流速1~3mL/min/g。
7.按照权利要求1-6的任一方法制备得到的蒙药漏芦花抗炎有效部位。
8.按照权利要求1-6的任一方法制备得到的蒙药漏芦花抗炎有效部位用于制备抗炎药物。
9.按照权利要求1-6的任一方法制备得到的蒙药漏芦花抗炎有效部位与辅料制成口服制剂。
10.按照权利要求1-6的任一方法制备得到的蒙药漏芦花抗炎有效部位与辅料制成口服片剂、口服胶囊剂。
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104997825A (zh) * | 2015-06-17 | 2015-10-28 | 内蒙古医科大学 | 从漏芦花中提取总生物碱的方法 |
CN105628834A (zh) * | 2016-01-29 | 2016-06-01 | 内蒙古医科大学 | 蒙药漏芦花指纹图谱建立方法以及质量评价方法 |
CN109535277A (zh) * | 2019-01-07 | 2019-03-29 | 内蒙古医科大学 | 蒙药漏芦花多糖的提取工艺及其含量测定方法 |
CN110859848A (zh) * | 2019-12-06 | 2020-03-06 | 中国农业大学 | 百蕊草素i在制备治疗全身重症脓毒症药物中的用途 |
CN112174976A (zh) * | 2020-11-19 | 2021-01-05 | 江西中医药大学 | 从水芹菜中分离的二苯并呋喃型木脂素及其方法和在抗痛风性关节炎上的用途 |
CN115737664A (zh) * | 2023-01-05 | 2023-03-07 | 中央民族大学 | 一种具有抗炎活性的元宝枫叶提取物及其制备方法 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1224023A (zh) * | 1998-01-19 | 1999-07-28 | 沈阳药科大学 | 中药漏芦中总甾酮的医药用途及制备方法 |
CN103520242A (zh) * | 2013-10-18 | 2014-01-22 | 内蒙古医科大学 | 超声提取蒙药漏芦花总皂苷的方法 |
-
2014
- 2014-12-10 CN CN201410758224.9A patent/CN104490969B/zh not_active Expired - Fee Related
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1224023A (zh) * | 1998-01-19 | 1999-07-28 | 沈阳药科大学 | 中药漏芦中总甾酮的医药用途及制备方法 |
CN103520242A (zh) * | 2013-10-18 | 2014-01-22 | 内蒙古医科大学 | 超声提取蒙药漏芦花总皂苷的方法 |
Non-Patent Citations (4)
Title |
---|
吴吉英等: "蒙药漏芦花中黄酮类化合物的研究", 《天然产物研究与开发》 * |
季宇彬: "《天然药物有效成分药理与应用》", 31 July 2007 * |
李英杰等: "催化动力学光度法测定漏芦花中槲皮素", 《内蒙古民族大学学报(自然科学版)》 * |
欧阳平凯: "《生物科技辞典》", 31 January 2004 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104997825A (zh) * | 2015-06-17 | 2015-10-28 | 内蒙古医科大学 | 从漏芦花中提取总生物碱的方法 |
CN105628834A (zh) * | 2016-01-29 | 2016-06-01 | 内蒙古医科大学 | 蒙药漏芦花指纹图谱建立方法以及质量评价方法 |
CN109535277A (zh) * | 2019-01-07 | 2019-03-29 | 内蒙古医科大学 | 蒙药漏芦花多糖的提取工艺及其含量测定方法 |
CN110859848A (zh) * | 2019-12-06 | 2020-03-06 | 中国农业大学 | 百蕊草素i在制备治疗全身重症脓毒症药物中的用途 |
CN112174976A (zh) * | 2020-11-19 | 2021-01-05 | 江西中医药大学 | 从水芹菜中分离的二苯并呋喃型木脂素及其方法和在抗痛风性关节炎上的用途 |
CN115737664A (zh) * | 2023-01-05 | 2023-03-07 | 中央民族大学 | 一种具有抗炎活性的元宝枫叶提取物及其制备方法 |
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