CN107929337A - 一种屏边三七抗炎有效部位的制备方法及应用 - Google Patents
一种屏边三七抗炎有效部位的制备方法及应用 Download PDFInfo
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Abstract
本发明涉及一种屏边三七抗炎有效部位的制备方法及应用,属于生物医药技术领域。将屏边三七经粉碎后,甲醇浸提,浸提液减压浓缩得到甲醇浸膏;将得到的甲醇浸膏用水分散,采用石油醚萃取得到去除脂溶性杂质的萃余水溶液;将得到的萃余水溶液用饱和正丁醇萃取,萃取有机溶液经减压浓缩得正丁醇浸膏;将得到的正丁醇浸膏通过硅胶柱色谱分离,用二氯甲烷/甲醇溶剂体系洗脱,洗脱液浓缩干燥后得到屏边三七有效部位。本发明可有效大量制备屏边三七有效部位,实现屏边三七在制备抗炎药物中的应用。
Description
技术领域
本发明涉及一种屏边三七抗炎有效部位的制备方法及应用,属于生物医药技术领域。
背景技术
屏边三七为五加科人参属植物屏边三七(Panax stipuleanatus H.T. Tsai etK.M. Feng)的根及根茎,主产于云南东南部屏边、马关、麻栗坡等地区。民间称之为野三七、竹节七、白三七、香刺、土三七。传统以根及根茎入药,民间认为其具有清除瘀血、镇痛、促进伤口愈合、止血、滋补之功效。目前,国内外学者对屏边三七的研究甚少,仅在初步化学成分(杨崇仁, 等. 云南植物研究, 1985, (01): 103-108.)、种植方法(发明专利CN201610036240.6、CN201610700439.4)、快速鉴定方法(发明专利CN201611218427.4)、保育方法(发明专利CN201610702302.2)、干燥方法(发明专利CN201510563209.3)以及降血脂降血糖活性(发明专利CN201610352339.7)等方面有少量的研究。但是对于屏边三七的抗炎作用尚未有报道。本发明从屏边三七的主根中提取分离出抗炎有效部位,并将其应用于治疗炎症相关的疾病。
发明内容
针对上述现有技术存在的问题及不足,本发明提供一种屏边三七抗炎有效部位的制备方法及应用。本发明可有效大量制备屏边三七有效部位,实现屏边三七在制备抗炎药物中的应用。本发明通过以下技术方案实现。
一种屏边三七抗炎有效部位的制备方法,其具体步骤如下:
步骤1、粉碎、浸提:将屏边三七经粉碎后,根据液料比为1:5~10 kg/L甲醇浸提,浸提液减压浓缩得到甲醇浸膏;
步骤2、石油醚脱脂:将步骤1得到的甲醇浸膏用水分散,根据相比为1:1~1.5采用石油醚萃取得到去除脂溶性杂质的萃余水溶液;
步骤3、正丁醇萃取:将步骤2得到的萃余水溶液根据相比为1:1~2用饱和正丁醇萃取,萃取有机溶液经减压浓缩得正丁醇浸膏;
步骤4、分离和纯化:将步骤3得到的正丁醇浸膏通过硅胶柱色谱分离,用二氯甲烷/甲醇溶剂体系洗脱,洗脱液浓缩干燥后得到屏边三七有效部位。
上述制备得到的屏边三七有效部位主要成分包括屏边三七皂苷R1和R2,所述2种成分的重量之和在该有效部位中的含量不低于50%,其结构式如下:
屏边三七皂苷R1
屏边三七皂苷R2。
一种屏边三七抗炎有效部位位在制备治疗炎症的药物中的应用。上述炎症主要包括关节炎、肺炎、肝炎、肾炎等。
本发明的屏边三七抗炎有效部位在制备治疗炎症的药物制剂时,可以加入药学上可接受的载体、稀释剂或赋形剂。
本发明药物制剂可以为固体制剂或液体制剂,主要包括片剂、散剂、颗粒剂、胶囊剂、滴丸剂、注射剂、栓剂、气雾剂等。
本发明的有益效果是:
1、本发明的制备方法操作简单,易于获得大量的屏边三七有效部位,适合工业化生产;
2、本发明制备的屏边三七有效部位化学成分清楚,已知成分含量高;该有效部位具有显著的治疗急性、慢性炎症的效果。
具体实施方式
下面结合具体实施方式,对本发明作进一步说明。
实施例1
该屏边三七抗炎有效部位的制备方法,其具体步骤如下:
步骤1、粉碎、浸提:将屏边三七经粉碎后,根据液料比为1:10kg/L甲醇浸提3次,合并后的浸提液减压浓缩得到250.6g甲醇浸膏;
步骤2、石油醚脱脂:将步骤1得到的甲醇浸膏用1L水分散,根据相比为1:1.5采用石油醚萃取得到去除脂溶性杂质的萃余水溶液;
步骤3、正丁醇萃取:将步骤2得到的萃余水溶液根据相比为1:2用饱和正丁醇萃取,萃取有机溶液经减压浓缩得正丁醇浸膏;
步骤4、分离和纯化:将步骤3得到的正丁醇浸膏通过硅胶柱色谱分离,用二氯甲烷/甲醇溶剂体系(体积比例为95:5,90:10,80:20,60:40,50:50)进行梯度洗脱,收集洗脱液(体积比为二氯甲烷:甲醇 = 80:20,60:40),浓缩干燥后得到11.7g屏边三七有效部位。
上述制备得到的屏边三七有效部位经ODS柱色谱分离(溶剂系统按体积比为甲醇:水=55:45,75:25,80:20,90:10,100:0)为流动相洗脱,得到屏边三七皂苷R1(1.5g),屏边三七皂苷R1(5.6g)。屏边三七皂苷R1和R2重量之和在屏边三七有效部位中的含量为60.7%。
R1和R2化特征及化学结构的定性鉴定如下:
屏边三七皂苷R1
屏边三七皂苷R1(stipuleanoside R1):淡黄色粉末。1H-NMR (CD3OD, 500 MHz) δ:0.80 (3H, s), 0.83 (3H, s), 0.90 (3H, s), 0.93 (3H, s), 0.94 (3H, s), 1.04(3H, s), 1.15 (3H, s); 13C-NMR (CD3OD, 125 MHz): δ:181.82 (C-28), 172.7 (C-6'), 145.2 (C-13), 123.67 (C-12), 108.2 (C-1'''), 106.5 (C-1'), 104.4(C-1''),91.2 (C-3), 87.0 (C-4'''), 82.3 (C-2'''), 81.2 (C-3'), 79.4 (C-4'), 78.2 (C-3''), 78.2(C-5''), 77.9 (C-5'), 76.3 (C-2'), 75.5 (C-2''), 74.5 (C-3'''),71.0 (C-4''), 63.0 (C-6''), 62.2 (C-5'''), 57.0 (C-5), 49.1 (C-9), 47.6 (C-17), 47.2 (C-19), 42.9 (C-14), 42.7 (C-18), 40.6 (C-8), 40.2 (C-4), 39.7 (C-1), 37.9 (C-10), 34.9 (C-21), 34.0 (C-7), 33.8 (C-29), 33.7 (C-22), 31.6 (C-20), 28.8 (C-15), 28.4 (C-23), 26.9 (C-2), 26.4 (C-27), 24.5 (C-11), 24.0 (C-16), 24.0 (C-30), 19.3 (C-6), 17.7 (C-26), 17.0 (C-24), 16.0 (C-25).
屏边三七皂苷R2
屏边三七皂苷R2(stipuleanoside R2):淡黄色粉末。1H-NMR (CD3OD, 500 MHz) δ:0.77 (3H, s), 0.78 (3H, s), 0.90 (3H, s), 0.92 (3H, s), 0.93 (3H, s), 1.03(3H, s), 1.14 (3H, s); 13C-NMR (CD3OD, 125 MHz):δ:178.0 (C-28), 176.5 (C-6'),144.6 (C-13), 123.8 (C-12), 107.9 (C-1'''), 106.4 (C-1'), 104.4 (C-1''), 95.7(C-1''''), 91.0 (C-3), 87.1 (C-4'''), 82.0 (C-2'''), 79.5 (C-3'), 79.5 (C-4'), 78.7 (C-5''''), 78.3 (C-5''), 78.2 (C-3''), 78.1 (C-5'), 78.0 (C-3''''),76.4 (C-2'), 75.6 (C-2''), 74.8 (C-3'''), 73.9 (C-2''''), 71.0 (C-4''), 71.0(C-4''''), 63.2 (C-6''), 62.4 (C-5'''), 62.2 (C-6''''), 57.0 (C-5), 49.1 (C-9), 48.0 (C-17), 47.2 (C-19), 42.9 (C-14), 42.6 (C-18), 40.7 (C-8), 40.2 (C-4), 39.8 (C-1), 37.9 (C-10), 34.9 (C-21), 33.9 (C-7), 33.5 (C-29), 33.1 (C-22), 31.5 (C-20), 28.9 (C-15), 28.5 (C-23), 26.9 (C-2), 26.3 (C-27), 24.5 (C-11), 24.0 (C-16), 24.0 (C-30), 19.3 (C-6), 17.7 (C-26), 17.0 (C-24), 16.0 (C-25).
该屏边三七抗炎有效部位在制备治疗炎症的药物中的应用。
(一)将本实施例制备得到的屏边三七抗炎有效部位的抗急性炎症活性
研究了屏边三七有效部位对二甲苯致小鼠耳肿胀的影响。
实验方法:
取60只昆明种小鼠(体重18~22 g,雌雄各半)适应性喂养2天,随机分成5组,每组12只,模型组、地塞米松组(10 mg/kg)及屏边三七有效部位低(25 mg/kg)、中(50 mg/kg)、高剂量组(100 mg/kg)。各组小鼠每天灌胃给药1次,给予模型组相同体积的蒸馏水,连续7 d。各组在于末次给药后在每只小鼠右耳内、外两侧各涂抹20uL二甲苯溶液。6h后将小鼠断颈处死,剪下左右耳,用直径6mm打孔器取双侧耳部同一部位的耳片,称重计算两耳片的重量差。小鼠耳肿胀度按以下公式计算:耳肿胀度=右耳重量-左耳重量。
实验结果:
由表1可知,屏边三七有效部位高、中剂量组及地塞米松组的耳肿胀度显著低于模型组(p<0.01)。屏边三七有效部位可显著抑制小鼠二甲苯致耳肿胀,使小鼠耳肿胀度显著降低。说明屏边三七有效部位具有抗急性炎症活性。
表 1 屏边三七有效部位对二甲苯致小鼠耳肿胀的影响
与模型组比较,* p<0.05,** p<0.01
(二)将本实施例制备得到的屏边三七抗炎有效部位的抗慢性炎症活性
研究屏边三七有效部位对小鼠棉球肉芽肿的影响。
实验方法:
取60只昆明种小鼠(体重18~22 g,雌雄各半)随机分成5组,每组12只,即模型组、地塞米松组(10 mg/kg)及屏边三七有效部位低(25 mg/kg)、中(50 mg/kg)、高剂量组(100 mg/kg)。各组均于腋下皮下植入同等大小无菌棉球(10 mg)。造模成功后于次日,各组小鼠每天灌胃给药1次,给予模型组相同体积的蒸馏水,连续7 d。末次给药12 h后将小鼠颈椎脱臼处死,取出棉球及周围的结缔组织,去除脂肪组织,在60 ℃下烘干,称重,按以下公式计算肉芽肿胀度:肉芽肿胀度=棉球肉芽肿干重-棉球原始质量
实验结果:
由表2可见,与模型组比较,屏边三七有效部位低、中、高剂量组及地塞米松组均能显著性降低小鼠棉球肉芽肿胀度(p<0.05,p<0.01),且呈剂量依赖关系。屏边三七有效部位对棉球导致的小鼠肉芽肿有一定的抑制作用,对慢性炎症具有一定的治疗作用。
表 2 屏边三七有效部位对小鼠棉球肉芽肿的影响(`x±s,n=12 )
与模型组比较,* p<0.05,** p<0.01。
实施例2
该屏边三七抗炎有效部位的制备方法,其具体步骤如下:
步骤1、粉碎、浸提:将屏边三七经粉碎后,根据液料比为1:5kg/L甲醇浸提3次,合并后的浸提液减压浓缩得到250.6g甲醇浸膏;
步骤2、石油醚脱脂:将步骤1得到的甲醇浸膏用1L水分散,根据相比为1:1采用石油醚萃取得到去除脂溶性杂质的萃余水溶液;
步骤3、正丁醇萃取:将步骤2得到的萃余水溶液根据相比为1:1用饱和正丁醇萃取,萃取有机溶液经减压浓缩得正丁醇浸膏;
步骤4、分离和纯化:将步骤3得到的正丁醇浸膏通过硅胶柱色谱分离,用二氯甲烷/甲醇溶剂体系(体积比例为95:5,90:10,80:20,60:40,50:50)进行梯度洗脱,收集洗脱液(体积比为二氯甲烷:甲醇 = 80:20,60:40),浓缩干燥后得到10.3 g屏边三七有效部位。
实施例3
该屏边三七抗炎有效部位的制备方法,其具体步骤如下:
步骤1、粉碎、浸提:将屏边三七经粉碎后,根据液料比为1:8kg/L甲醇浸提3次,合并后的浸提液减压浓缩得到245.4g甲醇浸膏;
步骤2、石油醚脱脂:将步骤1得到的甲醇浸膏用1L水分散,根据相比为1:1.2采用石油醚萃取得到去除脂溶性杂质的萃余水溶液;
步骤3、正丁醇萃取:将步骤2得到的萃余水溶液根据相比为1:1.2用饱和正丁醇萃取,萃取有机溶液经减压浓缩得正丁醇浸膏;
步骤4、分离和纯化:将步骤3得到的正丁醇浸膏通过硅胶柱色谱分离,用二氯甲烷/甲醇溶剂体系(体积比例为95:5,90:10,80:20,60:40,50:50)进行梯度洗脱,收集洗脱液(体积比为二氯甲烷:甲醇=80:20,60:40),浓缩干燥后得到10.8g屏边三七有效部位。
以上对本发明的具体实施方式作了详细说明,但是本发明并不限于上述实施方式,在本领域普通技术人员所具备的知识范围内,还可以在不脱离本发明宗旨的前提下作出各种变化。
Claims (2)
1.一种屏边三七抗炎有效部位的制备方法,其特征在于具体步骤如下:
步骤1、粉碎、浸提:将屏边三七经粉碎后,根据液料比为1:5~10 kg/L甲醇浸提,浸提液减压浓缩得到甲醇浸膏;
步骤2、石油醚脱脂:将步骤1得到的甲醇浸膏用水分散,根据相比为1:1~1.5采用石油醚萃取得到去除脂溶性杂质的萃余水溶液;
步骤3、正丁醇萃取:将步骤2得到的萃余水溶液根据相比为1:1~2用饱和正丁醇萃取,萃取有机溶液经减压浓缩得正丁醇浸膏;
步骤4、分离和纯化:将步骤3得到的正丁醇浸膏通过硅胶柱色谱分离,用二氯甲烷/甲醇溶剂体系洗脱,洗脱液浓缩干燥后得到屏边三七有效部位。
2.一种根据权利要求1所述的屏边三七抗炎有效部位在制备治疗炎症的药物中的应用。
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CN111494447A (zh) * | 2020-06-23 | 2020-08-07 | 云南中医药大学 | 一种抗耐药真菌药物组合物及其制剂 |
CN111494447B (zh) * | 2020-06-23 | 2022-05-27 | 云南中医药大学 | 一种抗耐药真菌药物组合物及其制剂 |
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