CN106198166A - 一种黑草总黄酮的提取与测定方法 - Google Patents
一种黑草总黄酮的提取与测定方法 Download PDFInfo
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- CN106198166A CN106198166A CN201610544766.5A CN201610544766A CN106198166A CN 106198166 A CN106198166 A CN 106198166A CN 201610544766 A CN201610544766 A CN 201610544766A CN 106198166 A CN106198166 A CN 106198166A
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- total flavones
- blackwort
- symphytum
- blackwort symphytum
- solution
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Abstract
本发明公开了一种黑草中总黄酮的提取和测定方法,用D101大孔树脂装柱,乙醇洗脱提取黑草总黄酮;以芦丁作为对照品,加入亚硝酸钠、硝酸铝、氢氧化钠测定吸光度,依次确定总黄酮含量,通过高效液相色谱法测定出黑草总黄酮中木犀草素、芹菜素及香叶木素的含量,流动相为乙腈‑0.1%磷酸,安捷伦ZORBAX Eclipse XDB‑C18为色谱柱,检测波长350nm。黑草总黄酮具有抗凝、抗炎、镇静和抗过敏作用,且毒性小,本发明为黑草总黄酮的药用价值开发提供了参考数据。
Description
技术领域
本发明涉及一种植物总黄酮的提取与测定方法,具体是一种黑草总黄酮提取物的提取方法与测定方法。
背景技术
自然界很多植物含有黄酮类化合物。据估计,我国传统中草药有 20%含有黄酮类化合物。黄酮一直是医药及食品工业的热点研究物质,因为黄酮类化合物具有多种生理作用,例如芦丁、槲皮素、槲皮苷能增强心脏收缩,减少心脏搏动次数;法尔杜鹃素、紫花杜鹃素等有止咳祛痰作用;牡荆素、汉黄芩素等具有抗肿瘤活性;水飞蓟素有保肝作用等。植物所含的黄酮一般不以单一成分存在,而以几种结构相似或相近的成分共同存在,其总量称为总黄酮含量。
黑草为玄参科鬼羽箭属植物Buchnera cruciata Hamilt.的干燥全草,又名幼克草、克草、黑骨草、羽箭草,分布于我国江西、福建、湖南、湖北、广东、广西、贵州和云南等地,在广西各地均有分布。其味微苦,寒,性凉,具有清热,凉血,解毒等功效,用于治疗流行感冒、中暑腹痛、蛛网膜下腔出血、暑热口渴、腹痛、荨麻疹等疾患。
目前已有相关黑草的研究,【文献】黑草醇提物的药理作用研究【作者】李燕婧,钟正贤,张颖,卢文杰,牙启康【刊名】广西中医药, 2012,35(1):49-51,通过黑草提取物进行相关药理试验,证明黑草提取物具有一定的抗凝、抗炎、镇痛和抗过敏作用,且毒性小,对于黑草成分的检测也已有相关研究,【文献】黑草药材 HPLC指纹图谱的研究【作者】刘吉成,陈大建【刊名】药物分析杂志, 2013, 33(3): 424-428,报道了以木犀草素素成分为参照峰,采用 HPLC法建立黑草药材的指纹图谱,对不同产地的黑草进行了检测,所建立的指纹图谱,为黑草药材的质量控制提供参考,进而对于其相应制剂的质量控制也会起积极的作用;色谱柱:Agilent Extend-C18(4.6 mm×250 mm,5µm),流动相:乙腈与 0.7%冰醋酸,梯度洗脱;流速1.0 mL·mi n-1;检测波长350 nm;柱温:35℃;进样量:10µl。【文献】黑草的化学成分研究【作者】卢文杰,牙启康,陈家源,谭晓,黄艳,李晓华,周斌【刊名】中草药, 2012,43(6): 1079-1081,对黑草的化学成分进行了检测,黑草提取物的提取方法为:将乙醇回流所得浸膏用水混悬,用石油醚萃取。所得石油醚萃取物经硅胶柱色谱分离,以石油醚-醋酸乙酯体积比分别为100:0和70:30进行梯度洗脱,TLC检测,合并相同组分,再经反复硅胶柱色谱得到化合物B-胡萝卜苷、谷甾醇、甘露醇、正二十七烷醇、植醇、二十九烷、棕榈酸、十八碳烯酸,醋酸乙酯萃取物经硅胶柱色谱分离,以醋酸乙酯-甲醇系统梯度洗脱,再经反复重结晶,得到化合物芹菜素、木犀草素、香叶木素。【文献】HPLC测定黑草中木犀草素和芹菜素的含量【作者】刘 吉成【刊名】中国实验方剂学杂志, 2012,18(20):72-74,黑草用甲醇回流提取,所得提取液用高效液相色谱测定黑草药材中木犀草素和芹菜素的含量,液相色谱条件为:Agilent Extend C18(4.6 mm×250 mm,5μm)色谱柱分离,以乙腈:0.7%冰醋酸 (30:70)为流动相,流速 1.0 mL·min-1,检测波长350 nm;柱温35℃,进样量l0µL。
黑草具有极高的药用价值,现有技术中还没有公开对黑草总黄酮含量及总黄酮中的成分含量,因此无法确定黑草总黄酮的药用机理,阻碍了黑草药用价值的开发。本发明提供了一种黑草总黄酮的提取方法和测定方法,同时对总黄酮中的木犀草素、芹菜素及香叶木素的含量进行了测定。
发明内容
本发明的目的是提供一种黑草总黄酮的提取方法和测定方法,同时用高效液相色谱法对总黄酮中的木犀草素、芹菜素及香叶木素的含量进行了准确测定,方法合理、稳定性好,为黑草药用价值的研究提供了参考数据,有利于黑草的进一步开发利用。
本发明所述的黑草总黄酮的提取方法,包括如下步骤:
(1)D101大孔树脂的预处理:以体积浓度95%乙醇浸泡D101大孔树脂20-30h,充分溶胀后,除去上浮树脂及杂物,用湿法装柱,继续用体积浓度95%乙醇不断洗涤,洗至流出液与水混合无白色浑浊现象,改用水洗至无醇味,备用;所述流出液与水混合的体积比为1:3;
(2)药材的提取、浓缩:取黑草药材,粉碎成粗粉,加入10倍于黑草重量的体积浓度60%乙醇;加热回流提取5次,每次2小时,提取液滤过,合并滤液;滤液回收乙醇,并浓缩至无醇味;
(3)将步骤(2)中的浓缩液上至(1)柱中,用体积浓度5%-95%乙醇洗至颜色较浅,收集并浓缩至干,即得黑草总黄酮。
所述的黑草总黄酮含量以芦丁计算不少于重量含量50.0%。
本发明提供了一种黑草总黄酮提取物的测定方法,采用分光光度法测定黑草总黄酮提取物,包括以下步骤:
(1)对照品储备液的制备:精密称取芦丁对照品,加甲醇溶解并稀释至刻度,摇匀,制得质量浓度为0.2074mg/mL的溶液;
(2)标准曲线的绘制:分别精密吸取对照品储备液0.0、1.0、2.0、3.0、4.0、5.0、6.0mL,分别至25mL量瓶中,分别加水至6.0mL,加5%亚硝酸钠溶液1.0 mL,摇匀,放置6分钟,加重量含量10%硝酸铝1.0mL,摇匀,放置6分钟;加重量含量4%氢氧化钠溶液10.0mL,再加水至刻度,摇匀,放置15分钟。于500nm波长处测定吸光度A,并进行空白对照,以对照品吸光度A为纵坐标,浓度C为横坐标绘制标准曲线;
(3)样品测定:精密吸取供试品溶液2mL,至25mL量瓶中,照标准曲线制备项下的方法,加水至6.0mL,加重量含量5%亚硝酸钠溶液1.0 mL,摇匀,放置6分钟,加10%硝酸铝1.0mL,摇匀,放置6分钟;加4%氢氧化钠溶液10.0mL,再加水至刻度,摇匀,放置15分钟。于500nm波长处测定吸光度,并进行空白对照,计算出总黄酮的含量,黑草中总黄酮含量以芦丁计算不少于重量含量50%。
本发明还提供了一种液相色谱法同时测定黑草总黄酮中木犀草素、芹菜素及香叶木素的含量的方法,其液相色谱条件为:
色谱柱:安捷伦ZORBAX Eclipse XDB-C18(4.6×250mm,5µm)色谱柱;
流动相:乙腈-0.1%磷酸(25:75);
流速为1mL/min;
检测波长为350nm,进样量为10µL。
含量测定方法包括以下步骤:
(1)对照品溶液的制备:分别精密称取木犀草素对照品14.53mg、芹菜素对照品4.32mg以及香叶木素对照品4.25mg,置于同一50mL容量瓶中,用甲醇溶解并稀释至刻度,摇匀,得木犀草素浓度为290.6µg/mL、芹菜素浓度为86.4µg/mL及香叶木素浓度为85µg/mL的对照品储备液。取对照品储备液2mL,置25mL容量瓶中,以甲醇稀释至刻度,摇匀,得木犀草素浓度为23.25µg/mL、芹菜素浓度为6.91µg/mL及香叶木素浓度为6.8µg/mL的对照品溶液。
(2)供试品溶液的制备:精密称取样品10mg,置25mL容量瓶中,用甲醇溶解并稀释至刻度,摇匀、滤过即得。
(3)样品的测定:对对照品溶液及供试品溶液进行高效液相色谱分析,记录峰面积,并基于所得到的液相色谱分析结果,采用外标法计算所述黑草总黄酮中木犀草素、芹菜素及香叶木素的含量。黑草总黄酮中木犀草素含量不少于重量含量5%、芹菜素含量不少于重量含量1.5%、香叶木素含量不少于重量含量0.5%;
本发明还提供了一种黑草中总黄酮的提取和测定方法,高效液相色谱法可有有效测定出黑草总黄酮中木犀草素、芹菜素及香叶木素的含量。黑草总黄酮具有抗凝、抗炎、镇静和抗过敏作用,且毒性小,本发明为黑草总黄酮的药用价值开发提供了参考数据。
测定过程包括以下步骤:
(1)色谱条件为:
色谱柱:安捷伦ZORBAX Eclipse XDB-C18:4.6×250mm,5µm色谱柱;
流动相:乙腈-0.1%磷酸;
流速为1mL/min;
检测波长为350nm,进样量为10µL;
所述流动相的体积比为25:75;
(2)步骤:
(A)对照品溶液的制备:分别精密称取木犀草素对照品14.53mg、芹菜素对照品4.32mg以及香叶木素对照品4.25mg,置于同一50mL容量瓶中,用甲醇溶解并稀释至刻度,摇匀,得木犀草素浓度为290.6µg/mL、芹菜素浓度为86.4µg/mL及香叶木素浓度为85µg/mL的对照品储备液。取对照品储备液2mL,置25mL容量瓶中,以甲醇稀释至刻度,摇匀,得木犀草素浓度为23.25µg/mL、芹菜素浓度为6.91µg/mL及香叶木素浓度为6.8µg/mL的对照品溶液;
(B)供试品溶液的制备:精密称取样品10mg,置25mL容量瓶中,用甲醇溶解并稀释至刻度,摇匀、滤过即得;
(C)样品的测定:对对照品溶液及供试品溶液进行高效液相色谱分析,记录峰面积,并基于所得到的液相色谱分析结果,采用外标法计算所述黑草总黄酮中木犀草素、芹菜素及香叶木素的含量。
上述黑草总黄酮的测定方法中,草总黄酮中木犀草素含量不少于重量含量5%、芹菜素含量不少于重量含量1.5%、香叶木素含量不少于重量含量0.5%。
附图说明
图1 对照品色谱图。
图2 供试品色谱图。
具体实施例
实施例1
(1)D101大孔树脂的预处理
以95%乙醇浸泡D101大孔树脂24h,充分溶胀后,除去上浮树脂及杂物,用湿法装柱,继续用体积浓度95%乙醇不断洗涤,洗至流出液与水体积比1:3混合无白色浑浊现象,改用水洗至无醇味,备用。
(2)药材的提取、浓缩
取黑草药材100g,粉碎成粗粉,加入1000mL体积浓度60%乙醇;加热回流提取5次,每次2小时,提取液滤过,合并滤液;滤液回收乙醇,并浓缩至无醇味。
(3)将步骤(2)中的浓缩液上至(1)柱中,分别用体积浓度10%,30%,50%,70%,95%乙醇洗至颜色较浅,收集并浓缩至干。
(4)洗脱液采用薄层色谱法进行检视,发现体积浓度70%乙醇洗脱部位所得黄酮种类最多,即得黑草总黄酮。
实施例2
(1)仪器与试药
①仪器:UV2550紫外-可见分光光度计(日本岛津公司)、XS205电子天平(瑞士梅特勒-托利多公司)
②试药:芦丁对照品(中检所,100080-200306)、甲醇、亚硝酸钠、硝酸铝、氢氧化钠
(2)溶液的制备
①对照品储备液的制备:精密称取芦丁对照品10.37mg,加甲醇溶解并稀释至刻度,摇匀即得(浓度为0.2074mg/mL)。
②标准曲线的绘制:分别精密吸取对照品储备液0.0、1.0、2.0、3.0、4.0、5.0、6.0mL,分别至25mL量瓶中,分别加水至6.0mL,加重量含量5%亚硝酸钠溶液1.0 mL,摇匀,放置6分钟,加重量含量10%硝酸铝1.0mL,摇匀,放置6分钟;加重量含量4%氢氧化钠溶液10.0mL,再加水至刻度,摇匀,放置15分钟。于500nm波长处测定吸收度,以对照品吸收度(A)对浓度(C,mg/mL)进行线性回归,得回归方程:
A=12.489C+0.0025(r2=0.9997)
③供试品溶液的制备:分别精密称取样品23.22mg、23.39mg,置25mL量瓶中,加甲醇溶解并定容至刻度,摇匀,即得。
④空白溶液的制备:以相应溶液作为空白。
(3)样品测定
精密吸取供试品溶液2mL,至25mL量瓶中,照标准曲线制备项下的方法,加水至6.0mL,加重量含量5%亚硝酸钠溶液1.0 mL,摇匀,放置6分钟,加重量含量10%硝酸铝1.0mL,摇匀,放置6分钟;加重量含量4%氢氧化钠溶液10.0mL,再加水至刻度,摇匀,放置15分钟。于500nm波长处测定吸收度,计算出总黄酮的含量(以芦丁计)
(4)结果计算
从回归方程计算出供试品溶液中总黄酮的浓度,进而计算出总黄酮的含量(以芦丁计)。黑草中总黄酮含量以芦丁计算不少于重量含量50%。
实施例3
(1)仪器与试药
仪器:高效液相色谱仪LC-10A(日本岛津公司)、威玛龙色谱工作站、XS205电子天平(瑞士梅特勒-托利多公司)
试药:木犀草素对照品(上海源叶生物科技有限公司,LOT:YA0408YA13)、芹菜素对照品(上海源叶生物科技有限公司,LOT:K18D5C1)、香叶木素对照品(上海源叶生物科技有限公司,批号:PM0509RA13)、甲醇、乙腈、磷酸。
(2)溶液的制备
①对照品溶液的制备:分别精密称取木犀草素对照品14.53mg、芹菜素对照品4.32mg以及香叶木素对照品4.25mg,置于同一50mL容量瓶中,用甲醇溶解并稀释至刻度,摇匀,得木犀草素浓度为290.6µg/mL、芹菜素浓度为86.4µg/mL及香叶木素浓度为85µg/mL的对照品储备液。取对照品储备液2mL,置25mL容量瓶中,以甲醇稀释至刻度,摇匀,得木犀草素浓度为23.25µg/mL、芹菜素浓度为6.91µg/mL及香叶木素浓度为6.8µg/mL的对照品溶液。
②供试品溶液的制备:精密称取样品10mg,置25mL容量瓶中,用甲醇溶解并稀释至刻度,摇匀、滤过即得。
(3)样品的测定
用高效液相色谱仪LC-10测定对照品溶液及供试品溶液。色谱条件为:采用安捷伦ZORBAX Eclipse XDB-C18(4.6×250mm,5µm)色谱柱,流动相为乙腈-0.1%磷酸的体积比为25:75,流速为1mL/min,检测波长为350,进样量为10µL。
(4)计算结果
根据得到的对照品及样品的色谱图,记录峰面积,采用外标法计算含量。本发明黑草总黄酮中木犀草素含量不少于5%、芹菜素含量不少于1.5%、香叶木素含量不少于0.5%。
实施例4 黑草总黄酮药理作用实验
(1)黑草总黄酮对小鼠凝血时间的影响
取18~22g小鼠50只,雌雄各半,随机分为空白对照组,黑草总黄酮高、中、低剂量组(10.0,5.0,3.0g/kg)和阳性阿司匹林组(0.2g/kg),每组10只小鼠。用药各组均予灌胃给药,空白对照组给予等量蒸馏水,每天1次,连续7d。末次给药1h后,用内径为1mm的玻璃毛细管插入小鼠眦球后静脉丛取血,至毛细玻璃管血柱达5cm为止,每隔30s折断毛细玻璃管一小段,同时用肉眼观察血柱是否移动,仔细检查有无血丝出现,计算从毛细管采血到凝血丝出现的时间,进行组间比较。结果见表1。
结果表明:与空白对照组比较,黑草总黄酮高、中、低剂量组及阿司匹林组均能明显延长小鼠凝血时间(P﹤0.05或P﹤0.01),提示黑草总黄酮具有抗凝血作用。
(2)黑草总黄酮对醋酸所致小鼠扭体次数的影响取18~22g小鼠50只,雌雄各半,随机分为空白对照组,黑草总黄酮高、中、低剂量组(10.0,5.0,3.0g/kg)和阳性阿司匹林组(0.2g/kg),每组10只小鼠。用药各组均予灌胃给药,空白对照组给予等量蒸馏水,每天1次,连续7d。末次给药1h后,每只小鼠腹腔注射0.6%醋酸溶液0.2mL,立即观察并记录20min内各小鼠的扭体次数。比较给药各组与空白对照组之间的差异,评价受试药的镇痛作用。结果见表2。
结果表明:与空白对照组比较,黑草总黄酮高、中、低剂量组及阿司匹林组均能明显减少小鼠扭体次数(P﹤0.05或P﹤0.01),提示黑草总黄酮具有镇痛作用。
(3)黑草总黄酮对巴豆油所致小鼠耳廓肿胀的抑制作用取18~22g小鼠50只,雌雄各半,随机分为空白对照组,黑草总黄酮高、中、低剂量组(10.0,5.0,3.0g/kg)和阳性阿司匹林组(0.2g/kg),每组10只小鼠。用药各组均予灌胃给药,空白对照组给予等量蒸馏水,每天1次,连续7d。末次给药0.5h后,用微量注射器取0.05mL2%巴豆油溶液涂于小鼠右耳。4h后处死小鼠,立即用口径为8mm的打孔器沿着小鼠左右耳廓相同部位打孔取材,然后在电子天平上分别称重,以两耳片重量(mg)的差值作为肿胀程度指标,并求出百分抑制率(%)。结果见表3。
结果表明:与空白对照组比较,黑草总黄酮高、中、低剂量组及阿司匹林组均能明显降低小鼠耳廓肿胀程度(P﹤0.05),提示黑草总黄酮具有抗炎作用。
(4)黑草总黄酮对小鼠被动皮肤过敏试验
抗原:医药天花粉。佐剂:4%氢氧化铝凝胶。临用时以3.75mg天花粉溶于1.5mL氢氧化铝凝胶中。致敏:健康小鼠10只,体重18~22g,将上述天花粉氢氧化铝凝胶混悬液予脚掌注射,每只小鼠的两只后脚掌各注入0.05mL,共注射0.1mL。15天后,断头取血,离心取抗血清备用。
被动皮肤致敏试验:取18~22g小鼠50只,雌雄各半,随机分为空白对照组,黑草总黄酮高、中、低剂量组(10.0,5.0,3.0g/kg)和阳性息斯敏组(0.01g/kg),每组10只小鼠。用药各组均予灌胃给药,空白对照组给予等量蒸馏水,每天1次,连续7d。取上述血清(10只致敏小鼠的混合抗血清)加生理盐水稀释成1:5,每鼠腹壁皮内注射两个点(相距0.2cm),每点0.03mL。再连续给药2天,于末次给药12h后,每只小鼠进行抗原攻击,尾静脉注射天花粉2.5mg/mL(用1%伊文思蓝-生理盐水配制),0.2mL/只,20min后处死动物,剪下腹部蓝斑皮肤(0.5g),剪碎,浸泡于丙酮生理盐水溶液(7:3)5mL中48小时,离心取上清液,用分光光度计于610nm波长处测定吸收度,并作组间比较,评价药物的抗过敏作用。结果见表4。
结果表明:与空白对照组比较,黑草总黄酮高、中剂量组及息斯敏组均能明显降低小鼠皮肤蓝斑吸光度(P﹤0.05或P﹤0.01),提示黑草总黄酮具有皮肤抗过敏作用。
(5)急性毒性试验表明,黑草总黄酮的最大给药量为410g生药/kg体重。
黑草总黄酮高、中、低剂量组及阿司匹林组均能明显延长小鼠凝血时间(P﹤0.05或P﹤0.01);黑草总黄酮高、中、低剂量组及阿司匹林组均能明显减少小鼠扭体次数(P﹤0.05或P﹤0.01);黑草总黄酮高、中、低剂量组及阿司匹林组均能明显降低小鼠耳廓肿胀程度(P﹤0.05);黑草总黄酮高、中剂量组及息斯敏组均能明显降低小鼠皮肤蓝斑吸光度(P﹤0.05或P﹤0.01)。急性毒性试验表明,黑草总黄酮的最大给药量为410g生药/kg体重。说明黑草总黄酮具有抗凝、抗炎、镇痛和抗过敏作用,且毒性小。
Claims (5)
1.一种黑草总黄酮的提取方法,其特征在于,包括如下步骤:
(1)D101大孔树脂的预处理:以体积浓度95%乙醇浸泡D101大孔树脂20-30h,充分溶胀后,除去上浮树脂及杂物,用湿法装柱,继续用95%乙醇不断洗涤,洗至流出液与水混合无白色浑浊现象,改用水洗至无醇味,备用;所述流出液与水混合的体积比为1:3;
(2)药材的提取、浓缩:取黑草药材,粉碎成粗粉,加入10倍于黑草重量的体积浓度60%乙醇;加热回流提取5次,每次2小时,提取液滤过,合并滤液;滤液回收乙醇,并浓缩至无醇味;
(3)将步骤(2)中的浓缩液上至(1)柱中,用体积浓度5%-95%乙醇洗至颜色较浅,收集并浓缩至干,即得黑草总黄酮;
所述的黑草总黄酮中含有木犀草素、芹菜素和香叶木素。
2.根据权利要求1所述的黑草总黄酮的提取方法,其特征在于:所述的黑草总黄酮含量以芦丁计算重量含量不少于50.0%。
3.权利要求1所述的黑草总黄酮的测定方法,其特征在于,所述的黑草总黄酮的测定方法采用分光光度法;测定过程包括以下步骤:
(1)对照品储备液的制备:精密称取芦丁对照品,加甲醇溶解并稀释至刻度,摇匀,制得质量浓度为0.2074mg/mL的溶液;
(2)标准曲线的绘制:分别精密吸取对照品储备液0.0、1.0、2.0、3.0、4.0、5.0、6.0mL,分别至25mL量瓶中,分别加水至6.0mL,加重量含量5%亚硝酸钠溶液1.0 mL,摇匀,放置6分钟,加重量含量10%硝酸铝1.0mL,摇匀,放置6分钟;加重量含量4%氢氧化钠溶液10.0mL,再加水至刻度,摇匀,放置15分钟;于500nm波长处测定吸光度A,并进行空白对照,以对照品吸光度A为纵坐标,浓度C为横坐标绘制标准曲线;
(3)样品测定:精密吸取供试品溶液2mL,至25mL量瓶中,照标准曲线制备项下的方法,加水至6.0mL,加重量含量5%亚硝酸钠溶液1.0 mL,摇匀,放置6分钟,加重量含量10%硝酸铝1.0mL,摇匀,放置6分钟;加4%氢氧化钠溶液10.0mL,再加水至刻度,摇匀,放置15分钟;于500nm波长处测定吸光度,并进行空白对照,计算出总黄酮的含量,黑草中总黄酮含量以芦丁计算不少于50%。
4.权利要求1所述的黑草总黄酮的测定方法,其特征在于,所述的黑草总黄酮中木犀草素、芹菜素及香叶木素含量的测定方法,采用高效液相色谱法,测定过程包括以下步骤:
(1)色谱条件为:
色谱柱:安捷伦ZORBAX Eclipse XDB-C18:4.6×250mm,5µm色谱柱;
流动相:乙腈-0.1%磷酸;
流速为1mL/min;
检测波长为350nm,进样量为10µL;
所述流动相的体积比为25:75;
(2)步骤:
(A)对照品溶液的制备:分别精密称取木犀草素对照品14.53mg、芹菜素对照品4.32mg以及香叶木素对照品4.25mg,置于同一50mL容量瓶中,用甲醇溶解并稀释至刻度,摇匀,得木犀草素浓度为290.6µg/mL、芹菜素浓度为86.4µg/mL及香叶木素浓度为85µg/mL的对照品储备液;取对照品储备液2mL,置25mL容量瓶中,以甲醇稀释至刻度,摇匀,得木犀草素浓度为23.25µg/mL、芹菜素浓度为6.91µg/mL及香叶木素浓度为6.8µg/mL的对照品溶液;
(B)供试品溶液的制备:精密称取样品10mg,置25mL容量瓶中,用甲醇溶解并稀释至刻度,摇匀、滤过即得;
(C)样品的测定:对对照品溶液及供试品溶液进行高效液相色谱分析,记录峰面积,并基于所得到的液相色谱分析结果,采用外标法计算所述黑草总黄酮中木犀草素、芹菜素及香叶木素的含量。
5.根据权利要求4所述的黑草总黄酮的测定方法,其特征在于,黑草总黄酮中木犀草素含量不少于重量含量5%、芹菜素含量不少于重量含量1.5%、香叶木素含量不少于重量含量0.5%。
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CN106769964A (zh) * | 2016-12-09 | 2017-05-31 | 中国科学院新疆理化技术研究所 | 一枝蒿提取物的质量控制方法 |
CN106769964B (zh) * | 2016-12-09 | 2019-05-14 | 中国科学院新疆理化技术研究所 | 一枝蒿提取物的质量控制方法 |
CN110057646A (zh) * | 2019-05-29 | 2019-07-26 | 江苏永健医药科技有限公司 | 一种黄酮对照品的制备方法 |
CN114452333A (zh) * | 2022-01-19 | 2022-05-10 | 广西壮族自治区中医药研究院 | 黑草或其提取物在制备抗肿瘤药物中的应用 |
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