CN113262274A - 一种黑米提取物及其制备方法和用途 - Google Patents
一种黑米提取物及其制备方法和用途 Download PDFInfo
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- CN113262274A CN113262274A CN202110522682.2A CN202110522682A CN113262274A CN 113262274 A CN113262274 A CN 113262274A CN 202110522682 A CN202110522682 A CN 202110522682A CN 113262274 A CN113262274 A CN 113262274A
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- black rice
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- rice extract
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- uric acid
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- A61K2236/55—Liquid-liquid separation; Phase separation
Abstract
本发明公开了一种黑米提取物的制备方法,包括乙醇浸提、大孔吸附树脂纯化、色谱层析分离等步骤,提取物中的主要成分是矢车菊素‑3‑O‑葡萄糖苷,含量达20‑70%。通过建立高尿酸血症小鼠模型,分析评价了提取物对小鼠体内尿酸水平、脏器功能损伤、尿酸代谢通路关键酶的影响,结果表明该提取物具有降尿酸和预防痛风发生的积极作用,可应用于制备黄嘌呤氧化酶抑制剂或降尿酸药物,在保健食品方面也具有很大的应用前景。
Description
技术领域
本发明涉及一种黑米提取物及其制备方法和用途。
背景技术
尿酸嘌呤类代谢的最终产物,其生成受内源性和外源性因素双重调控。外源性因素主要是人体饮食消化后,氧化分解的代谢产物嘌呤转运到肝脏,经黄嘌呤氧化酶(XO)的氧化作用,随血浆流经肾脏,经肾小球滤过和近曲小管的重吸收,再分泌到远端小管二次吸收,最后约占尿酸总量10%经肾脏排出体外。肝脏、肾脏、肠道是尿酸代谢的重要部位,其中肝脏XO在尿酸的代谢过程中发挥着重要作用。当人体内尿酸生成过多和排泄不足时,出现系统调控失衡,导致高尿酸血症的出现。研究发现,尿酸在关节沉积易发痛风。痛风是一种单钠尿酸盐沉积所致的晶体相关性关节病,为嘌呤代谢紊乱及(或)尿酸排泄减少所致,受遗传因素和环境因素影响,临床上表现特点为高尿酸血症、反复发作的急性关节炎、痛风石沉积、痛风石性慢性关节炎和关节畸形等,累及肾脏,引起慢性间质性肾炎和肾结石。
XO是尿酸代谢途径中的关键酶之一。它是一种多功能的钼黄素蛋白,广泛分布于从细菌到人类的各种生物中。XO催化次黄嘌呤氧化为黄嘌呤,随后黄嘌呤氧化为尿酸,并在嘌呤代谢中将O2还原为超氧阴离子自由基或过氧化氢,抑制XO被认为是治疗HUA及相关疾病的重要手段之一。以XO为靶点,寻找或开发一种具有安全高效且无毒副作用,天然物质来源的XO抑制剂,通过抑制其活性,调控尿酸水平以达到降低尿酸的目的,对于干预高尿酸血症和痛风具有很重要的意义。
黑米一直享誉“黑珍珠”的称号,凭借价格低廉和获取途径广泛的优势,在水稻作物中占有无可替代的角色。作为一种功能稻和全谷物资源,在其果皮、种皮和胚芽中含有大量的生物活性成分,包括酚类化合物、谷维素、氨基酸、多种维生素、黄酮类化合物、膳食纤维、花青素、不饱和脂肪酸及微量元素等。研究表明黑米花色苷作为一种天然活性物质,具有抗炎、抗氧化、抗癌、预防心血管疾病、调节脂肪代谢等作用,许多国内外研究主要集中在黑米花色苷的提取分离和纯化利用方面,目前国内外没有对黑米提取物干预尿酸代谢的相关文献报道,且在分子层面发挥作用的机理仍不明确,近几年成为研究的热点内容。
发明内容
针对上述问题,本发明提供了一种黑米提取物的制备方法,并研究了该提取物在干预基础性代谢疾病方面的应用前景,尤其是在抑制黄嘌呤氧化酶或降尿酸中的应用。
上述目的是通过以下技术方案实现的:
一种黑米提取物的制备方法,包括以下步骤:
1)提取:将黑米碾碎成粉末,加入含有盐酸的乙醇溶液进行浸提,提取液浓缩后用乙酸乙酯萃取,收集水相,浓缩干燥得到黑米粗提物;
2)纯化:将黑米粗提物用水溶解后上大孔吸附树脂柱,先用0.01-0.1%HCl洗脱,再用含有柠檬酸-磷酸氢二钠缓冲液的乙醇溶液洗脱,收集乙醇洗脱液,浓缩干燥得到初级黑米提取纯化物;
3)层析分离:将十八烷基硅烷键合硅胶色谱填料活化后装入层析柱,将初级黑米提取纯化物用水溶解后上柱,待其被完全吸附后,用含0.01-0.1%三氟乙酸的水和不同浓度甲醇依次洗脱,收集10-30%甲醇洗脱液,浓缩干燥即得。
优选地,步骤1)中,所述乙醇溶液中含有盐酸0.01-0.1mol/L,所述乙醇溶液的浓度为30-70%。所述浸提的温度为50-80℃。
优选地,步骤2)中,所述含有柠檬酸-磷酸氢二钠缓冲液的乙醇洗脱,是将无水乙醇与pH 3.0的柠檬酸-磷酸氢二钠缓冲液按体积比40-70:60-30混合后配制而成。
优选地,所述大孔吸附树脂的型号为AB-8。
优选地,步骤2)中所述的洗脱,其流速为2-6mL/min。
优选地,所述十八烷基硅烷键合硅胶色谱填料的孔径为100-150A,粒径为30-50μm。
经HPLC液相检测,以上所制备的黑米提取物中,主要成分是矢车菊素-3-O-葡萄糖苷,含量达20-70%。
根据本发明的一个具体实施例,较佳的一个制备方法如下:
1)提取:将黑米碾碎成粉末,加入含有0.05mol/L盐酸的50%乙醇溶液,60℃条件下进行浸提,提取液浓缩后用乙酸乙酯萃取,收集水相,浓缩干燥得到黑米粗提物;
2)纯化:将黑米粗提物用水溶解后上AB-8型大孔吸附树脂柱,先用0.1%HCl洗脱,再用含有柠檬酸-磷酸氢二钠缓冲液的乙醇溶液(无水乙醇:pH 3.0的柠檬酸-磷酸氢二钠缓冲液=60:40,v/v)洗脱,洗脱流速4mL/min,收集乙醇洗脱液,浓缩干燥得到初级黑米提取纯化物;
3)层析分离:将十八烷基硅烷键合硅胶色谱填料(孔径120A,粒径30-50μm)活化后装入层析柱,将初级黑米提取纯化物用水溶解后上柱,待其被完全吸附后,用含0.1%三氟乙酸的水和不同浓度甲醇依次洗脱,收集20%甲醇洗脱液,浓缩干燥即得。
本发明中所涉及到的盐酸、三氟乙酸、乙醇、甲醇百分比浓度均为体积浓度,如20%甲醇指的是每100mL甲醇水溶液中含有甲醇20mL。
体内动物实验表明:
(1)对高尿酸小鼠体内尿酸水平和肾脏功能的影响;连续灌胃7天的氧嗪酸钾混悬液来诱导高尿酸血症小鼠造模之后,灌胃黑米提取物的各组小鼠均较好的降低了高尿酸小鼠体内血清尿酸(UA)水平,同时降低血清中尿素氮(BUN)和肌酐(Cr),削弱由氧嗪酸钾造模产生的肾脏损伤和降低带来小鼠肾脏损伤的负面影响。
(2)对小鼠脏器功能损伤情况:各组小鼠的体重、肝肾质量及脏器指数的数据间均未表现出显著性差异。由此说明黑米提取物在实验期间均未对小鼠的内脏器官造成明显的毒性损伤。
(3)对小鼠体内尿酸代谢通路关键酶和抗氧化水平的影响:小鼠灌胃氧嗪酸钾后,其肝脏中XO的活性极显著地升高,灌胃给予黑米提取物后均明显下调了高尿酸小鼠体内肝脏中的XO水平。同时,增加了体内的超氧化物歧化酶(SOD)、还原型谷胱甘肽(GSH)、过氧化氢酶(CAT)的活力,可很好地改善高尿酸血症小鼠体内过氧化反应,具有更强抗氧化能力。
综上所述:本发明制备的黑米提取物具有降尿酸和预防痛风发生的积极作用,可应用于制备黄嘌呤氧化酶抑制剂或降尿酸药物,在保健食品如代餐粉方面也具有很大的应用前景。
附图说明
图1:实施例中样品1-4的HPLC图谱。
具体实施方式
下面结合附图和具体实施例对本发明做进一步说明。
实施例1
(1)将黑米碾碎成粉末并冻干,称取100g黑米粉,加入1000mL 50%乙醇(含有盐酸0.05mol/L),混合均匀后将其60℃条件下磁力搅拌浸提30min,4000r/min离心10min以获取上清液。离心后沉淀物按照上述相同工艺再次浸提。将所有上清液合并,并利用旋转蒸发仪除去大量水分。采用乙酸乙酯萃取法对黑米提取浓缩液中的脂溶性杂质进行除杂,除杂开始前,乙酸乙酯进行水饱和处理,并按照浓缩液:乙酸乙酯体积比1:3进行混合萃取。收集下层目标水溶液,经真空冷冻干燥后得到黑米粗提物。
(2)依次利用95%乙醇溶液、5%HCl溶液及2%NaOH溶液活化AB-8大孔吸附树脂,并装入50cm×5cm的树脂柱,用3BV的双蒸水冲洗,去除残留溶剂。将收集到的黑米粗提物,按料液比1:15(m:V)用水溶解后依照柱体积按比例装柱,装载量为1.5BV。待其被完全吸附在树脂后,用约5BV的0.1%的HCl不断冲洗树脂,以去除样品中残留的糖类、蛋白质类及其他多余的可溶性成分。之后,加入4BV的洗脱液(无水乙醇:pH 3.0的柠檬酸-磷酸氢二钠=60:40)进行洗脱,洗脱流速控制在4mL/min,收集洗脱液,经旋转蒸发浓缩、真空冷冻干燥后得到初级黑米提取纯化物,将其置于密封的铝箔袋中,于-20℃条件下储存备用。
(3)将初级黑米提取纯化物加水充分搅拌至完全溶解,在4000r/min条件下4℃低温离心10min以获得上清液,等待上样。利用SP-120-30/50-ODS-B反向制备柱经纯甲醇浸泡2h充分活化后,装入25cm×2.5cm的普通层析柱中,整个填装过程保持层析柱的平整、紧密、均匀和无气泡。填装完成后,用5倍柱体积(BV)双蒸水冲洗除去残余甲醇,并将上清液装柱。待其被完全吸附后,先利用5BV含0.1%三氟乙酸的双蒸水不断冲洗层析柱以除去杂质化合物,后依次用5BV含0.1%三氟乙酸的10%、20%、30%、40%甲醇水洗脱,收集各浓度甲醇洗脱液并浓缩干燥,得到样品1-4。
HPLC液相检测:
将实施例1得出的四个浓度甲醇样品进行分析。HPLC分析采用岛津LC-20ADXR系统,DAD检测器,分析柱选用Diamonsil C18柱(4.6mm×250mm,5μm),流速为1.0mL/min,柱温25℃±5℃。流动相A为5%甲酸水溶液(v/v)、流动相B为甲醇,进样量为10μL,DAD检测器检测波长为280nm和520nm。
得率=各浓度甲醇洗脱后的产物重量/初级黑米提取纯化物重量×100%
表1黑米提取物的得率和主要的物质含量组成
从以上结果可以看出,20%甲醇洗脱产物不仅得率最高,而且矢车菊素-3-O-葡萄糖苷含量也最高。利用本实验室前期建立的黑米花色苷HPLC定量测定分析方法,利用UPLC-QTOF-MS/MS对样品进行结构成分分析,根据Masslynx v4.1软件预测分子式,结合液相色谱出峰顺序、一级二级质谱离子碎片和文献对比进行化学结构的确定。
实施例2提取物的降尿酸功效评价
1.试验方法
将70只昆明雄性小鼠进行一周的适应性饲养,动物自由摄食和饮水,两天换一次垫料,之后将小鼠随机分成7组,分别为:空白对照组(NC)、高尿酸模型组(HUA)、别嘌呤醇阳性组(AP)、样品1-4组,每组均含有10只小鼠。在保证正常饮食饮水的条件下,每天上午8点对所有小鼠进行禁食处理,断粮1h之后开始灌胃。每组小鼠的给药情况如表2所示:NC组小鼠灌胃0.5%CMC-Na溶液,对除NC以外的其它组全部进行氧嗪酸钾混悬液进行灌胃造模,按250mg/kg·d的剂量对小鼠连续灌胃7天,建立小鼠高尿酸血症动物模型。在当天结束氧嗪酸钾混悬液灌胃1h后,给HUA组小鼠灌胃5mg/kg的别嘌呤醇混悬溶液,试验组的小鼠均灌胃100mg/kg的黑米提取物样品混悬溶液,NC组与HUA组小鼠灌胃蒸馏水。第6日结束小鼠灌胃后,对其进行12h上禁食不禁水处理,以便第7日结束灌胃后对小鼠进行血液和脏器的样本提取工作。
表2实验小鼠的分组及给药情况
在第7天小鼠灌胃结束之后,对小鼠进行摘取眼球处理并从眼眶处进行小鼠血液的采集,小鼠血液样本在室温条件下自然凝血1h后,以3000r/min离心15min离心分离出血清,迅速收集并分装后置于-20℃下保存,避免发生溶血现象。小鼠在完成眼球取血后拉断颈椎处死,将死亡小鼠固定于解剖板上。分离其肝脏、肾脏组织,将得到的小鼠脏器组织在4℃预冷生理盐水中漂洗,除去血液及黏连的结缔组织,经滤纸拭干后称重并按照以下公式计算脏器指数。
采集的小鼠血液样本在室温条件下自然凝血1h后,将其置于4℃条件下经3000r/min离心10min,取上清液,即为实验用血清样本。将其分装后置于-80℃条件下保存,用试剂盒检测项目包括UA、BUN、Cr和SOD。测定前于4℃条件下自然解冻后使用,避免反复冻融。测定过程严格按照相关测定试剂盒的操作说明进行,每项测定重复三次以上,以保证实验结果的准确可靠性。
选取小鼠新鲜肝脏组织样本,准确称取0.50g组织,按照质量比1:9的比例加入4℃的预冷生理盐水,于冰浴条件下进行机械匀浆。匀浆液置于4℃低温条件下经3500r/min离心10min,轻轻吸掉表面脂肪组织后,缓慢吸取上清液,即为实验用10%肝组织匀浆待测样本。将其分装后置于-20℃条件下保存待测。测定前于4℃条件下自然解冻后使用,避免反复冻融。测定过程严格按照相关测定试剂盒的操作说明进行,测定项目包括XO、CAT和CSH,每项测定均重复三次以上,以保证实验结果的准确可靠性。
2.实验结果
①对小鼠脏器功能损伤情况
在动物实验的各项生化指标中,体重、脏器质量与脏器指数能够最直观地反应出动物各个脏器的损伤状态,以表征其毒性损伤作用。持续灌胃7天后小鼠体重、肝肾重量及肝肾的脏器指数变化经过单因素差异性分析显示,各组小鼠的体重、肝肾质量及脏器指数的数据间均未表现出显著性差异,由此说明灌胃黑米提取物在实验期间均未对小鼠的内脏器官造成明显的毒性损伤。
②对高尿酸小鼠体内尿酸(UA)水平影响
能直观反映高尿酸血症模型造模成功的关键在于小鼠体内血清尿酸(UA)水平的变化,黑米提取物对高尿酸血症小鼠的血清UA水平影响表3所示,连续灌胃7d的氧嗪酸钾混悬液来诱导高尿酸血症小鼠造模之后,与NC组小鼠相比,HUA组小鼠的血清UA值极显著上升,表明高尿酸血症小鼠动物模型造模成功。与HUA组相比,AP组可极显著降低HUA小鼠体内的血清UA含量,而样品2组的小鼠血清UA值相比于模型组下降了约68%,变化趋势最为明显。在该动物模型下,样品2中的黑米提取物其降尿酸能力要强于其他样品。
灌胃组别 | 血清UA(μmol/L) |
空白对照组 | 56.3809±6.2102 |
高尿酸模型组 | 98.4995±7.1728 |
别嘌呤醇阳性组 | 23.3325±3.3314 |
样品1 | 45.3285±3.9813 |
样品2 | 31.6100±2.1999 |
样品3 | 37.2648±5.7891 |
样品4 | 42.4895±1.5451 |
③对高尿酸小鼠体内肾脏功能(BUN、Cr)的评价
高尿酸血症及尿酸代谢异常被认为与慢性肾病的发生发展息息相关,其中,肾脏最初发生病变的即表现为肾小球的滤过率下降,主要通过血清中尿素氮(BUN)和肌酐(Cr)的含量间接表征。BUN和Cr是排查影响肾脏功能的有效手段之一。两者经由肾小球滤过和重吸收作用,将其而排泄至外,而肾脏最初发生病变的表现为肾小球的工作负荷下降,因此依据通过检测患者血清BUN和Cr变化,了解肾功能是否出现异常而做出判断。当血清BUN和Cr在血清中含量明显增加时,说明肾脏的肾小球滤过率出现下降的趋势意味着肾脏器官可能出现了功能性损害。经过7天灌胃之后,各组小鼠的血清BUN和Cr水平变化如表4和表5所示。
经过高尿酸血症小鼠造模之后,与NC组相比,HUA组小鼠的血清BUN和Cr水平出现明显的升高现象,与NC组之间呈现出显著性差异,这证明高尿酸血症小鼠的肾脏受到损伤致其功能有所下降。而与HUA组相比,样品组均能降低高尿酸血症小鼠的血清BUN和Cr水平,对高尿酸血症小鼠血清尿素氮水平的影响较小,可以推测灌胃黑米提取物对小鼠的肾脏损伤较轻,并能够显著调节高尿酸血症小鼠血清Cr恢复至接近正常水平。同时,改善高尿酸血症小鼠所致的肾脏损伤。
灌胃组别 | 血清BUN(mmol/L) |
空白对照组 | 3.067±0.474 |
高尿酸模型组 | 3.579±0.722 |
别嘌呤醇阳性组 | 3.282±0.489 |
样品1 | 3.494±0.764 |
样品2 | 3.305±0.078 |
样品3 | 3.375±0.592 |
样品4 | 3.361±0.095 |
灌胃组别 | 血清Cr(μmol/L) |
空白对照组 | 22.9511±7.5548 |
高尿酸模型组 | 25.7667±1.6513 |
别嘌呤醇阳性组 | 19.3378±4.2638 |
样品1 | 22.9011±6.1573 |
样品2 | 20.0056±2.1977 |
样品3 | 21.2145±4.9842 |
样品4 | 21.8456±9.2584 |
④对小鼠体内尿酸代谢通路关键酶(XO)的影响
在尿酸代谢途径中,腺嘌呤核苷酸、次黄嘌呤核苷酸和鸟嘌呤核苷酸都可以被酶催化成相对应的核苷,在腺苷脱氨酶催化作用下生成次黄嘌呤核苷,次黄嘌呤在XO作用下生成底物黄嘌呤,随后继续氧化形成终产物尿酸。因此XO成为尿酸合成通路的关键生物活性酶之一,而XO也一直被作为干预尿酸代谢异常及高尿酸血症的关键研究靶点。连续7天灌胃后各组小鼠肝脏中XO活性结果如表6所示。与空白组相比,模型组的小鼠灌胃氧嗪酸钾后,其肝脏中XO的活性极显著地升高。与模型组相比,作为临床治疗的阳性药别嘌呤醇,给予治疗后可明显地降低患有HUA小鼠体内肝脏中XO的活性,并低于空白组的小鼠。而试验组中均可下调了HUA小鼠体内肝脏中XO活性,从而减少尿酸的生成。该结果与表5中各组小鼠血清UA水平结果相呼应。
灌胃组别 | 肝脏XO(U/g prot) |
空白对照组 | 10.4286±1.2965 |
高尿酸模型组 | 16.5760±1.9860 |
别嘌呤醇阳性组 | 7.5633±1.2135 |
样品1 | 11.7300±0.8317 |
样品2 | 9.8267±1.1863 |
样品3 | 10.2923±3.5482 |
样品4 | 12.5846±2.1237 |
⑤对小鼠体内抗氧化水平(SOD、CAT和GSH)的影响
正常情况下机体内存在着氧化剂-抗氧化剂的动态平衡。在压力、心血管疾病、糖尿病、抑郁症等各种状态下,这种平衡受到干扰。当平衡被打破,可能导致超出生理极限的过量自由基产生,包括活性氧和活性氮,这一现象称为氧化应激。正常情况下,机体内存在酶促抗氧化剂,如超氧化物歧化酶(SOD)、谷胱甘肽还原酶和非酶促抗氧化剂,如GSH、CAT等,它们的存在可帮助清除生理状态下机体产生的氧化物。这类酶能清除O2-、H2O2等,具有很强的抗氧化作用,可以保护机体细胞免受损伤。
经过7d氧嗪酸钾诱导建造高尿酸血症模型之后,结果由表7-9所示,与NC组相比,HUA组小鼠血清中的SOD,肝脏中的GSH和CAT的活性均显著降低,表明高尿酸血症小鼠体内氧化应激反应加剧,抗氧化能力降低。与HUA组相比,AP组别嘌呤醇使得其活性有明显地上升,呈现出显著性差异,但是只是有所缓解,并不能使其恢复至NC组的水平。但灌胃样品1-4组有明显的变化,不仅提高活性水平,还可改善机体内的氧化应激反应,增强抗氧化效果。
灌胃组别 | 血清SOD(U/g prot) |
空白对照组 | 89.47±3.77 |
高尿酸模型组 | 68.79±5.05 |
别嘌呤醇阳性组 | 79.73±6.18 |
样品1 | 90.78±3.84 |
样品2 | 82.70±13.31 |
样品3 | 85.43±9.82 |
样品4 | 86.16±8.15 |
灌胃组别 | 肝脏GSH(μmol/g prot) |
空白对照组 | 11.57±0.6630 |
高尿酸模型组 | 4.63±1.2571 |
别嘌呤醇阳性组 | 9.20±3.4611 |
样品1 | 7.95±1.4038 |
样品2 | 8.14±4.8219 |
样品3 | 7.8659±2.1646 |
样品4 | 6.1578±2.1262 |
灌胃组别 | 肝脏CAT(U/g prot) |
空白对照组 | 243.596±5.2887 |
高尿酸模型组 | 180.7733±8.1900 |
别嘌呤醇阳性组 | 233.68±9.7913 |
样品1 | 230.3767±9.9328 |
样品2 | 232.8267±9.8811 |
样品3 | 228.5231±3.77263 |
样品4 | 212.5846±7.1237 |
实施例3
1)提取:将黑米碾碎成粉末,加入含有0.1mol/L盐酸的30%乙醇溶液,80℃条件下进行浸提,提取液浓缩后用乙酸乙酯萃取,收集水相,浓缩干燥得到黑米粗提物;
2)纯化:将黑米粗提物用水溶解后上AB-8型大孔吸附树脂柱,先用0.05%HCl洗脱,再用含有柠檬酸-磷酸氢二钠缓冲液的乙醇溶液(无水乙醇:pH 3.0的柠檬酸-磷酸氢二钠缓冲液=50:50,v/v)洗脱,洗脱流速2mL/min,收集乙醇洗脱液,浓缩干燥得到初级黑米提取纯化物;
3)层析分离:将十八烷基硅烷键合硅胶色谱填料(孔径100A,粒径30-50μm)活化后装入层析柱,将初级黑米提取纯化物用水溶解后上柱,待其被完全吸附后,用含0.01%三氟乙酸的水和不同浓度甲醇依次洗脱,收集20%甲醇洗脱液,浓缩干燥即得。
实施例4
1)提取:将黑米碾碎成粉末,加入含有0.01mol/L盐酸的70%乙醇溶液,50℃条件下进行浸提,提取液浓缩后用乙酸乙酯萃取,收集水相,浓缩干燥得到黑米粗提物;
2)纯化:将黑米粗提物用水溶解后上AB-8型大孔吸附树脂柱,先用0.01%HCl洗脱,再用含有柠檬酸-磷酸氢二钠缓冲液的乙醇溶液(无水乙醇:pH 3.0的柠檬酸-磷酸氢二钠缓冲液=40:60,v/v)洗脱,洗脱流速6mL/min,收集乙醇洗脱液,浓缩干燥得到初级黑米提取纯化物;
3)层析分离:将十八烷基硅烷键合硅胶色谱填料(孔径150A,粒径30-50μm)活化后装入层析柱,将初级黑米提取纯化物用水溶解后上柱,待其被完全吸附后,用含0.05%三氟乙酸的水和不同浓度甲醇依次洗脱,收集20%甲醇洗脱液,浓缩干燥即得。
得率(%) | C3G含量(%) | |
实施例3 | 55.3 | 42.0 |
实施例4 | 47.2 | 38.5 |
Claims (9)
1.一种黑米提取物的制备方法,其特征在于包括以下步骤:
1)提取:将黑米碾碎成粉末,加入含有盐酸的乙醇溶液进行浸提,提取液浓缩后用乙酸乙酯萃取,收集水相,浓缩干燥得到黑米粗提物;
2)纯化:将黑米粗提物用水溶解后上大孔吸附树脂柱,先用0.01-0.1%HCl洗脱,再用含有柠檬酸-磷酸氢二钠缓冲液的乙醇溶液洗脱,收集乙醇洗脱液,浓缩干燥得到初级黑米提取纯化物;
3)层析分离:将十八烷基硅烷键合硅胶色谱填料活化后装入层析柱,将初级黑米提取纯化物用水溶解后上柱,待其被完全吸附后,用含0.01-0.1%三氟乙酸的水及不同浓度甲醇依次洗脱,收集10-30%甲醇洗脱液,浓缩干燥即得。
2.如权利要求1所述黑米提取物的制备方法,其特征在于:步骤1)中,所述乙醇溶液中含有盐酸0.01-0.1mol/L,所述乙醇溶液的浓度为30-70%。
3.如权利要求1所述黑米提取物的制备方法,其特征在于:步骤1)中,所述浸提的温度为50-80℃。
4.如权利要求1所述黑米提取物的制备方法,其特征在于:步骤2)中,所述含有柠檬酸-磷酸氢二钠缓冲液的乙醇洗脱,是将无水乙醇与pH 3.0的柠檬酸-磷酸氢二钠缓冲液按体积比40-70:60-30混合后配制而成。
5.如权利要求1所述黑米提取物的制备方法,其特征在于:所述大孔吸附树脂的型号为AB-8。
6.如权利要求1所述黑米提取物的制备方法,其特征在于:步骤2)中所述的洗脱,其流速为2-6mL/min。
7.如权利要求1所述黑米提取物的制备方法,其特征在于:所述十八烷基硅烷键合硅胶色谱填料的孔径为100-150A,粒径为30-50μm。
8.一种黑米提取物,其特征在于:它是按权利要求1-7任一项所述方法制备的,该提取物中含有20-70%的矢车菊素-3-O-葡萄糖苷。
9.权利要求8所述的黑米提取物在制备黄嘌呤氧化酶抑制剂或降尿酸药物中的用途。
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