CN117476126A - 一种靶向制备降尿酸活性的蜂胶黄酮提取方法及应用 - Google Patents
一种靶向制备降尿酸活性的蜂胶黄酮提取方法及应用 Download PDFInfo
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- CN117476126A CN117476126A CN202311830237.8A CN202311830237A CN117476126A CN 117476126 A CN117476126 A CN 117476126A CN 202311830237 A CN202311830237 A CN 202311830237A CN 117476126 A CN117476126 A CN 117476126A
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- uric acid
- propolis
- flavone
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Abstract
本发明提供一种靶向制备降尿酸活性的蜂胶黄酮提取方法及应用,涉及蜂胶深加工技术领域。所述靶向制备降尿酸活性的蜂胶黄酮提取方法主要包括构建黄酮的降尿酸活性预测方法、针对蜂胶黄酮进行预测,探索高活性的黄酮化合物,并采用COSMO‑RS模型定向设计新型绿色提取溶剂低共熔溶剂DES、筛选最佳DES提取配方获得定向提取的高含量且具有较好降尿酸活性的蜂胶黄酮提取物等步骤。本发明克服了现有技术的不足,能够以降尿酸活性为导向,快速、高效且定向制备降尿酸活性的蜂胶黄酮提取物,为开发具有降尿酸活性的蜂胶功能产品提供了有力支持。
Description
技术领域
本发明涉及蜂胶深加工技术领域,具体涉及一种靶向制备降尿酸活性的蜂胶黄酮提取方法及应用。
背景技术
蜂胶是蜜蜂科昆虫意大利蜂Apis mellifera L、工蜂采集的植物树脂与其上颚腺、蜡腺等分泌物混合形成的具有黏性的固体胶状物。它在世界具有悠久的使用历史,人类研究和应用蜂胶的历史可以追溯到公元前三千多年。此外,中国卫生部主编的我国具有权威性的中医药大典《中华本草》第九卷认证了蜂胶的八大医学功效;2002年卫法监发[2002]5l号文件公布:蜂胶是可用于保健食品的物品。2005年,蜂胶被正式收录于《中华人民共和国药典》;此外,从1996年起到2018年1月,原卫生部和国家食品药品监督管理局(总局)已批准蜂胶类保健食品1328件。蜂胶作为一种有吸引力的天然产物,其促进健康的功效受到越来越多的关注。
蜂胶是自然界总黄酮类化合物含量最高的天然物质之一。目前,从蜂胶中鉴定出来的黄酮类化合物包括:白杨素、芹菜素、金合欢素、槲皮素、高良姜素、芦丁、山奈酚、鼠李素、松属素等。正由于复杂的化学成分,使得蜂胶具有广泛的生物学活性,例如抗菌消炎、抗病毒、抗氧化、抗肿瘤、降血糖、改善微循环、促进组织再生等。
目前,对于蜂胶的研究主要集中在蜂胶中黄酮类化合物的提取,蜂胶提取物主要由乙醇制备,但也可以使用其他溶剂,如水、二甲基亚砜、甲醇、丙酮、乙酸乙酯等。尽管已报道了超声、微波等多动态辅助有机溶剂联合提取方法用于蜂胶黄酮的提取,但是仅停留在提高蜂胶黄酮的提取率层面,并没有考虑以降尿酸活性为导向,对蜂胶黄酮进行定向提取。
高尿酸血症(HUA)是一种由嘌呤代谢紊乱或尿酸排泄受阻而引起的代谢性疾病,以血尿酸浓度升高为特征,使尿酸盐晶体沉积,进一步导致痛风性关节炎、心血管和肾脏疾病的风险增加。黄嘌呤氧化酶(Xanthine oxidase, XOD)是嘌呤分解代谢途径中的关键限速酶,它可依次催化次黄嘌呤和黄嘌呤氧化为尿酸,导致痛风的发生。因此,XOD也被研究为许多天然产物降低尿酸合成的抑制靶点。有研究报道黄酮类化合物,特别是黄酮醇以及皂苷、萜类化合物等具有降尿酸作用,这些化合物可以通过抑制XOD和调节尿酸转运体来降低血尿酸。蜂胶是作为自然界总黄酮类化合物含量最高的天然物质之一,丰富的黄酮类化合物使其具有作为高效、理想的降尿酸功能食品的潜力。
发明内容
针对现有技术不足,本发明提供一种靶向制备降尿酸活性的蜂胶黄酮提取方法及应用,以降尿酸活性为导向,快速、高效且定向制备降尿酸活性的蜂胶黄酮提取物,为开发具有降尿酸活性的蜂胶功能的产品提供了有力支持。
为实现以上目的,本发明的技术方案通过以下技术方案予以实现:
一种靶向制备降尿酸活性的蜂胶黄酮提取方法,所述提取方法包括以下步骤:
S1、构建黄酮的降尿酸活性预测方法,且具体构建方法包括以下步骤:
S1-1、获取具有降尿酸活性的黄酮类化合物及其SMILES结构序列和pIC50值;
S1-2、对目标分子进行MACCA、ECFP4分子指纹相似性聚类分析;
S1-3、进行特征相关性分析和结合机器学习模型构建膳食黄酮类化合物定量构效关系QSAR模型;
S2、筛选出具有降尿酸活性的蜂胶黄酮化合物,并采用COSMO-RS模型定向设计新型绿色提取溶剂-低共熔溶剂DES;
S3、筛选最佳DES提取配方获得定向提取的高含量且具有高降尿酸活性的蜂胶黄酮提取物。
优选的,所述步骤S1-1中黄酮类化合物的获取方法为从中、英文文献报道的公开数据以及团队内部实验结果数据集中搜集具有降尿酸活性的黄酮类化合物,且步骤S1-2中对数据集的3D描述符和Rdkit基础理化描述进行计算。
优选的,所述步骤S1-1中pIC50值的获取方法为根据IC50半抑制率浓度值计算出pIC50值,且计算公式如下所示:
。
优选的,所述步骤S2的具体方法包括以下步骤:
S2-1、采用QSAR模型对蜂胶黄酮进行降尿酸活性预测,筛选出具有降尿酸活性的蜂胶黄酮化合物;
S2-2、针对高活性目标黄酮类化合物,进行能量优化;
S2-3、经优化后,得到具有最佳分子表面屏蔽电荷的模型DESs的表面各片段的密度分布,表示为σ-Profiles,并分析计算多种DESs表面段的化学势;
S2-4、利用COSMO-thermX软件,计算蜂胶黄酮单体在无限稀释条件下在DES中的相对溶解度。
优选的,所述步骤S2-2中能量优化的具体方式为:从PubChem中下载,通过MMFF94力场获得各化合物的初始构象,然后,利用密度泛函理论和TZVP基组计算并优化了氢键受体HBAs、氢键供体HBDs、蜂胶黄酮化合物模型的最佳分子几何形状和电荷密度。
优选的,所述步骤S2-4中在无限稀释条件下计算相对溶解度的具体方式为:将最佳溶解度的对数设为0,所有其他溶剂相对于最佳溶剂或参考溶剂排序;
相对溶解度计算公式如下:
;
式中:表示溶质的相对溶解度;/>和/>分别表示纯化合物的化学势和纯化合物在溶剂中无限稀释时的化学势;/>为j的自由能;其中溶质即蜂胶黄酮化合物。
优选的,所述步骤S3中,筛选最佳DES提取配方获得定向提取的高含量且具有较好降尿酸活性的蜂胶黄酮提取物包括以下步骤:
S3-1、对定向设计的多种DES溶剂进行化学合成;
S3-2、DES对蜂胶黄酮的提取方法的建立,采用超声辅助提取的方法对蜂胶黄酮进行高效富集,定向提取;
S3-3、通过对不同蜂胶黄酮提取物的总黄酮含量以及主要蜂胶黄酮含量进行测定,从而对COSMO-RS筛选出的DES进行实验验证。
优选的,所述步骤S3-1中具体的方法为将HBAs和HBDs按适当的摩尔比混合,并添加10~30%的去离子水,然后将混合物置于磁力搅拌装置中,在60~80℃下连续搅拌直到混合物呈现均一透明液体,将混合物冷却至室温,避光保存备用。
优选的,所述步骤S3-2中为筛选最佳DES的方法为:将0.1~10 g蜂胶粉与3~300 mL不同的DES混合,置于离心管中,将混合物在超声功率为200~600 W、提取温度为40~60℃,提取处理10~30 min;然后,将混合物在4℃下6000~12000 rpm离心10~30 min,收集上清液;最后,取100μL上清液用甲醇稀释10倍,经0.22 μm微孔尼龙膜过滤后,用于HPLC进行定量分析。
上述获得的蜂胶黄酮提取物应用于抑制黄嘌呤氧化酶及降尿酸产品的开发生产以及实验室研究。
本发明提供一种靶向制备降尿酸活性的蜂胶黄酮提取方法及应用,与现有技术相比优点在于:
本发明首次开发并提供了一种靶向制备降尿酸活性的蜂胶黄酮提取方法及应用,采用自下而上的定向提取方法,即以降尿酸活性为导向,通过COSMO-RS模型定向设计DES提取溶剂,实现快速、高效、定向制备具有降尿酸活性的蜂胶黄酮提取物,为开发具有降尿酸功能的蜂胶功能产品提供了有力支持。
附图说明
图1为本发明中靶向制备降尿酸活性的蜂胶黄酮提取方法的流程图;
图2为本发明为基于xgboost算法开发的pIC50 QSAR预测模型;
图3为本发明不同DES和乙醇提取的蜂胶提取物总黄酮含量测定;
图4为本发明市售蜂胶乙醇提取物和蜂胶DES提取物的高效液相色谱图,其中,HPLC谱图中各化合物成分分别为:1、咖啡酸;2、p-香豆酸;3、阿魏酸;4、异阿魏酸;5、3, 4-二甲氧基肉桂酸;6、肉桂酸;7、4-甲氧基肉桂酸;8、5-甲氧基短叶松素;9、短叶松素;10、松属素;11、咖啡酸苄酯;12、短叶松素-3-乙酸酯;13、柯因;14、咖啡酸苯乙酯;15、6-甲氧基柯因;16、p-香豆酸苄酯;17、阿魏酸苄酯;18、7-甲氧基短叶松素-3-乙酸酯;19、咖啡酸肉桂酯;20、球松素;21短叶松素-3-O-丙酸酯;22、柚木柯因;23、短叶松素-3-O-丁酸酯;24、p-香豆酸肉桂酯;25、短叶松素-3-O-己酸酯;26、肉桂酸肉桂酯;
图5为本发明不同DES和乙醇提取溶剂提取主要蜂胶黄酮柯因、松属素的含量。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面结合本发明实施例对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1:
1、构建黄酮的降尿酸活性预测方法:
(1)从中、英文文献报道的公开数据以及团队内部实验结果数据集中搜集到93种具有降尿酸活性的黄酮类化合物,并下载这些黄酮化合物的SMILES结构序列,及其报道的IC50半抑制率浓度值,并通过以下公式计算出pIC50值:
且所述93种黄酮类化合物具体英文名称、Compound CID如下表1所示:
表1
(2)对目标分子进行MACCA、ECFP4分子指纹相似性聚类分析;
(3)计算数据集431个3D描述符和208个Rdkit基础理化描述符,并进行特征相关性分析和结合机器学习模型构建膳食黄酮类化合物定量构效关系QSAR模型。通过以下公式评估QSAR模型的预测能力和鲁棒性。
式中,,/>,/>分别表示为真实值、预测值和平均值;/>是残差的平方和,也叫残差平方和;/>是总平方和;n是样本数;平均绝对误差(MAE)表示真实值和预测值之间的差值,预测值通过对数据集的绝对差的平均值反映真实误差;均方误差(MSE)表示平方误差的期望值;均方根误差(RMSE)表示预测误差的标准差。
如图2所示为基于xgboost算法开发的pIC50 QSAR预测模型,可用于快速预测黄酮的XOD抑制活性。从图中可以看出,训练集和测试集以及真实的pIC50与预测的pIC50均表现出良好的相关性,其R2分别为0.717和0.754。此外,两组的残差都很小,说明所构建的QSAR模型具有较好的预测能力。
为了进一步评估模型的稳定性,我们计算了模型的RMSE、MSE和MAE参数。训练集的MSE、MAE、RMSE分别为0.221、0.360、0.471。这些结果表明,预测值与实际值的差异较小,偏差误差较低,进一步验证了模型具有较好的稳定性和预测能力。
2、针对蜂胶黄酮进行预测,探索高活性的黄酮化合物,并采用COSMO-RS模型定向设计新型绿色提取溶剂-低共熔溶剂DES:
(1)采用QSAR模型对50种蜂胶黄酮进行降尿酸活性预测,以筛选出具有降尿酸活性的蜂胶黄酮化合物。通过对比预测结果,我们筛选出蜂胶提取物中含量较高,且具有较强XOD抑制活性的蜂胶黄酮,柯因和松属素。
且QSAR模型对蜂胶黄酮中柯因、松属素的XOD抑制活性的预测结果见下表2:
表2
如表2所示,柯因和松属素对XOD抑制活性的IC50值分别为1.2675 μM和13.0571 μM,两者具有较强的XOD抑制活性。
(2)针对高活性目标黄酮类化合物,进行能量优化。从PubChem中下载,通过MMFF94力场获得各化合物的初始构象。然后,利用密度泛函理论(DFT)和TZVP基组计算并优化了氢键受体HBAs、氢键供体HBDs、蜂胶黄酮化合物模型的最佳分子几何形状和电荷密度;
(3)经几何优化后,得到了具有最佳分子表面屏蔽电荷的模型DESs的表面各片段的密度分布,表示为σ-Profiles,并分析计算了多种DESs表面段的化学势(σ-Potentials);
(4)利用COSMO-thermX软件,计算蜂胶黄酮单体在无限稀释条件下在DES中的相对溶解度。在无限稀释条件下计算相对溶解度,最佳溶解度的对数设为0,所有其他溶剂相对于最佳溶剂或参考溶剂排序。
且目标黄酮化合物在COSMO-RS设计的不同DES溶剂中的相对溶解度如下表3所示:
表3
如表3所示,目标黄酮在不同的DES溶剂中,溶解度不同,其中,在氯化胆碱和1, 4-丁二醇组成的DES中预测的相对溶解度最大,推测目标黄酮在此溶剂中的提取效率最高。
3、筛选最佳DES提取配方获得定向提取的高含量且具有较强降尿酸活性的蜂胶黄酮提取物:
(1)对定向设计的多种DES溶剂进行化学合成,具体不同DES溶剂的组成如下表4所示:
表4
如表4所示,将HBAs和HBDs按适当的摩尔比混合,并添加10~30%的去离子水(v/v),然后将混合物置于磁力搅拌装置中,在60~80℃下连续搅拌直到混合物呈现均一透明液体,将混合物冷却至室温,避光保存备用。
(2)DES对蜂胶黄酮的提取方法的建立,采用超声辅助提取的方法对蜂胶黄酮进行高效富集,定向提取。
为了对COSMO-RS预测的DES进行实验验证,将0.1 g蜂胶粉与3 mL不同的DES混合,置于离心管中,将混合物在超声功率为300 W、提取温度为50℃,提取处理30 min,然后,将混合物在4℃下8000 rpm离心20 min,收集上清液;
通过采用硝酸铝比色法测定不同蜂胶黄酮提取物的总黄酮含量进行测定,结果如图3所示,图中为不同DES和乙醇提取蜂胶中的总黄酮化合物含量,可以看出DES-4(氯化胆碱-1, 4-丁二醇)制备的蜂胶提取物的总黄酮含量最高为82.67 mg/g蜂胶,远高于蜂胶乙醇提取物的总黄酮含量31.35 mg/g蜂胶;这一结果证明了COSMO-RS筛选DES作为蜂胶提取溶剂的准确性。
(3)采用高效液相色谱法测定不同提取物的主要蜂胶黄酮含量。取100 μL不同DES蜂胶提取液,用甲醇稀释10倍,经0.22 μm微孔尼龙膜过滤后,用于HPLC进行定量分析。如图4所示为市售蜂胶乙醇提取物和氯化胆碱-1, 4-丁二醇为代表的DES蜂胶提取物的高效液相色谱图,从图中可以看出,DES和乙醇的蜂胶提取物中的主要黄酮均为柯因和松属素,但是两者含量却有较大差别,说明了DES可以实现蜂胶黄酮的高效定向提取。
图5为不同DES和乙醇提取溶剂提取柯因和松属素的含量(mg/g蜂胶),结果表明,氯化胆碱-1, 4-丁二醇制备的DES-4对柯因和松属素的提取率最高,提取物中的含量分别为26.11±0.23、23.4±0.16(mg/g蜂胶),DES-1次之,而市售的蜂胶乙醇提取物中柯因和松属素的含量较低,分别为13.6±0.26、9.06±0.44 mg/g蜂胶。采用定向设计DES提取的蜂胶黄酮含量比市售的蜂胶黄酮含量高2~3倍,证明了DES设计提取溶剂可以实现蜂胶黄酮的靶向提取。
4、最佳蜂胶黄酮提取物的XOD抑制实验验证,评估其降尿酸活性:
(1)将蜂胶DES提取物、市售蜂胶乙醇提取物分别溶解在10%的二甲基亚砜(DMSO)中,制备1 mg/mL蜂胶提取物原液,然后用pH 7.4的磷酸缓冲溶液稀释至所需浓度,分别制备0.1、0.2、0.4、0.6、0.8、1.0 mg/mL溶液,用于体外黄嘌呤氧化酶抑制试验。
将柯因、松属素分别溶解在10%的二甲基亚砜(DMSO)中,制备1 mg/mL蜂胶黄酮单体原液。然后用pH 7.4的磷酸缓冲溶液稀释至所需浓度,分别制备0.1、0.5、1、2、4、6、8、10μg/mL溶液,用于体外黄嘌呤氧化酶抑制试验。
式中,A为有样品溶液的吸光度,B为不含样品溶液的吸光度,C为空白溶液(不含XOD溶液和样品)的吸光度。
(2)针对步骤(1)的体外XOD活性抑制结果,进行XOD抑制率的测定,并计算其IC50值。具体见下表5:
表5
由表5可知,蜂胶DES提取物的降尿酸活性高于市售蜂胶乙醇提取物,IC50值为220.13±1.43 μg/mL。此外,蜂胶黄酮化合物单体柯因、松属素也具有较高的降尿酸活性,IC50值分别为0.325μg/mL、3.075±0.18 μg/mL,与预测结果大致相同,表明了QSAR模型预测的准确性高以及靶向提取的效率高。
以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (10)
1.一种靶向制备降尿酸活性的蜂胶黄酮提取方法,其特征在于,所述提取方法包括以下步骤:
S1、构建黄酮的降尿酸活性预测方法,且具体构建方法包括以下步骤:
S1-1、获取具有降尿酸活性的黄酮类化合物及其SMILES结构序列和pIC50值;
S1-2、对目标分子进行MACCA、ECFP4分子指纹相似性聚类分析;
S1-3、进行特征相关性分析和结合机器学习模型构建膳食黄酮类化合物定量构效关系QSAR模型;
S2、筛选出具有降尿酸活性的蜂胶黄酮化合物,并采用COSMO-RS模型定向设计新型绿色提取溶剂-低共熔溶剂DES;
S3、筛选最佳DES提取配方获得定向提取的高含量且具有高降尿酸活性的蜂胶黄酮提取物。
2.根据权利要求1所述的一种靶向制备降尿酸活性的蜂胶黄酮提取方法,其特征在于:所述步骤S1-1中黄酮类化合物的获取方法为从中、英文文献报道的公开数据以及团队内部实验结果数据集中搜集具有降尿酸活性的黄酮类化合物,且步骤S1-2中对数据集的3D描述符和Rdkit基础理化描述进行计算。
3.根据权利要求1所述的一种靶向制备降尿酸活性的蜂胶黄酮提取方法,其特征在于:所述步骤S1-1中pIC50值的获取方法为根据IC50半抑制率浓度值计算出pIC50值,且计算公式如下所示:
。
4.根据权利要求1所述的一种靶向制备降尿酸活性的蜂胶黄酮提取方法,其特征在于,所述步骤S2的具体方法包括以下步骤:
S2-1、采用QSAR模型对蜂胶黄酮进行降尿酸活性预测,筛选出具有降尿酸活性的蜂胶黄酮化合物;
S2-2、针对高活性目标黄酮类化合物,进行能量优化;
S2-3、经优化后,得到具有最佳分子表面屏蔽电荷的模型DESs的表面各片段的密度分布,表示为σ-Profiles,并分析计算多种DESs表面段的化学势;
S2-4、利用COSMO-thermX软件,计算蜂胶黄酮单体在无限稀释条件下在DES中的相对溶解度。
5.根据权利要求4所述的一种靶向制备降尿酸活性的蜂胶黄酮提取方法,其特征在于,所述步骤S2-2中能量优化的具体方式为:从PubChem中下载,通过MMFF94力场获得各化合物的初始构象,然后,利用密度泛函理论和TZVP基组计算并优化了氢键受体HBAs、氢键供体HBDs、蜂胶黄酮化合物模型的最佳分子几何形状和电荷密度。
6.根据权利要求4所述的一种靶向制备降尿酸活性的蜂胶黄酮提取方法,其特征在于,所述步骤S2-4中在无限稀释条件下计算相对溶解度的具体方式为:将最佳溶解度的对数设为0,所有其他溶剂相对于最佳溶剂或参考溶剂排序;
相对溶解度计算公式如下:
;
式中:表示溶质的相对溶解度;/>和/>分别表示纯化合物的化学势和纯化合物在溶剂中无限稀释时的化学势;/>为j的自由能;其中溶质即蜂胶黄酮化合物。
7.根据权利要求1所述的一种靶向制备降尿酸活性的蜂胶黄酮提取方法,其特征在于,所述步骤S3中,筛选最佳DES提取配方获得定向提取的高含量且具有较好降尿酸活性的蜂胶黄酮提取物包括以下步骤:
S3-1、对定向设计的多种DES溶剂进行化学合成;
S3-2、DES对蜂胶黄酮的提取方法的建立,采用超声辅助提取的方法对蜂胶黄酮进行高效富集,定向提取;
S3-3、通过对不同蜂胶黄酮提取物的总黄酮含量以及主要蜂胶黄酮含量进行测定,从而对COSMO-RS筛选出的DES进行实验验证。
8.根据权利要求7所述的一种靶向制备降尿酸活性的蜂胶黄酮提取方法,其特征在于,所述步骤S3-1中具体的方法为将HBAs和HBDs按适当的摩尔比混合,并添加10~30%的去离子水,然后将混合物置于磁力搅拌装置中,在60~80℃下连续搅拌直到混合物呈现均一透明液体,将混合物冷却至室温,避光保存备用。
9.根据权利要求7所述的一种靶向制备降尿酸活性的蜂胶黄酮提取方法,其特征在于,所述步骤S3-2中为筛选最佳DES的方法为:将0.1~10 g蜂胶粉与3~300 mL不同的DES混合,置于离心管中,将混合物在超声功率为200~600 W、提取温度为40~60℃,提取处理10~30min;然后,将混合物在4℃下6000~12000 rpm离心10~30 min,收集上清液;最后,取100μL上清液用甲醇稀释10倍,经0.22 μm微孔尼龙膜过滤后,用于HPLC进行定量分析。
10.一种采用上述权利要求1-9任一所述方法提取的蜂胶黄酮提取物应用于抑制黄嘌呤氧化酶及降尿酸产品的开发生产以及实验室研究。
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