CN110585221A - 一种改善脂肪变性的合欢皮新木脂体化合物及其应用 - Google Patents
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Abstract
本发明公开了一种改善脂肪变性的合欢皮新木脂体化合物及其应用，属于生物制药技术领域。本发明制备的化合物(‑)‑Lyoniresinol‑9'‑O‑glucoside可改善因FFAs诱导的HepG2细胞脂滴累积及脂肪变性，在给药24h，给药浓度为20μM的条件下，可将脂滴面积由954.125％降低至脂滴平均面积为134.19％，具有重要的应用前景。
Description
技术领域
本发明涉及一种改善脂肪变性的合欢皮新木脂体化合物及其应用，属于生物制药技术领域。
背景技术
合欢皮(Albiziae Cortex)是豆科植物合欢(Albzia julibrissin Durazz)的树皮，是一种较为常用的中药，性味甘、平，具有解郁、和血、宁心和消肿之功效。近年来，随着学者对合欢皮的深入研究，其化学成分和药理药效活性不断的被发现。
迄今为止,已从合欢属植物中分离出多种化合物,其中包括三萜、黄酮、木脂素类等。有文献表明，合欢皮中的木脂素类成分含量较低，对于综合评价合欢皮水溶性木脂素类成分的提取纯化工艺造成了一定的困难。同时，合欢皮常作为复方药物的一剂组分，极少单独应用，因而其含有的各类化学成分的药理活性尚不明确。
发明内容
本发明的第一个目的是提供一种式1所示化合物在制备改善脂肪变性的药物中的应用，其中，
在一种实施方式中，所述木脂体化合物的有效剂量为10μM。
本发明的第二个目的是提供一种药物组合物，含有式1所示的化合物。
在一种实施方式中，所述组合物为药物组合物。
在一种实施方式中，所述组合物还含有药学上可接受的载体。
在一种实施方式中，所述药学上可接受的载体包括稀释剂、赋形剂或溶剂化物。
在一种实施方式中，所述药物组合物的剂型为片剂、胶囊、颗粒剂、散剂、糖浆剂、口服液或注射剂
式1所示化合物在制备药物中的应用。
本发明的第三个目的是提供所述化合物在制备预防或治疗脂代谢紊乱的药物中的应用。
在一种实施方式中，所述药物包括但不限于治疗糖尿病、高血脂或脂肪肝的药物。
本发明还要求保护所述化合物在制备缓解高血糖、高血脂或糖脂代谢紊乱的保健品方面的应用。
有益效果：本发明制备的化合物可改善因FFAs诱导的HepG2细胞脂滴累积及脂肪变性，在给药24h，给药浓度为20μM的条件下，可将脂滴面积由954.125％(*P<0.05，n＝3/组)，降低至脂滴平均面积为134.19％(与FFAs诱导组相比#P<0.05，n＝3/组)，表明(-)-Lyoniresinol-9'-O-glucoside可有效治疗FFAs诱导的脂代谢紊乱及脂肪变性。
附图说明
图1为8:1洗脱段的成分在分析型高效液相色谱检测下的色谱图。
图2为Davisil C18反向柱32％CH3OH洗脱段的成分在半备型高效液相色谱检测下的色谱图。
图3为(-)-Lyoniresinol-9'-O-glucoside的ESI-MS色谱图。
图4为(-)-Lyoniresinol-9'-O-glucoside的1HNMR谱图。
图5为为(-)-Lyoniresinol-9'-O-glucoside的13CNMR谱图。
图6A为(-)-Lyoniresinol-9'-O-glucoside抑制FFAs诱导的脂滴生成(×400)；B为脂滴堆积面积值。*P<0.05与空白对照组相比，#P<0.05与FFAs模型组相比，n＝3/组。
具体实施方式
实施例1
(1)提取：取干燥合欢皮20kg,粉碎，用5倍75％乙醇(水)即每次100L,80℃回流提取2次，每次2小时。过滤除去合欢皮残渣，合并合欢皮75％乙醇提取液，冷冻干燥，得合欢皮乙醇粗提物1.6kg。将粗提物研碎，混悬与2L去离子水中，使其尽可能溶解。混悬后依次用乙酸乙酯及饱和正丁醇萃取，萃取采用每次添加乙酸乙酯和饱和正丁醇少量多次的原则，分别合并乙酸乙酯相及饱和正丁醇相的萃取液，得乙酸乙酯部位和正丁醇部位提取物。
(2)分离：取正丁醇部位254g，用去离子水溶解混悬，采用D101大孔吸附树脂进行分离纯化，分别用2～3倍柱体积的乙醇-水混合溶液为流动相进行梯度洗脱，富集的30％乙醇洗脱段、50％乙醇洗脱段、70％乙醇洗脱段、95％乙醇洗脱段。大孔吸脂30％乙醇洗脱段经硅胶硅胶柱层析，采用梯度溶剂洗脱，以二氯甲烷(CH2Cl2)：甲醇(CH3OH)＝(40:1,20:1,16:1,10:1,8:1和6:1)的混合液为流动相，分别以4倍柱体积左右的流动相进行梯度洗脱，用250ml锥形瓶分别收集洗脱液，经TLC检测合并相同组分，得各洗脱段。对硅胶8:1洗脱段进行分析型高效液相(HPLC)检测(色谱柱为X-Bridge C18,5μm，4.6×250mm，流速为1ml/min，柱温30℃),紫外检测波长为254nm，洗脱条件：
其检测结果如图1所示，根据图1的分析型高效液相色谱图中各组分的保留时间，确定对CH2Cl2:CH3OH＝8:1洗脱段进行过反向硅胶柱(Davisil C18,50μm)的洗脱条件为tR＝22min、24min、26min，60min时所对应的CH3OH浓度39.8％、42.9％、46.2％、100％，因为图1为分析型液相色谱图，色谱柱为X-Bridge C18(5μm,4.6×250mm),而Davisil C18反向柱填料为50μm，所以流动相甲醇浓度均减去10％，即29.8％、32.9％，36.2％，100％。以甲醇和水为洗脱相进行洗脱。根据图1确定洗脱相组合为29％CH3OH(H2O),32％CH3OH(H2O),36％CH3OH(H2O),100％CH3OH。对32％CH3OH洗脱组分进行分析型HPLC检测，摸索分离条件，以确定最佳分离方法(如表一所示)。对32％CH3OH洗脱段进行半制备液相分离(色谱柱为X-Bridge C18,5μm，10×250mm，流速为4ml/min，柱温30℃)，如图2所示(UV检测波长290nm),保留时间tR＝35.20min处的化合物为(-)-Lyoniresinol-9'-O-glucoside(Aj6)。
表1液相色谱条件
在获得前述实施例所描述的液相分离方法的过程中，还尝试过条件一至条件四的分离条件，这些分离条件虽然也能分离出目标单体化合物，但受杂质峰的干扰，所得产物Aj6纯度较差，难以进行下一步的分离鉴定。在此基础上所优化得到的最佳分离条件可以排除杂质峰对目标产物的干扰，分离得到高纯度单体化合物，半制备分离色谱图如图2所示。收集保留时间tR＝35.20min处的化合物，75℃减压旋蒸，回收溶剂，真空干燥箱干燥得Aj6单体化合物。
(3)结构鉴定
Aj6为淡黄色粉末，通过UPLC-ESI-MS检测，其质谱图如图3～5所示。ESI-MS m/z617[M+Cl-]，通过Monoisotopic Mass,Even Electron Ions确定其分子式为C28H38O13。
将样品Aj5用氘代甲醇溶解于核磁管中，采用全数字化核磁共振波谱仪测定1HNMR、13CNMR，其结果如下:
1H NMR(400MHz,CD3OD)δ6.59(1H,s,H-8),6.43(2H,s,H-2’,6’),4.24(1H,d,J＝5.3Hz,H-4),4.15(1H,d,J＝7.7Hz,H-1”),3.87(3H,s,-OCH3),3.84(3H,d,J＝3.6Hz,-OCH3),3.77(6H,s,3’,5’-OCH3),3.72(1H,d,J＝5.2Hz),3.69(1H,d,J＝5.1Hz),3.62(5H,dt,J＝22.8,6.5Hz),3.49(1H,dd,J＝12.5,7.2Hz),3.44–3.35(2H,m),3.30–3.13(3H,m),2.70(2H,t,J＝8.0Hz,H),2.13(1H,dd,J＝13.1,6.9Hz,H-3),1.70(1H,d,J＝6.3Hz,H-1).13C NMR(101MHz,CD3OD)δ147.61(C-3’,5’),147.29(C-5),146.15(C-7),138.06(C-1’),137.49(C-6),133.21(C-4’),128.83(C-9),124.84(C-10),106.41(C-8),105.72(C-2’,6’),102.85(C-1”),76.79(C-5”),76.58(C-3”),73.67(C-2”),70.61(C-3α),70.16(C-4”),64.83(C-2α),61.31(C-6”),58.72(5-OMe),55.53(7-OMe),55.23(3’,5’-OMe),45.19(C-3),41.83(C-4),39.85(C-2),32.43(C-1).
根据质谱及1HNMR、13CNMR最终确定其结构如下，化学名(-)-Lyoniresinol-9'-O-glucoside。
实施例2改善脂肪变性的活性研究
(1)在含有25％葡萄糖、10％FBS(胎牛血清、Gibco)、100U/ml青霉素和100U/ml链霉素的DMEM中培养HepG2细胞(American Type Culture collection,USA)。在含有5％CO2的37℃恒温培养箱中培养。每1-2天更换培养基，将85-90％汇合的细胞以1：3汇合的比例传代。在所有实验中，细胞在第2代和第5代之间使用。取对数生长期的HepG2细胞，铺12孔板，每孔铺10万。待12h细胞贴壁生长后，弃去正常DMEM高糖培养基，加入含有0.3mM FFAs(油酸:棕榈酸＝2:1)的DMEM高糖培养基继续培养24h，即HepG2细胞被造模成为脂代谢紊乱模型后，每孔给药浓度设置5个浓度梯度(其中Control组为正常DMEM培养基培养的HepG2细胞)，即以5μM、10μM、20μM、40μM、80μM的终浓度继续培养24h.
(2)弃去12孔板的培养基，用PBS洗三遍，4％多聚甲醛固定30min，固定结束后以PBS充分洗涤，随后以60％异丙醇浸洗10～15秒，再以PBS洗三遍。以油红O(油红储存液：去离子水＝3：2)室温染色20～30min，显微镜下观察细胞着色情况，弃去油红染液，以60％异丙醇分化至间质清晰(分化约5～10秒即可，分化过度会导致油红O褪色)，最后以PBS洗三遍，每孔加1ml PBS封片、拍照。
(3)油红O染色结果如图6所示(图6B，脂质堆积值面积使用Image-Pro Plus 6.0软件在相同参数下进行处理)。与空白对照组脂滴面积100％相比，高脂培养基培养24h后，FFAs可显著诱导HepG2细胞脂滴累积及脂肪变性，此时脂滴面积与空白对照组相比平均为954.125％(*P<0.05，n＝3/组)，给药24h后，给药浓度为10μM时可显著改善FFAs诱导的HepG2细胞脂代谢紊乱，改善脂肪变性。给药浓度为20μM时，与空白对照组相比脂滴平均面积为134.19％(与FFAs诱导组相比#P<0.05，n＝3/组)，说明(-)-Lyoniresinol-9'-O-glucoside可有效治疗FFAs诱导的脂代谢紊乱及脂肪变性。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (10)
1.化合物在制备改善脂肪变性的药物中的应用，其特征在于，所述化合物的结构式为
2.根据权利要求1所述的应用，其特征在于，所述化合物的有效剂量≥10μM。
3.化合物在制备预防或治疗脂代谢紊乱相关疾病的药物中的应用。
4.根据权利要求3所述的应用，其特征在于，包括糖尿病、高血脂或脂肪肝。
5.化合物在在制备缓解高血糖、高血脂、糖脂代谢紊乱，或改善肥胖的食品或保健品方面的应用。
6.根据权利要求5所述的应用，其特征在于，将所述化合物与食品载体混合，制备食疗产品。
7.一种药物组合物，其特征在于，含有≥10μM的化合物
8.根据权利要求7所述的药物组合物，其特征在于，还含有药学上可接受的载体。
9.根据权利要求7或8所述的药物组合物，其特征在于，所述药学上可接受的载体包括稀释剂、赋形剂或溶剂化物。
10.根据权利要求7或8所述的药物组合物，其特征在于，所述药物组合物的剂型为片剂、胶囊、颗粒剂、散剂、糖浆剂、口服液或注射剂。
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