CN113912654A - 胡芦巴叶提取物及其制备方法与应用 - Google Patents
胡芦巴叶提取物及其制备方法与应用 Download PDFInfo
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- CN113912654A CN113912654A CN202111283368.XA CN202111283368A CN113912654A CN 113912654 A CN113912654 A CN 113912654A CN 202111283368 A CN202111283368 A CN 202111283368A CN 113912654 A CN113912654 A CN 113912654A
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- beta
- fenugreek
- extraction
- glucose
- glucoside
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Abstract
本发明利用超声醇提、溶剂萃取法对胡芦巴叶中的黄酮组分进行了富集制备，采用高效液相色谱‑四极杆飞行时间串联质谱(HPLC‑Q‑TOF‑MS/MS)对其中的黄酮类化合物进行了表征；同时考察了胡芦巴黄酮提取物对H2O2诱导的L02肝细胞应激损伤的保护作用，结果表明胡芦巴叶提取物可以用于制备抗氧化、护肝产品。
Description
技术领域
本发明属于植物提取领域，具体涉及胡芦巴叶提取物及其制备方法与应用。
背景技术
氧化应激损伤是指机体在受到各种有害刺激时，体内活性氧自由基(ReactiveOxygen Species，ROS)的产生和抗氧化能力失衡，过量的ROS造成机体细胞和组织不同程度损伤的病理和生理学过程。氧化应激与肝病的发生发展密切相关。ROS可通过直接或间接方式引起肝细胞内生物大分子如蛋白质、脂质和核酸等的化学修饰，导致肝细胞的结构和功能发生异常。氧化应激损伤还可导致中性粒细胞炎性浸润，蛋白酶分泌增加，产生大量氧化中间产物，及机体存在的一些抗氧化酶的变化，主要包括超氧化物歧化酶超氧化物歧化酶(Superoxide Dismutase,SOD)和过氧化氢酶(Catalase,CAT)等。因此研究肝细胞抗氧化应激损伤，抑制肝细胞凋亡对于治疗肝脏疾病具有重要意义。
胡芦巴(Trigonella foenum-graecum L.)为豆科胡芦巴属一年生草本植物。胡芦巴属药食同源之品，其干燥成熟的种子作为常用中药收入《中华人民共和国药典》。目前已从胡芦巴中分离和研究了多种有效成分，主要包括生物碱、皂苷和黄酮类化合物，这些成分已被证实其潜在的药用价值。除了其已知的药用价值，如胃兴奋剂、抗糖尿病和半乳糖(乳糖诱导)效应外，有研究还表明其具有降低胆固醇血症、抗脂血症、抗氧化剂、护肝、抗炎、抗菌、抗真菌、抗溃疡、抗结石、抗癌药和其他杂酚的药物作用。这些研究大多使用了胡芦巴种子粉末或不同形式的提取物，而对其叶子提取物研究的很少。因此，对胡芦巴叶有效成分的提取、组分分析及其生物活性的研究很有必要。
发明内容
针对上述现有技术的不足，本发明的目的在于提供胡芦巴叶提取物及其制备方法，并通过试验验证胡芦巴叶提取物可以用于制备预防或治疗肝损伤产品，对氧化应激所致的肝细胞损伤具有保护作用。
在本发明的实验方案中，从胡芦巴叶提取8种化合物的制备方法包括以下步骤：
(一)胡芦巴叶用乙醇提取；
(二)浓缩提取液，得胡芦巴叶醇提物浓缩物；
(三)将浓缩物加水混合，用有机溶剂萃取；
(四)水相组分浓缩、干燥，得胡芦巴叶提取物。
在本发明的实施方案中，最终得到的胡芦巴叶提取物：槲皮素3-O-β-D-葡萄糖-(1→2)-β-D半乳糖7-O-β-D-葡萄糖苷、山奈酚3-O-β-D-葡萄糖-(1→2)-β-D-半乳糖7-O-β-D-葡萄糖苷、槲皮素3-O-β-D-(6”-乙酰基)葡萄糖-(1→2)-β-D-半乳糖7-O-β-D-葡萄糖苷、山奈酚3-O-β-D-(6”-乙酰基)葡萄糖-(1→2)-β-D-半乳糖7-O-β-D-葡萄糖苷、槲皮素3-O-β-D-葡萄糖-(1→2)-β-D-葡萄糖苷、山奈酚3-O-β-D-葡萄糖-(1→2)-β-D-半乳糖苷、槲皮素3-O-β-D-(6”'-乙酰基)葡萄糖-(1→2)-β-D-半乳糖苷、山奈酚3-O-β-D-(6”'-乙酰基)葡萄糖-(1→2)-β-D-半乳糖苷。
进一步地，步骤(一)中胡芦巴叶可以先经干燥、粉碎、过40目筛。
进一步地，步骤(一)中提取乙醇浓度为100％～90％。
本发明乙醇提取的方式，可以采用常规的天然药物提取方法，例如超声、回流等提取方法。
在本发明的具体实施方案中，其中超声频率可以选择30～80kHz、提取温度、提取时间0.5～4h、提取次数可以为2～6次，以及提取时间可以为0.5～3h。
其中，在本发明的具体实施方案中，步骤(一)中采用超声提取法，超声频率为30～50kHz，提取料液比为1:5～1:15(g/mL)，提取温度为50～70℃、超声提取次数为4～6次，每次提取时间为0.5～2h。
进一步地，步骤(三)中，采用的有机溶剂为石油醚、乙酸乙酯。
其中，依次使用石油醚、乙酸乙酯对水相进行萃取，最终收取水相。
萃取用有机溶剂的量和萃取次数，可以采用常规的检测手段进行跟踪、确定，例如，有机溶剂与水的体积比可以选自1～10:1，萃取次数1-10次。
例如，先采用2～4倍体积的石油醚萃取2～5次，再使用2～4倍体积的乙酸乙酯萃取2～5次。
其中，步骤(四)中水相组分的浓缩方式为减压浓缩，干燥方式为冷冻干燥。
本发明首次从胡芦巴叶提取物中，检测到以下物质：槲皮素3-O-β-D-葡萄糖-(1→2)-β-D半乳糖7-O-β-D-葡萄糖苷、山奈酚3-O-β-D-葡萄糖-(1→2)-β-D-半乳糖7-O-β-D-葡萄糖苷、槲皮素3-O-β-D-(6”-乙酰基)葡萄糖-(1→2)-β-D-半乳糖7-O-β-D-葡萄糖苷、山奈酚3-O-β-D-(6”-乙酰基)葡萄糖-(1→2)-β-D-半乳糖7-O-β-D-葡萄糖苷、槲皮素3-O-β-D-葡萄糖-(1→2)-β-D-葡萄糖苷、山奈酚3-O-β-D-葡萄糖-(1→2)-β-D-半乳糖苷、槲皮素3-O-β-D-(6”'-乙酰基)葡萄糖-(1→2)-β-D-半乳糖苷、山奈酚3-O-β-D-(6”'-乙酰基)葡萄糖-(1→2)-β-D-半乳糖苷。
最后，本发明通过上述技术方案制备的胡芦巴叶黄酮组分(fenugreek leafflavonoids,FLFs)，用于H2O2诱导L02建立氧化损伤细胞模型的建立，考察FLFs对L02细胞的抗氧化、抗凋亡作用，试验研究表明胡芦巴叶提取物在制备对肝细胞损伤的保护作用产品中具有一定的应用价值，尤其是氧化应激所致的肝细胞损伤。
本发明的有益效果在于：
1、本发明胡芦巴叶提取物的原料易得，提取方法简单高效。
2、本发明的研究结果表明胡芦巴叶提取物对氧化应激所致的肝细胞损伤具有保护作用，可用于制备氧化应激所致的肝细胞损伤的保护产品。
3、本发明利用采用高效液相色谱-四极杆飞行时间串联质谱HPLC-Q-TOF-MS/MS)对胡芦巴叶提取物的黄酮类化合物进行了表征，首次从胡芦巴叶提取物中鉴定出了8种黄酮类化合物，对胡芦巴叶提取物所含成分更加明确，有利于后续胡芦巴叶提取物在药品、保健品等方面的研究。
4、本发明研究表明胡芦巴可能通过调控MAPKs/Nrf2/HO-1途径对H2O2诱导L02细胞损伤发挥保护作用，从而逆转H2O2诱导的肝细胞凋亡，证明胡芦巴叶提取物具有应用于护肝产品中的潜力。
附图说明
图1为FLFs的HPLC图；
图2为FLFs的UPLC-Triple-TOF/MS的总离子流图；
图3为FLFs对L02细胞活力的影响图；
图4为不同浓度H2O2对L02细胞活力的影响图；
图5为FLFs对H2O2损伤L02细胞活力的影响图；
图6中A、B、C、D、E分别为FLFs对H2O2损伤L02细胞LDH、MDA、SOD、GSH和CAT的影响图；
图7中A、B、C、D分别为FLFs对氧化损伤的L02细胞中JNK、ERK1/2、Nrf2和HO-1蛋白表达的影响图；
图8为Hoechst33258染色检测FLFs对L02细胞凋亡的影响图。
具体实施方式
下面结合具体的实施方式来对本发明的技术方案做说明。
实施例1
胡芦巴叶黄酮组分(Fenugreek Leaf flavonoids,FLFs)的制备：胡芦巴叶阴干、粉碎，经95％乙醇超声提取，提取条件为：料液比1:10(g/mL)，提取5次，每次1h，提取温度60℃，超声频率40kHz。提取后合并上清液，减压浓缩，得到胡芦巴叶醇提物浸膏。将醇提物浸膏加水混悬，依次用2倍体积石油醚和乙酸乙酯各萃取5次，将水萃取组分经减压浓缩、冷冻干燥，得FLFs，提取率为：7.8％。
实施例2
胡芦巴叶提取物成分分析及生物活性研究：
1材料
1.1实验药品
胡芦巴采自青海省西宁市湟中县，取新鲜胡芦巴叶片阴干后粉碎，过40目筛，待用。L02人正常肝细胞购自中国科学院上海细胞所。RPMI1640培养基(厂商：BiologicalIndustries公司)；胎牛血清(浙江天杭生物技术股份有限公司，)；磷酸盐缓冲液(PBS；厂商：北京索莱宝科技有限公司)、MTT(厂商：北京索莱宝科技有限公司)；超氧化物歧化酶(Superoxide Orgotein Dismutase,SOD)、谷胱甘肽(glutathione,GSH；厂商：南京建成生物工程研究所)、乳酸脱氢酶(Lactic Dehydrogenase,LDH；厂商：南京建成生物工程研究所)等检测试剂盒；丙二醛(Malondialdehyde,MDA)、过氧化氢酶检测试剂盒(catalase,CAT)、蛋白浓度测定试剂盒、Hoechst33258染色液(厂商：碧云天生物技术有限公司)；c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK；厂商：Cell Signaling Technology公司)、细胞外调节蛋白激酶(extracellular regulated protein kinases,ERK1/2；厂商：CellSignaling Technology公司)、核因子E2相关因子(2nuclear factor erythroid-2related factor,2Nrf2；厂商：Cell Signaling Technology公司),血红素加氧酶(1hemeoxygenase 1,HO-1；厂商：Cell Signaling Technology公司)等抗体；兔二抗(厂商：武汉爱博泰克生物科技有限公司)；30％H2O2、乙醇、乙腈、甲酸(厂商：国药集团化学试剂有限公司)。
1.2实验仪器
二氧化碳培养箱(Memmert ICP500，德国)；酶标仪(BioTek Synergy Epoch2，美国)；倒置显微镜(Leica DMi1，德国)；高速冷冻离心机(Eppendorf5430R，德国)；Tanon5200化学发光成像系统(上海天能科技有限公司)；电泳仪(Tetra Cell，美国伯乐公司)；PL303电子天平(梅特勒-托利多仪器有限公司)；冷冻干燥机(LGJ-10，北京四环科学仪器厂有限公司)；激光共聚焦显微镜(Leica，德国)；AcquityTM ultra型高效液相色谱仪(Waters，美国)，Triple TOF 5600+型飞行时间质谱，配有电喷雾离子源(AB SCIEX，美国)；Eppendorf minispan离心机(Eppendorf，德国)。
2实验方法
2.1 FLFs的表征
取适量胡芦巴黄酮提取物溶于甲醇，离心取上清，进样。液相条件：流动相：A：0.1％甲酸水溶液B：0.1％甲酸乙腈流速：0.3mL·min-1检测波长：254nm色谱柱：BEH-C18column 1.7μm,2.1×150mm；Waters Corp.)；进样量：3μL柱温箱：50℃，梯度洗脱程序如表1。质谱条件：UPLC-Triple-TOF 5600+飞行时间液质联用仪：负离子扫描模式；扫描范围：m/z 100-1500；离子源温度TEM)：550℃；离子源电压IS)：-4500V(负；一级扫描：去簇电压DP)：100V；聚焦电压CE)：10V；二级扫描：使用TOF MS～Product Ion～IDA模式采集质谱数据，CID能量为-20、-40和-60V，进样前，用CDS泵做质量轴校正，使质量轴误差小于2ppm。
表1液相梯度洗脱程序
2.2 FLFs储备液的配置
取水萃取物进行冷冻干燥，将FLFs采用DMSO溶解，配置成10mg·mL-1的储备液，0.22μm滤膜过滤除菌，使用时采用RPMI 1640细胞培养基稀释至所需浓度。
2.3 L02细胞培养
将L02细胞以含10％胎牛血清、1％青链霉素的RPMI 1640培养基，置于37℃、5％CO2细胞培养箱中培养，用含EDTA的胰酶进行消化传代，选取对数生长期的细胞进行后续实验。
2.4 MTT检测FLFs对L02细胞存活率的影响
取对数生长期L02细胞调整浓度至5×104个/孔，每孔150μL接种于96孔板中培养24h。用含0～100μg·mL-1不同浓度的FLFs溶液替换原有培养基，继续培养12h后，MTT法检测490nm波长处的吸光值(A)。并按公式1计算细胞存活率。
公式1：细胞存活率/％＝A处理组/A空白组×100％
2.5 L02细胞氧化损伤模型的建立
按2.4的方法将L02细胞接种于96孔培养板。用无血清培养基将H2O2配成0～1000μmol·L-1等10个不同浓度，分别加入96孔板中，作用12h后，MTT法检测490nm波长处的吸光值(A)。计算细胞存活率，确定H2O2的最佳造模浓度。
2.6 FLFs对损伤后L02细胞存活率的影响
取对数期细胞调整浓度至5×104个/孔，每孔150μL接种于96孔板，接种24h后细胞完全贴壁。实验分为空白对照组(Con)、H2O2组和H2O2+不同浓度的FLFs组。处理12h后，MTT法检测各组细胞在490nm波长处的吸光度值(A)，并计算细胞存活率。
2.7 L02细胞内LDH泄漏量、MDA含量、GSH、SOD和CAT活性的测定
取对数期L02细胞接种于6孔板，接种24h后细胞完全贴壁。实验分为Con、H2O2组和H2O2+不同浓度的FLFs组。孵育12h后，严格按照试剂盒说明书进行操作，检测细胞内LDH泄漏量，SOD和CAT的活性以及GSH和MDA的含量。
2.8 Hoechst 33258荧光核染色
取无菌盖玻片置于六孔板内，同2.7方法培养细胞。刺激细胞发生凋亡并加FLFs处理后，吸尽培养液，加入试剂盒中的固定液10min，吸弃固定液，用PBS洗2遍，吸弃液体后加入Hoechst 33258染色液，染色5min，吸弃染色液，用PBS洗两遍，每次3min，吸尽液体，加一滴抗荧光淬灭封片液于载玻片上，盖上贴有细胞的盖玻片，用激光共聚焦显微镜观察细胞状态。
2.9 Western blot检测蛋白的表达
同2.7方法培养细胞。L02细胞加FLFs处理12h，按照试剂盒说明书抽提细胞总蛋白，用BCA法测定蛋白浓度。样品蛋白进行变性处理后，用10％的SDS-PAGE分离胶、5％的浓缩胶分离蛋白，转膜，5％脱脂牛奶封闭1h，用1:1000稀释的兔抗JNK、p-JNK、ERK1/2、p-ERK1/2、β-Actin、Nrf2和HO-1等一抗在4℃条件下孵育过夜，TBST洗3次，再用1:5000稀释的辣根过氧化物酶标记的二抗室温孵育1h，TBST洗3次，显影后拍照，并对蛋白条带进行灰度分析。
2.10数据处理
Western blot数据采用Image J软件采集处理，采用Graphpad prism 8.0统计软件进行统计学分析。数据用平均数±标准差表述。各组均数之间采用one-way ANOVA，以P<0.05认为差异具有统计学意义。
3实验结果
3.1 FLFs成分分析
利用HPLC-Q-TOF-MS/MS对FLFs中的特征成分进行了分析，其液相色谱见图1，总离子流图见图2。所得分离组分的色谱和质谱数据经计算机数据处理系统自动检索和与质谱库对照，结合分子离子峰和二级质谱碎片离子峰，经查阅文献对照和人工检索解析，鉴定出FLFs主要含有8种黄酮类化合物。黄酮苷类化合物的糖基多结合在3,5,7-位，依据正、负离子的二级质谱碎片峰信息，推定其结构关系，结合黄酮类裂解规律并与对照品比较，最终推定化合物结构。
经Q-TOF-MS/MS检测，FLFs中的化合物1、3、5和7其准分子离子[M-1]-分别为m/z787.196 3、829.205 5、625.141 4、667.153 2，其二级质谱均产生了m/z 301的离子，表明其苷元均为槲皮素(quercetin)。化合物1的二级质谱中产生了m/z 625.148 8[M－H－162]-和462.083 3[M－H－324]-的离子，表明分子中存在3个六碳糖结构，并推测其通过1个单糖链和1个二糖链结构连接于母核，根据高分辨质谱结果拟合，其分子式为C33H40O22，再结合Scifinder和Reaxy数据库检索结果，推测化合物1为槲皮素3-O-β-D-葡萄糖-(1→2)-β-D-半乳糖7-O-β-D-葡萄糖苷(quercetin 3-O-β-D-glucosyl-(1→2)-β-D-galactoside 7-O-β-D-glucoside)。化合物3的二级质谱中产生了m/z 667.158 7[M－H－162]-和462.0823[M－H－324－43]-的离子，较化合物1多1个乙酰基，推测该化合物为槲皮素3-O-β-D-(6”-乙酰基)葡萄糖-(1→2)-β-D-半乳糖7-O-β-D-葡萄糖苷(quercetin 3-O-β-D-(6”-acetyl)glucosyl-(1→2)-β-D-galactoside 7-O-β-D-glucoside)。化合物5的二级质谱中产生了m/z 301.035 63[M－H－324]-离子，表明分子中存在2个六碳糖结构，根据数据库检索结果，推测该化合物为槲皮素3-O-β-D-葡萄糖-(1→2)-β-D-葡萄糖苷(quercetin 3-O-β-D-glucosyl-(1→2)-β-D-glucoside)。化合物7的二级质谱中产生了m/z 625.148 0[M－H－43]-和301.035 4[M－H－324－43]-离子，较化合物5多1个乙酰基，推测该化合物为槲皮素3-O-β-D-(6”'-乙酰基)葡萄糖-(1→2)-β-D-半乳糖苷(quercetin 3-O-β-D-(6”'-acetyl)glucosyl-(1→2)-β-D-galactoside)。
经Q-TOF-MS/MS检测，FLFs中的化合物2、4、6和8其准分子离子[M－1]-分别为m/z771.200 4、813.211 0、609.146 4、651.156 8，4个化合物的二级质谱均产生了m/z 285的离子，表明其苷元均为山柰酚(kaempferol)。化合物2的二级质谱中产生了m/z609.152 7[M－H－162]-和446.087 6[M－H－324]-的离子，表明分子中存在3个六碳糖结构，并推测其通过1个单糖链和1个二糖链结构连接于母核，根据高分辨质谱结果拟合，其分子式为C33H40O21，再结合数据库检索结果，推测化合物2为山奈酚3-O-β-D-葡萄糖-(1→2)-β-D-半乳糖7-O-β-D-葡萄糖苷(kaempferol 3-O-β-D-glucosyl-(1→2)-β-D-galactoside 7-O-β-D-glucoside)。化合物4的二级质谱中产生了m/z 651.166 5[M－H－162]-和446.089 7[M－H－324－43]-的离子，较化合物2多1个乙酰基，推测该化合物为山奈酚3-O-β-D-(6”-乙酰基)葡萄糖-(1→2)-β-D-半乳糖7-O-β-D-葡萄糖苷(kaempferol 3-O-β-D-(6”-acetyl)glucosyl-(1→2)-β-D-galactoside 7-O-β-D-glucoside)。化合物6的二级质谱中产生了m/z 285.040 7[M－H－324]-离子，表明分子中存在2个六碳糖结构，同时产生的m/z255.029 3[A－H－CO]-和227.0341[A－H－2CO]-离子，符合山柰酚的裂解规律，该化合物为山奈酚3-O-β-D-葡萄糖-(1→2)-β-D-半乳糖苷(kaempferol 3-O-β-D-glucosyl(1→2)-β-D-galactoside)。化合物8的二级质谱中产生了m/z 285.040 2[M－H－324－43]-离子，较化合物6多1个乙酰基，推测该化合物为山奈酚3-O-β-D-(6”'-乙酰基)葡萄糖-(1→2)-β-D-半乳糖苷(kaempferol 3-O-β-D-(6”'-acetyl)glucosyl-(1→2)-β-D-galactoside)。各化合物裂解的准分子离子峰、碎片峰质核比结果见表2。
表2 FLFs的LC-MS数据信息与指认
3.2不同浓度FLFs对L02细胞活力的影响
FLFs以不同浓度作用L02细胞12h后，FLFs在3.125～25μg·mL-1时能增强L02细胞的活力，在浓度大于50μg·mL-1时抑制L02细胞的增殖活性，并且随着FLFs浓度的增加抑制作用增强(与空白组比较，*P<0.05，**P<0.01；图3)，基于上述结果，本实验采用3.125～25μg·mL-1的浓度梯度进行后续试验。
3.3 L02细胞损伤模型的建立
为筛选出合适的H2O2建模浓度，用不同浓度的H2O2刺激L02细胞12h。随着H2O2浓度的增加，L02细胞的存活率也明显下降(与空白组比较，*P<0.05；**P<0.01图4)。以半数致死量(IC50)为原则，本研究选择H2O2的造模浓度为650μmol·L-1。
3.4 FLFs对H2O2损伤L02细胞活力的影响
由MTT结果可见，H2O2处理组细胞存活率与空白组相比差异显著降低(P<0.01)；与H2O2组相比，3.125-25μg·mL-1浓度下FLFs能显著提高H2O2损伤后L02细胞的存活率(P<0.01)(与空白组比较，**P<0.01；与H2O2组相比较，##P<0.0；图5)。
3.5 FLFs对H2O2损伤L02细胞LDH、SOD、GSH、MDA和CAT的影响
如图6A和图6B(与空白组比较，*P<0.05，**P<0.01；与H2O2组相比较，#P<0.05，##P<0.01)所示，与空白组相比，H2O2组的LDH和MDA水平显著升高(P<0.01)，说明肝细胞被损伤。经FLFs处理后，低浓度的FLFs就能显著降低LDH和MDA水平(P<0.01)，且随FLFs浓度的增大，效果越明显。如图6C-6E(与空白组比较，*P<0.05，**P<0.01；与H2O2组相比较，#P<0.05，##P<0.01)所示，与空白组相比，H2O2组的SOD、GSH和CAT水平显著降低，FLFs浓度大于6.25μg·mL-1时，SOD、GSH和CAT活性水平均有所升高。上述结果表明，FLFs能够改善H2O2对L02细胞的氧化损伤。
3.6 FLFs对氧化损伤的L02细胞中JNK、ERK1/2、Nrf2、HO-1蛋白表达的影响
为了明确FLFs对于H2O2损伤的L02细胞的所起的保护作用，本发明利用Westernblot检测H2O2损伤的L02细胞内JNK、ERK1/2、Nrf2、HO-1等的表达水平及其磷酸化表达水平。H2O2刺激后引起L02细胞中p-JNK/JNK和p-ERK1/2/ERK1/2水平上调，与对照组相比差异具有统计学意义(P<0.01)，低浓度(3.125μg·mL-1)的FLFs作用后就能显著抑制p-JNK/JNK和p-ERK1/2/ERK1/2比值的增加(P<0.05，P<0.01)，且随着FLFs浓度逐渐增加，抑制作用越显著，结果如图7A和7B(与空白组比较，**P<0.01；与H2O2组相比较，#P<0.05,##P<0.01)所示。H2O2刺激后引起L02细胞中Nrf2和HO-1蛋白表达受到抑制，FLFs作用后上调了Nrf2和HO-1的表达水平(图7C和7D；与空白组比较，**P<0.01；与H2O2组相比较，#P<0.05,##P<0.01)。
3.7 Hoechst 33258凋亡染色观察FLFs对H2O2诱导L02细胞凋亡的影响
采用Hoechst 33258染色后(图8)，空白组细胞呈均匀微弱的荧光，H2O2作用L02细胞后，细胞核出现高强度集中的荧光，且胞核变小、浓集，呈现出凋亡细胞的特征，用不同浓度FLFs处理后，细胞核荧光强度随剂量增加而减弱、凋亡细胞的数量也减少，胞核逐渐恢复正常形态。
4结果分析
本发明通过制备FLFs，考察了FLFs对H2O2刺激后L02细胞氧化应激损伤的保护作用探究了作用机制。H2O2是一种重要的活性氧成分，过量的H2O2可以导致肝细胞发生损伤和凋亡。当肝细胞受到H2O2诱导的氧化应激损伤时，细胞存活率和细胞毒性是反映细胞受损程度的重要指标。首先采用650μmol·L-1H2O2，作用12h后成功构建了L02细胞氧化应激损伤模型。并通过MTT实验发现FLFs(3.125～25μg·mL-1)能显著提高H2O2损伤后L02细胞的存活率。体内抗氧化剂的消耗与自由基的增加有关。SOD、CAT和GSH均为内源性清除有毒自由基的主要抗氧化酶，可以直接清除活性氧ROS。检测结果显示，FLFs可提高了GSH、SOD和CAT活性。LDH作为胞浆内含酶之一，在正常情况下不能透过细胞膜，当靶细胞受到损伤时，细胞膜通透性改变，LDH可释放至介质中，因此检测LDH的泄漏量，可反映细胞损伤程度，而检测丙二醛的量可反映机体脂质过氧化的程度。该研究结果发现，FLFs可明显减少氧化损伤后LDH释放量和MDA含量。上述结果均表明FLFs可以提高机体清除氧自由基和抗氧化能力，能有效保护由H2O2导致的肝细胞损伤。
MAPK信号通路广泛参与氧化应激所致的肝细胞损伤与凋亡，p38、ERK和JNK是MAPK家族的3个亚群，精细调控细胞的增殖、分化和凋亡。Nrf2/HO-1途径是参与机体氧化应激调控的另一重要信号通路，有效调控Nrf2/HO-1信号轴可成为治疗氧化应激性疾病的重要靶点。Nrf2/HO-1途径已被证实在H2O2诱导的肝细胞中被激活，其激活是启动细胞内源性抗氧化途径之一。此外，MAPKs可协同激活Nrf2/HO-1通路，而Nrf2又可通过调节基因表达影响JNK活化。而本发明的研究结果表明FLFs处理组的ERK1/2和JNK蛋白的磷酸化水平显著低于H2O2组，且FLFS能浓度依赖性地促进Nrf2和HO-1的表达水平。综上所述，FLFs可能通过调控MAPKs/Nrf2/HO-1途径对H2O2诱导L02细胞损伤发挥保护作用，从而逆转H2O2诱导的肝细胞凋亡。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。
Claims (10)
1.一种从胡芦巴叶中提取8种化合物的方法,其特征在于，包括以下步骤：(一)胡芦巴叶用乙醇提取；(二)浓缩提取液，得胡芦巴叶醇提物浓缩物；(三)将浓缩物加水混合，用有机溶剂萃取；(四)水相组分浓缩、干燥，得含有如下8种化合物的胡芦巴叶提取物；所述化合物为：槲皮素3-O-β-D-葡萄糖-(1→2)-β-D半乳糖7-O-β-D-葡萄糖苷、山奈酚3-O-β-D-葡萄糖-(1→2)-β-D-半乳糖7-O-β-D-葡萄糖苷、槲皮素3-O-β-D-(6”-乙酰基)葡萄糖-(1→2)-β-D-半乳糖7-O-β-D-葡萄糖苷、山奈酚3-O-β-D-(6”-乙酰基)葡萄糖-(1→2)-β-D-半乳糖7-O-β-D-葡萄糖苷、槲皮素3-O-β-D-葡萄糖-(1→2)-β-D-葡萄糖苷、山奈酚3-O-β-D-葡萄糖-(1→2)-β-D-半乳糖苷、槲皮素3-O-β-D-(6”'-乙酰基)葡萄糖-(1→2)-β-D-半乳糖苷、山奈酚3-O-β-D-(6”'-乙酰基)葡萄糖-(1→2)-β-D-半乳糖苷。
2.如权利要求1所述的提取方法，其特征在于，步骤(一)中胡芦巴叶经干燥、粉碎、过40目筛。
3.如权利要求1所述的提取方法，其特征在于，步骤(一)中提取乙醇浓度为100％～90％。
4.如权利要求1所述的提取方法，其特征在于，步骤(一)中采用超声提取；进一步地，超声提取料液比为1:5～1:15(g/mL)，提取温度50～70℃，超声提取次数为4～6次，每次提取时间为0.5～2h，超声频率30～50kHz。
5.如权利要求1所述的提取方法，其特征在于，如权利要求1所述的提取方法，其特征在于，步骤(三)中采用的有机溶剂为石油醚、乙酸乙酯。
6.如权利要求1所述的提取方法，其特征在于，步骤(三)中有机溶剂与水的体积比为2:1，萃取次数为4～6次。
7.如权利要求1所述的提取方法，其特征在于，步骤(四)中水相组分的浓缩方式为减压浓缩，干燥方式为冷冻干燥。
8.一种胡芦巴叶提取物，其特征在于由权利要求1～7任一所述的制备方法制备。
9.胡芦巴叶提取物在制备保护肝细胞损伤产品中的应用，进一步地，所述胡芦巴叶提取物如权利要求8所述。
10.如权利要求9所述的胡芦巴叶提取物在制备预防或治疗肝损伤产品中的应用，进一步地，所述肝细胞损伤为氧化应激所致的肝细胞损伤。
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