Screening Quality Markers of Regulating Lipid Metabolism Activity of Rubus suavissimus S. Lee Based on Spectral Effect Relationship
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摘要: 目的：基于谱效关系筛选广西甜茶调节脂代谢活性部位中的质量标志物。方法：采用95%乙醇提取广西甜茶，依次用石油醚、氯仿、乙酸乙酯、正丁醇进行萃取得到不同萃取部位，将不同萃取部位溶液作用于诱导分化后的3T3-L1前脂肪细胞，以油红O染色法和细胞甘油三酯释放量为指标，筛选活性部位。采用超高效液相色谱-四极杆飞行时间质谱（UPLC-Q-TOF-MS/MS）对活性部位进行成分分析和解析，基于灰色关联度法和偏最小二乘回归法分析活性部位共有峰与其调节脂代谢药效之间的谱效关系，筛选质量标志物。结果：乙酸乙酯及正丁醇萃取部位为调节脂代谢的活性部位，鞣花酸、矢车菊素-3-O-芸香糖苷、甜茶素和对映-贝壳杉-16-烯-19-羧酸-13-O-β-D-葡萄糖苷4个成分与调节脂代谢活性作用高度相关，可作为甜茶调节脂代谢活性的质量标志物。结论：基于谱效关系的广西甜茶质量标志物的研究，对阐明药效物质基础、筛选与药效相关的核心质量标志物、保证药材质量的可控性和合理应用有重要意义。Abstract: Objective: The study was designed to test solvent extracts of Rubus suavissimus (RS) for compounds that regulate lipid metabolism and to measure their relative activity as quantitative markers of lipid metabolism-regulating activities based on the spectrum-effect relationship. Method: RS was extracted with 95% ethanol, the different extraction parts were successively extracted with petroleum ether, methylene chloride, ethyl acetate and n-butanol, respectively. The solution of different extraction parts was applied to the induced differentiated 3T3-L1 preadipocytes, and the oil red O staining and triglyceride release from the cells were determined to screen the best metabolism-regulating site. Analysis of different extraction sited by UPLC-Q-TOF-MS/MS technique to separate and identify the components. Based on specific assays, image intensity analysis, grey correlation and partial least squares regressions, the spectrum-effect relationship of the most abundant active components and their ability to regulate lipid metabolism were determined, and the quality markers were screened. Results: Ethyl acetate and n-butanol extraction site was the best metabolism-regulating site. Four components, including ellagic acid, centaurin-3-O-rutinoside, rubusoside and enantio-kauri-16-ene-19-carboxylic acid-13-O-β-D-glucoside were highly associated with metabolism-regulating effects. It could be used as quality markers for regulating lipid metabolism. Conclusion: Investigation of the quality markers of RS extracts based on the spectrum-effect relationship is of great importance for elucidating the pharmacodynamics, screening the core quality markers for therapeutic activity, and ensuring the safety and rational application of traditional medicines.
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Keywords:
- Rubus suavissimus /
- 3T3-L1 cells /
- lipid metabolism /
- spectrum-effect relationship /
- quality marker
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广西甜茶（以下称甜茶）为蔷薇科悬钩子属植物甜叶悬钩子Rubus suavissimus S. Lee[1]或Rubus chingii var. suavissimus （S. K. Lee） L. T. Lu [2]的叶，是广西特色珍稀甜味植物[3]，收录于《广西壮族自治区壮药质量标准（第二卷）》[4]。甜茶在民间可代茶饮用，因其无毒、高甜度、低热量、具有保健功能，于2020年被纳入广西食品安全地方标准[5]。甜茶中富含甜茶素[6]、多酚类[7]、萜类[8]及黄酮类[9]等成分，具有改善糖脂代谢异常[10−14]、降血压[15]等药理活性。甜茶作为一种非糖的天然低热量甜味剂，可满足肥胖症、高血压和糖尿病患者对甜味的需求[16]。因其甜度高和热量低，已应用于酸奶[17]、糕点[18−20]、保健饮料[21]和低糖饮料[22]等食品。利用甜茶代替部分蔗糖加工食品，不仅可以降低成本，同时也符合食品逐渐向低糖化发展的要求。可见甜茶因高甜度、低热量和广泛的药理活性而日益受到国内外高度关注，在食品、医药及保健品等领域极具开发前景。
目前已有关于甜茶提取物或甜茶中活性成分调节脂代谢的研究，广西甜茶醇提物对葡萄糖致小鼠高血糖有抑制作用[12]；甜茶素能降低高脂小鼠的血糖和血清甘油三酯水平，抑制小鼠的糖异生，减少胆固醇的含量，从而调节脂代谢紊乱[15，23−25]。但未针对具体的调节脂代谢活性部位进行深入研究，且未阐明其作用的物质基础。如何全面反映甜茶的调节脂代谢成分是研究甜茶调节脂代谢作用物质基础的难点。中药质量标志物（Q-marker）可以为传统医药科学合理的质量控制提供一定的研究方向和理论依据[26]。谱效关系在中药的药效物质研究中广泛应用，通过数理统计模型对图谱和药效指标进行相关性分析，确定与药效相关成分，从而筛选出Q-Marker[27−30]，该方法可以明确地反映Q-Marker与药效活性的关联，可寻找潜在的药效物质基础。
综上，本研究制备甜茶不同溶剂萃取部位，通过3T3-L1细胞模型筛选调节细胞脂质代谢的活性部位，并采用UPLC-Q-TOF-MS/MS鉴定化学成分；基于灰色关联度法和偏最小二乘回归法的谱效关系筛选出甜茶调节脂代谢活性的质量标志物，为甜茶调节脂代谢药效物质的研发和质量控制提供参考。
1. 材料与方法
1.1 材料与仪器
甜茶样品 采集于广西壮族自治区来宾市金秀瑶族自治县，经广西中医药大学药学院蔡毅教授鉴定为蔷薇科悬钩子属植物甜叶悬钩子Rubus suavissimus S. Lee的叶，标本保存于广西中医药大学药学院中药鉴定教研室；小鼠胚胎成纤维3T3-L1细胞株（前脂肪细胞） 美国ATCC公司；95%乙醇、石油醚、二氯甲烷、乙酸乙酯、正丁醇 分析纯，上海阿拉丁生化科技股份有限公司；3-异丁基-1-甲基黄嘌呤（IBMX） 美国Sigma公司；胰岛素注射液 江苏万邦生化医药股份有限公司；胎牛血清（FBS）、高糖DMEM 美国Gibco公司；地塞米松（Dex）、二甲基亚枫（DMSO）、噻唑兰（MTT） 北京索莱宝科技有限公司；罗格列酮片 成都恒瑞制药有限公司；甘油三酯（TG）测定试剂盒 南京建成生物工程研究所；没食子酸、芦丁、甜茶素、甜菊醇、异甜菊醇 成都普菲德生物技术有限公司；咖啡酸、鞣花酸、金丝桃苷、异槲皮苷、槲皮苷、熊果酸、齐墩果酸 成都曼斯特生物科技有限公司；山柰酚、槲皮素 上海中药标准化研究中心，所有对照品纯度均≥98%。
Bnider C150 CO2培养箱 德国Bnider公司；Infinite 200 PRO酶标仪 瑞士Tecan公司；N-1300旋转蒸发仪 上海爱朗仪器有限公司；CKX53倒置显微镜 日本Olympus公司；DXL-D代谢笼 苏州市冯氏实验动物设备有限公司；Bruker impact HD型Q-TOF-MS/MS 德国Bruker公司；InertSil ODS-3（2 µm，2.1×150 mm）色谱柱 岛津（上海）实验器材有限公司。
1.2 实验方法
1.2.1 甜茶不同溶剂萃取物的制备
将甜茶干燥粉碎后过二号筛，取10 g粉末分别用100、80和60 mL的95%乙醇超声（300 W，40 kHz）1 h，提取3次，抽滤后合并滤液，减压回收乙醇，即得甜茶总提取物；将甜茶总提取物干膏加入100 mL超纯水超声成混悬液后，依次用石油醚、二氯甲烷、乙酸乙酯和正丁醇进行萃取，萃取溶剂与浓缩液体积比为1:1，萃取3次，合并同部位萃取液，浓缩挥干溶剂分别得到总提取物（出膏率30.51%）、石油醚部位（出膏率5.58%）、二氯甲烷部位（出膏率1.32%）、乙酸乙酯部位（出膏率10.94%）和正丁醇部位（出膏率12.21%）。
1.2.2 活性部位的筛选
1.2.2.1 MTT实验
采用经典的“鸡尾酒法”对3T3-L1细胞进行培养及诱导[31]。以浓度为5000 cell/孔的标准将对数生长期的3T3-L1细胞接种于96孔板，分为空白组、对照组、给药组。待细胞贴壁后，空白组为只含有细胞的完全培养基，对照组即体积分数为0.1%的DMSO的细胞培养液。给药组加入不同浓度的甜茶萃取部位作用48 h。吸弃孔内培养液，加入5 mg/mL MTT溶液避光孵育4 h，最后加入150 μL DMSO，使用酶标仪于570 nm处测量各孔吸光度值。
细胞存活率按下式计算：
细胞存活率(%)=A1−A2A3−A2×100 式中：A1、A2、A3分别为给药组、对照组、空白组的吸光度值。
1.2.2.2 3T3-L1细胞诱导分化
将细胞接种于24孔板，分为空白组、对照组、罗格列酮组、给药组。待孔内细胞密度≥90%后，空白组加入含10% FBS的高糖DMEM培养基，其他组使用含10% FBS、0.5 mmol/L IBMX、1 μmol/L Dex、10 μg/mL胰岛素的高糖DMEM培养基开始诱导，给药组诱导培养基中分别加入甜茶不同萃取部位的低、中、高浓度溶液，罗格列酮组加入5 μmol/L罗格列酮的药液，对照组加入等量DMSO（≤0.1%），持续诱导2 d。诱导结束后换成含10% FBS、10 μg/mL胰岛素的高糖DMEM培养基进行分化，每3 d更换一次培养基，共分化6 d。
1.2.2.3 TG释放量测定及油红O染色
分化结束后，吸取孔板内培养液，1000 r/min离心10 min，取上清液按试剂盒说明测定细胞TG释放量。同时，用PBS清洗孔板内细胞2次，4%多聚甲醛固定细胞15 min，使用PBS清洗2次后加入0.3%油红O染液染色1 h，吸弃染液，清洗后于倒置显微镜下观察并拍照。
1.2.3 UPLC-Q-TOF-MS/MS测定活性部位化学成分
1.2.3.1 供试品溶液的制备
采用UPLC-Q-TOF-MS/MS测定甜茶调节脂代谢活性部位中的化学成分，样品使用DMSO溶解，浓度为100.00 mg/mL，过0.22 μm微孔滤膜，上机检测。
1.2.3.2 对照品溶液的配制
精密称定咖啡酸、芦丁、金丝桃苷、槲皮苷、异槲皮苷、槲皮素、甜茶素、山柰酚、甜菊醇、异甜菊醇、熊果酸和齐墩果酸对照品，加色谱甲醇分别配制成1.54、1.55、1.19、1.39、1.26、1.19、4.96、0.93、1.05、1.29、1.12和1.02 mg/mL的对照品储备液1；分别精密称定没食子酸和鞣花酸，分别以纯水和DMSO配制成1.12和2.11 mg/mL的对照品储备液2和3，保存于4 ℃冰箱中备用。
混合对照品溶液的制备：取上述对照品储备液各100 μL混合，再加入1 mL甲醇，超声混匀，12000 r/min离心5 min，取上清液过0.22 μm微孔滤膜，即得。
1.2.3.3 色谱条件
流动相：0.1%甲酸水溶液（A）-乙腈（B），洗脱梯度（0~1 min，5% B；1~6 min，5%~15% B；6~20 min，15%~24% B；20~25 min，24%~33% B；25~30 min，33%~54% B；30~35 min，54%~80% B；35~38 min，80% B；38~40 min，80%~95% B；40~43 min，95% B；43~44 min，95%~5% B；44~50 min，5% B）；流速：0.3 mL/min；柱温40 ℃；进样量5 μL。
1.2.3.4 质谱条件
负离子模式电喷雾电离离子源（ESI−），质量扫描范围m/z 50~1000，干燥气温度200 ℃，流速8 L/min，雾化气压力29 psi，毛细管电压3.5 kV，采用MS/MS automatic模式采集样品质谱数据，设置能量梯度为10、25、40 eV。
1.2.4 谱效关系分析
1.2.4.1 灰色关联度法
灰色关联度法是构建谱效关系的常用方法，其对模糊系统分析有效。取峰面积>500万的活性部位共有峰进行计算。以峰面积为比较序列Xi，细胞TG释放量为参考序列X0，通过均值法无量纲化峰面积，计算绝对差序列、两极最小差、两极最大差，并计算关联系数，最后取序列平均值，计算共有峰对细胞TG释放量的灰色关联系度。
1.2.4.2 偏最小二乘回归法
偏最小二乘回归分析法是一种多元统计回归建模方法，主要研究的是多自变量对多因变量的回归建模，能在样本数较少情况下建立精度较高的回归模型。以归一化后的峰面积为自变量，细胞TG释放量为因变量，导入SIMCA 10.0计算VIP值及标准化回归系数，当VIP>1、回归系数为正相关且≥0.0600时，认为峰代表的化合物显著影响3T3-L1细胞TG释放量。
1.3 数据处理
利用SPSS 24.0软件进行数据分析，数据以均数±标准差（¯x±s）的形式表示。数据均进行正态性和方差齐性检验，采用单因素方差分析进行组间比较，后用LSD检验进行多重比较。利用SPSSAU在线平台进行灰色关联法分析，运用SIMCA 10.0软件进行偏最小二乘回归分析。
2. 结果与分析
2.1 甜茶调节3T3-L1细胞脂代谢活性部位筛选
2.1.1 甜茶不同萃取部位对3T3-L1细胞活力的影响
如图1所示，甜茶总提取物、乙酸乙酯和正丁醇萃取部位在浓度为12.5~50.0 mg/L时对细胞活力无显著影响（P>0.05）；石油醚部位在浓度为12.5 mg/L对细胞活力无显著影响（P>0.05），浓度为25.0~50.0 mg/L时可显著抑制3T3-L1细胞活性（P<0.05）；二氯甲烷部位在浓度为0.3125~1.2500 mg/L时对细胞活力无显著影响（P>0.05）。
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图 1 甜茶不同萃取部位对3T3-L1细胞活力的影响（n=6）注：与空白组相比，*表示差异显著P<0.05，**表示差异极显著P<0.01。Figure 1. Effect of different fractions of RS on the viability of 3T3-L1 cells (n=6)2.1.2 甜茶不同萃取部位对3T3-L1细胞分化及TG释放量的影响
3T3-L1前脂肪细胞在PPARγ激动剂的作用下向成熟脂肪分化，表现为细胞内脂滴增加[32]，促使脂质累积在脂肪组织中以减轻其他组织的脂毒性[33]。如图2、图3所示，与空白组相比，对照组细胞内脂滴增多，TG含量极显著上升（P<0.01）；相较于对照组，罗格列酮组细胞脂滴和TG释放量极显著增加（P<0.01），表明罗格列酮显著促进3T3-L1细胞分化；石油醚部位对细胞分化无明显影响（P>0.05）；甜茶总提取物及二氯甲烷部位仅在高剂量时表现出促进脂滴形成和TG累积作用（P<0.05）；乙酸乙酯部位中、高剂量组细胞脂滴及TG含量显著增加（P<0.05，P<0.01）；正丁醇部位各剂量组细胞脂滴及TG释放量显著增多（P<0.05，P<0.01）；表明甜茶正丁醇及乙酸乙酯部位具有显著促进3T3-L1细胞分化的作用，提示甜茶调节3T3-L1细胞脂代谢有效成分多集中于正丁醇和乙酸乙酯部位，可作为甜茶调节3T3-L1细胞脂代谢的活性部位。
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图 2 甜茶不同萃取部位对3T3-L1细胞TG释放量的影响（n=6）注：与空白组相比，##表示差异极显著P<0.01；与对照组相比，*表示差异显著P<0.05，**表示差异极显著P<0.01。Figure 2. Effect of different fractions of RS on 3T3-L1 cell TG release (n=6)![]()
图 3 甜茶不同萃取部位对3T3-L1细胞分化的影响（20×）注：a空白组；b对照组；c罗格列酮组；d~f石油醚部位低、中、高剂量组；g~i二氯甲烷部位低、中、高剂量组；j~l乙酸乙酯部位低、中、高剂量组；m~o正丁醇部位低、中、高剂量组；p~r甜茶总提取物低、中、高剂量组。Figure 3. Effect of different fractions of RS on 3T3-L1 cell differentiation (20×)2.2 活性部位化学成分的鉴定
运用UPLC-Q-TOF-MS/MS对甜茶正丁醇、乙酸乙酯萃取部位进行化学成分分析，负离子模式下的混合对照品及各活性部位溶液的总离子流图（TIC）如图4所示，鉴定结果见表1。通过保留时间、多级离子碎片及信号强度，并结合文献信息，共确认化学成分70个，包括12个多酚类、21个黄酮类、18个二萜类、17个三萜类和2个木脂素类。指认活性部位共有峰63个。峰1、10、18、19、21、22、28、34、48、50、65、67、68和70通过与对照品对比分别被鉴定为没食子酸、咖啡酸、鞣花酸、芦丁、金丝桃苷、异槲皮苷、槲皮苷、槲皮素、山奈酚、甜茶素、甜菊醇、异甜菊醇、熊果酸和齐墩果酸。
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图 4 负离子模式下混合对照品及甜茶正丁醇、乙酸乙酯萃取部位总离子流图注：a.混合对照品；b.正丁醇萃取部位；c.乙酸乙酯萃取部位；1-没食子酸；10-咖啡酸；18-鞣花酸；19-芦丁；21-金丝桃苷；22-异槲皮苷；28-槲皮苷；34-槲皮素；48-山奈酚；50-甜茶素；65-甜菊醇；67-异甜菊醇；68-熊果酸；70-齐墩果酸。Figure 4. TIC chromatogram of mixed references, n-butanol fraction and ethyl acetate fraction of RS under negative ion mode表 1 甜茶乙酸乙酯和正丁醇部位中鉴定的化合物Table 1. Compounds identified in ethyl acetate and n-butanol parts of RS编号 tR
（min）分子式 理论值（m/z） 实测值（m/z） 误差（ppm） 特征碎片离子（m/z） 中文名称 英文名称 乙酸乙酯部位 正丁醇部位 化合物类型 参考
文献1* 2.0 C7H6O5 169.0142 169.0145 –1.3 125.0251 没食子酸 gallic acid + + P [34] 2 3.1 C7H4O6 182.9935 182.9947 –6.3 139.0040, 95.0148 2-吡喃酮-4,
6-二羧酸2-pyrone-4,6-dicarboxylic acid + + P [35] 3 4.7 C27H22O18 633.0733 633.074 –1.0 481.0517, 300.9995, 257.0090, 229.0139, 169.0146 柯里拉京 corilagin + + P [36] 4 5.1 C34H24O22 783.0686 783.0692 –0.7 481.0592, 300.9984, 275.0188 长庚马兜铃素 pedunculagin + + P [36] 5 5.3 C21H20O9 415.1035 415.1025 2.3 161.0429, 151.0398, 123.0444 三叶海棠素 toringin – + F [37] 6 5.6 C8H8O5 183.0299 183.0302 –1.9 169.0135, 125.0225 没食子酸甲酯 methyl gallate + + P [34] 7 5.9 C15H18O9 341.0878 341.0881 –0.8 179.0347, 161.0458, 161.0247, 135.0451 咖啡酸-O-己糖苷 caffeic acid-O-hexoside + + P [34] 8 6.2 C27H22O18 633.0733 633.0724 1.5 481.0592, 300.9987, 257.0088, 229.0152, 169.0146 / 1-α–galloyl-2,3-（S）-hexahydroxy-diphenoyl-D-glucose + + P [38] 9 6.5 C27H30O17 625.141 625.1413 –0.4 463.0864, 301.0342, 283.0245, 271.0244, 255.0288, 151.0043 槲皮素-3,4’-O-D–β–吡喃葡萄糖苷 quercetin-3,4’-O-D-β–glucopyranoside – + F [39] 10* 6.6 C9H8O4 179.0350 179.0354 –2.2 135.0451 咖啡酸 caffeic acid + + P [34] 11 6.8 C33H40O21 771.1989 771.1985 0.9 609.1466, 301.0344, 283.0245, 271.0234, 255.0305, 151.0044 槲皮素3-O-葡萄糖基芸香糖苷 quercetin 3-O-glucosylrutinoside – + F [40] 12 7.2 C13H8O8 291.0146 291.0141 2.0 247.0245, 203.0345, 175.0406, 147.0455 短叶苏木酚酸 brevifolincarboxylic acid + + P [41] 13 8.0 C21H10O13 469.0049 469.0041 1.6 316.9910, 300.9995 地榆酸双内酯 sanguisorbic acid dilactone + + P [34] 14 8.0 C16H20O9 355.1035 355.1037 –0.8 193.0511, 161.0438, 161.0243, 133.0301 阿魏酸-O-己糖苷 ferulic acid-O-hexoside + + P [34] 15 10.0 C26H28O16 595.1305 595.1311 –1.1 301.0331, 271.0245, 255.0298, 151.0032 槲皮素-3-O-木糖基葡萄糖苷 1uercetin-3-O-xylosyl glucoside + + F [42] 16 10.5 C16H14O4 269.0819 269.0818 0.4 151.0043, 145.0297 5-羟基-7-甲氧基二氢黄酮 5-hydroxy-7-methoxydihydroflavone + + F [43] 17 11.1 C27H30O16 609.1461 609.1469 –1.3 447.0900, 285.0396, 255.0301 矢车菊-3-O-槐糖苷 cyanidin-3-O-sophoroside + + F [44] 18* 11.2 C14H6O8 300.9990 300.9992 –0.2 282.9952, 257.0090, 239.0046 鞣花酸 ellagic acid + + P [36] 19* 11.5 C27H30O16 609.1461 609.1473 –1.9 301.0349, 283.0245, 271.0242, 255.0302, 151.0039 芦丁 rutin + + F [34] 20 11.6 C27H30O16 609.1461 609.1472 –1.8 447.0917, 285.0391, 255.0302 矢车菊素-3,5-二葡萄糖苷 cyanidin-3,5-diglucoside + + F [44] 21* 11.8 C21H20O12 463.0882 463.0891 –1.9 301.0347, 283.0240, 271.0239, 255.0301, 151.0031 金丝桃苷 hyperoside + + F [36] 22* 12.1 C21H20O12 463.0882 463.0888 –1.3 301.0333, 283.0252, 271.0237, 255.0301 异槲皮苷 isoquercitrin + + F [45] 23 12.7 C27H30O15 593.1512 593.1524 –2.0 447.0910, 285.0402, 255.0304, 151.0036 矢车菊素-3-O-芸香糖苷 cyanidin 3-O-rutinoside + + F [44] 24 13.5 C20H18O11 433.0776 433.0785 –2.0 301.0335, 283.0243, 255.0302, 227.0347, 151.0041 番石榴苷 guaijaverin + + F [46] 25 13.7 C21H20O11 447.0933 447.094 –1.6 285.0388, 255.0306, 239.0349, 151.0040 山奈酚-O-己糖苷 kaempferol-O-hexoside + + F [34] 26 14.0 C27H30O15 593.1512 593.1523 –1.9 285.0408, 255.0302, 239.0351, 151.0043 山奈酚-3-O-芸香糖苷 kaempferol-3-O-rutinoside + + F [34] 27 14.7 C21H20O11 447.0933 447.0941 –1.8 285.0395, 255.0304, 151.0042 木犀草苷 luteoloside + + F [46] 28* 14.9 C21H20O11 447.0933 447.0931 0.4 283.0261, 271.0237, 255.0295, 151.0042 槲皮苷 quercitrin + + F [47] 29 17.9 C30H26O15 625.1199 625.1213 –2.2 463.0880, 301.0344, 271.0256, 255.0295, 151.0032 槲皮素-3-O-槐糖苷 quercetin-3-O-sophoroside + + F [48] 30 18.2 C30H26O15 625.1199 625.1207 –2.4 463.0894, 301.0358, 271.0252, 255.0301, 161.0248 山奈酚-O-咖啡酰基-己糖苷 quercetin-O-caffeoyl-hexoside + + F [34] 31 18.7 C26H44O9 499.2913 499.2911 0.3 337.2352, 277.2189 3β,16α,17,19-四羟基-对映贝壳杉-17-O–β-D-葡萄糖苷 3β,16α,17,19-tetrahydroxy-ent-kaurane -17-O-β–D-glucoside + + D [49] 32 20.1 C30H26O14 609.1250 609.1264 –2.4 447.0936, 285.0413, 255.0303, 161.0251 山奈酚-O-酰基-己糖苷 kaempferol-O-caffeoyl-hexoside + + F [34] 33 20.6 C26H42O10 513.2705 513.2701 0.8 351.2188, 333.2042, 161.0455 / glaucocalyxin G + + D [50] 34* 21.7 C15H10O7 301.0354 301.0356 –0.8 283.0255, 271.0252, 255.0298, 151.0037 槲皮素 quercetin + + F [36] 35 22.8 C31H38O11 585.2341 585.2358 –2.9 497.1550, 493.1864, 221.0455 / erythro-7,8-dihydro-Buddlenol B + + L [51] 36 23.0 C26H42O10 513.2705 513.2708 –0.6 351.2177, 333.2075 / cussovantoside A + + D [52] 37 23.5 C36H58O11 665.3906 665.3905 0.2 503.3376, 473.3349 / nigaichigoside F1 + + T [53] 38 23.8 C26H44O8 483.2963 483.2969 –1.2 321.2434, 285.2180 甜茶苷A suavisoside A + + D [51] 39 24.1 C20H32O4 335.2228 335.2228 –0.1 317.2114, 291.2284, 243.2121 16β,17-二羟基-贝壳杉-19-羧酸 16β,17-dihdyroxy-kauran-19-oic acid + + D [54] 40 24.1 C31H38O11 585.2341 585.2362 –3.5 497.1536, 493.1870, 221.0461 / threo-7,8-dihydro-Buddlenol B + + L [51] 41 24.3 C26H40O9 495.2600 495.2600 –0.1 333.2086, 303.2064 / 7β,17-dihydroxy-ent-kaur-15-en-19-oic acid 19-O-β–D-glucopyranoside ester + + D [55] 42 24.3 C20H30O4 333.2071 333.207 –1.1 315.1967, 289.2157, 271.2070 7β–羟基斯替维醇 7β–hydroxysteviol + + D [51] 43 24.7 C32H52O14 659.3284 659.3276 0.5 497.2756, 335.2219 / β–D-glucopyranosyl -17-hydroxy-ent-kauran-19-oate-16-O-β–D-glucopyranoside – + D [56] 44 24.7 C20H30O4 333.2071 333.2073 –0.4 315.1966, 289.2131, 271.2049 对映-13,17-二羟基-贝壳杉-15-烯-19-
羧酸ent-13,17-dihdyroxy-kauran-15-en-19-oic acid + + D [57] 45 24.9 C26H42O8 481.2807 481.2815 –1.7 319.2283, 301.2193, 275.2156 舒格罗克苷 sugereoside + + D [51] 46 25.2 C26H42O9 497.2756 497.2767 –2.2 335.2233, 317.2124 / 7β,17-dihydroxy-16β–ent-kauran-19-oic acid 19-O-β–D-glucopyranoside ester + + D [55] 47 25.6 C38H60O18 803.3707 803.3712 –0.7 641.3176, 479.2663, 317.2128 / 13-[（O-β–D-glucopyranosyl）oxy] ent-kaur-16-en-19- oic acid-2-O-β–D-glucopyranosyl-β–D- glucopyranosyl ester + + D [58] 48* 25.9 C15H10O6 285.0405 285.0407 –0.9 255.0311, 239.0365, 151.0026 山柰酚 kaempferol + + F [36] 49 27.0 C30H48O7 519.3327 519.3333 –1.2 501.3225, 471.3115, 459.3121, 441.2982 / 2β,3β,19α,23,24-pentahydroxy-urs-12-en-28-oic acid + + T [59] 50* 27.7 C32H50O13 641.3179 641.3197 –2.8 479.2676, 317.2127, 273.2224 甜茶素 rubusoside + + D [60] 51 28.2 C30H48O7 519.3327 519.3346 –3.6 501.3240, 471.3119, 459.3133, 441.3015 桔梗皂苷元 platycodigenin + + T [61] 52 28.2 C30H46O7 517.3171 517.3186 –3.0 499.3074, 473.3253, 469.2974 灵芝酸C2 ganoderic acid C2 + + T [62] 53 28.6 C30H48O6 503.3378 503.3395 –3.3 485.3290, 473.3275, 455.3116, 437.3059 / terminolic acid + + T [63] 54 29.5 C36H56O11 663.3750 663.3743 1.0 501.3236, 457.3343, 429.3301 甜茶苷J rubuside J + + T [64] 55 29.6 C26H40O8 479.2650 479.2661 –2.2 317.2110, 255.2388 对映-贝壳杉-16-烯-19-羧酸-13-O–β-D-葡萄糖苷 ent-kauran-16-en-19-oic-13-O-β–D-glucoside + + D [65] 56 29.7 C26H42O9 497.2757 497.276 –0.8 335.2214, 317.2119 / paniculoside Ⅳ + + D [51] 57 29.9 C30H48O6 503.3378 503.3395 –3.3 485.3281, 473.3251, 455.3125 2α,3β,19α,23-四羟基-12-烯-28-乌苏酸 2α,3β,19α,23-tetrahydroxy-urs-12-
en-28-oic acid+ + T [51] 58 30.0 C30H46O6 501.3222 501.3237 –3.1 483.3132, 467.3198 冬青素A ilexgenin A + + T [66] 59 31.1 C26H40O8 479.2650 479.2653 –0.5 317.2129, 255.2349 / cussoracosides E + + D [67] 60 31.3 C30H48O5 487.3429 487.3442 –2.7 457.3311, 443.3410, 421.3113 蔷薇酸 euscaphic acid + + T [63] 61 32.2 C30H50O5 489.3585 489.3579 1.4 429.3278, 423.3265, 393.3135 山茶苷C camelliagenin C + + T [68] 62 33.0 C30H48O5 487.3429 487.3441 –2.4 457.3323, 443.3435, 421.3097 / 2α,3β,24-trihydroxylup-20（29）-en-28-oic acid + + T [63] 63 33.5 C30H46O5 485.3272 485.3276 –0.8 455.3192, 411.3260 / 2α,3α,23-trihydroxyurs-12,18-dien-28-oic acid + + T [63] 64 33.8 C30H44O5 483.3116 483.3111 1.1 439.3384, 403.3233 覆盆子酸 fupenzic acid + + T [69] 65 33.9 C20H30O3 317.2122 317.2124 –0.7 273.2227, 255.2342 甜菊醇 steviol + + D [34] 66 34.1 C30H46O5 485.3272 485.3278 –1.1 455.3165, 411.3270 / 2α,19α–dihydroxy-
3-oxo-12-ursen-28-
oic acid+ + T [70] 67* 34.6 C20H30O3 317.2122 317.2114 2.7 273.2253, 255.2341 异甜菊醇 isosteviol + – D [71] 68* 37.6 C30H48O3 455.3531 455.3540 –2.1 411.3627, 393.3541 熊果酸 ursolic acid + – T [34] 69 37.6 C30H50O4 473.3636 473.3631 1.2 443.3581, 407.3347 山茶苷A camelliagenin A + – T [72] 70* 37.9 C30H48O3 455.3531 455.3527 –1.7 411.3660, 393.3552 齐墩果酸 oleanolic acid + + T [36] 注：*为通过与对照品比较进行鉴定，F：黄酮类化合物，D：二萜类化合物，T：三萜类化合物，P：多酚类化合物，L：木脂素类化合物。 2.3 谱效关系研究
2.3.1 灰色关联度法分析
运用灰色关联度法分析甜茶活性部位共有峰对细胞TG释放量灰色关联系度。关联度越大则峰代表的化合物与细胞TG释放关联密切，见表2。40个共有峰与甜茶调节脂代谢活性能力关联度均大于0.5，有较高的关联度，故表明甜茶的调节脂代谢活性作用是多成分协同作用发挥的效果。对调节脂代谢活性能力贡献最大的色谱峰是70、66、60、50、55、59、18、54、51、23、58、45（关联度>0.7）。
表 2 灰色关联度计算结果Table 2. Calculation results of grey correlation degree峰号 灰色关联度 70 0.9183 66 0.8189 60 0.8074 50 0.7988 55 0.7667 59 0.7634 18 0.7509 54 0.7363 51 0.7337 23 0.7301 58 0.7232 45 0.7055 2.3.2 偏最小二乘回归法分析
偏最小二乘回归法分析结果见图5。VIP值越大，表示贡献率越高；VIP>1，表示有显著贡献，共30个共有峰。回归系数绝对值越大则表示自变量的贡献度越大，回归系数为正值说明其与调节脂代谢活性呈正相关，负值与药效呈负相关。
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图 5 共有峰对细胞TG释放量的VIP图（a）及标准化回归系数（b）Figure 5. VIP plot (a) and standardized regression coefficient (b) of common peaks on cellular TG release其中14个峰与脂代谢活性能力呈正相关，贡献度依次为55>62>23>50>18>14>47>45>38>7>14>53>66>21。与脂代谢活性能力呈负相关的峰有：59>27>32>34>25。
2.3.3 灰色关联度法和偏最小二乘回归法综合分析
综合灰色关联度法和偏最小二乘回归法的计算结果，进一步对结果综合分析。以灰色关联度值>0.7、VIP值>1、回归系数正相关且≥0.0600为筛选条件，筛选出峰18（鞣花酸）、23（矢车菊素-3-O-芸香糖苷）、50（甜茶素）、55（对映-贝壳杉-16-烯-19-羧酸-13-O-β-D-葡萄糖苷）可显著影响3T3-L1细胞TG释放，具有调节3T3-L1细胞脂代谢作用（表3）。故鞣花酸、矢车菊素-3-O-芸香糖苷、甜茶素和对映-贝壳杉-16-烯-19-羧酸-13-O-β-D-葡萄糖苷可能是甜茶调节3T3-L1细胞脂代谢活性的潜在质量标志物。
表 3 灰色关联度法及偏最小二乘回归法计算结果Table 3. Gray correlation and partial least squares regression method calculation results峰号 tR
（min）分子式 理论值
（m/z）实测值
（m/z）误差
（ppm）特征碎片离子
（m/z）化合物名称 化合物类型 灰色关联度 VIP值 回归
系数18 11.2 C14H6O8 300.9990 300.9992 -0.2 282.9952,
257.0090,
239.0046鞣花酸 多酚 0.7509 1.0871 0.0673 23 12.7 C27H30O15 593.1512 593.1524 -2.0 447.0910,
285.0402,
255.0304,
151.0036矢车菊素-3-O-芸香糖苷 黄酮 0.7301 1.0798 0.0665 50 27.7 C32H50O13 641.3179 641.3197 -2.8 479.2676,
317.2127,
273.2224甜茶素 二萜 0.7988 1.0062 0.0618 55 29.6 C26H40O8 479.2650 479.2661 -2.2 317.2110,
255.2388对映-贝壳杉-16-烯-19-羧酸-13-O-β-D-葡萄糖苷 二萜 0.7667 1.0812 0.0663 3. 讨论与结论
甜茶乙酸乙酯、正丁醇部位能够显著促进3T3-L1细胞分化与TG释放，提示甜茶调节3T3-L1细胞脂代谢活性成分主要集中于乙酸乙酯和正丁醇部位。采用正离子模式和负离子模式分别对乙酸乙酯和正丁醇部位进行UPLC-Q-TOF-MS/MS分析，结果各峰在负离子模式下的灵敏强度和响应值高于正离子模式，最终采用负离子模式进行分析。两个部位共指认化学成分70个，其中活性部位共有峰63个。
鞣花酸、矢车菊素-3-O-芸香糖苷、甜茶素和对映-贝壳杉-16-烯-19-羧酸-13-O-β-D-葡萄糖苷可作为调节细胞脂质代谢活性的质量标志物。鞣花酸是一种天然多酚类化合物，是富含鞣质类成分中药的药效学物质基础。鞣花酸能够抑制脂肪细胞增殖、分化，具有降脂的作用[73−75]，还可以通过调控AKT/SREBP-1c/FASN信号通路抑制脂肪酸从头合成逆转肝细胞脂质沉积从而改善脂肪变性[76]。而鞣花酸由肠道微生物代谢产生的尿石素也被认为是其在体内发挥生物活性的物质基础[77]。矢车菊素-3-O-芸香糖苷为黄酮类成分，具有良好的健康功效[78]。矢车菊素-3-O-芸香糖苷通过抑制脂质消化和吸收从而发挥降脂的作用[79]，还可以通过激活Akt-ERK-MAPK信号通路，上调脂肪酸氧化相关基因蛋白表达水平，提高产热，促进碳水化合物和脂肪代谢[80]。甜茶素和对映-贝壳杉-16-烯-19-羧酸-13-O-β-D-葡萄糖苷为贝壳杉烷型二萜类成分，这类的生物活性多样，主要是调血糖血脂、抗过敏、抗肿瘤、抗炎等作用。研究表明甜茶素具调节脂代谢紊乱作用[15,23−25]。课题组通过1H-NMR代谢组学技术分析，甜茶素的抗高甘油三酯血症作用可能与干预氨基酸代谢、酮体合成、胆碱代谢等途径有关[81]。对映-贝壳杉-16-烯-19-羧酸-13-O-β-D-葡萄糖苷的母核与甜茶素相似，故可能产生与甜茶素相似的药理活性。研究结果表明甜茶的乙酸乙酯部位和正丁醇部位具丰富的多酚、黄酮类和二萜类化合物，因而能促进3T3-L1前脂肪细胞分化。因此，甜茶活性部位中的这4种成分可能通过协同作用发挥调节脂代谢的作用。
综上，本研究基于谱效关系发现鞣花酸、矢车菊素-3-O-芸香糖苷、甜茶素和对映-贝壳杉-16-烯-19-羧酸-13-O-β-D-葡萄糖苷可作为甜茶调节脂代谢活性的质量标志物，研究结果为深入发掘甜茶调节脂代谢药效物质和质量研究提供理论支持。
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图 1 甜茶不同萃取部位对3T3-L1细胞活力的影响（n=6）
注：与空白组相比，*表示差异显著P<0.05，**表示差异极显著P<0.01。
Figure 1. Effect of different fractions of RS on the viability of 3T3-L1 cells (n=6)
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图 2 甜茶不同萃取部位对3T3-L1细胞TG释放量的影响（n=6）
注：与空白组相比，##表示差异极显著P<0.01；与对照组相比，*表示差异显著P<0.05，**表示差异极显著P<0.01。
Figure 2. Effect of different fractions of RS on 3T3-L1 cell TG release (n=6)
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图 3 甜茶不同萃取部位对3T3-L1细胞分化的影响（20×）
注：a空白组；b对照组；c罗格列酮组；d~f石油醚部位低、中、高剂量组；g~i二氯甲烷部位低、中、高剂量组；j~l乙酸乙酯部位低、中、高剂量组；m~o正丁醇部位低、中、高剂量组；p~r甜茶总提取物低、中、高剂量组。
Figure 3. Effect of different fractions of RS on 3T3-L1 cell differentiation (20×)
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图 4 负离子模式下混合对照品及甜茶正丁醇、乙酸乙酯萃取部位总离子流图
注：a.混合对照品；b.正丁醇萃取部位；c.乙酸乙酯萃取部位；1-没食子酸；10-咖啡酸；18-鞣花酸；19-芦丁；21-金丝桃苷；22-异槲皮苷；28-槲皮苷；34-槲皮素；48-山奈酚；50-甜茶素；65-甜菊醇；67-异甜菊醇；68-熊果酸；70-齐墩果酸。
Figure 4. TIC chromatogram of mixed references, n-butanol fraction and ethyl acetate fraction of RS under negative ion mode
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图 5 共有峰对细胞TG释放量的VIP图（a）及标准化回归系数（b）
Figure 5. VIP plot (a) and standardized regression coefficient (b) of common peaks on cellular TG release
表 1 甜茶乙酸乙酯和正丁醇部位中鉴定的化合物
Table 1 Compounds identified in ethyl acetate and n-butanol parts of RS
编号 tR
（min）分子式 理论值（m/z） 实测值（m/z） 误差（ppm） 特征碎片离子（m/z） 中文名称 英文名称 乙酸乙酯部位 正丁醇部位 化合物类型 参考
文献1* 2.0 C7H6O5 169.0142 169.0145 –1.3 125.0251 没食子酸 gallic acid + + P [34] 2 3.1 C7H4O6 182.9935 182.9947 –6.3 139.0040, 95.0148 2-吡喃酮-4,
6-二羧酸2-pyrone-4,6-dicarboxylic acid + + P [35] 3 4.7 C27H22O18 633.0733 633.074 –1.0 481.0517, 300.9995, 257.0090, 229.0139, 169.0146 柯里拉京 corilagin + + P [36] 4 5.1 C34H24O22 783.0686 783.0692 –0.7 481.0592, 300.9984, 275.0188 长庚马兜铃素 pedunculagin + + P [36] 5 5.3 C21H20O9 415.1035 415.1025 2.3 161.0429, 151.0398, 123.0444 三叶海棠素 toringin – + F [37] 6 5.6 C8H8O5 183.0299 183.0302 –1.9 169.0135, 125.0225 没食子酸甲酯 methyl gallate + + P [34] 7 5.9 C15H18O9 341.0878 341.0881 –0.8 179.0347, 161.0458, 161.0247, 135.0451 咖啡酸-O-己糖苷 caffeic acid-O-hexoside + + P [34] 8 6.2 C27H22O18 633.0733 633.0724 1.5 481.0592, 300.9987, 257.0088, 229.0152, 169.0146 / 1-α–galloyl-2,3-（S）-hexahydroxy-diphenoyl-D-glucose + + P [38] 9 6.5 C27H30O17 625.141 625.1413 –0.4 463.0864, 301.0342, 283.0245, 271.0244, 255.0288, 151.0043 槲皮素-3,4’-O-D–β–吡喃葡萄糖苷 quercetin-3,4’-O-D-β–glucopyranoside – + F [39] 10* 6.6 C9H8O4 179.0350 179.0354 –2.2 135.0451 咖啡酸 caffeic acid + + P [34] 11 6.8 C33H40O21 771.1989 771.1985 0.9 609.1466, 301.0344, 283.0245, 271.0234, 255.0305, 151.0044 槲皮素3-O-葡萄糖基芸香糖苷 quercetin 3-O-glucosylrutinoside – + F [40] 12 7.2 C13H8O8 291.0146 291.0141 2.0 247.0245, 203.0345, 175.0406, 147.0455 短叶苏木酚酸 brevifolincarboxylic acid + + P [41] 13 8.0 C21H10O13 469.0049 469.0041 1.6 316.9910, 300.9995 地榆酸双内酯 sanguisorbic acid dilactone + + P [34] 14 8.0 C16H20O9 355.1035 355.1037 –0.8 193.0511, 161.0438, 161.0243, 133.0301 阿魏酸-O-己糖苷 ferulic acid-O-hexoside + + P [34] 15 10.0 C26H28O16 595.1305 595.1311 –1.1 301.0331, 271.0245, 255.0298, 151.0032 槲皮素-3-O-木糖基葡萄糖苷 1uercetin-3-O-xylosyl glucoside + + F [42] 16 10.5 C16H14O4 269.0819 269.0818 0.4 151.0043, 145.0297 5-羟基-7-甲氧基二氢黄酮 5-hydroxy-7-methoxydihydroflavone + + F [43] 17 11.1 C27H30O16 609.1461 609.1469 –1.3 447.0900, 285.0396, 255.0301 矢车菊-3-O-槐糖苷 cyanidin-3-O-sophoroside + + F [44] 18* 11.2 C14H6O8 300.9990 300.9992 –0.2 282.9952, 257.0090, 239.0046 鞣花酸 ellagic acid + + P [36] 19* 11.5 C27H30O16 609.1461 609.1473 –1.9 301.0349, 283.0245, 271.0242, 255.0302, 151.0039 芦丁 rutin + + F [34] 20 11.6 C27H30O16 609.1461 609.1472 –1.8 447.0917, 285.0391, 255.0302 矢车菊素-3,5-二葡萄糖苷 cyanidin-3,5-diglucoside + + F [44] 21* 11.8 C21H20O12 463.0882 463.0891 –1.9 301.0347, 283.0240, 271.0239, 255.0301, 151.0031 金丝桃苷 hyperoside + + F [36] 22* 12.1 C21H20O12 463.0882 463.0888 –1.3 301.0333, 283.0252, 271.0237, 255.0301 异槲皮苷 isoquercitrin + + F [45] 23 12.7 C27H30O15 593.1512 593.1524 –2.0 447.0910, 285.0402, 255.0304, 151.0036 矢车菊素-3-O-芸香糖苷 cyanidin 3-O-rutinoside + + F [44] 24 13.5 C20H18O11 433.0776 433.0785 –2.0 301.0335, 283.0243, 255.0302, 227.0347, 151.0041 番石榴苷 guaijaverin + + F [46] 25 13.7 C21H20O11 447.0933 447.094 –1.6 285.0388, 255.0306, 239.0349, 151.0040 山奈酚-O-己糖苷 kaempferol-O-hexoside + + F [34] 26 14.0 C27H30O15 593.1512 593.1523 –1.9 285.0408, 255.0302, 239.0351, 151.0043 山奈酚-3-O-芸香糖苷 kaempferol-3-O-rutinoside + + F [34] 27 14.7 C21H20O11 447.0933 447.0941 –1.8 285.0395, 255.0304, 151.0042 木犀草苷 luteoloside + + F [46] 28* 14.9 C21H20O11 447.0933 447.0931 0.4 283.0261, 271.0237, 255.0295, 151.0042 槲皮苷 quercitrin + + F [47] 29 17.9 C30H26O15 625.1199 625.1213 –2.2 463.0880, 301.0344, 271.0256, 255.0295, 151.0032 槲皮素-3-O-槐糖苷 quercetin-3-O-sophoroside + + F [48] 30 18.2 C30H26O15 625.1199 625.1207 –2.4 463.0894, 301.0358, 271.0252, 255.0301, 161.0248 山奈酚-O-咖啡酰基-己糖苷 quercetin-O-caffeoyl-hexoside + + F [34] 31 18.7 C26H44O9 499.2913 499.2911 0.3 337.2352, 277.2189 3β,16α,17,19-四羟基-对映贝壳杉-17-O–β-D-葡萄糖苷 3β,16α,17,19-tetrahydroxy-ent-kaurane -17-O-β–D-glucoside + + D [49] 32 20.1 C30H26O14 609.1250 609.1264 –2.4 447.0936, 285.0413, 255.0303, 161.0251 山奈酚-O-酰基-己糖苷 kaempferol-O-caffeoyl-hexoside + + F [34] 33 20.6 C26H42O10 513.2705 513.2701 0.8 351.2188, 333.2042, 161.0455 / glaucocalyxin G + + D [50] 34* 21.7 C15H10O7 301.0354 301.0356 –0.8 283.0255, 271.0252, 255.0298, 151.0037 槲皮素 quercetin + + F [36] 35 22.8 C31H38O11 585.2341 585.2358 –2.9 497.1550, 493.1864, 221.0455 / erythro-7,8-dihydro-Buddlenol B + + L [51] 36 23.0 C26H42O10 513.2705 513.2708 –0.6 351.2177, 333.2075 / cussovantoside A + + D [52] 37 23.5 C36H58O11 665.3906 665.3905 0.2 503.3376, 473.3349 / nigaichigoside F1 + + T [53] 38 23.8 C26H44O8 483.2963 483.2969 –1.2 321.2434, 285.2180 甜茶苷A suavisoside A + + D [51] 39 24.1 C20H32O4 335.2228 335.2228 –0.1 317.2114, 291.2284, 243.2121 16β,17-二羟基-贝壳杉-19-羧酸 16β,17-dihdyroxy-kauran-19-oic acid + + D [54] 40 24.1 C31H38O11 585.2341 585.2362 –3.5 497.1536, 493.1870, 221.0461 / threo-7,8-dihydro-Buddlenol B + + L [51] 41 24.3 C26H40O9 495.2600 495.2600 –0.1 333.2086, 303.2064 / 7β,17-dihydroxy-ent-kaur-15-en-19-oic acid 19-O-β–D-glucopyranoside ester + + D [55] 42 24.3 C20H30O4 333.2071 333.207 –1.1 315.1967, 289.2157, 271.2070 7β–羟基斯替维醇 7β–hydroxysteviol + + D [51] 43 24.7 C32H52O14 659.3284 659.3276 0.5 497.2756, 335.2219 / β–D-glucopyranosyl -17-hydroxy-ent-kauran-19-oate-16-O-β–D-glucopyranoside – + D [56] 44 24.7 C20H30O4 333.2071 333.2073 –0.4 315.1966, 289.2131, 271.2049 对映-13,17-二羟基-贝壳杉-15-烯-19-
羧酸ent-13,17-dihdyroxy-kauran-15-en-19-oic acid + + D [57] 45 24.9 C26H42O8 481.2807 481.2815 –1.7 319.2283, 301.2193, 275.2156 舒格罗克苷 sugereoside + + D [51] 46 25.2 C26H42O9 497.2756 497.2767 –2.2 335.2233, 317.2124 / 7β,17-dihydroxy-16β–ent-kauran-19-oic acid 19-O-β–D-glucopyranoside ester + + D [55] 47 25.6 C38H60O18 803.3707 803.3712 –0.7 641.3176, 479.2663, 317.2128 / 13-[（O-β–D-glucopyranosyl）oxy] ent-kaur-16-en-19- oic acid-2-O-β–D-glucopyranosyl-β–D- glucopyranosyl ester + + D [58] 48* 25.9 C15H10O6 285.0405 285.0407 –0.9 255.0311, 239.0365, 151.0026 山柰酚 kaempferol + + F [36] 49 27.0 C30H48O7 519.3327 519.3333 –1.2 501.3225, 471.3115, 459.3121, 441.2982 / 2β,3β,19α,23,24-pentahydroxy-urs-12-en-28-oic acid + + T [59] 50* 27.7 C32H50O13 641.3179 641.3197 –2.8 479.2676, 317.2127, 273.2224 甜茶素 rubusoside + + D [60] 51 28.2 C30H48O7 519.3327 519.3346 –3.6 501.3240, 471.3119, 459.3133, 441.3015 桔梗皂苷元 platycodigenin + + T [61] 52 28.2 C30H46O7 517.3171 517.3186 –3.0 499.3074, 473.3253, 469.2974 灵芝酸C2 ganoderic acid C2 + + T [62] 53 28.6 C30H48O6 503.3378 503.3395 –3.3 485.3290, 473.3275, 455.3116, 437.3059 / terminolic acid + + T [63] 54 29.5 C36H56O11 663.3750 663.3743 1.0 501.3236, 457.3343, 429.3301 甜茶苷J rubuside J + + T [64] 55 29.6 C26H40O8 479.2650 479.2661 –2.2 317.2110, 255.2388 对映-贝壳杉-16-烯-19-羧酸-13-O–β-D-葡萄糖苷 ent-kauran-16-en-19-oic-13-O-β–D-glucoside + + D [65] 56 29.7 C26H42O9 497.2757 497.276 –0.8 335.2214, 317.2119 / paniculoside Ⅳ + + D [51] 57 29.9 C30H48O6 503.3378 503.3395 –3.3 485.3281, 473.3251, 455.3125 2α,3β,19α,23-四羟基-12-烯-28-乌苏酸 2α,3β,19α,23-tetrahydroxy-urs-12-
en-28-oic acid+ + T [51] 58 30.0 C30H46O6 501.3222 501.3237 –3.1 483.3132, 467.3198 冬青素A ilexgenin A + + T [66] 59 31.1 C26H40O8 479.2650 479.2653 –0.5 317.2129, 255.2349 / cussoracosides E + + D [67] 60 31.3 C30H48O5 487.3429 487.3442 –2.7 457.3311, 443.3410, 421.3113 蔷薇酸 euscaphic acid + + T [63] 61 32.2 C30H50O5 489.3585 489.3579 1.4 429.3278, 423.3265, 393.3135 山茶苷C camelliagenin C + + T [68] 62 33.0 C30H48O5 487.3429 487.3441 –2.4 457.3323, 443.3435, 421.3097 / 2α,3β,24-trihydroxylup-20（29）-en-28-oic acid + + T [63] 63 33.5 C30H46O5 485.3272 485.3276 –0.8 455.3192, 411.3260 / 2α,3α,23-trihydroxyurs-12,18-dien-28-oic acid + + T [63] 64 33.8 C30H44O5 483.3116 483.3111 1.1 439.3384, 403.3233 覆盆子酸 fupenzic acid + + T [69] 65 33.9 C20H30O3 317.2122 317.2124 –0.7 273.2227, 255.2342 甜菊醇 steviol + + D [34] 66 34.1 C30H46O5 485.3272 485.3278 –1.1 455.3165, 411.3270 / 2α,19α–dihydroxy-
3-oxo-12-ursen-28-
oic acid+ + T [70] 67* 34.6 C20H30O3 317.2122 317.2114 2.7 273.2253, 255.2341 异甜菊醇 isosteviol + – D [71] 68* 37.6 C30H48O3 455.3531 455.3540 –2.1 411.3627, 393.3541 熊果酸 ursolic acid + – T [34] 69 37.6 C30H50O4 473.3636 473.3631 1.2 443.3581, 407.3347 山茶苷A camelliagenin A + – T [72] 70* 37.9 C30H48O3 455.3531 455.3527 –1.7 411.3660, 393.3552 齐墩果酸 oleanolic acid + + T [36] 注：*为通过与对照品比较进行鉴定，F：黄酮类化合物，D：二萜类化合物，T：三萜类化合物，P：多酚类化合物，L：木脂素类化合物。 
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表 2 灰色关联度计算结果
Table 2 Calculation results of grey correlation degree
峰号 灰色关联度 70 0.9183 66 0.8189 60 0.8074 50 0.7988 55 0.7667 59 0.7634 18 0.7509 54 0.7363 51 0.7337 23 0.7301 58 0.7232 45 0.7055
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表 3 灰色关联度法及偏最小二乘回归法计算结果
Table 3 Gray correlation and partial least squares regression method calculation results
峰号 tR
（min）分子式 理论值
（m/z）实测值
（m/z）误差
（ppm）特征碎片离子
（m/z）化合物名称 化合物类型 灰色关联度 VIP值 回归
系数18 11.2 C14H6O8 300.9990 300.9992 -0.2 282.9952,
257.0090,
239.0046鞣花酸 多酚 0.7509 1.0871 0.0673 23 12.7 C27H30O15 593.1512 593.1524 -2.0 447.0910,
285.0402,
255.0304,
151.0036矢车菊素-3-O-芸香糖苷 黄酮 0.7301 1.0798 0.0665 50 27.7 C32H50O13 641.3179 641.3197 -2.8 479.2676,
317.2127,
273.2224甜茶素 二萜 0.7988 1.0062 0.0618 55 29.6 C26H40O8 479.2650 479.2661 -2.2 317.2110,
255.2388对映-贝壳杉-16-烯-19-羧酸-13-O-β-D-葡萄糖苷 二萜 0.7667 1.0812 0.0663
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