沙棘叶中鞣质类成分及其抗炎和抗肥胖活性研究

廿氏春秋 2021-12-20

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摘要：目的 研究沙棘Hippophae rhamnoides叶的化学成分及其抗炎和抗肥胖活性。方法采用硅胶、ODS、Sephadex LH-20、MCI CHP20柱色谱和半制备液相等方法进行分离纯化，通过波谱分析鉴定化合物的结构。对分离得到的化合物进行体外抑制RAW264.7巨噬细胞一氧化氮生成的抗炎活性和抑制3T3-L1脂肪细胞三酰甘油蓄积的抗肥胖活性评价。结果从沙棘叶中分离得到18个鞣质类化合物，分别鉴定为1,2,6-三-O-没食子酰基-β-D-吡喃葡萄糖（1）、1,3,6-三-O-没食子酰基-β-D-吡喃葡萄糖（2）、1,4,6-三-O-没食子酰基-β-D-吡喃葡萄糖（3）、1,3,4,6-四-O-没食子酰基-β-D-吡喃葡萄糖（4）、1,2,3,6-四-O-没食子酰基-β-D-吡喃葡萄糖（5）、1,2,3,4,6-五-O-没食子酰基-β-D-吡喃葡萄糖（6）、1-O-没食子酰基-4,6-(S)-六羟基联苯甲酰基-β-D-吡喃型葡萄糖（7）、1-O-没食子酰基-2,3-(S)-六羟基联苯甲酰基-β-D-吡喃型葡萄糖（8）、1,3-二-O-没食子酰基-4,6-(S)-六羟基联苯甲酰基-β-D-吡喃型葡萄糖（9）、1,6-二-O-没食子酰基-2,3-(S)-六羟基联苯甲酰基-β-D-吡喃型葡萄糖（10）、木麻黄鞣亭（11）、1,2,3-三-O-没食子酰基-4,6-(S)-六羟基联苯二甲酰基-β-D-吡喃型葡萄糖（12）、1,4,6-三-O-没食子酰基-2,3-(S)-六羟基联苯二甲酰基- β-D-吡喃化合物235681012和13为首次从该属植物中分离得到，中的鞣质类化合物具有较强的抗炎活性和抗肥胖活性。

沙棘Hippophae rhamnoides L. 为胡颓子科沙棘属的落叶灌木或小乔木，主要分布在丝绸之路沿线国家。我国是世界上沙棘种质资源和蕴藏量最丰富的国家，共有7种7亚种，广泛分布于我国西北、华北、东北、西南等地，其蕴藏量约占世界沙棘资源的90%^[1]。

《中国药典》2015年版收载有沙棘，其果实具有健脾消食、止咳祛痰、活血散瘀的功效，系蒙古族、藏族习用药材。沙棘叶虽然未被《中国药典》所收载，但是国家卫生和计划生育委员会已于2013年将沙棘叶作为新资源食品管理。沙棘叶主要含有黄酮类^[2-3]、萜类^[3-4]、鞣质类^[5-6]化合物。沙棘叶具有抗炎^[3]、抗肥胖^[3]、调血脂^[7]、抗氧化^[8]等生物活性。但是沙棘叶的药效物质基础尚未完全清楚，为了进一步明确沙棘叶抗炎和抗肥胖药效物质基础研究，本实验从沙棘叶中分离得到了18个化合物，分别鉴定为1,2,6-三-O-没食子酰基-β-D-吡喃葡萄糖（1,2,6-tri-O-galloyl-β-D-glucopyranose，1）、1,3,6-三-O-没食子酰基-β-D-吡喃葡萄糖（1,3,6-tri-O- galloyl-β-D-glucopyranose，2）、1,4,6-三-O-没食子酰基-β-D-吡喃葡萄糖（1,4,6-tri-O-galloyl-β-D- glucopyranose，3）、1,3,4,6-四-O-没食子酰基-β-D-吡喃葡萄糖（1,3,4,6-tetra-O-galloyl-β-D-gluco- pyranose，4）、1,2,3,6-四-O-没食子酰基-β-D-吡喃葡萄糖（1,2,3,6-tetra-O-galloyl-β-D-glucopyranose，5）、1,2,3,4,6-五-O-没食子酰基-β-D-吡喃葡萄糖（1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose，6）、1-O-没食子酰基-4,6-(S)-六羟基联苯甲酰基-β-D-吡喃型葡萄糖[1-O-galloyl-4,6-(S)-HHDP-β-D-glucopyranose，7]、1-O-没食子酰基-2,3-(S)-六羟基联苯甲酰基-β-D-吡喃型葡萄糖 [1-O-galloyl-2,3- (S)-HHDP-β-D-glucopyranose，8]、1,3-二-O-没食子酰基-4,6-(S)-六羟基联苯甲酰基-β-D-吡喃型葡萄糖[1,3-di-O-galloyl-4,6-(S)-HHDP-β-D-glucopyranose，9]β-D-吡喃型葡萄糖 [1,6-di-O-galloyl-2,3-(S)-HHDP-β-D-glucopyranose，10]、木麻黄鞣亭（，11）、1,2,3-三-O-没食子酰基-4,6-(S)-六羟基联苯二甲酰基-β-D-吡喃型葡萄糖 [S)-HHDP-β-D-glucopyranose，12]、1,4,6-三-O-没食子酰基-2,3-(S)-六羟基联苯二甲酰基-β-D-吡喃型葡萄糖[1,4,6-tri-O-galloyl-2,3-(S)-HHDP-β-D-13]、hippophaenin B（14）、

1 仪器与材料

JEOL ECA 600型核磁共振波谱仪，JEOL GC mate质谱仪，JASCO半制备型高效液相色谱仪，柱色谱硅胶、ODS、Sephadex LH-20葡聚糖凝胶、MCI CHP20吸附树脂等，半制备色谱柱YMK Pak Ph10 mm）、Cosmosil 5C₁₈π-NAP（250 mm×10 mm）等，试剂均为分析纯。

实验用沙棘叶采于内蒙古，经兰州大学药学院生药学研究所马志刚教授鉴定为沙棘Hippophae rhamnoides L. 的干燥叶，标本（SJ20171001）保存于兰州大学药学院。

2 提取与分离

沙棘叶6 kg用80%甲醇浸泡提取，反复3次，滤过合并提取液，减压浓缩得浸膏1.4 kg。浸膏用水分散后，用氯仿、醋酸乙酯、正丁醇分别萃取3次，减压回收溶剂，得相应的提取物，其中醋酸乙酯层和水层分别为60 g和933 g。取醋酸乙酯部位57 g浸膏，20%甲醇溶解后，进行Sephadex LH-20柱色谱分离，以甲醇-水（20∶80→100∶0）溶剂系统梯度洗脱，得到4个部分Fr. 1～4，Fr. 4（20 g）经过硅胶、MCI CHP-20柱色谱和半制备高效液相色谱分离纯化，得到化合物1（180.0 mg）、2（8.0 mg）、3（38 mg）、4（10 mg）、5（37 mg）、6（50 mg）、7（140.0 mg）、8（180.0 mg）、9（10 mg）、10（68 mg）、11（560 mg）、12（82 mg）、13（290 mg）、15（120.5 mg）、16（170.1 mg）、17（130.1 mg）。取水层910 g浸膏，用Sephadex LH-20柱色谱进行分离，以甲醇-水（0∶100→100∶0）溶剂系统梯度洗脱，得到6个部分Fr. 1～6，Fr. 3（15 g）经过MCI CHP-20柱色谱分离纯化，得到化合物14（560 mg）、16（700.0 mg）。Fr. 1（580 g）经过MCI CHP-20柱色谱分离纯化，得到化合物18（1 400 mg）。

3 结构鉴定

化合物1：浅褐色粉末，[α]25D −92.1° (c 0.2, MeOH)；FAB-MS m/z: 635 [M－H]⁻，HR-FAB-MS m/z: 635.088 3 [M－H]⁻ (计算值635.088 3，C₂₇H₂₃O₁₈)；¹H-NMR (600 MHz, acetone-d₆)δ: 5.99 (1H, d, J = 8.4 Hz, glu-H-1), 5.26 (1H, t, J = 8.8 Hz, glu-H-2), 3.99 (1H, t, J = 9.5 Hz, glu-H-3), 3.77 (1H, t, J = 9.5 Hz, glu-H-4), 3.93 (1H, m, glu H-5), 4.58 (1H, dd, J = 12.1, 1.8 Hz, glu-H-6α), 4.49 (1H,dd, J = 12.1, 4.7 Hz, glu-H-6β), 7.16(2H, s, galloyl-H-2, 6), 7.10 (2H, s, galloyl-H-2, 6), 7.08 (2H, s, galloyl-H-2,6)；¹³C-NMR (150 MHz, acetone-d₆)δ: 93.6 (glu-C-1), 73.9 (glu-C-2),75.6 (glu-C-3), 71.1 (glu-C-4), 76.1 (glu-C-5), 63.9 (glu-C-6), 121.7, 121.5,120.3 (galloyl-C-1), 110.3, 110.1, 110.0 (galloyl C-2, 6), 146.1, 146.0, 145.9(galloyl-C-3, 5), 139.6, 139.0, 138.9 (galloyl-C-4), 166.7, 166.0, 165.2(galloyl-C = O)。以上数据与文献报道基本一致^[9]，故鉴定化合物1为1,2,6-三-O-没食子酰基-β-D-吡喃葡萄糖。

化合物2：浅褐色粉末，[α]25D +22.5° (c, 0.5, MeOH)；FAB-MS m/z: 635 [M－H]⁻，HR-FAB-MS m/z: 635.088 4 [M－H]⁻ (计算值635.088 3，C₂₇H₂₃O₁₈)；¹H-NMR (600 MHz, acetone-d₆)δ: 5.90 (1H, d, J = 7.9 Hz, glu-H-1), 3.89 (1H, t, J = 8.3 Hz, glu-H-2), 5.36 (1H, t, J = 9.2 Hz, glu-H-3), 3.92 (1H, t, J = 9.3 Hz, glu-H-4), 3.98 (1H, m, glu-H-5), 4.58 (1H, dd, J = 12.1, 1.7 Hz, glu-H-6α), 4.49 (1H,dd, J = 12.1, 4.7 Hz, glu-H-6β), 7.19(2H, s, galloyl-H-2, 6), 7.19 (2H, s, galloyl-H-2, 6), 7.16 (2H, s, galloyl-H-2,6)；¹³C-NMR (150 MHz, acetone-d₆)δ: 95.7 (glu- C-1), 72.4 (glu-C-2),78.8 (glu-C-3), 69.3 (glu-C-4), 75.9 (glu-C-5), 63.9 (glu-C-6), 122.1, 121.7,120.9 (galloyl-C-1), 110.4, 110.2, 110.0 (galloyl-C-2, 6), 146.1, 146.1, 146.0(galloyl-C-3, 5), 139.4, 139.0, 138.7 (galloyl-C-4), 166.7, 166.6, 165.4(galloyl-C = O)。以上数据与文献报道基本一致^[10]，故鉴定化合物2为1,3,6-三-O-没食子酰基-β-D-吡喃葡萄糖。

化合物3：浅褐色粉末，[α]25D +28.9° (c, 0.5, MeOH)；FAB-MS m/z: 635 [M－H]⁻，HR-FAB-MS m/z: 635.088 4 [M－H]⁻ (计算值635.088 3，C₂₇H₂₃O₁₈)；¹H-NMR (600 MHz, acetone-d₆)δ: 5.86 (1H, d, J = 8.3 Hz, glu-H-1), 3.74 (1H, t, J = 8.2 Hz, glu-H-2), 4.00 (1H, t, J = 9.3 Hz, glu-H-3), 5.27 (1H, t, J = 9.0 Hz, glu-H-4), 4.15 (1H, m, glu-H-5), 4.46 (1H, dd, J = 12.4, 1.7 Hz, glu-H-6α), 4.20 (1H,dd, J = 12.4, 5.2 Hz, glu-H-6β), 7.20(2H, s, galloyl-H-2, 6), 7.17 (2H, s, galloyl-H-2, 6), 7.14 (2H, s, galloyl-H-2,6)；¹³C-NMR (150 MHz, Acetone-d₆)δ: 95.4 (glu C-1), 74.1 (glu-C-2),75.5 (glu-C-3), 71.6 (glu-C-4), 73.9 (glu-C-5), 63.4 (glu-C-6), 121.5, 121.4,120.8 (galloyl-C-1), 110.3, 110.2, 110.0 (galloyl-C-2, 6), 146.0, 146.0, 145.9(galloyl-C-3, 5), 139.4, 139.0, 1以上数据与文献报道基本一致^[11]，故鉴定化合物3为1,4,6-三-O-没食子酰基-β-D-吡喃葡萄糖。

化合物4：浅褐色粉末，[α]25D +30.8° (c, 0.5, MeOH)；FAB-MS m/z: 787 [M－H]⁻，HR-FAB-MS m/z: 787.099 3 [M－H]⁻ (计算值787.099 2，C₃₄H₂₇O₂₂)；¹H-NMR (600 MHz, acetone-d₆)δ: 6.01 (1H, d, J = 8.3 Hz, glu-H-1), 4.09 (1H, t, J = 8.6 Hz, glu-H-2), 5.70 (1H, t, J = 9.6 Hz, glu-H-3), 5.51 (1H, t, J = 9.6 Hz, glu-H-4), 4.41 (1H, m, glu-H-5), 4.58 (1H, dd, J = 12.4, 1.7 Hz, glu-H-6α), 4.49 (1H,dd, J = 12.4, 5.2 Hz, glu-H-6β), 7.21(2H, s, galloyl-H-2, 6), 7.15 (2H, s, galloyl-H-2, 6), 7.10 (2H, s, galloyl-H-2,6), 7.07 (2H, s, galloyl-H-2, 6)；¹³C-NMR (150 MHz, acetone-d₆) δ: 94.8 (glu-C-1), 71.2 (glu-C-2), 75.1 (glu-C-3), 68.9 (glu-C-4),73.0 (glu-C-5), 62.6 (glu-C-6), 120.2, 120.1, 119.4, 119.4 (galloyl-C-1),109.6, 109.4, 109.4, 109.3 (galloyl-C-2, 6), 145.5, 145.4, 145.4, 145.3(galloyl-C-3, 5), 139.2, 139.0, 138.5, 138.5 (galloyl-C-4), 166.21, 166.2,165.7, 165.3 (galloyl-C = O)。以上数据与文献报道基本一致^[12]，故鉴定化合物4为1,3,4,6-四-O-没食子酰基- β-D-吡喃葡萄糖。

化合物5：浅褐色粉末，[α]25D +28.1° (c, 0.5, MeOH)；FAB-MS m/z: 787 [M－H]⁻，HR-FAB-MS m/z: 787.099 3 [M－H]⁻ (计算值787.099 2，C₃₄H₂₇O₂₂)；¹H-NMR (600 MHz, acetone-d₆)δ: 6.19 (1H, d, J = 8.6 Hz, glu-H-1), 5.48 (1H, t, J = 8.6 Hz, glu-H-2), 5.69 (1H, t, J = 8.9 Hz, glu-H-3), 4.11 (1H, m, glu-H-4), 4.16 (1H, m, glu-H-5),4.62 (1H, dd, J = 12.1, 1.8 Hz, glu-H-6α),4.58 (1H, dd, J = 12.1, 4.7 Hz, glu-H-6β),7.19 (2H, s, galloyl-H-2, 6), 7.10 (2H, .9, 145.9 (galloyl- C-3, 5), 139.7, 139.2, 139.0,138.9 (galloyl-C-4), 166.6, 166.2, 165.8, 165.0 (galloyl-C = O)。以上数据与文献报道基本一致^[13]，故鉴定化合物5为1,2,3,6-四-O-没食子酰基-β-D-吡喃葡萄糖。

化合物6：浅褐色粉末，[α]25D +23.0° (c, 0.5, MeOH)；FAB-MS m/z: 939 [M－H]⁻，HR-FAB-MS m/z: 939.110 3 [M－H]⁻ (计算值939.110 2，C₄₁H₃₁O₂₆)；¹H-NMR (600 MHz, acetone-d₆)δ: 6.34 (1H, d, J = 8.5 Hz, glu H-1), 5.62 (1H, t, J = 8.4 Hz, glu-H-2), 6.02 (1H, t, J = 9.5 Hz, glu-H-3), 5.67 (1H, t, J = 9.5 Hz, glu-H-4), 4.58 (1H, m, glu-H-5), 4.55 (1H, dd, J = 12.4, 1.9 Hz, glu-H-6α), 4.42 (1H,dd, J = 12.4, 4.4 Hz, glu-H-6β), 7.19(2H, s, galloyl-H-2, 6), 7.12 (2H, s, galloyl-H-2, 6), 7.06 (2H, s, galloyl-H-2,6), 7.02 (2H, s, galloyl-H-2, 6), 6.98 (2H, s, galloyl- H-2, 6)；¹³C-NMR (150 MHz, acetone-d₆)δ: 93.3 (glu-C-1), 71.8 (glu-C-2),73.3 (glu-C-3), 69.3 (glu-C-4), 74.0 (glu-C-5), 62.8 (glu-C-6), 121.4, 120.7,120.6, 120.6, 119.9 (galloyl-C-1), 110.4, 110.3, 110.2, 110.1, 110.1 (galloyl-C-2,6), 146.1, 146.03, 145.98, 145.94, 145.86 (C-3, 5), 139.8, 139.4, 139.3, 139.2,139.0 (galloyl-C-4), 166.4, 165.9, 165.7, 165.6, 165.0 (galloyl-C = O)。以上数据与文献报道基本一致^[13]，故鉴定化合物6为1,2,3,4,6-五-O-没食子酰基-β-D-吡喃葡萄糖。

化合物7：浅褐色粉末，[α]25D −26.3° (c 0.3, MeOH)；FAB-MS m/z: 633 [M－H]⁻，HR-FAB-MS m/z: 633.072 9 [M－H]⁻ (计算值633.072 7，C₂₇H₂₁O₁₈)；¹H-NMR (600 MHz, acetone-d₆)δ: 5.75 (1H, d, J = 7.9 Hz, glu-H-1), 3.79 (1H, t, J = 8.3 Hz, glu-H-2), 3.93 (1H, t, J = 9.6 Hz, glu-H-3), 4.91 (1H, t, J = 9.6 Hz, glu-H-4), 4.19 (1H, m, glu-H-5), 5.22 (1H, dd, J = 12.7, 6.2 Hz, glu-H-6α), 3.86 (1H,d, J = 12.7 Hz, glu-H-6β), 7.24 (2H,s, galloyl-H-2, 6), 6.81, 6.66 (各1H, s, HHDP H-3, 3′)；¹³C-NMR (150 MHz, acetone-d₆) δ: 95.1 (glu-C-1), 73.1 (glu-C-2), 74.0 (glu-C-3), 71.8 (glu-C-4),72.1 (glu-C-5), 63.0 (glu- C-6), 119.1 (galloyl-C-1), 109.6 (galloyl-C-2, 6),145.2 (galloyl-C-3, 5), 139.2 (galloyl-C-4), 165.6 (galloyl C = O), 115.5,115.2 (HHDP C-1, 1′), 125.4, 125.1 (HHDP C-2, 2′), 107.0, 107.4 (HHDP C-3, 3′),144.3, 144.3 (HHDP C-4, 4′), 135.9, 135.9 (HHDP C-5, 5′), 144.0, 143.9 (HHDPC-6, 6′), 168.5, 168.2 (HHDP-C = O)。以上数据与文献报道基本一致^[14]，故鉴定化合物7为1-O-没食子酰基-4,6-(S)-六羟基联苯甲酰基-β-D-吡喃型葡萄糖。

化合物8：浅褐色粉末，[α]25D −31.8° (c, 0.3, MeOH)；FAB-MS m/z: 633 [M－H]⁻，HR-FAB-MS m/z: 633.072 5 [M－H]⁻(计算值633.072 7，C₂₇H₂₁O)；¹H-NMR (600 MHz, acetone-d₆)δ: 6.13 (1H, d, J = 8.2 Hz, glu H-1), 5.01 (1H, t, J = 8.6 Hz, glu-H-2), 5.22 (1H, t, J = 9.6 Hz, glu-H-3), 3.96 (1H, t, J = 9.6 Hz, glu-H-4), 3.75 (1H, m, glu-H-5), 3.82 (1H, dd, J = 12.1, 12.4 Hz, glu-H-6α), 2.92 (1H,dd, J= 12.1, 4.8 Hz, glu-H-6β), 7.15 (2H, s, galloyl-H-2, 6), 6.71, 6.43 (各1H, s, HHDP H-3, 3′)；¹³C-NMR (150 MHz, acetone-d₆)δ: 92.1 (glu-C-1), 75.4 (glu-C-2),80.3 (glu-C-3), 67.8 (glu-C-4), 75.4 (glu-C-5), 61.7 (glu-C-6), 120.2 (galloyl-C-1),110.1 (galloyl-C-2, 6), 146.2 (galloyl-C-3, 5), 139.7 (galloyl- C-4), 165.0(galloyl-C = O), 114.53, 114.45 (HHDP C-1, 1′), 127.0, 126.5 (HHDP C-2, 2′),107.6, 107.1 (HHDP C-3, 3′), 145.2, 145.0 (HHDP C-4, 4′), 136.3, 136.2 (HHDPC-5, 5′), 144.5, 144.4 (HHDP C-6, 6′), 169.4, 168.7 (HHDP C = O)。以上数据与文献报道基本一致^[15]，故鉴定化合物8为1-O-没食子酰基-2,3- (S)-六羟基联苯甲酰基-β-D-吡喃型葡萄糖。

化合物9：浅褐色粉末，[α]25D −45.4° (c, 0.3, MeOH)；FAB-MS m/z: 785 [M－H]⁻，HR-FAB-MS m/z: 785.083 0 [M－H]⁻ (计算值 785.083 6，C₃₄H₂₅O₂₂)；¹H-NMR (600 MHz, acetone-d₆)δ: 5.92 (1H, d, J = 8.3 Hz, glu-H-1), 3.74 (1H, t, J = 8.2 Hz, glu-H-2), 5.51 (1H, t, J = 9.2 Hz, glu-H-3), 5.07 (1H, t, J = 10.0 Hz, glu-H-4), 4.37 (1H, m, glu-H-5), 5.33 (1H, dd, J = 13.4, 6.5 Hz, glu-H-6α), 4.20 (1H,d, J = 13.4 Hz, glu-H-6β), 7.22 (2H,s, galloyl-H-2, 6), 7.05 (2H, s, galloyl-H-2, 6), 6.64, 6.44 (各1H, s, HHDP H-3, 3′)；¹³C-NMR (150 MHz, acetone-d₆)δ: 95.9 (glu-C-1), 72.5 (glu-C-2),75.6 (glu-C-3), 70.8 (glu-C-4), 72.9 (glu-C-5), 63.3 (glu-C-6), 121.4, 120.6(galloyl-C-1), 110.4, 110.2 (galloyl-C-2, 6), 146.1, 145.8 (galloyl-C-3, 5),139.6, 138.9 (galloyl-C-4), 166.6, 165.3 (galloyl-C = O), 115.9, 115.5 (HHDPC-1, 1′), 126.6, 126.2 (HHDP C-2, 2′), 108.1, 107.8 (HHDP C-3, 3′), 145.3,145.2 (HHDP C-4, 4′), 136.5, 136.5 (HHDP C-5, 5′), 144.5, 144.5 (HHDP C-6, 6′),168.0, 167.7 (HHDP C = O)。以上数据与文献报道基本一致^[16]。故鉴定化合物9为1,3-二-O-没食子酰基-4,6-(S)-六羟基联苯甲酰基-β-D-吡喃型葡萄糖。

化合物10：浅褐色粉末，[α]25D −53.0° (c, 0.2, MeOH)；FAB-MS m/z: 785 [M－H]⁻，HR-FAB-MS m/z: 785.084 0 [M－H]⁻ (计算值785.083 6，C₃₄H₂₅O₂₂)；¹H-NMR (600 MHz, acetone-d₆)δ: 6.22 (1H, d, J = 8.4 Hz, glu-H-1), 5.07 (1H, t, J = 8.4 Hz, glu-H-2), 5.29 (1H, t, J = 9.5 Hz, glu-H-3), 4.08 (1H, t, J = 9.5 Hz, glu-H-4), 4.09 (1H, m, glu-H-5), 4.59 (1H, dd, J = 12.1, 1.5 Hz, glu-H-6α), 4.54 (1H,d, J = 12.1, 4.0 Hz, glu-H-6β), 7.17(2H, s, galloyl-H-2, 6), 7.15 (2H, s, galloyl-H-2, 6), 6.71, 6.44 (各1H, s, HHDP H-3, 3′)；¹³C-NMR (150 MHz, acetone-d₆)δ: 92.3 (glu-C-1), 75.5 (glu-C-2),80.0 (glu-C-3), 76.3 (glu-C-4), 67.9 (glu-C-5), 63.5 (glu-C-6), 121.5, 120.2(galloyl-C-1), 110.2, 110.0 (galloyl-C-2, 6), 146.2, 146.1 (galloyl-C-3, 5),139.8, 138.9 (galloyl-C-4), 166.6, 165.0 (galloyl-C = O), 114.7, 114.5 (HHDPC-1, 1′), 127.0, 126.5 (HHDP C-2, 2′), 107.7, 107.1 (HHDP C-3, 3′), 145.2,145.0 (HHDP C-4, 4′), 136.4, 136.3 (HHDP C-5, 5′), 144.6, 144.6 (HHDP C-6, 6′),169.4, 168.8 (HHDP C = O)。以上数据与文献报道基本一致^[17]，故鉴定化合物10为1,6-二-O-没食子酰基-2,3-(S)-六羟基联苯甲酰基-β-D-吡喃型葡萄糖。

化合物11：浅褐色粉末，[α]25D +31.2° (c, 1.0, MeOH)；FAB-MS m/z: 935 [M－H]⁻，HR-FAB-MS ¹H-NMR (600 MHz, acetone-d₆) δ: 6.22 (1H, d, J = 8.8Hz, glu-H-1), 5.19 (1H, t, J = 8.8Hz, glu-H-2), 5.46 (1H, t, J = 9.2Hz, glu-H-3), 5.17 (1H, t, J = 9.9Hz, glu-H-4), 4.51 (1H, m, glu-H-5), 5.37 (1H, dd, J = 12.1, 6.6 Hz, glu-H-6α), 4.42 (1H, d, J = 12.1 Hz, glu-H-6β), 7.18 (2H, s, galloyl-H-2, 6), 6.68, 6.55,6.47, 6.37 (1H, s, HHDP H-3, 3′)；¹³C-NMR (150 MHz, acetone-d₆) δ: 92.3 (glu-C-1), 75.9 (glu-C-2), 77.2 (glu-C-3), 69.2 (glu-C-4),73.5 (glu-C-5), 63.1 (glu-C-6), 120.0 (galloyl-C-1), 110.3 (galloyl-C-2, 6),146.3 (galloyl-C-3, 5), 140.0 (galloyl-C-4), 165.0 (galloyl-C = O)；116.0, 115.8, 115.0, 114.2 (HHDPC-1, 1′), 126.5, 126.5, 126. 1, 125.9 (HHDP C-2, 2′), 108.3, 107.6, 107.3,107.3 (HHDP C-3, 3′), 145.3, 145.1 (HHDP C-4, 4′), 136.63, 136.56, 136.50,136.2 (HHDP C-5, 5′), 144.62, 144.58, 144.58, 144.58, 144.5 (HHDP C-6, 6′),169.2, 168.6, 168.0, 167.8 (HHDP C = O)。以上数据与文献报道基本一致^[18]，故鉴定化合物11为木麻黄鞣亭。

化合物12：淡棕色粉末，[α]25D +46.7° (c, 1.0, MeOH)；FAB-MS m/z: 937 [M－H]⁻，HR-FAB-MS m/z: 937.094 8 [M－H]⁻ (计算值937.094 6，C₄₁H₂₉O₂₆)；¹H-NMR (600 MHz, acetone-d₆)δ: 6.22 (1H, d, J = 8.3 Hz, glu-H-1), 5.62 (1H, t, J = 8.3 Hz, glu-H-2), 5.87 (1H, t, J = 9.3 Hz, glu-H-3), 5.24 (1H, t, J = 10.0 Hz, glu-H-4), 4.57 (m, glu-H-5), 5.37 (1H, dd, J = 13.4, 6.5 Hz, glu-H-6α), 3.91 (1H,d, J = 13.4 Hz, glu-H-6β), 7.14 (2H,s, galloyl-H-2, 6), 7.03 (2H, s, galloyl-H-2, 6), 7.00 (2H, s, galloyl-H-2, 6),6.68, 6.49 (各1H, s, HHDPH-3, 3′)；¹³C-NMR (150 MHz, acetone-d₆)δ: 93.6 (glu-C-1), 71.7 (glu-C-2),73.1 (glu-C-3), 70.6 (glu-C-4), 73.0 (glu-C-5), 63.0 (glu- C-6)；120.5, 120.4, 119.8 (galloyl-C-1),110.3, 110.1, 110.0 (galloyl-C-2, 6), 146.1, 145.9, 145.7 (galloyl- C-3, 5),139.8, 139.2, 139.1 (galloyl-C-4), 166.2, 165.4, 164.9 (galloyl-C = O), 115.6,115.5 (HHDP C-1, 1′

化合物13：淡棕色粉末[α]25D +51.0° (c, 1.0, MeOH)；FAB-MS m/z: 937 [M－H]⁻，HR-FAB-MS m/z: 937.095 0 [M－H]⁻ (计算值937.094 6，C₄₁H₂₉O₂₆)；¹H-NMR (600 MHz, acetone-d₆)δ: 6.37 (1H, d, J = 8.3 Hz, glu-H-1), 5.22 (1H, t, J = 8.9 Hz, glu-H-2), 5.61 (1H, t, J = 10.0 Hz, glu-H-3), 5.63 (1H, t, J = 10.0 Hz, glu-H-4), 5.54 (m, glu-H-5), 4.56 (1H, dd, J = 12.4, 2.1 Hz, glu-H-6α), 4.40 (1H,dd, J = 12.4, 4.5 Hz, glu-H-6β), 7.18(2H, s, galloyl-H-2, 6), 7.17 (2H, s, galloyl-H-2, 6), 7.15 (2H, s, galloyl-H-2,6), 6.68, 6.45 (各1H, s, HHDPH-3, 3′)；¹³C-NMR (150 MHz, acetone-d₆)δ: 92.1 (glu-C-1), 75.4 (glu-C-2),67.9 (glu-C-3), 77.4 (glu-C-4), 74.2 (glu-C-5), 62.8 (glu-C-6)；121.3, 120.6, 120.0 (galloyl-C-1),110.3, 110.2, 110.1 (galloyl-C-2, 6), 146.3, 146.1, 146.0 (galloyl-C-3, 5),139.8, 139.5, 114.6, 114.4 (HHDP C-1, 1′), 126.4, 126.1 (HHDP C-2, 2′), 107.4,107.2 (HHDP C-3, 3′), 144.6, 144.6 (HHDP C-4, 4′), 136.4 (HHDP C-5, 5′), 145.2(HHDP C-6, 6′), 169.0, 168.6 (HHDP C = O)。以上数据与文献报道的数据一致^[17]，故鉴定化合物13为1,4,6-三-O-没食子酰基-2,3-(S)-六羟基联苯二甲酰基- β-D-吡喃型葡萄糖。

化合物14：淡棕色粉末，[α]25D +28.0° (c, 0.2, MeOH)；FAB-MS m/z: 1 103 [M－H]⁻，HR-FAB-MS m/z: 1 103.085 5 [M－H]⁻ (计算值1 103.084 8，C₄₈H₃₁O₃₁)；¹H-NMR (600 MHz, Acetone-d₆-D₂O)δ: 5.64 (1H, d, J = 4.8 Hz, glu-H-1), 4.66 (1H, dd, J = 5.2, 2.0 Hz, glu-H-2), 5.44 (1H, dd, J = 2.1, 2.0 Hz, glu-H-3), 5.42 (1H, dd, J = 8.6, 2.1 Hz, glu-H-4), 5.29 (1H, dd, J = 8.9, 3.4 Hz, glu-H-4), 4.80 (1H, dd, J = 13.0, 3.8 Hz, glu-H-6α), 4.02 (1H, d, J = 13.0 Hz, glu- H-6β), 7.06 (2H, s, galloyl-H-2, 6), 7.11, 6.84,6.51, 6.24 (各1H, s, HHDP-H,valoneoyl-H)；¹³C-NMR (150 MHz, acetone-d₆-D₂O)δ: 67.4 (glu-C-1), 77.0 (glu-C-2),69.8 (glu-C-3), 74.2 (glu-C-4), 71.0 (glu- C-5), 64.7 (glu-C-6), 120.9 (galloylC-1), 110.1 (galloyl C-2, 6), 146.0 (galloyl C-3, 5), 139.3 (galloyl C-4),166.0 (galloyl C = O), 115.7, 120.9, 124.5 (valoneoyl C-1, 1′, 1′′), 116.4,117.1, 138.7 (valoneoyl C-2, 2′, 2′′), 145.3, 145.3, 145.9 (valoneoyl C-3, 3′,3′′), 135.0, 136.9, 137.6 (valoneoyl C-4, 4′, 4′′), 145.0, 147.1, 140.2(valoneoyl C-5, 5′, 5′′), 108.3, 105.0, 109.8 (valoneoyl C-6, 6′, 6′′), 167.6,168.9, 168.9 (valoneoyl C = O), 120.3, 126.7 (HHDP C-1, 1′), 116.1, 116.3 (HHDPC-2, 2′), 145.9, 146.3 (HHDP C-3, 3′), 127.5, 137.0 (HHDP C-4, 4′), 143.1,143.7 (HHDP C-5, 5′), 117.2, 105.4 (HHDP C-6, 6′), 165.0, 169.7 (HHDP C = O)。

化合物15：淡棕色粉末，[α]25D +89.0° (c, 0.8, MeOH)；FAB-MS m/z: 783 [M－H]⁻，HR-FAB-MS m/z: 783.068 1 [M－H]⁻ (计算值783.067 9，C₃₄H₂₃O₂₂)；¹H-NMR (600 MHz, acetone-d₆)δ: 5.48 (1H, t, J = 3.7 Hz, α-glu-H-1), 5.08 (1H, dd, J = 9.7, 3.7 Hz, α-glu-H-2), 5.47 (1H, t, J = 9.7 Hz, α-glu- H-3), 5.10 (1H, t, J = 8.6 Hz, α-glu-H-4), 4.62 (1H, ddd, J = 10.1, 7.2, 1.7 Hz, α-glu-H-5), 5.29 (1H, dd, J =12.1, 6.8 Hz, α-glu-H-6α), 3.78 (1H, dd, J= 12.1, 1.7 Hz, α-glu-H-6β), 5.08 (1H, d, J= 8.2 Hz, β-glu- H-1), 4.85 (1H, t, J= 8.2 Hz, β-glu-H-2), 5.24 (1H, dd, J= 9.2, 8.9 Hz, β-glu-H-3), 5.08 (1H, t, J= 9.6 Hz, β-glu-H-4), 4.23 (1H, ddd, J= 9.6, 6.9, 1.4 Hz, β-glu-H-5), 5.31 (1H, dd, J = 13.4, 6.9 Hz, β-glu- H-6α), 3.85 (1H, dd, J = 13.4, 1.0 Hz, β-glu-H-6β), 6.68, 6.67, 6.60, 6.59, 6.56, 6.51,6.33, 6.32 (各1H, s, HHDPH-3, 3′)；¹³C-NMR (150 MHz, acetone-d₆)δ: 91.8 (α-glu-C-1), 75.6 (α-glu-C-2),75.8 (α-glu-C-3), 69.9 (α-glu-C-4), 67.4 (α-glu-C-5), 63.5 (α-glu-C-6), 95.5(β-glu-C-1), 78.4 (β-glu-C-2), 77.6 (β-glu-C-3), 69.6 (β-glu-C-4), 72.5 (β-glu-C-5),63.5 (β-glu-C-6), 115.9, 115.9, 115.8, 115.7, 115.0, 115.0, 114.0, 114.0 (HHDP C-1,1′), 127.0, 126.8, 126.7, 126.7, 126.7, 126.6, 126.2, 126.1 (HHDP C-2, 2′),108.4, 108.3, 107.8, 107.7, 107.6, 107.5, 107.3, 107.2 (HHDP C-3, 3′), 145.2,145.2, 145.1, 145.1, 145.1 (HHDP C-4, 4′), 136.5, 136.5, 136.0, 136.0 (HHDPC-5, 5′), 144.6, 144.5, 144.5, 144.4, 144.3, 144.3 (HHDP C-6, 6′), 169.3,169.3, 168.8, 168.7, 168.2, 168.1, 167.8, 167.8 (H故鉴定为pedunculagin。

化合物16：淡棕色粉末[α]25D +38.0° (c, 0.8, MeOH)；FAB-MS m/z: 935 [M－H]⁻，HR-FAB-MS m/z: 935.079 1 [M－H]⁻ (计算值935.078 9，C₄₁H₂₇O₂₆)；¹H-NMR (600 MHz, acetone-d₆-D₂O)δ: 5.62 (1H, d, J = 5.2 Hz, glu-H-1), 4.68 (1H, dd, J = 5.2, 5.0 Hz, glu-H-2), 5.47 (1H, t, J = 2.0 Hz, glu-H-3), 5.49 (1H, dd, J = 8.0, 2.0 Hz, glu-H-4), 5.36 (1H, dd, J = 8.0, 2.8 Hz, glu-H-5), 4.90 (1H, dd, J = 13.1, 3.5 Hz, glu-H-6α), 4.09 (1H, d, J = 13.1 Hz, glu-H-6β), 7.13 (2H, s, galloyl-H-2, 6), 6.88, 6.58,6.53 (各1H, s, HHDPH-3, 3′)；¹³C-NMR (150 MHz, acetone-d₆-D₂O)δ: 67.1 (glu-C-1), 77.0 (glu-C-2),69.8 (glu-C-3), 71.2 (glu-C-4), 74.1 (glu-C-5), 64.6 (glu-C-6), 120.6 (galloylC-1), 110.1 (galloyl C-2, 6), 146.1 (galloyl C-3, 5), 139.6 (galloyl C-4),166.2, 165.4, 166.4 (galloyl C = O), 116.6, 116.4, 116.2, 115.4 (HHDP C-1, 1′),127.4, 126.8, 124.8, 119.8 (HHDP C-2, 2′), 117.3, 108.6, 107.2, 105.4 (HHDPC-3, 3′), 146.2, 145.9, 145.3, 143.9 (HHDP C-4, 4′), 139.0, 137.0, 136.1, 135.1(HHDP C-5, 5′), 145.3, 144.8, 144.8, 143.7 (HHDP C-6, 6′), 169.9, 169.3, 168.9,165.6 (HHDP C = O)。以上数据与文献报道的数据一致^[14]，故鉴定化合物16为木麻黄鞣宁。

H]⁻，HR-FAB-MS m/z: 300.998 8 [M－H]⁻（计算值 300.998 4，C₁₄H₅O₈）；¹H-NMR (600 MHz, DMSO-d₆)δ: 7.46 (2H, s, H-5, 5′)；¹³C-NMR (150 MHz, DMSO-d₆)δ: 107.4 (C-6，6′), 110.1 (C-5，5′), 112.3 (C-1，1′), 136.4 (C-2，2′), 139.7 (C-3，3′), 148.1 (C-4，4′), 159.1 (C-7，7′)。以上数据与文献报道的数据一致^[21]，故鉴定化合物17为鞣花酸。

化合物18：无色粉末，FAB-MS m/z: 193 [M－H]⁻，HR-FAB-MS m/z: 193.071 0 [M－H]⁻ (计算值193.071 1，C₉H₁₃O₆)；¹H-NMR (600 MHz, DMSO-d₆)δ: 3.31(3H, s, OMe), 3.87 (1H, t, J = 3.4Hz, H-1), 3.10 (1H, dd, J = 9.6, 3.1Hz, H-2), 3.38 (1H, t, J = 9.3 Hz,H-3), 3.29 (1H, t, J = 9.3 Hz, H-4),3.43 (1H, dd, J = 9.6, 3.9 Hz, H-5), 3.68 (1H, t, J = 3.8 Hz, H-6)；¹³C-NMR (150 MHz, DMSO-d₆) δ: 57.0 (OCH₃), 68.1 (C-1), 70.5 (C-5), 72.0 (C-6), 72.2(C-3), 73.3 (C-4), 81.1 (C-2)。以上数据与文献报道的数据一致^[22]，故鉴定化合物18为松醇。

4 体外抗炎和抗肥胖活性评价

4.1 抑制RAW264.7巨噬细胞一氧化氮（NO）生成的抗炎活性评价

用含10%胎牛血清的Ham’s F12培养基，在37 ℃、5% CO₂条件下培养RAW264.7巨噬细胞。将溶解于DMSO的不同浓度的供试品、LPS（100 ng/mL）和IFN-γ（10 U/mL）一同加入到细胞培养液中。培养16 h后，取细胞上清培养液100 μL置于新的96孔板中，加入Griess试剂（含5%磷酸的1%对氨基苯磺酰胺，0.1%萘乙二胺盐酸盐各50 μL）。室温下避光放置10 min，用酶标仪测定反应产物在570 nm的吸光度，并扣除在655 nm的背景吸光度值。

实验结果（表1）显示，与阳性对照氨基胍相比，多数鞣质类化合物具有较强的抑制一氧化氮生成的作用。化合物11、16和6的活性较强，IC₅₀值分别为10.8、11.4、11.5 μmol/L，说明鞣质的抗炎活性可能随着没食子酰基和六羟基联苯二甲酰基的数目的增多而增强。

4.2 抑制3T3L-1脂肪细胞三酰甘油（TG）蓄积的抗肥胖活性评价

用含10%新生小牛血清的高糖DMEM培养基，在37 ℃、5% CO₂条件下培养3T3-L1脂肪细胞，待细胞生长至完全融合后2 d（第0天）时，更换成诱导分化培养基（含10%胎牛血清、10 μg/mL胰岛素、1.0 μmol/L地塞米松、500 μmol/L 1-甲基-3-异丁基黄嘌呤）并添加不同浓度的样品培养3 d，换用促进分化培养基（含10%胎牛血清、5 μg/mL胰岛素）并添加不同浓度的样品培养3 d，换用同样培养基并添加不同浓度的样品培养至第8天。3T3-L1脂肪细胞用PBS (−) 清洗后，用含1 mmol/L EDTA的25 mmol/L Tris-HCl缓冲液收集细胞，超声匀浆，用LabAssay^TMTriglyceride试剂盒（WAKO Pure ChemicalIndustries Ltd.）测定细胞中蓄积的TG含量，同时用甘油磷酸脱氢酶（GPDH）试剂盒（TaKaRa Bio Inc.）测定GPDH活性，分别定量分析脂肪蓄积和分化程度，评价供试品的抗肥胖活性。