劉宏棟,高余英,高 琦,周小青,陳 杰,朱金華,李 飛,李 斌*
窄葉鮮卑花地上部分化學(xué)成分及降血脂活性研究
劉宏棟1,高余英2,高 琦2,周小青3,陳 杰4,朱金華5,李 飛6,李 斌1*
1. 江西中醫(yī)藥大學(xué) 院士工作站,江西 南昌 330004 2. 宜春市第三人民醫(yī)院,江西 宜春 336000 3. 江西中醫(yī)藥大學(xué),江西 南昌 330004 4. 江西中醫(yī)藥大學(xué)藥學(xué)院,江西 南昌 330004 5. 江西中醫(yī)藥大學(xué)中醫(yī)學(xué)院,江西 南昌 330004 6. 四川大學(xué)華西醫(yī)院 四川大學(xué)-牛津大學(xué)消化道腫瘤中心,四川 成都 610065
研究窄葉鮮卑花地上部分的化學(xué)成分及其體外降血脂活性。采用硅膠柱色譜、ODS反相柱色譜、半制備液相色譜等方法進行分離純化,利用NMR、MS等技術(shù)鑒定化合物結(jié)構(gòu),再通過建立人肝癌HepG2細(xì)胞高脂模型測試各化合物調(diào)脂活性。從窄葉鮮卑花的95%乙醇提取物中分離得到了21個化合物,分別鑒定為藜蘆酸甲酯(1)、阿魏酸甲酯(2)、3-羥基-4-甲氧基肉桂酸甲酯(3)、3,4,5-三甲氧基肉桂醇(4)、對羥基桂皮酸甲酯-4β吡喃葡萄糖苷(5)、藜蘆酸葡萄糖苷(6)、aglycone(7)、aruncide A(8)、3-[(2)-4-hydroxyl -4-methyl-2-pentenyl)]furan-5-2-one(9)、吐葉醇(10)、蚱蜢酮(11)、3,7-dimethyl-3(),6-octadien-5-one-1βglucoside(12)、(2,3,4)-4-(4-hydroxy-3- methoxybenzyl)-2-(5-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-tetrahydrofuran-3-ol(13)、1,2-bis(4-hydroxy-3-methoxyphenyl)- 1,3-propanediol(14)、salicifoneoliganol(15)、herpetriol(16)、達(dá)瑪烯二醇-II(17)、山楂酸甲酯(18)、麥珠子酸(19)、阿魏酸二十四烷酯(20)、對羥基反式肉桂酸二十四烷基酯(21)?;衔?~8、10、11、13~21為首次從鮮卑花屬植物中分離得到,化合物7、8、17、18在體外實驗中顯示出降脂活性。
窄葉鮮卑花;藜蘆酸甲酯;阿魏酸甲酯;達(dá)瑪烯二醇-II;山楂酸甲酯;降血脂活性;人肝癌HepG2細(xì)胞
窄葉鮮卑花(Rehd.) Hand. -Mazz.是薔薇科(Rosaceae)鮮卑花屬Maxim.灌木,廣泛分布于我國中西部地區(qū),包括青海、甘肅、云南、四川和西藏等地,生長于海拔3000~4000 m的山坡或砂石攤上。藏族民間通常以其葉和嫩枝入藥,俗稱“柳茶”,用于治療消化不良及腹部不適[1-2],《中華本草》記載其功效為消食、理氣的功效[3]。藥理學(xué)研究發(fā)現(xiàn)窄葉鮮卑花提取物能顯著降低高脂動物模型體內(nèi)的總膽固醇、三酰甘油、低密度脂蛋白和丙二醛的含量,調(diào)節(jié)脂質(zhì)代謝,抑制體質(zhì)量增加[2-4]。前期本課題組已從窄葉鮮卑花中分離得到多個單萜苷和黃酮等化合物[5-13],其中單萜類為其調(diào)血脂作用的主要藥效成分[7-13]。為進一步闡明窄葉鮮卑花中的化學(xué)成分,并尋找結(jié)構(gòu)新穎、活性強的單體化合物,本實驗采用多種色譜技術(shù)對窄葉鮮卑花地上部分的95%乙醇提物進行研究,分離純化得到21個化合物,分別鑒定為藜蘆酸甲酯(methyl veratrate,1)、阿魏酸甲酯[()-3,4- dimethoxycinnamic acid methyl ester,2]、3-羥基-4-甲氧基肉桂酸甲酯[()-methyl 3-(3-hydroxy-4- methoxyphenyl) acrylate,3]、3,4,5-三甲氧基肉桂醇(3,4,5-trimethoxycinnamyl alcohol,4)、對羥基桂皮酸甲酯-4β吡喃葡萄糖苷(linocinnamarin,5)、藜蘆酸葡萄糖苷(tecomin,6)、aglycone(7)、aruncide A(8)、3-[(2)-4-hydroxyl-4-methyl-2-pentenyl)] furan-5-2-one(9)、吐葉醇(vomifoliol,10)、蚱蜢酮(grasshopper ketone,11)、3,7-dimethyl-3(),6-octadien-5-one-1βglucoside(12)、(2,3,4)-4-(4-hydroxy-3-methoxybenzyl)-2-(5-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-tetrahydrofuran-3-ol(13)、1,2-bis(4-hydroxy-3-methoxyphenyl)-1,3- propanediol(14)、salicifoneoliganol(15)、herpetriol(16)、達(dá)瑪烯二醇-II(dammarenediol-II,17)、山楂酸甲酯(methyl maslinat,18)、麥珠子酸(alphitolic acid,19)、阿魏酸二十四烷酯(tetracosyl ferulate,20)、對羥基反式肉桂酸二十四烷基酯(-tetracosyl trans--coumarate,21)。其中19個化合物(1~8、10、11、13~21)為首次從鮮卑花屬植物中分離得到。同時,對分離獲得的化合物進行了體外降血脂藥理活性評價,化合物7、8、17、18在人肝癌HepG2細(xì)胞高脂模型中顯示出降血脂活性,其中化合物8、17作用最強。
Bruker AV600型核磁共振儀(TMS為內(nèi)標(biāo),德國布魯克公司);安捷倫UPLC-Q-TOF-MS超高效液相質(zhì)譜聯(lián)用儀質(zhì)譜儀(美國安捷倫科技有限公司);LC-52制備型液相色譜儀(北京賽譜銳思科技有限公司);YMC-Pack ODS-A型半制備型反相色譜柱(250 mm×20 mm,5 μm,日本YMC株式會社);W2-100SP型旋轉(zhuǎn)蒸發(fā)儀(上海申生科技有限公司);BSA224S-CW型電子天平(德國賽多利斯公司)。超凈工作臺(Heraguard ECO,賽默飛世爾科技有限公司);細(xì)胞培養(yǎng)箱(3111型二氧化碳培養(yǎng)箱,賽默飛世爾科技有限公司);酶標(biāo)儀(ELx808吸收光酶標(biāo)儀,美國伯騰儀器有限公司);顯微鏡(TS2倒置生物顯微鏡,尼康儀器有限公司)。
硅膠(200~300目,青島海洋化工廠);薄層色譜硅膠板(GF254,50 mm×100 mm,青島海洋化工廠);Sephadex LH-20葡聚糖凝膠(Amersham Pharmacia Biotech AB);YMC ODS-A-HG反相硅膠(日本YMC株式會社);色譜甲醇(上海星可高純?nèi)軇┯邢薰荆秽邕蛩{(lán)(MTT,大連美倫公司),二甲基亞砜(DMSO,德國BioFroxx公司),DMEM高糖培養(yǎng)基(Dulbecco′s Modified Eagle Medium,Biological Industries),胰蛋白酶(Biological Industries),磷酸緩沖鹽溶液(PBS,Biological Industries),油紅O(北京索萊寶科技有限公司),胎牛血清(FBS,美國Gibco公司),青霉素、鏈霉素雙抗(Biological Industries),油酸鈉(上海源葉生物科技有限公司)。非諾貝特(A01316179),北京百靈威科技有限公司。
實驗用窄葉鮮卑花藥材于2012年采自四川省,經(jīng)四川大學(xué)華西藥學(xué)院王天志教授鑒定為薔薇科繡線菊亞科鮮卑花屬植物窄葉鮮卑花(Rehd.) Hand. -Mazz.。植物樣本(編號001612)保存于江西中醫(yī)藥大學(xué)植物標(biāo)本館。
窄葉鮮卑花地上部分(50 kg)經(jīng)晾曬干燥、粉碎后,用95%乙醇加熱回流提取2次(每次150 L,2 h),合并提取液并減壓回收乙醇,濃縮后地粗浸膏。將粗浸膏用純化水充分混懸,依次使用石油醚、二氯甲烷、醋酸乙酯、正丁醇萃取。各萃取溶液經(jīng)減壓回收,分別得到石油醚部位(0.1 kg)、二氯甲烷部位(1.7 kg)、醋酸乙酯部位(0.2 kg)、正丁醇部位(1.4 kg)和水部位。
正丁醇部位(1.0 kg)用硅膠柱色譜(200~300目)分離,以二氯甲烷-甲醇(95∶5~0∶100)梯度洗脫,TLC檢測合并相同部分,得到6個部位(A~F)。其中C部位(237.0 g)經(jīng)硅膠柱色譜(200~300目)分離,二氯甲烷-甲醇(98∶2~0∶100)梯度洗脫,TLC檢測合并相同部分,得到4部分(C1~C4);C3部分用硅膠柱色譜(200~300目)分離,醋酸乙酯-丙酮(100∶0~0∶100)梯度洗脫,TLC檢測合并得到5個流分(C3-1~C3-5),C3-1流分(3.0 g)經(jīng)常壓柱色譜及制備HPLC純化等色譜、化學(xué)分離手段得到化合物1(43 mg)、2(15 mg)、3(11 mg)、5(9 mg)、15(9 mg)。C3-3流分(48.0 g)經(jīng)常壓柱色譜及制備HPLC純化等色譜、化學(xué)分離手段得到化合物6(200 mg)、8(2.0 g)、12(10 mg)。
二氯甲烷部位(1.6 kg)用硅膠柱色譜(200~300目)分離,以二氯甲烷-醋酸乙酯(100∶0~0∶100~甲醇)梯度洗脫,TLC檢測合并相同部分,得到7個部位(Fr. 1~7)。流分Fr. 2(110.0 g)經(jīng)硅膠柱色譜得到化合物17(15 mg)、20(500 mg)、21(300 mg)。流分Fr. 5(110.0 g)用硅膠柱色譜分離,以二氯甲烷-丙酮(100∶0~0∶100)梯度洗脫,TLC檢測合并得到5個流分(Fr. 5-1~5-5),所得流分Fr. 5-1(2.0 g)經(jīng)硅膠柱色譜及制備HPLC純化得到化合物4(10 mg)、7(8 mg)、9(12 mg)。Fr. 5-2(10.0 g)經(jīng)硅膠柱色譜和反相柱色譜及得到化合物18(6.5 g)、19(80 mg)。流分Fr. 5-3(35.0 g)經(jīng)硅膠柱色譜及制備HPLC純化得到化合物10(4 mg)、11(6 mg)、13(8 mg)、14(5 mg)、16(20 mg)。
化合物1:白色無定形粉末;HR-ESI-MS/: 219.064 3 [M+Na]+(計算值219.063 4,C10H12O4Na),/197.082 0 [M+H]+(計算值197.081 5,C10H13O4)。1H-NMR (600 MHz, CDCl3): 7.67 (1H, d,= 8.3 Hz, H-5), 7.53 (1H, s, H-2), 6.88 (1H, d,= 8.3 Hz, H-6), 3.93 (3H, s, 9-OCH3), 3.92 (3H, s, 10-OCH3), 3.88 (3H, s, 8-OCH3);13C-NMR (150 MHz, CDCl3): 166.8 (C-7), 152.8 (C-3), 148.5 (C-4), 123.5 (C-5), 122.6 (C-1), 111.8 (C-2), 110.1 (C-6), 56.1 (9-OCH3), 56.0 (10-OCH3), 52.1 (8-OCH3)。以上數(shù)據(jù)與文獻(xiàn)報道一致[14],故鑒定化合物1為藜蘆酸甲酯。
化合物2:白色無定形粉末;HR-ESI-MS/: 231.063 5 [M+Na]+(計算值231.063 2,C11H12O4Na) 和/209.081 8 [M+H]+(計算值209.081 3,C11H13O4);1H-NMR (600 MHz, CDCl3):7.61 (1H, d,= 15.8 Hz, H-7), 7.06 (1H, d,= 8.1 Hz, H-6), 7.01(1H, s, H-2), 6.91 (1H, d,= 8.2 Hz, H-5), 6.28 (1H, d,= 15.8 Hz, H-8), 3.91 (3H, s, 11-OCH3), 3.78 (3H, s, 10-OCH3);13C-NMR (150 MHz, CDCl3): 167.6 (C-9), 148.0 (C-4), 146.6 (C-3), 144.8 (C-7), 126.8 (C-1), 123.0 (C-6), 115.0 (C-8), 114.6 (C-5), 109.2 (C-2), 55.8 (11-OCH3), 51.5 (10-OCH3)。以上數(shù)據(jù)與文獻(xiàn)報道一致[15],故鑒定化合物2為阿魏酸甲酯。
化合物3:白色無定形粉末;HR-ESI-MS/: 231.063 5 [M+Na]+(計算值231.062 1,C11H12O4Na) 和/209.080 8 [M+H]+(計算值209.081 3,C11H13O4);1H-NMR (600 MHz, CDCl3):7.61 (1H, d,= 15.8 Hz, H-7), 7.13(1H, d,= 1.8 Hz, H-2), 7.02 (1H, dd,= 1.8, 8.2 Hz, H-6), 6.83 (1H, d,= 8.2 Hz, H-5), 6.28 (1H, d,= 15.8 Hz, H-8), 3.91 (3H, s, 11-OCH3), 3.78 (3H, s, 10-OCH3);13C-NMR (150 MHz, CDCl3): 167.6 (C-9), 148.4 (C-4), 145.7 (C-3), 144.6 (C-7), 128.0 (C-1), 121.7 (C-6), 115.7 (C-2), 115.7 (C-8), 113.0 (C-5), 56.1 (11-OCH3), 52.5 (10-OCH3)。以上數(shù)據(jù)與文獻(xiàn)報道一致[16],故鑒定化合物3為3-羥基-4-甲氧基肉桂酸甲酯。
化合物4:白色針晶(二氯甲烷),mp 76~78 ℃;HR-ESI-MS/: 247.093 1 [M+Na]+(計算值247.094 4,C12H16O4Na) 和 225.112 7 [M+H]+(計算值225.112 5,C12H17O4);1H-NMR (600 MHz, CDCl3):6.62(2H, s, H-2, 6), 6.52 (1H, d,= 15.6 Hz, H-7), 6.27 (1H, d,= 15.6 Hz, H-8), 4.31 (2H, d,= 5.1 Hz, H-9), 3.85 (6H, s, 10, 12-OCH3), 3.83 (3H, s, 11-OCH3);13C-NMR (150 MHz, CDCl3): 153.3 (C-3, 5), 138.1 (C-4), 132.4 (C-1), 131.1 (C-7), 128.0 (C-8), 103.5 (C-2, 6), 63.6 (C-9), 61.1 (11-OCH3), 56.0 (10, 12-OCH3)。以上數(shù)據(jù)與文獻(xiàn)報道一致[17],故鑒定化合物4為3,4,5-三甲氧基肉桂醇。
化合物5:淡黃色粉末;HR-ESI-MS/: 363.106 2 [M+Na]+(計算值363.105 5,C16H20O8Na) 和/341.123 2 [M+H]+(計算值 341.123 5,C16H21O8);1H-NMR (600 MHz, CDCl3):7.75 (1H, d,= 15.8 Hz, H-7), 7.57 (2H, d,= 8.6 Hz, H-2, 6), 6.96 (2H, d,= 8.6 Hz, H-3, 5), 6.42 (1H, d,= 15.8 Hz, H-8), 5.58 (1H, d,= 7.7 Hz, H-1′), 3.85 (1H, m, H-6′a), 3.83 (3H, s, 10-OCH3), 3.71 (1H, m, H-6′b), 3.31~3.51 (4H, m, H-2′~5′);13C-NMR (150 MHz, CDCl3): 167.4 (C-9), 163.3 (C-4), 147.4 (C-7), 131.1 (C-2, 6), 128.1 (C-1), 115.4 (C-3, 5), 115.3 (C-8), 95.7 (C-1′), 78.7 (C-5′), 78.1 (C-3′), 74.1 (C-2′), 71.0 (C-4′), 62.2 (C-6′), 55.8 (10-OCH3)。以上數(shù)據(jù)與文獻(xiàn)報道一致[18],故鑒定化合物5為對羥基桂皮酸甲酯-4β吡喃葡萄糖苷。
化合物6:白色無定形粉末;HR-ESI-MS/: 367.101 0 [M+Na]+(計算值367.100 5,C15H20O9Na)和/345.117 3 [M+H]+(計算值345.118 6,C15H21O9);1H-NMR (600 MHz, DMSO-6):7.65 (1H, dd,= 1.8, 8.5 Hz, H-6), 7.48 (1H, d,= 1.8 Hz, H-2), 7.08 (1H, d,= 8.5 Hz, H-5), 5.54 (1H, d,= 7.3 Hz, H-1′), 3.83 (3H, s, 9-OCH3), 3.81 (3H, s, 8-OCH3), 3.81 (1H, m, H-6′a), 3.66 (1H, m, H-6′b), 3.12~3.50 (4H, m, H-2′~5′);13C-NMR (150 MHz, DMSO-6):164.4 (C-7), 148.4 (C-3), 153.3 (C-4), 123.8 (C-1), 121.2 (C-6), 112.1 (C-2), 111.1 (C-5), 94.8 (C-1′), 78.1 (C-5′), 76.3 (C-3′), 72.5 (C-2′), 69.5 (C-4′), 60.5 (C-6′), 55.7 (9-OCH3), 55.6 (8-OCH3)。以上數(shù)據(jù)與文獻(xiàn)報道一致[19],故鑒定化合物6為藜蘆酸葡萄糖苷。
化合物7:無色膠狀物;HR-ESI-MS/: 205.083 3 [M+Na]+(計算值205.084 1,C10H14O3Na) 和/183.102 2 [M+H]+(計算值183.102 1,C10H15O3);1H-NMR (600 MHz, CDCl3):6.41 (1H, m, H-2), 5.26 (1H, m, H-6), 5.24 (1H, m, H-5), 4.56 (2H, m, H-1), 3.07 (1H, m, H-4b), 2.65 (1H, m, H-4a), 1.76 (3H, s, 8-CH3), 1.73 (3H, s, 9-CH3);13C-NMR (150 MHz, CDCl3):170.7 (C-10), 141.5 (C-2), 140.6 (C-7), 127.7 (C-3), 123.1 (C-6), 75.6 (C-5), 59.1 (C-1), 36.2 (C-4), 25.8 (8-CH3), 18.5(9-CH3)。以上數(shù)據(jù)與文獻(xiàn)報道一致[20],故鑒定化合物7為aglycone。
化合物8:淡黃色膠狀物;HR-ESI-MS/: 367.136 4 [M+Na]+(計算值367.136 9,C16H24O8Na) 和/345.154 5 [M+H]+(計算值345.154 9,C16H25O8);1H-NMR (600 MHz, DMSO-6):6.32 (1H, m, H-2), 5.28 (1H, m, H-6), 5.24 (1H, d,= 8.9 Hz, H-5), 4.73 (1H, m, H-1b), 4.67 (1H, m, H-1a), 4.17 (1H, d,= 7.7 Hz, H-1′), 3.65 (1H, dt,= 3.2, 11.3 Hz, H-6′a), 3.43 (1H, dt,= 5.0, 11.0 Hz, H-6′b), 3.01~3.21 (4H, m, H-2′~5′), 2.95 (1H, t,= 8.3 Hz, H-4b), 2.60 (1H, m, H-4a), 1.71 (3H, s, 8-CH3), 1.71 (3H, s, 9-CH3);13C-NMR (150 MHz, DMSO-6):169.2 (C-10), 139.0 (C-7), 138.7 (C-2), 126.1 (C-3), 123.5 (C-6), 102.7 (C-1′), 76.8 (C-5′), 76.6 (C-3′), 74.6 (C-5), 73.3 (C-2′), 70.0 (C-4′), 64.8 (C-1), 61.0 (C-6′), 35.0 (C-4), 25.3 (8-CH3), 18.1 (9-CH3)。以上數(shù)據(jù)與文獻(xiàn)報道一致[21],故鑒定化合物8為aruncide A。
化合物9:無色膠狀物;HR-ESI-MS/: 207.098 6 [M+Na]+(計算值207.099 7,C10H16O3Na) 和/185.116 3 [M+H]+(計算值185.117 8,C10H17O3);1H-NMR (600 MHz, CDCl3):5.71 (1H, m, H-7), 5.60 (1H, dt,= 6.8, 15.4 Hz, H-6), 4.31 (1H, dt,= 3.1, 8.7 Hz, H-4b), 4.21 (1H, dt,= 7.0, 9.0 Hz, H-4a), 2.61 (1H, m, H-5b), 2.55 (1H, m, H-5a), 2.34 (1H, m, H-2), 2.23 (1H, m, H-3b), 1.96 (1H, m, H-3a), 1.31 (6H, s, 9, 10-CH3);13C-NMR (150 MHz, CDCl3): 179.8 (C-1), 141.4 (C-7), 122.5 (C-6), 70.6 (C-8), 66.6 (C-4), 39.2 (C-2), 32.8 (C-5), 30.0 (9, 10-CH3), 27.8 (C-3)。以上數(shù)據(jù)與文獻(xiàn)報道一致[22],故鑒定化合物9為3-[(2)-4-hydroxyl-4-methyl-2-pentenyl)] furan-5-2-one。
化合物10:無色膠狀物;HR-ESI-MS/: 247.131 4 [M+Na]+(計算值247.131 0,C13H20O3Na) 和/225.149 3 [M+H]+(計算值225.149 1,C13H21O3);1H-NMR (600 MHz, MeOD):5.87 (1H, s, H-5), 5.81 (1H, d,= 15.8 Hz, H-8), 5.76 (1H, d,= 15.7 Hz, H-7), 4.31 (1H, m, H-9), 2.47 (1H, d,= 16.9 Hz, H-3b), 2.15 (1H, m, H-3a), 1.88 (3H, s, 13-OCH3), 1.24 (3H, d,= 6.4 Hz, 10-OCH3), 1.03 (3H, s, 12-OCH3), 1.01 (3H, s, 11-OCH3);13C-NMR (150 MHz, MeOD): 201.1 (C-4), 167.4 (C-6), 136.8 (C-8), 130.0 (C-7), 127.0 (C-5), 78.3 (C-1), 68.5 (C-9), 50.6 (C-3), 42.3 (C-2), 24.4 (10-CH3), 23.7 (11-CH3), 23.4 (12-CH3), 19.5 (13-CH3)。以上數(shù)據(jù)與文獻(xiàn)報道一致[23],故鑒定化合物10為吐葉醇。
化合物11:無色膠狀物;HR-ESI-MS/: 247.132 4 [M+Na]+(計算值247.131 0,C13H20O3Na) 和/225.148 5 [M+H]+(計算值225.149 1,C13H21O3);1H-NMR (600 MHz, MeOD): 5.82 (1H, s, H-8), 4.21 (1H, m, H-4), 2.19 (1H, m, H-5b), 2.18 (3H, s, 10-CH3), 1.92 (1H, m, H-5a), 1.36 (1H, m, H-3b), 1.35 (3H, s, 11-CH3), 1.35 (3H, s, 12-CH3), 1.33 (1H, m, H-3a), 1.15 (3H, s, 13-CH3);13C-NMR (150 MHz, MeOD): 211.5 (C-7), 200.8 (C-9), 120.1 (C-1), 101.1 (C-7), 72.3 (C-6), 64.3 (C-4), 49.8 (C-5), 49.6 (C-3), 37.1 (C-2), 32.2 (13-CH3), 30.7 (11-CH3), 29.2 (12-CH3), 26.4 (10-CH3)。以上數(shù)據(jù)與文獻(xiàn)報道一致[24],故鑒定化合物11為蚱蜢酮。
化合物12:無色膠狀物;HR-ESI-MS/: 353.158 2 [M+Na]+(計算值353.157 6,C16H26O7Na)和/331.174 5 [M+H]+(計算值331.175 7,C16H27O7);1H-NMR (600 MHz, MeOD):6.18 (1H, s, H-4), 6.14 (1H, s, H-6), 4.31 (1H, d,= 7.8 Hz, H-1′), 3.97 (1H, dt,= 7.0, 9.4 Hz, H-1b), 3.86 (1H, m, H-6′a), 3.73 (1H, m, H-1a), 3.67 (1H, m, H-6′b), 3.10~3.39 (4H, m, H-2′~5′), 2.94 (2H, m, H-2), 2.13 (3H, s, 9-CH3), 1.97 (3H, s, 10-CH3), 1.91 (3H, s, 8-CH3);13C-NMR (150 MHz, MeOD): 192.9 (C-5), 157.0 (C-7), 156.6 (C-3), 128.3 (C-4), 127.1 (C-6), 104.2 (C-1′), 78.0 (C-5′), 77.8 (C-3′), 75.0 (C-2′), 71.5 (C-4′), 69.2 (C-1), 62.7 (C-6′), 35.1 (C-2), 27.7 (8-CH3), 26.2 (10-CH3), 20.7 (9-CH3)。以上數(shù)據(jù)與文獻(xiàn)報道一致[25],故鑒定化合物12為3,7-dimethyl- 3(),6-octadien-5-one-1βglucoside。
化合物13:無色膠狀物;HR-ESI-MS/: 399.142 3 [M+Na]+(計算值399.142 0, C20H24O7Na,) 和/377.161 7 [M+H]+(計算值377.160 0,C20H25O7);1H-NMR (600 MHz, MeOD):6.96 (1H, s, H-6′), 6.81 (1H, d,= 1.6 Hz, H-2′′), 6.76 (1H, s, H-2′), 6.76 (1H, s, H-4′), 6.71 (1H, d,= 8.1 Hz, H-5′′), 6.70 (1H, dd,= 1.5, 8.1 Hz, H-6′′), 4.82 (1H, m, H-2), 3.85 (3H, s, 7′-OCH3), 3.84 (3H, s, 7′′-OCH3), 3.80 (2H, d,= 11.3 Hz, H-3a), 3.66 (2H, dd,= 5.5, 8.3 Hz, H-5), 3.11 (1H, dd,= 3.3, 13.0 Hz, H-4a2), 2.58 (1H, m, H-4), 2.48 (1H, t,= 12.7 Hz, H-4a1);13C-NMR (150 MHz, MeOD):149.1 (C-3′′), 148.5 (C-5′), 147.2 (C-3′), 145.8 (C-4′′), 133.2 (C-1′′), 130.6 (C-1′), 122.1 (C-6′′), 121.4 (C-2′), 116.1 (C-4′), 115.5 (C-5′′), 113.3 (C-2′′), 112.6 (C-6′), 85.5 (C-2), 83.1 (C-3), 71.8 (C-5), 64.4 (C-3a), 56.2 (7′-OCH3), 56.2 (7′′-OCH3), 51.8 (C-4), 35.1 (C-4a)。以上數(shù)據(jù)與文獻(xiàn)報道一致[26],故鑒定化合物13為(2,3,4)-4-(4-hydroxy-3-methoxybenzyl)-2-(5-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-tetra-hydrofuran-3-ol。
化合物14:白色無定形粉末;HR-ESI-MS/: 357.132 3 [M+Na]+(計算值357.131 4,C18H22O6Na) 和/335.148 2 [M+H]+(計算值335.149 5,C18H23O6);1H-NMR (600 MHz, MeOD): 6.68 (1H, d,= 8.0 Hz, H-5′), 6.67 (1H, d,= 8.0 Hz, H-5′′), 6.65 (1H, m, H-6′′), 6.64 (1H, d,= 1.5 Hz, H-2′′), 6.61 (1H, d,= 1.6 Hz, H-2′), 6.61 (1H, dd,= 1.7, 8.0 Hz, H-6′), 4.91 (1H, m, H-1), 3.80 (1H, dd,= 5.0, 11.1 Hz, H-4b), 3.74 (3H, s, 7′-OCH3), 3.68 (3H, m, 7′′-OCH3), 3.67 (1H, m, H-2), 3.51 (1H, dd,= 6.0, 11.1 Hz, H-4a), 2.91 (2H, m, H-3);13C-NMR (150 MHz, MeOD): 148.4 (C-3′), 148.3 (C-3′′), 146.5 (C-4′), 146.1 (C-4′′), 136.3 (C-1′), 132.1 (C-1′′), 123.2 (C-6′′), 120.2 (C-6′), 115.6 (C-5′), 115.3 (C-5′′), 114.4 (C-2′′), 111.5 (C-2′), 75.4 (C-1), 64.4 (C-3), 56.7 (C-2), 56.2 (7′′-OCH3), 56.1 (7′-OCH3), 54.7 (C-4)。以上數(shù)據(jù)與文獻(xiàn)報道一致[27],故鑒定化合物14為1,2-bis(4-hydroxy-3-methoxyphenyl)-1,3-propanediol。
化合物15:白色無定形粉末;HR-ESI-MS/: 399.142 1 [M+Na]+(計算值399.142 0,C20H24O7Na) 和/377.161 3 [M+H]+(計算值377.160 0,C20H25O7);1H-NMR (600 MHz, DMSO-6):6.64 (2H, s, H-2, 6), 6.52 (1H, s, H-2′), 6.47 (1H, s, H-6′), 5.36 (1H, d,= 7.0 Hz, H-7), 3.72 (1H, m, H-9b), 3.72 (6H, s, 10, 11-OCH3), 3.68 (1H, m, H-9a), 2.43 (2H, m, H-7′), 1.63 (2H, m, H-8′);13C-NMR (150 MHz, DMSO-6): 147.8 (C-3, 5), 144.5 (C-4′), 140.6 (C-3′), 135.1 (C-1′), 134.8 (C-4), 131.7 (C-1), 129.1 (C-5′), 115.6 (C-6′), 114.8 (C-2′), 103.6 (C-2, 6), 86.7 (C-7), 63.0 (C-9), 60.1 (C-9′), 56.1 (10, 11-OCH3), 53.5 (C-8), 34.6 (C-8′), 31.2 (C-7′)。以上數(shù)據(jù)與文獻(xiàn)報道一致[28],故鑒定化合物15為salicifoneoliganol。
化合物16:白色無定形粉末;HR-ESI-MS/: 561.211 5 [M+Na]+(計算值561.210 0,C30H34O9Na) 和/539.226 4 [M+H]+(計算值539.228 1,C30H35O9);1H-NMR (600 MHz, MeOD):6.93 (1H, d,= 1.5 Hz, H-6′), 6.91 (1H, d,= 1.4 Hz, H-2′), 6.81 (1H, dd,= 1.5, 8.1 Hz, H-6′′), 6.71~6.74 (5H, m, H-2, 5, 6, 2′′, 5′′), 5.48 (1H, d,= 6.2 Hz, H-7′′), 4.73 (1H, dd,= 6.4, 8.1 Hz, H-7′), 3.84 (3H, s, 10′-OCH3), 3.83 (3H, s, 10′′-OCH3), 3.80 (3H, s, 10-OCH3), 3.62 (1H, m, H-8′′);13C-NMR (150 MHz, MeOD): 149.0 (C-3′), 149.0 (C-3′′), 147.7 (C-3), 147.5 (C-4′), 147.0 (C-4′′), 145.3 (C-4), 135.6 (C-1′′), 135.4 (C-1′), 134.6 (C-1), 130.0 (C-5′), 119.7 (C-6), 119.7 (C-6′′), 118.2 (C-5′′), 116.1 (C-6′), 116.0 (C-5), 114.2 (C-2), 110.5 (C-2′′), 110.4 (C-2′), 89.0 (C-7′′), 84.0 (C-7′), 73.4 (C-9), 64.8 (C-9′′), 60.4 (C-9′), 56.7 (10-OCH3), 56.7 (10′-OCH3), 56.3 (10′′-OCH3), 55.3 (C-8′′), 54.0 (C-8′), 44.0 (C-8), 33.8 (C-7)。以上數(shù)據(jù)與文獻(xiàn)報道一致[29],故鑒定化合物16為herpetriol。
化合物17:無色針晶(二氯甲烷),mp 130~133 ℃;HR-ESI-MS/: 467.387 5 [M+Na]+(計算值467.386 5,C30H52O2Na) 和/445.403 4 [M+H]+(計算值445.404 5,C30H53O2);1H-NMR (600 MHz, MeOD):5.08 (1H, t,= 7.0 Hz, H-24), 3.16 (1H, dd,= 11.1, 5.0 Hz, H-3), 1.65 (3H, s, 26-CH3), 1.61 (3H, s, 27-CH3), 1.09 (3H, s, 21-CH3), 0.93 (3H, s, 18-CH3), 0.92 (3H, s, 19-CH3), 0.83 (3H, s, 28-CH3), 0.82 (3H, s, 29-CH3), 0.73 (3H, s, 30-CH3);13C-NMR (150 MHz, CDCl3): 131.5 (C-25), 124.6 (C-24), 78.9 (C-3), 75.3 (C-20), 55.7 (C-5), 50.5 (C-9), 50.2 (C-14), 49.8 (C-17), 42.2 (C-13), 40.4 (C-22), 40.3 (C-8), 38.9 (C-1), 38.9 (C-4), 37.0 (C-10), 35.1 (C-7), 31.1 (C-15), 28.1 (28-CH3), 27.4 (C-12), 27.3 (C-2), 25.6 (26-CH3), 25.3 (21-CH3), 24.7 (C-16), 22.4 (C-23), 21.4 (C-11), 18.2 (C-6), 17.6 (27-CH3), 16.3 (30-CH3), 16.1 (19-CH3), 15.4 (18-CH3), 15.3 (29-CH3)。以上數(shù)據(jù)與文獻(xiàn)報道一致[30],故鑒定化合物17為達(dá)瑪烯二醇-II。
化合物18:白色無定形粉末;HR-ESI-MS/: 509.361 5 [M+Na]+(計算值509.360 7,C31H50O4Na) 和/487.377 2 [M+H]+(計算值487.378 7,C31H51O4);1H-NMR (600 MHz, MeOD):5.26 (1H, brs, H-12), 3.68 (1H, m, H-2), 3.61 (3H, s, 31-OCH3), 3.01 (1H, d,= 9.3 Hz, H-3), 2.85 (1H, m, H-18), 1.11 (3H, s, 30-CH3), 1.02 (3H, s, 24-CH3), 0.96 (3H, s, 25-CH3), 0.91 (3H, s, 27-CH3), 0.90 (3H, s, 23-CH3), 0.81 (3H, s, 26-CH3), 0.70 (3H, s, 29-CH3);13C-NMR (150 MHz, CDCl3):180.3 (C-28), 143.5 (C-13), 122.4 (C-12), 84.1 (C-3), 68.9 (C-2), 55.2 (C-5), 50.8 (31-OCH3), 47.5 (C-9), 46.3 (C-17), 46.2 (C-1), 45.7 (C-19), 41.6 (C-14), 41.0 (C-18), 39.2 (C-8), 39.0 (C-4), 38.2 (C-10), 33.7 (C-21), 33.0 (29-CH3), 32.4 (C-7), 32.3 (C-22), 30.6 (C-20), 28.5 (23-CH3), 27.5 (C-15), 25.8 (27-CH3), 23.4 (30-CH3), 23.3 (C-16), 23.0 (C-11), 18.2 (C-6), 17.0 (26-CH3), 16.6 (24-CH3), 16.6 (25-CH3)。以上數(shù)據(jù)與文獻(xiàn)報道一致[31],故鑒定化合物18為山楂酸甲酯。
化合物19:無色晶體(二氯甲烷),mp 232~234 ℃;HR-ESI-MS/: 495.344 5 [M+Na]+(計算值495.345 0,C30H48O4Na) 和/473.362 3 [M+H]+(計算值473.363 1,C30H49O4);1H-NMR (600 MHz, MeOD): 4.67 (1H, d,= 1.5 Hz, H-29a), 4.55 (1H, d,= 1.5 Hz, H-29b), 3.61 (1H, m, H-2), 2.98 (1H, m, H-19), 2.96 (1H, m, H-3), 1.65 (3H, s, 30-CH3), 0.96 (3H, s, 24-CH3), 0.95 (3H, s, 25-CH3), 0.90 (3H, s, 27-CH3), 0.87 (3H, s, 23-CH3), 0.76 (3H, s, 26-CH3);13C-NMR (150 MHz, CDCl3): 180.4 (C-28), 150.3 (C-20), 109.8 (C-29), 84.0 (C-3), 69.3 (C-2), 56.4 (C-7), 56.3 (C-17), 55.5 (C-5), 50.5 (C-9), 49.3 (C-18), 47.1 (C-19), 46.8 (C-1), 42.5 (C-14), 40.8 (C-8), 39.2 (C-4), 38.6 (C-13), 38.4 (C-10), 37.1 (C-22), 32.2 (C-16), 30.6 (C-15), 29.7 (C-21), 28.5 (23-CH3), 25.4 (C-12), 21.0 (C-11), 19.4 (30-CH3), 18.4 (C-6), 17.4 (25-CH3), 16.6 (24-CH3), 16.1 (26-CH3), 14.7 (27-CH3)。以上數(shù)據(jù)與文獻(xiàn)報道一致[32],故鑒定化合物19為麥珠子酸。
化合物20:白色無定形粉末;EI-MS/: 530 [M]+;1H-NMR (600 MHz, CDCl3):7.61 (1H, d,= 15.8 Hz, H-7), 7.06 (1H, dd,= 1.8, 8.1 Hz, H-6), 7.02 (1H, d,= 1.8 Hz, H-2), 6.90 (1H, d,= 8.1 Hz, H-5), 6.28 (1H, d,= 15.8 Hz, H-8), 4.17 (2H, t,= 6.7 Hz, H-10), 3.92 (3H, s, 34-OCH3), 1.00~2.00 (22H, m, H-11~32), 0.87 (3H, t,= 7.0 Hz, H-33);13C-NMR (150 MHz, CDCl3): 167.4 (C-9), 148.1 (C-3), 146.8 (C-4), 144.7 (C-7), 127.1 (C-1), 123.1 (C-6), 115.7 (C-5), 114.7 (C-8), 109.3 (C-2), 64.7 (C-10), 56.0 (34-OCH3), 28.1~30.0 (C-13~31), 26.0 (C-12), 22.7 (C-32), 14.2 (C-33)。以上數(shù)據(jù)與文獻(xiàn)報道基本一致[33-34],故鑒定化合物20為阿魏酸二十四烷酯。
化合物21:白色無定形粉末;EI-MS/: 500 [M]+;1H-NMR (600 MHz, CDCl3):7.60 (1H, d,= 15.9 Hz, H-7), 7.42 (2H, d,= 8.5 Hz, H-2, 6), 6.82 (2H, d,= 8.6 Hz, H-3, 5), 6.29 (1H, d,= 15.9 Hz, H-8), 4.16 (2H, t,= 6.7 Hz, -OCH2), 3.92 (3H, s, 34-OCH3), 1.69 (2H, m, OCH2CH2), 1.25 [(42H, br, (CH2)21], 0.87 (3H, t,= 7.0 Hz, 33-CH3);13C-NMR (150 MHz, CDCl3): 167.6 (C-9), 157.6 (C-4), 144.3 (C-7), 130.0 (C-2, 6), 127.4 (C-1), 116.1 (C-3, 5), 115.8 (C-8), 64.7 (C-10), 29.4~30.0 (C-13~31), 28.8 (C-11), 26.0 (C-12), 22.7 (C-32), 14.2 (33-CH3)。以上數(shù)據(jù)與文獻(xiàn)報道基本一致[35],故鑒定化合物21為對羥基反式肉桂酸二十四烷基酯。
采用MTT法測定化合物對HepG2細(xì)胞株的毒性作用。HepG2細(xì)胞株(美國菌種保藏管理中心)使用DMEM高糖培養(yǎng)基(含10%胎牛血清、100 U/mL青霉素和100 μg/mL鏈霉素)于37 ℃、5% CO2的環(huán)境下培養(yǎng)。當(dāng)細(xì)胞密度達(dá)到70%~80%,用胰蛋白酶消化后將細(xì)胞分散,使成為密度為5×104個/mL的細(xì)胞懸液,以每孔200 μL接種于96孔板。待細(xì)胞貼壁后,按對照組、實驗組,分別給予5% DMSO溶液和化合物溶液(各化合物用5% DMSO溶液配制成100、10、1 μmol/L),放入培養(yǎng)箱中培養(yǎng)24 h后取出,避光環(huán)境下在每孔中加入MTT試劑(5 mg/mL)10 μL,搖勻后于培養(yǎng)箱中靜置4 h,取出,棄上清,在每孔中加入DMSO液200 μL,振搖(37 ℃、10 min、600 r/min),使用酶標(biāo)儀在25 ℃,570 nm下測定吸光度()值。研究結(jié)果顯示,化合物17(達(dá)瑪烯二醇-II)、18(山楂酸甲酯)在100 μmol/L濃度下對HepG2細(xì)胞有顯著的毒性作用,其余化合物在100、10、1 μmol/L濃度下對HepG2細(xì)胞24 h存活率均大于90%,綜合考慮選取10 μmol/L濃度用于后續(xù)的活性研究,結(jié)果見表1。
細(xì)胞存活率=(實驗-空白基質(zhì))/(對照-空白基質(zhì))
HepG2細(xì)胞使用DMEM高糖培養(yǎng)基(含10%胎牛血清,100 U/mL青霉素和100 μg/mL鏈霉素)于37 ℃、5% CO2的環(huán)境下培養(yǎng)。當(dāng)細(xì)胞密度達(dá)到70%~80%,用胰蛋白酶消化后將細(xì)胞分散,使成為密度為5×104個/mL的細(xì)胞懸液,以每孔200 μL接種于96孔板。待細(xì)胞貼壁后,將每孔中培養(yǎng)基換為200 μL無血清DMEM培養(yǎng)基,饑餓培養(yǎng)12 h后,除對照組外,模型組、陽性對照組和樣品組的無血清培養(yǎng)基換為200 μL含0.5 mmol/L油酸鈉的DMEM培養(yǎng)基(無血清),樣品組給予各化合物樣品(10 μmol/L)、陽性對照組給予非諾貝特(10 μmol/L),繼續(xù)培養(yǎng)24 h后棄去上清液,PBS清洗2次,每次0.5 mL;每孔加入4%多聚甲醛0.5 mL,室溫下靜置15 min后棄去,PBS清洗2次,每次0.5 mL;在每孔中加入油紅O工作液0.5 mL,染色15 min后棄去;0.5 mL PBS清洗2次后,加DMSO 200 μL溶解,震搖(25 ℃、5 min、600 r/min),使用酶標(biāo)儀在25 ℃,515 nm波長下測定吸光度()值。
表1 不同濃度的化合物對HepG2細(xì)胞存活率的影響()
Table 1 Effect of different compound concentrations on the survival rate of HepG2 cells ()
組別細(xì)胞存活率/%100 μmol·L?110 μmol·L?11 μmol·L?1 對照100.00±1.41 2103.32±3.1998.08±2.3498.28±5.71 7103.81±3.0093.95±3.0293.22±2.10 8102.93±13.1694.31±4.7594.16±2.88 1724.92±2.22***103.18±5.84106.88±8.13 1827.73±5.35***105.47±5.96101.22±7.63 19109.83±8.20108.35±3.44102.96±14.19
與對照組比較:***<0.001
???< 0.001control group
油酸(oleic acid,OA)是一種單不飽和Omega-9脂肪酸,是脂肪酸合成的最終產(chǎn)物,人體的肝脂肪變性與過量油酸的積累有關(guān)[36-37],HepG2細(xì)胞是人肝癌細(xì)胞系,但具有糖脂代謝等正常的生理功能,是國際公認(rèn)的研究降血脂作用的細(xì)胞系,且易于培養(yǎng)[38-40],油酸誘導(dǎo)HepG2細(xì)胞與脂肪性肝細(xì)胞的形態(tài)相似[41-42]。本實驗根據(jù)課題組前期體外高脂模型研究結(jié)果[13],使用0.5 mmol/L油酸誘導(dǎo)的HepG2高脂模型測定化合物的降脂活性,體外活性測定結(jié)果顯示(表2):單萜類化合物7(aglycone)、8(aruncide A)與三萜類化合物17(達(dá)瑪烯二醇-II)、18(山楂酸甲酯)在10 μmol/L濃度下均顯示出較好的降脂活性,數(shù)據(jù)結(jié)果與模型組對比具有顯著性差異,其中化合物8、17在所篩選化合物中降脂作用最好?;衔?(阿魏酸甲酯)和19(麥珠子酸)則沒有顯示出降脂活性。
表2 化合物在油酸誘導(dǎo)的HepG2高脂模型中的降脂作用結(jié)果()
Table 2 Results of lipid-lowering action of the compound in a model of HepG2 hyperlipidemia induced by oleic acid ()
組別A值 對照0.40±0.01 模型1.00±0.06### 陽性對照(非諾貝特)0.78±0.05** 20.93±0.06 70.77±0.04*** 80.81±0.03** 170.69±0.06*** 180.83±0.08* 190.93±0.11
與對照組比較:###<0.001;與模型組比較:*<0.05**<0.01***<0.001
###<0.001control group;*<0.05**<0.01***<0.001model group
利益沖突 所有作者均聲明不存在利益沖突
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Chemical constituents from the aerial part ofand their hypolipidemic activity
LIU Hong-dong1, GAO Yu-ying2, GAO Qi2, ZHOU Xiao-qing3, CHEN Jie4, ZHU Jin-hua5, LI Fei6, LI Bin1
1. Academician Workstation, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China 2. Yichun Third People?s Hospital,Yichun 336000, China 3. Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China 4. School of Pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China 5. School of Traditional Chinese Medical, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China 6. Gastroenterology and Hepatology, Sichuan University-Oxford University Huaxi Gastrointestinal Cancer Centre, West China Hospital, Sichuan University, Chengdu 610065, China
To study the chemical composition from the aerial part ofand their activity.Silica gel column chromatography, ODS reversed-phase column chromatography, semi-preparative liquid chromatography were used for separation and purification. NMR, MS and other techniques were used to identify the structure of the isolated compounds, and the lipid-lowering activities of each compound were tested by establishing HepG2 cell hyperlipid model.Twenty-one compounds were separated from 95% ethanol extract of, which were identified as methyl veratrate (1), ()-3,4-dimethoxycinnamic acid methyl ester (2), ()-methyl 3-(3-hydroxy-4-methoxyphenyl) acrylate (3), 3,4,5-trimethoxycinnamyl alcohol (4), linocinnamarin (5), tecomin (6), aglycone (7), aruncide A (8), 3-[(2)-4-hydroxyl - 4-methyl-2-pentenyl)]furan-5-2-one (9), vomifoliol (10), grasshopper ketone (11), 3,7-dimethyl-3(),6-octadien-5-one-1βglucoside (12), (2,3,4)-4-(4-hydroxy-3-methoxybenzyl)-2-(5-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-tetrahydrofuran-3-ol (13), 1,2-bis(4-hydroxy-3-methoxyphenyl)-1,3-propanediol (14), salicifoneoliganol (15), herpetriol (16), dammarenediol- II (17), methyl maslinat (18), alphitolic acid (19), tetracosyl ferulate (20),-tetracosyl trans--coumarate (21).19 compounds (1—8, 10, 11, 13—21) are isolated from Sibiraea genus for the first time; Compounds 7, 8, 17 and 18 showed lipid-lowering activity.
(Rehd.) Hand. -Mazz.; methyl veratrate; ()-3,4-dimethoxycinnamic acid methyl ester; dammarenediol-II; methyl maslinat; hypolipidemic; HepG2
R284.1
A
0253 - 2670(2022)14 - 4276 - 09
10.7501/j.issn.0253-2670.2022.14.007
2021-12-08
國家自然科學(xué)基金資助項目(81760708);江西省主要學(xué)科學(xué)術(shù)和技術(shù)帶頭人資助計劃項目(20162BCB22016);江西省教育廳重點項目(GJJ160809)
劉宏棟,男,講師,研究方向為中藥藥效物質(zhì)基礎(chǔ)研究。E-mail: lhd1567@163.com
李 斌,女,教授,研究方向為中藥資源開發(fā)與利用。E-mail: lbin@crjz.com
[責(zé)任編輯 王文倩]