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甜茶(Rubus suavissimus),为蔷薇科悬钩子属植物(Liu et al.,2020),主要分布于广西桂林、柳州、梧州等地区,故又被称为广西甜茶(闫志刚等,2017),与罗汉果、甜叶菊并称广西三大甜味植物。壮族和瑶族人民将其作为茶饮用于治疗糖尿病,被誉为瑶药中的“神茶”(郑华等,2019)。壮医记载其具有解热毒,通龙路,调气道、水道的功效(广西壮族自治区壮药质量标准,2011)。甜茶集糖、茶、药于一体,极具食品及药品开发的潜力。
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随着生活水平的提高和老龄化进程的加快,糖尿病已经成为严重影响人类身体健康和生活质量的慢性疾病。为了维持血糖处于正常水平,糖尿病患者需要长期服用降血糖药物,以避免因高血糖而导致的并发症,如器官损伤、衰竭等。α-葡萄糖苷酶抑制剂是一种重要的降血糖药物,临床一线药物有阿卡波糖、伏格列波糖等,但是这类药物易导致胃肠道紊乱、肝功能受损等(朱月霞等,2021)。因此,研发安全的新型α-葡萄糖苷酶抑制剂对糖尿病的治疗具有重要意义。现代化学和药理学研究表明,甜茶的主要化学成分为萜类、黄酮类、酚酸类,具有降血糖、抗过敏、抗炎等生物活性(吴家超等,2021)。当前,对甜茶降血糖作用的相关研究多见于其提取物(蒙淑洁等,2019;Su et al.,2020;吴婕和宫江宁,2021),而对甜茶中的α-葡萄糖苷酶抑制作用的物质基础研究较少(Liu et al.,2019),潜在的活性物质尚待开发。甜茶作为降糖茶饮的历史悠久,为了丰富其具有α-葡萄糖苷酶抑制作用的活性物质基础,本研究综合运用现代色谱分离技术对甜茶叶进行系统分离,进而对分离得到的化合物单体进行活性研究,以期发现更多具有α-葡萄糖苷酶抑制活性的化合物,为后续相关降血糖产品的开发提供科学的理论依据。
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1 仪器与方法
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1.1 材料
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样品于2019年7月采集于广西壮族自治区桂林市灌阳县,经广西植物研究所唐辉研究员鉴定为甜茶(Rubus suavissimus)的叶子,样品的标本保存于广西植物功能物质与资源持续利用重点实验室(标本号:20190753)。
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1.2 仪器和试剂
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XS205 DualRange分析天平(瑞士苏黎世的梅特勒-托利多集团),LCMS-IT-TOF高分辨质谱仪(日本岛津公司),Avance Ⅲ HD 500 MHz核磁共振波谱仪(德国布鲁克公司),LC-20AT高效液相色谱仪(日本岛津公司),旋转蒸发仪(日本东京理化公司),CF810C冷却水循环仪(日本雅马拓公司),SP-MAX3500FL多功能酶标仪(上海闪谱生物科技有限公司)。
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阿卡波糖(上海源叶生物科技有限公司),对硝基苯-α-D-吡喃葡萄糖苷(pNPG,上海源叶生物科技有限公司),α-葡萄糖苷酶(美国西格玛奥德里奇公司),无水碳酸钠(西陇化工股份有限公司),磷酸缓冲液(PBS,北京索莱宝科技有限公司),分析甲醇(西陇化工股份有限公司),分析乙醇(西陇化工股份有限公司),色谱甲醇(美国斯百全化学公司),色谱乙腈(美国斯百全化学公司)。
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1.3 提取和分离
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取甜茶的干燥叶(5.5 kg),加入95%乙醇溶液于室温下浸泡提取3次,每次7 d,合并提取液,减压回收溶剂后得到总浸膏(432.2 g)。总浸膏中加入40%乙醇水溶液,充分溶解,静置分层,弃去下层沉淀物,将上清液减压回收溶剂至无醇味后,经凝胶柱Sephadex LH-20(10 cm × 30 cm),以甲醇-水溶液(0%~100%,V/V)为洗脱剂进行梯度洗脱,在薄层色谱分析指导下合并洗脱液,得到11个组分Fr.1~Fr.11。
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将Fr.4(21.9 g)以树脂DIAION HP20SS色谱柱(4 cm × 30 cm)进行分离,以甲醇-水(0%~100%,V/V)为洗脱剂进行梯度洗脱,得到化合物1(5.5 g)。Fr.6(8.1 g)经过MCI柱(3 cm × 23 cm),以甲醇-水溶液(0%~100%,V/V)进行梯度洗脱,得到Fr.61~Fr.66。Fr.61经Sephadex LH-20柱,甲醇-水溶剂分离,得到化合物3(66.0 mg)。Fr.62经甲醇溶剂反复结晶得到化合物6(46.2 mg)。Fr.63依次经MCI柱(甲醇-水溶液,0%~100%,V/V)、HPLC液相色谱柱(50%甲醇-水溶液,V/V)纯化得到化合物 2(5.9 mg)。Fr.64经HPLC液相色谱柱,以25%乙腈-水溶液等度洗脱(V/V),纯化得到化合物4(30.7 mg)和化合物5(6.0 mg)。Fr.65经ODS柱色谱分离(甲醇-水溶液,0%~100%,V/V),得到化合物7(78.3 mg)。Fr.66经ODS色谱柱,以甲醇-水溶液进行梯度洗脱(0%~100%,V/V),得到化合物8(26.7 mg)。Fr.5(4.6 g)以ODS色谱柱进行分离,甲醇-水(0%~100%,V/V)为洗脱剂进行梯度洗脱,经Sephadex LH-20色谱柱纯化,得到化合物9(59.8 mg)。Fr.8(5.4 g)以MCI色谱柱进行分离,以甲醇-水(0%~100%,V/V)为洗脱剂进行梯度洗脱,得到化合物10(23.6 mg)。
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1.4 α-葡萄糖苷酶抑制活性测试
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α-葡萄糖苷酶抑制活性测试参考文献(Pan et al.,2020;梁森林等,2022)的方法,并作适当调整,以阿卡波糖为阳性对照药,对硝基苯-α-D-吡喃葡萄糖苷(PNPG,1 mmol·L-1)为底物,磷酸缓冲液(PBS,50 mmol·L-1)为溶剂系统,α-葡萄糖苷酶配置成0.25 U·mL-1。实验设置4个组,即样品组、样品背景对照组、空白组和空白对照组。按表1的反应体系进行活性测试,具体步骤如下。首先,取96孔板,样品组依次加入样品溶液40 μL、α-葡萄糖苷酶溶液20 μL,样品背景对照组依次加入样品溶液40 μL、PBS缓冲液20 μL,空白组依次加入α-葡萄糖苷酶溶液20 μL、PBS缓冲液40 μL,空白对照组加入PBS缓冲液60 μL;然后,将加液后的96孔板置于恒温箱中于37℃条件下平衡5 min后取出;接着,各实验组加入PNPG溶液50 μL,并将其置于恒温箱中于37℃条件下反应30 min,取出;最后,向各实验组加入Na2CO3溶液50 μL终止反应,于405 nm波长下测定并读取吸光度值。样品组的吸光度值记为A1,样品背景对照组的吸光度值记为A2,空白组的吸光度值记为B1,空白对照组的吸光度值记为B2。按如下公式计算抑制率:抑制率 = [1-(A1-A2)/(B1-B2)] × 100%。所有数据均平行测试3次,测试结果以平均值±标准偏差表示。
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2 结果与分析
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2.1 化合物的结构鉴定(图1)
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化合物1 白色粉末。HR-ESI-MS m/z: 665.311 5 [M + Na]+(calcd for C32H50O13Na,665.314 4)。1H NMR(500 MHz,D2O)δ: 5.38(1H,d,J = 7.8 Hz,19-glc-H-1′′′),5.10(1H,s,H-17a),4.89(1H,s,H-17b),4.58(1H,d,J = 7.5 Hz,13-glc-H-1′),3.22~3.86(sugar proton),1.22(3H,s,H-18),0.90(3H,s,H-20); 13C NMR(125 MHz,D2O)δ: 178.6(C-19),153. 0(C-16),104.8(C-17),97.4(13-glc-C-1′),94.1(19-glc-C-1″),86.5(C-13),76.8(19-glc-C-5″),76.3(13-glc-C-3′),76.0(19-glc-C-3″),75.8(13-glc-C-5′),73.3(13-glc-C-2′),72.0(19-glc-C-2″),69.8(13-glc-C-4′),69.4(19-glc-C-4″),60.9(13-glc-C-6′),60.7(19-glc-C-6″),57.1(C-5),53.5(C-9),47.2(C-15),44.0(C-14),43.9(C-4),42.1(C-8),41.0(C-7),40.5(C-1),39.3(C-10),37.7(C-3),36.3(C-12),28.1(C-18),21.4(C-6),20.4(C-11),18.8(C-2),15.0(C-20)。以上数据与文献(王剑霞和吕华冲,2008)报道的基本一致,故化合物1鉴定为甜茶苷。
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图1 化合物1-10的化学结构式
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Fig.1 Chemical structures of compounds 1-10
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化合物2 黄色粉末。HR-ESI-MS m/z: 593.146 4 [M-H]-(calcd for C27H31O15,593.151 2)。1H NMR(500 MHz,methanol-d4)δ: 8.04(2H,d,J = 8.8 Hz,H-2′,6′),6.83(2H,d,J = 8.8 Hz,H-3′,5′),6.31(1H,s,H-8),6.12(1H,s,H-6),4.95(1H,d,J = 7.8 Hz,gal-H-1″),4.47(1H,brs,rha-H-1′′′),1.14(3H,d,J = 6.2 Hz,rha-H-6′′′),3.22~3.76(sugar proton); 13C NMR(125 MHz,methanol-d4)δ: 180.0(C-4),168.4(C-7),163.4(C-5),162.2(C-4′),159.7(C-9),159.2(C-2),136.6(C-3),133.0(C-2′,6′),123.3(C-1′),116.8(C-3′,5′),106.3(gal-C-1″),105.7(C-10),102.5(rha-C-1′′′),101.2(C-6),94.0(C-8),76.0(gal-C-5″),75.7(gal-C-3″),74.5(rha-C-4′′′),73.6(rha-C-2′′′),72.9(rha-C-3′′′),72.7(gal-C-2″),70.8(gal-C-4″),70.3(rha-C-5′′′),68.1(gal-C-6″),18.6(rha-C-6′′′)。以上数据与文献(Hou et al.,2005)报道的基本一致,故化合物2鉴定为山奈酚-3-O-洋槐糖苷。
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化合物3 白色粉末。HR-ESI-MS m/z: 169.014 1 [M-H]-(calcd for C7H5O5,169.014 2)。1H NMR(500 MHz,methanol-d4)δ: 7.06(2H,s,H-2,6); 13C NMR(125 MHz,methanol-d4)δ: 170.5(C-7),146.4(C-3,5),139.8(C-4),121.9(C-1),110.5(C-2,6)。以上数据与文献(吕闪闪等,2018)报道的基本一致,故化合物3鉴定为没食子酸。
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化合物4 淡黄色油状物。HR-ESI-MS m/z: 359.150 7 [M-H]-(calcd for C20H23O6,359.150 0)。1H NMR(500 MHz,methanol-d4)δ: 6.91(1H,d,J = 1.8 Hz,H-2),6.78(1H,dd,J = 8.2,1.8 Hz,H-6),6.72(1H,d,J = 8.2 Hz,H-5),6.68(2H,s,H-2′,6′),5.45(1H,d,J = 6.2 Hz,H-7),3.80(3H,s,3-OCH3),3.76(3H,s,3′-OCH3),3.71(2H,m,H-9),3.53(2H,t,J = 6.5 Hz,H-9′),3.43(1H,m,H-8),2.58(2H,m,H-7′),1.77(2H,m,H-8′); 13C NMR(125 MHz,methanol-d4)δ: 149.0(C-3),147.6(C-4),147.4(C-2′),145.2(C-3′),136.9(C-5′),134.8(C-1),129.8(C-1′),119.7(C-6),117.9(C-6′),116.1(C-5),114.0(C-4′),110.5(C-2),88.9(C-7),64.9(C-9),62.2(C-9′),56.7(3-OCH3),56.3(3′-OCH3),55.4(C-8),35.8(C-8′),32.9(C-7′)。以上数据与文献(汪青青,2013)报道的基本一致,故化合物4鉴定为二聚松柏醇。
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化合物5 淡黄色油状物。HR-ESI-MS m/z: 413.151 1 [M + Na]+(calcd for C21H26O7Na,413.157 1)。1H NMR(500 MHz,methanol-d4)δ: 6.73(2H,d,J = 2.2 Hz,H-2,6),6.68(2H,s,H-2′,6′),5.50(1H,d,J = 6.2 Hz,H-7),3.86(3H,s,3′-OCH3),3.85(2H,m,H-9),3.81(6H,s,3-OCH3,5-OCH3),3.57(2H,t,J = 6.4 Hz,H-9′),3.47(1H,m,H-8),2.63(2H,m,H-7′),1.82(2H,m,H-8′); 13C NMR(125 MHz,methanol-d4)δ: 149.3(C-3),149.3(C-5),147.5(C-3′),145.2(C-4′),137.0(C-4),134.0(C-1),134.0(C-1′),129.8(C-5′),117.9(C-6′),114.1(C-2′),104.1(C-2,6),89.1(C-7),65.0(C-9),62.6(C-9′),56.8(C-8),56.7(3-OCH3),56.7(5-OCH3),55.6(3′-OCH3),35.8(C-8′),32.9(C-7′)。以上数据与文献(汪青青,2013)报道的基本一致,故化合物5鉴定为5-甲氧基二聚松柏醇。
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化合物6 黄色粉末。HR-ESI-MS m/z: 293.031 7 [M + H]+(calcd for C13H9O8,293.029 2)。1H NMR(500 MHz,DMSO-d6)δ: 10.92(1H,s,-OH),10.10(2H,s,-OH × 2),7.28(1H,s,H-3′),4.34(1H,brs,H-4),2.98(1H,dd,J = 18.7,7.6 Hz,H-5a),2.42(1H,d,J = 18.7 Hz,H-5b);13C NMR(125 MHz,DMSO-d6)δ: 193.5(C-1),173.7(C-6),160.4(C-7′),149.7(C-2),145.8(C-4′),143.9(C-6′),140.3(C-3),139.2(C-5′),115.3(C-2′),113.2(C-1′),108.1(C-3′),41.1(C-4),37.6(C-5)。以上数据与文献(Tanaka et al.,1990)报道的基本一致,故化合物6鉴定为云实酸。
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化合物7 白色粉末。HR-ESI-MS m/z: 479.258 3 [M-H]-(calcd for C26H39O8,479.265 0)。1H NMR(500 MHz,methanol-d4)δ: 5.20(1H,s,H-17a),4.87(1H,s,H-17b),4.51(1H,d,J = 7.8 Hz,13-glc-H-1′),1.20(3H,s,H-18),0.99(3H,s,H-20); 13C NMR(125 MHz,methanol-d4)δ: 181.6(C-19),154.0(C-16),105.5(C-17),99.2(13-glc-C-1′),87.6(C-13),78.1(13-glc-C-3′),77.6(13-glc-C-5′),75.2(13-glc-C-2′),71.6(glc-C-4′),62.7(glc-C-6′),58.1(C-5),55.2(C-9),49.0(C-15),45.1(C-14),44.6(C-4),43.2(C-8),42.6(C-7),41.9(C-1),40.6(C-10),39.1(C-3),38.7(C-12),29.5(C-18),23.0(C-6),21.4(C-11),20.3(C-2),16.2(C-20)。以上数据与文献(Ohtani et al.,1992)报道的基本一致,故化合物7鉴定为斯替维单糖苷。
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化合物8 白色粉末。HR-ESI-MS m/z: 317.211 0 [M-H]-(calcd for C20H29O3,317.212 2)。1H NMR(500 MHz,chloroform-d)δ: 4.98(1H,s,H-17a),4.81(1H,s,H-17b),1.23(3H,s,H-18),0.95(3H,s,H-20); 13C NMR(125 MHz,chloroform-d)δ: 183.4(C-19),155.8(C-16),103.2(C-17),80.5(C-13),57.0(C-5),54.0(C-9),47.6(C-15),47.1(C-14),43.7(C-4),41.9(C-8),41.4(C-7),40.6(C-1),39.6(C-12),39.5(C-10),37.9(C-3),29.0(C-18),21.9(C-6),20.6(C-11),19.2(C-2),15.6(C-20)。以上数据与文献(Ohtani et al.,1992)报道的基本一致,故化合物8鉴定为斯替维醇。
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化合物9 黄色粉末。HR-ESI-MS m/z: 329.246 4 [M + Na]+(calcd for C20H34O2Na,329.244 2)。1H NMR(500 MHz,methanol-d4)δ: 3.71(1H,d,J = 11.3 Hz,H-17a),3.61(1H,d,J = 11.3 Hz,H-17b),1.10(3H,s Me-20),1.07(3H,s Me-19),1.03(3H,s Me-18); 13C NMR(125 MHz,methanol-d4)δ: 82.8(C-16),66.8(C-17),56.8(C-5),56.8(C-9),53.4(C-15),46.2(C-13),45.5(C-8),42.1(C-1),42.1(C-3),40.3(C-14),39.7(C-10),37.8(C-7),34.9(C-4),34.9(C-18),27.1(C-12),22.7(C-19),21.3(C-6),19.8(C-2,11),18.4(C-20)。以上数据与文献(Etse et al.,1987)报道的基本一致,故化合物9鉴定为16α,17-二羟基对映贝壳杉烷。
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化合物10 黄色粉末。HR-ESI-MS m/z: 463.086 0 [M-H]-(calcd for C21H19O12,463.088 2)。1H NMR(500 MHz,methanol-d4)δ: 7.85(1H,d,J = 2.3 Hz,H-2′),7.60(1H,dd,J = 8.4,2.3 Hz,H-6′),6.88(1H,d,J = 8.4 Hz,H-5′),6.41(1H,d,J = 2.2 Hz,H-8),6.22(1H,d,J = 2.2 Hz,H-6),5.17(1H,d,J = 7.8 Hz,gal-H-1″),3.48~3.87(6H,m,gal-H-2″-6″); 13C NMR(125 MHz,methanol-d4)δ: 179.6(C-4),166.0(C-7),163.0(C-5),158.8(C-2),158.4(C-9),145.0(C-4′),145.8(C-3′),135.8(C-3),123.0(C-6′),122.9(C-1′),117.8(C-5′),116.1(C-2′),105.6(C-10),105.4(gal-C-1″),99.9(C-6),94.7(C-8),77.2(gal-C-5″),75.1(gal-C-3″),73.2(gal-C-2″),70.0(gal-C-4″),62.0(gal-C-6″)。以上数据与文献(张维库等,2007)报道的基本一致,故化合物10鉴定为槲皮素-3-O-β-D-吡喃半乳糖苷。
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2.2 α-葡萄糖苷酶抑制活性测试结果
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α-葡萄糖苷酶的抑制活性测试结果显示,化合物2、3、5、6、10具有较强的活性,IC50值分别为(0.14 ± 0.03)mg · mL-1、(0.36 ± 0.02)mg · mL-1、(0.44 ± 0.01)mg · mL-1、(0.53 ± 0.04)mg · mL-1和(0.14 ± 0.03)mg · mL-1,均优于阳性对照[阿卡波糖,IC50值为(0.69 ± 0.02)mg·mL-1]。具体活性测试结果见表2。
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3 讨论与结论
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α-葡萄糖苷酶抑制剂通过抑制小肠黏膜细胞的α-葡萄糖苷酶的活性,降低葡萄糖的生成速度,从而减少小肠对葡萄糖的吸收以降低血糖,然而当前临床使用的该类药物具有较严重的副作用(朱月霞等,2021)。因此,开发新型、安全、有效的α-葡萄糖苷酶抑制剂对糖尿病的治疗具有重要意义,寻找天然的α-葡萄糖苷酶抑制剂成为研究的热点(朱运平等,2011; Quan et al.,2020; Yuca et al.,2021)。本研究基于甜茶提取物对α-葡萄糖苷酶具有抑制作用(吴婕等,2021),对其开展化学成分及生物活性研究,从甜茶叶中分离得到10个化合物,化合物2、4、5、9为首次从甜茶中分离得到。其中,化合物2和10为黄酮苷类,化合物3和6为酚酸类,活性测试结果显示化合物2、3、6和10均具有较强的α-葡萄糖苷酶抑制活性,与文献报道一致(Liu et al.,2019;岳丹伟,2021;Lin et al.,2022;薛深等,2023),化合物5为木脂素类,具有较强的α-葡萄糖苷酶抑制活性,为首次报道。化合物5的结构与化合物4的相比,仅在C-5为多了一个甲氧基,化合物5表现出较强的活性,而化合物4在相同测试浓度下无活性,推测C-5位的甲氧基是关键的活性基团。
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注: a表示阳性对照;* 和**表示与阳性对照组比较,*P<0.05,** P<0.01。
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Note: a indicates positive control; * and ** indicate comparisons with positive control group, * P<0.05, ** P<0.01.
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甜茶作为茶饮有悠久的历史,其主要成分甜茶苷的甜度是蔗糖的300倍,热量仅为蔗糖的1%(马建春等,2008),具有高甜度、低热量的特点,是糖尿病患者理想的甜味剂,发达国家正大力开发相关产品,如日本已有多种饮料、糖果和药品已上市(朱明婧等,2015)。本研究从甜茶中发现了具有较好的α-葡萄糖苷酶抑制活性的化学成分,进一步证实了其具有降血糖作用,为甜茶开发降血糖功能食品或降血糖药物提供了科学依据。
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参考文献
-
ETSE JT, GRAY A, WATERMAN PG, 1987. Chemistry in the Annonacesa, XXIV. Kaurane and kaur-16-ene diterpenes from the stem bark of Annona reticulate [J]. J Nat Prod, 50(5): 979-983.
-
Guangxi Food and Drug Administration, 2011. Zhuang medicine quality standards of Guangxi Autonomous Region: Vol. 2 [S]. Nanning: Guangxi Science & Technology Press: 269. [广西壮族自治区食品药品监督管理局, 2011. 广西壮族自治区壮药质量标准: 第二卷 [S]. 南宁: 广西科学技术出版社: 269. ]
-
HOU WC, LIN RD, LEE TH, et al. , 2005. The phenolic constituents and free radical scavenging activities of Gynura formosana Kiamnra [J]. J Sci Food Agric, 85(4): 615-621.
-
LIANG SL, HUANG YL, HE RJ, et al. , 2022. Separation and preparation of alkaloids with inhibitory activity of α-glucosidase from Sophora tonkinensis by pH-zone-refining counter-current chromatography [J]. Guihaia, 42(9): 1459-1465. [梁森林, 黄永林, 何瑞杰, 等, 2022. pH区带精制逆流色谱法分离制备越南槐中具有抑制α-糖苷酶活性的生物碱 [J]. 广西植物, 42(9): 1459-1465. ]
-
LIN YT, LIN HR, YANG CS, et al. , 2022. Antioxidant and anti-α-glucosidase activities of various solvent extracts and major bioactive components from the fruits of Crataegus pinnatifida [J]. Antioxidants, 11: 320.
-
LIU MZ, HUANG XQ, LIU Q, et al. , 2019. Separation of α-glucosidase inhibitors from Potentilla kleiniana Wight et Arn using solvent and flow-rate gradient high-speed counter-current chromatography target-guided by ultrafiltration HPLC-MS screening [J]. Phytochem Anal, 30: 661-668.
-
LIU MZ, LI XJ, LIU Q, et al. , 2020. Comprehensive profiling of α-glucosidase inhibitors from the leaves of Rubus suavissimus using an off-line hyphenation of HSCCC, ultrafiltration HPLC-UV-MS and prep-HPLC [J]. J Food Compost Anal, 85: 103336.
-
LÜ SS, WANG X, YE QZ, et al. , 2018. Chemical constituents from the leaves of Terminalia catappa and their hypoglycemic activities [J]. Chin Trad Pat Med, 40(12): 2693-2697. [吕闪闪, 王璇, 叶全知, 等, 2018. 大叶榄仁叶化学成分及其降糖活性 [J]. 中成药, 40(12): 2693-2697. ]
-
MA JC, HE W, WU ZF, 2008. Progress on rubusoside in Rubus suavissimu S. Lee [J]. Food Drug, 10(5): 73-75. [马建春, 何伟, 伍振峰, 2008. 甜茶素的研究进展 [J]. 食品与药品, 10(5): 73-75. ]
-
MENG SJ, YAN ZG, XU YL, et al. , 2019. Effects of extract of Rubus suavissimus S. Lee on blood sugar and glucose tolerance in mice [J]. Hubei Agric Sci, 58(20): 118-120. [蒙淑洁, 闫志刚, 徐永莉, 等, 2019. 广西甜茶醇提物对小鼠血糖及糖耐量的影响 [J]. 湖北农业科学, 58(20): 118-120. ]
-
OHTANI K, AIKAWA Y, KASAI R, et al. , 1992. Minor diterpene glycosides from sweet leaves of Rubus suavissimus [J]. Phytochemistry, 31(5): 1553-1559.
-
PAN ZH, NING DS, FU YX, et al. , 2020. Preparative isolation of piceatannol derivatives from passion fruit (Passiflora edulis) seeds by high-speed countercurrent chromatography combined with high-performance liquid chromatography and screening for α-glucosidase inhibitory activities [J]. J Agric Food Chem, 68(6): 1555-1562.
-
QUAN YS, ZHANG XY, YIN XM, et al. , 2020. Potential α-glucosidase inhibitor from Hylotelephium erythrostictum [J]. Bioorg Med Chem Lett, 30(24): 127665.
-
SU ZH, LING X, JI KW, et al. , 2020. 1H NMR-based urinary metabonomic study of the antidiabetic effects of Rubus suavissimus S. Lee in STZ-induced T1DM rats [J]. J Chromatogr B, 1158: 122347.
-
TANAKA T, NONAKA G, NISHIOKA I, 1990. Tannins and related compounds. C. 1) Reaction of dehydrohexahydroxy-diphenic acid esters with bases, and its application to the structure determination of pomegranate tannins, granatins A and B [J]. Chem Pharm Bull, 38(9): 2424-2428.
-
WANG JX, LÜ HC, 2008. Studies on the diterpenoids of Rubus suavissimus S. Lee [J]. Lishizhen Med Mat Med Res, 19(3): 664-665. [王剑霞, 吕华冲, 2008. 广西甜茶二萜类成分的研究 [J]. 时珍国医国药, 19(3): 664-665. ]
-
WANG QQ, 2013. Studies on chemical constituents of Rhododenron seniavinii Maxim [D]. Guangzhou: Jinan University. [汪青青, 2013. 满山白化学成分研究 [D]. 广州: 暨南大学. ]
-
WU JC, LI SP, ZHANG YY, et al. , 2021. Research progress on chemical constituents and pharmacological effects of Rubus suavissimus [J]. Chin J Trad Chin Med Pharm, 36(6): 3504-3508. [吴家超, 李水萍, 张永怡, 等, 2021. 民族药甜茶化学成分与药理作用研究进展 [J]. 中华中医药杂志, 36(6): 3504-3508. ]
-
WU J, GONG JN, 2021. Purification of polyphenols from sweet tea by the macroporous resins and its inhibitory activity on α-glucosidase and DPPH [J]. J Agric Sci Technol, 23(6): 113-119. [吴婕, 宫江宁, 2021. 大孔树脂纯化甜茶多酚及其对α-葡萄糖苷酶抑制活性和DPPH抗氧化性的研究 [J]. 中国农业科技导报, 23(6): 113-119. ]
-
XUE S, WANG YJ, LIU FF, et al. , 2023. Study on the inhibition mechanisms of gallic acid derivatives on α-glucosidase [J]. J Zhejiang Norm Univ (Nat Sci Ed), 46(1): 74-80. [薛琛, 王怡婷, 刘芬芬, 等, 2023. 没食子酸衍生物抑制α-葡萄糖苷酶的机理研究 [J]. 浙江师范大学学报(自然科学版), 46(1): 74-80. ]
-
YAN ZG, MENG SJ, WEI RC, et al. , 2017. Current situation and application of Rubus suavissimus [J]. Chin Trad Herb Drugs, 48(12): 2572-2578. [闫志刚, 蒙淑洁, 韦荣昌, 等, 2017. 广西甜茶研究与应用现状 [J]. 中草药, 48(12): 2572-2578. ]
-
YUCA H, ÖZBEK H, DEMIREZER LÖ, et al. , 2021. trans-Tiliroside: a potent α-glucosidase inhibitor from the leaves of Elaeagnus angustifolia L. [J]. Phytochemistry, 188: 112795.
-
YUE DW, 2021. Study on chemical constituents, antioxidant and hypoglycemic activities of flavonoids from Flos. Dolichoris lablab L. [D]. Wuhu: Anhui Polytechnic University. [岳丹伟, 2021. 白扁豆花黄酮类化合物分离鉴定及其抗氧化、降糖活性研究 [D]. 芜湖: 安徽工程大学. ]
-
ZHANG WK, ZHANG XQ, YE WC, et al. , 2007. Chemical constituents of the aerial parts of Euphorbia sosoria [J]. J Chin Pharm Univ, 38(4): 315-319. [张维库, 张晓琦, 叶文才, 2007. 对叶大戟地上部分的化学成分 [J]. 中国药科大学学报, 38(4): 315-319. ]
-
ZHENG H, WEI QM, MENG CM, et al. , 2019. The study of Rubus suavissimus S. Lee (RS) improving the diabetic complications of the streptozotocin (STZ) rats by promoting mitophagy [J]. Chin J Ethnomed Ethnopharm, 28(10): 22-26. [郑华, 魏秋梅, 孟春梅, 等, 2019. 瑶山甜茶通过调节线粒体自噬改善糖尿病大鼠并发症的实验研究 [J]. 中国民族民间医药, 28(10): 22-26. ]
-
ZHU MJ, LIU B, LI FF, 2015. Research progress and development prospect of natural sweeteners [J]. Chin Cond, 40(11): 136-140. [朱明婧, 刘博, 李飞飞, 2015. 天然甜味剂研究进展与开发前景分析 [J]. 中国调味品, 40(11): 136-140. ]
-
ZHU YP, LI XT, LI LT, 2011. The source of natural α-glucosidase inhibitor and its application research situation [J]. J Chin Inst Food Sci Technol, 11(4): 154-160. [朱运平, 李秀婷, 李里特, 2011. 天然α-葡萄糖苷酶抑制剂来源及应用研究现状 [J]. 中国食品学报, 11(4): 154-160. ]
-
ZHU YX, SHAO ZB, WU XX, et al. , 2021. Research progress of α-glucosidase inhibitors from marine natural products [J]. J Nanjing Univ Trad Chin Med, 37(2): 311-320. [朱月霞, 邵仲柏, 吴小小, 等, 2021. 海洋天然产物中α-葡萄糖苷酶抑制剂研究进展 [J]. 南京中医药大学学报, 37(2): 311-320. ]
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摘要
为探究甜茶(Rubus suavissimus)中具有α-葡萄糖苷酶抑制活性的次级代谢产物,该文利用多种现代色谱分离技术对其干燥叶进行提取分离纯化,综合运用质谱、核磁共振波谱分析方法确定了单体化合物的结构,并对分离得到的化合物进行了α-葡萄糖苷酶抑制活性的测试。结果表明:(1)从甜茶的干燥叶中分离鉴定出10个化合物,分别为甜茶苷(1)、山奈酚-3-O-洋槐糖苷(2)、没食子酸(3)、二聚松柏醇(4)、5-甲氧基二聚松柏醇(5)、云实酸(6)、斯替维单糖苷(7)、斯替维醇(8)、16α,17-二羟基对映贝壳杉烷(9)、槲皮素-3-O-β-D-吡喃半乳糖苷(10),其中化合物2、4、5、9均为首次从甜茶中分离得到。(2)α-葡萄糖苷酶抑制活性测试结果显示,化合物2、3、5、6、10具有较强的α-葡萄糖苷酶抑制活性。该研究结果丰富了甜茶中具有α-葡萄糖苷酶抑制活性的化合物,并为降血糖相关产品的开发提供了理论依据。
Abstract
Rubus suavissimus is mainly distributed in Guilin, Liuzhou, Wuzhou and other regions in Guangxi Zhuang Autonomous Region, so it is called as “Guangxi tiancha” in China. R. suavissimus, together with Siraitia grosvenorii and Stevia rebaudianastevia are praised as three famous sweet plants in Guangxi Zhuang Autonomous Region. Zhuang and Yao people use the leaves of R. suavissimus as a tea to treat diabetes, and it is known as the “divine tea” of Yao medicine. Therefore, R. suavissimus is a combination of sugar, tea and medicine, which has great potential for the development of food and medicine. In order to investigate the secondary metabolites with α-glucosidase inhibitory activity from R. suavissimus, herein, the extraction, separation and purification of secondary metabolites were performed on the leaves of R. suavissimus. The structures of purified compounds were determined based on the data of mass spectrometry (HR-ESI-MS) and nuclear magnetic resonance (1H NMR and 13C NMR). In addition, the α-glucosidase inhibitory activity of the purified compounds were evaluated by pharmacological methods simultaneously. The results were as follows: (1) Ten compounds were purified and their structures were elucidated as rubusoside (1), kaempferol 3-O-robinobioside (2), gallic acid (3), dihydrodehydroconiferyl alcohol (4), 5-methoxydihydrodehydroconiferyl alcohol (5), brevifolincarboxylic acid (6), steviolmonoside (7), steviol (8), 16α, 17-dihydroxykaurane (9), and quercetin 3-O-β-D-galactopyranoside (10). Among them, compounds 2, 4, 5 and 9 were isolated from R. suavissimus for the first time. (2) Compounds 2, 3, 5, 6 and 10 showed strong inhibitory activity on α-glucosidase. The IC50 values of compounds 2, 3, 5, 6 and 10 were (0.14 ± 0.03) mg·mL-1, (0.36 ± 0.02) mg·mL-1, (0.44 ± 0.01) mg·mL-1, (0.53 ± 0.04) mg·mL-1 and (0.14 ± 0.03) mg·mL-1 respectively, which were stronger than the positive control acarbose with the IC50 values as (0.69 ± 0.02) mg·mL-1. Thus, compounds 2, 3, 5, 6 and 10, which were isolated from the leaves of R. suavissimus, could be a potential α-glucosidase inhibitors based on their bioactivity results. Compounds with α-glucosidase inhibitory activity from R. suavissimus will provide the basis for development of related hypoglycemic products.
Keywords
Rubus suavissimus ; α-glucosidase ; Rubus ; Rosaceae ; diabetes