賈 莉，張 弓

(1.延安市人民醫(yī)院呼吸科，陜西 延安 716000；2.延安大學(xué)附屬醫(yī)院臨床藥物科，陜西 延安 716000）

茶藨子是虎耳草科茶藨屬灌木植物,全世界約有160 多個(gè)種［1］,茶藨子是漿果資源,被稱為第3代新型果樹,具有很高的營(yíng)養(yǎng)價(jià)值,其果實(shí)富含維生素、糖類和不飽和脂肪酸等,具有良好的抗氧化活性,被廣泛用于食品加工行業(yè)(果醬、飲料、釀酒和糖果等)［2］,此外,茶藨子果實(shí)還具有治療感冒、月經(jīng)不調(diào)、降血脂、關(guān)節(jié)炎、腎炎等多種藥理活性［3］；其芽、花和嫩葉可用沸水沖泡代茶用,具有清熱、潤(rùn)肝、利尿等功效［4］；民間常將其根泡酒作為藥酒,用于治療風(fēng)濕?。?］。

目前研究表明茶藨子含有黃酮類［4-6］、β-咔啉生物堿類［7］、萜類［8］、多酚類［9］和甾體類［4,10］等成分。其中,黃酮和生物堿類被認(rèn)為是茶藨子產(chǎn)生多種藥理活性的主要功能性成分［11］。五裂茶藨子Ribes meyeriMaxim.是眾多茶藨子中的一種,主要分布于青海、四川、甘肅、新疆等地區(qū)。其性甘、微寒,具有滋補(bǔ)、止瀉、斂毒、除黃水之功效。李靜等［12］對(duì)五裂茶藨子葉的化學(xué)成分進(jìn)行研究,從中分離純化出8 個(gè)單體化合物,其中6 個(gè)黃酮、2個(gè)其他化合物,并對(duì)所得化合物的抗炎活性進(jìn)行了測(cè)試。該藥材在我國(guó)西部地區(qū)一直被廣泛使用,但是有關(guān)其物質(zhì)基礎(chǔ)研究嚴(yán)重不足。本實(shí)驗(yàn)擬通過現(xiàn)代色譜系統(tǒng)(中壓制備色譜系統(tǒng)和DAC-50 系統(tǒng))結(jié)合核磁波譜解析的方法對(duì)五裂茶藨子葉的化學(xué)成分進(jìn)行分離純化和結(jié)構(gòu)鑒定,以期為其進(jìn)一步開發(fā)應(yīng)用提供理論依據(jù)。

1 材料

R-1020 旋轉(zhuǎn)蒸發(fā)儀(鄭州長(zhǎng)城科工貿(mào)有限公司)；TY-20 L 中藥超微粉碎機(jī)(濟(jì)南天宇專用設(shè)備有限公司)；HZ-TQG 提取濃縮機(jī)組(上海輝展實(shí)驗(yàn)設(shè)備有限公司)；大孔樹脂中壓柱色譜體系、反相C18DAC-50 制備色譜體系(江蘇漢邦科技有限公司)；硅膠常壓柱色譜(青島海洋化工有限公司)；分析純乙醇、石油醚、乙酸乙酯和制備級(jí)甲醇(南京化學(xué)試劑有限公司)。藥材于2017 年購(gòu)買于青海省康樂藥材市場(chǎng),經(jīng)中國(guó)科學(xué)院西北高原生物研究所盧學(xué)峰研究員鑒定為正品。

2 提取與分離

取10 kg 五裂茶藨子葉,粉碎,過篩后平均分為5 份分別用紗布包裹,放入提取濃縮機(jī)組中80%乙醇提取,料液比1 ∶8,提取溫度70 ℃,提取時(shí)間3 h,共提取3 次。所得提取液進(jìn)行濃縮至藥液濃度足夠大但無(wú)沉淀析出。將濃縮液上樣于中壓大孔樹脂柱色譜中,首先以水進(jìn)行充分洗脫去除蛋白和多糖類等物質(zhì)。然后分別用30%、60%、90%乙醇分別進(jìn)行梯度洗脫,每一梯度所用溶液體積為4倍柱體積。將60%乙醇洗脫組分濃縮至藥液濃度足夠大且無(wú)沉淀析出后上樣于常壓硅膠柱色譜,用石油醚-乙酸乙酯(20 ∶1～15 ∶1～10 ∶1～5 ∶1～1 ∶1) 梯度洗脫,每一梯度所用洗脫液體積為3倍柱體積,5 個(gè)組分分別為Fr.1～Fr.5,對(duì)所得5個(gè)組分通過旋轉(zhuǎn)蒸發(fā)儀進(jìn)行減壓濃縮,濃縮至藥液濃度足夠大然后作為DAC-50 的制備樣品。反相DAC-50 制備液相系統(tǒng)采用甲醇-水作為洗脫溶劑,Fr.1 組分經(jīng)65%甲醇-水洗脫,分離純化得化合物1 (8 mg)、4 (13 mg)、5 (10 mg)、6 (21 mg)；Fr.2 組分經(jīng)55% 甲醇-水洗脫,純化得化合物10(7 mg)、14 (11 mg)、15 (14 mg)。Fr.3 經(jīng)45%甲醇-水洗脫,分離純化得化合物12 (22 mg)、13(15 mg)；Fr.4 組分經(jīng)35% 甲醇-水洗脫,分離純化得化合物2 (9 mg)、3 (16 mg)、9 (19 mg)、11 (13 mg)；Fr.5 組分經(jīng)25%甲醇-水洗脫,分離純化得化合物7 (12 mg)、8 (14 mg)。

3 結(jié)構(gòu)鑒定

化合物1:黃色粉末。ESI-MSm/z:333.1［M+H］+。1H-NMR (600 MHz,DMSO-d6)δ:3.83(6H,s,C3′,C5′-OCH3),6.18 (1H,d,J=1.2 Hz,H-6),6.54 (1H,d,J=1.2 Hz,H-8),6.91(1H,s,H-3),7.34 (1H,m,H-2′,6′),9.27(1H,s,4-OH),10.67 (1H,s,7-OH),12.98(1H,s,5-OH)；13C-NMR (150 MHz,DMSO-d6)δ:181.9 (C-4),164.2 (C-2),164.2 (C-4′),163.8 (C-7),161.5 (C-9),157.5 (C-5),148.5(C-3′,5′),139.8 (C-1′),120.6 (C-10),104.6(C-2′,6′),103.8 (C-3),98.9 (C-6),94.3 (C-8),56.5 (C3′,C5′-OCH3)。以上數(shù)據(jù)與文獻(xiàn)［12］ 報(bào)道基本一致,故鑒定為小麥黃素。

化合物2:黃色粉末。ESI-MSm/z:493.1［M+H］+。1H-NMR (600 MHz,DMSO-d6)δ:12.93(1H,s,5-OH),9.38 (1H,s,4′-OH),7.31 (2H,s,H-2,6),6.82 (1H,d,J=1.9 Hz,H-8),6.41(1H,d,J=1.9 Hz,H-6),6.93 (1H,d,J=1.9 Hz,H-3),3.64 (6H,s),5.26 (1H,s),3.32(1H,m),3.30 (1H,m),3.02 (1H,m),3.66(1H,m),3.52 (1H,d),3.31 (1H,m)；13C-NMR(150 MHz,DMSO-d6)δ:183.5 (C-4),165.6 (C-2),164.4 (C-7),162.5 (C-5),158.3 (C-9),149.7 (C-3′,5′),141.5 (C-4′),121.7 (C-1′),106.8 (C-10),105.9 (C-2′,6′),105.5 (C-3),99.8 (C-6),96.7 (C-8),101.5 (C-1″),78.7 (C-5″),74.6 (C-2″),77.8 (C-3″),68.9 (C-4″),61.9 (C-6″)。以上數(shù)據(jù)與文獻(xiàn)［13］ 報(bào)道基本一致,故鑒定為小麥黃素-7-O-β-D-吡喃葡萄糖。

化合物3:黃色粉末。ESI-MSm/z:431.2［M-H］-。1H-NMR (600 MHz,DMSO-d6)δ:7.64(d,2H,J=8.7 Hz,H-2′,6′),6.87 (d,2H,J=8.7 Hz,H-3′,5′),6.43 (d,1H,J=1.6 Hz,H-8),6.23 (d,1H,J=1.6 Hz,H-6),5.25 (d,1H,J=1.3 Hz,Rha-H-1),0.77 (d,3H,J=5.7 Hz,Rha-H-6)；13C-NMR (150 MHz,DMSOd6)δ:176.6 (C-4),163.4 (C-7),160.2 (C-5),158.9 (C-4′),156.1 (C-2),155.5 (C-9),133.1(C-3),129.5 (C-2′,6′),121.5 (C-1′),116.4(C-3′,5′),105.0 (C-10),99.7 (C-6),94.7 (C-8),102.8 (Rha-C-1),71.6 (Rha-C-2),71.3(Rha-C-3),72.1 (Rha-C-4),71.0 (Rha-C-5),17.8 (Rha-C-6)。以上數(shù)據(jù)與文獻(xiàn)［14］ 報(bào)道基本一致,故鑒定為山柰酚-3-O-α-L-吡喃鼠李糖。

化合物4:黃色針晶。ESI-MSm/z:331.0［M+H］+。1H-NMR (600 MHz,DMSO-d6)δ:12.42(1H,s,5-OH),7.84 (1H,d,J=2.0 Hz,H-2′),7.82 (1H,dd,J=2.0,8.5 Hz,H-6′),6.83 (1H,d,J=8.5 Hz,H-5′),6.86 (1H,d,J=2.0 Hz,H-8),6.43 (1H,d,J=2.0 Hz,H-6),3.75 (3H,s,3′-OCH3),3.76 (3H,s,7-OCH3)；13C-NMR (150 MHz,DMSO-d6)δ:148.1 (C-2),135.1 (C-3),177.1 (C-4),161.4 (C-5),96.4 (C-6),164.9(C-7),91.5 (C-8),155.7 (C-9),104.4 (C-10),122.0 (C-1′),112.6 (C-2′),146.4 (C-3′),148.0 (C-4′),114.6 (C-5′),123.0 (C-6′),55.8(-OCH3),55.4 (-OCH3)。以上數(shù)據(jù)與文獻(xiàn)［15］報(bào)道基本一致,故鑒定為甲基鼠李素。

化合物5:黃色粉末。ESI-MSm/z:285.1［M-H］-。1H-NMR (600 MHz,CD3OD)δ:8.19(2H,d,J=8.7 Hz,H-2′ and H-4′),6.87 (2H,d,J=8.7 Hz,H-3′ and H-5′),6.42 (1H,d,J=1.3 Hz,H-8),6.21 (1H,d,J=1.6 Hz,H-6)；13CNMR (150 MHz,CD3OD)δ:176.9 (C-4),165.2(C-7),162.1 (C-5),158.1 (C-4′),155.8 (C-9),145.6 (C-2),134.7 (C-3),128.3 (C-2′ and C-6′),121.3 (C-1′),113.9 (C-3′ and C-5′),102.1(C-10),96.9 (C-6),94.1 (C-8)。以上數(shù)據(jù)與文獻(xiàn)［16］ 報(bào)道基本一致,故鑒定為山柰酚。

化合物6:黃色針晶。ESI-MSm/z:303.5 ［M+H］+。1H-NMR (600 MHz,CD3OD)δ:7.64 (1H,d,J=1.8 Hz,H-2′),7.54 (1H,dd,J=8.5,1.9 Hz,H-6′),6.79 (1H,d,J=8.5 Hz,H-5′),6.29(1H,d,J=1.5 Hz,H-8),6.09 (1H,d,J=1.7 Hz,H-6)；13C-NMR (150 MHz,CD3OD)δ:174.9(C-4),163.1 (C-7),160.1 (C-5),155.8 (C-9),146.3 (C-2),145.5 (C-4′),143.8 (C-3′),134.8(C-3),121.7 (C-1′),120.3 (C-6′),113.8 (C-5′),113.6 (C-2′),102.1 (C-10),96.8 (C-6),92.0 (C-8)。以上數(shù)據(jù)與文獻(xiàn)［17］ 報(bào)道基本一致,故鑒定為槲皮素。

化合物7:白色晶體。ESI-MSm/z:435.3［M-H］-。1H-NMR (600 MHz,CD3OD)δ:6.86(1H,s,H-2′),6.74 (1H,d,J=8.2 Hz,H-6′),6.69 (1H,d,J=8.1 Hz,H-5′),5.82 (1H,s,H-6),5.88 (1H,s,H-8),5.13 (1H,d,J=10.6 Hz,H-2),4.78 (1H,d,J=10.6 Hz,H-3),3.90～3.32 (6H,m,Ara-H)；13C-NMR (150 MHz,CD3OD)δ:193.6 (C-4),166.9 (C-7),163.6 (C-5),161.8 (C-9),145.6 (C-4′),144.9 (C-3′),126.9 (C-1′),118.8 (C-6′),113.9 (C-5′),113.8(C-2′),100.6 (C-10),98.8 (C-1″),95.7 (C-6),94.9 (C-8),81.8 (C-2),73.9 (C-3),71.4 (C-2″),68.9 (C-4″),64.8 (C-3″),60.9 (C-5″)。以上數(shù)據(jù)與文獻(xiàn) ［18］ 報(bào)道基本一致,故鑒定為(2R,3R) -二氫槲皮素-3-O-α-L-吡喃阿拉伯糖。

化合物8:白色非晶粉末。ESI-MSm/z:463.2［M-H］-。1H-NMR (600 MHz,CD3OD)δ:7.75(1H,d,J=1.7 Hz,H-2′),7.52 (1H,dd,J=8.5,1.8 Hz,H-6′),6.79 (1H,d,J=8.5 Hz,H-5′),6.40 (1H,s,H-8),6.21 (1H,d,J=1.3 Hz,H-6),5.22 (1H,d,J=7.8 Hz,H-1″),3.87～3.51(6H,m,Gal-H)；13C-NMR (150 MHz,CD3OD)δ:177.8 (C-4),163.9 (C-7),160.9 (C-5),156.8(C-2),156.7 (C-9),147.8 (C-4′),143.9 (C-3′),133.8 (C-3),120.9 (C-1′),120.9 (C-6′),115.8 (C-5′),113.9 (C-2′),103.8 (C-10),102.8 (C-1′),97.7 (C-6),92.7 (C-8),74.9 (C-5″),72.8 (C-3″),70.9 (C-2″),67.8 (C-4″),60.6 (C-6″)。以上數(shù)據(jù)與文獻(xiàn)［19］ 報(bào)道基本一致,故鑒定為槲皮素-3-O-β-D-半乳糖。

化合物9:黃色粉末。ESI-MSm/z:499.2［M-H］-。1H-NMR (600 MHz,CD3OD)δ:7.89(1H,d,J=1.9 Hz,H-2′),7.67 (1H,dd,J=8.4,1.9 Hz,H-6′),6.92 (1H,d,J=8.5 Hz,H-5′),6.32 (1H,s,H-6),5.19 (1H,d,J=7.8 Hz,H-1″),3.91 (1H,d,J=3.7 Hz,H-2″),3.79 (1H,d,J=8.0 Hz,H-3″),3.68 (1H,dd,J=11.2,6.1 Hz,H-4″),3.58-3.49 (2H,m,H-6α″ and H-6β″),3.51 (1H,t,J=6.2 Hz,H-5′)；13C-NMR (150 MHz,CD3OD)δ:178.8 (C-4),157.5 (C-2),153.75 (C-7),153.6 (C-5),148.8 (C-4′),145.5(C-9),144.7 (C-3′),134.8 (C-3),124.9 (C-8),121.54 (C-1′),121.2 (C-6′),116.7 (C-5′),114.7 (C-2′),104.5 (C-10),103.5 (C-1″),75.4(C-5″),73.6 (C-3″),71.5 (C-2″),68.2 (C-4″),60.1 (C-6″)。以上數(shù)據(jù)與文獻(xiàn)［20］ 報(bào)道基本一致,故鑒定為棉花素-3-O-β-D-吡喃半乳糖苷。

化合物10:黃色粉末。ESI-MSm/z:465.1［M+H］+。1H-NMR (600 MHz,CD3OD)δ:7.65(1H,s,H-2′),7.75 (1H,d,J=8.1 Hz,H-5′),6.96 (1H,d,J=7.9 Hz,H-6′),6.45 (1H,s,H-8),6.32 (1H,s,H-6),5.33 (1H,d,J=7.5 Hz,H-1″),3.82-3.42 (6H,m,Glu-H)；13C-NMR (150 MHz,CD3OD)δ:178.4 (C-4),164.9 (C-7),161.7 (C-5),157.6 (C-9),157.2 (C-2),148.54(C-4′),144.6 (C-3′),134.2 (C-3),121.8 (C-6′),121.7 (C-1′),116.3 (C-5′),114.6 (C-2′),104.3 (C-10),102.9 (C-1″),98.6 (C-6′),93.4(C-8),77.3 (C-3),76.8 (C-5),74.4 (C-2),69.9 (C-4),61.4 (C-6)。以上數(shù)據(jù)與文獻(xiàn)［21］報(bào)道基本一致,故鑒定為異槲皮素。

化合物11:黃色粉末。ESI-MSm/z:435.3［M+H］+。1H-NMR (600 MHz,CD3OD)δ:7.81(1H,s,H-2′),7.62 (1H,d,J=5.7 Hz,H-6′),6.93 (1H,s,H-5′),6.45 (1H,s,H-8),6.23(1H,s,H-6),5.21 (1H,d,J=6.0 Hz,H-1″),3.87～3.42 (6H,m,Ara-H)；13C-NMR (150 MHz,CD3OD)δ:177.8 (C-4),165.1 (C-7),162.1 (C-5),156.9 (C-9),157.1 (C-2),147.9 (C-4′),143.9 (C-3′),133.8 (C-3),120.9 (C-6′),120.9(C-1′),115.8 (C-5′),115.1 (C-2′),103.9 (C-1′),102.8 (C-10),97.9 (C-6),92.8 (C-8),71.9 (C-3″),70.9 (C-2″),68.1 (C-4″),66.0 (C-5″)。以上數(shù)據(jù)與文獻(xiàn)［22］ 報(bào)道基本一致,故鑒定為槲皮素-3-O-α-L-吡喃阿拉伯糖苷。

化合物12:黃色粉末。ESI-MSm/z:451.3［M+H］+。1H-NMR (600 MHz,CD3OD)δ:7.76(1H,s,H-2′),7.65 (1H,s,H-6′),6.47 (1H,s,H-8),6.26 (1H,s,H-6),4.92 (1H,d,J=7.5 Hz,H-1″),4.11～3.97 (1H,m,H-5″α),3.62(1H,s,H-4″),3.61 (1H,d,J=7.8 Hz,H-2″),3.52 (1H,d,J=8.9 Hz,H-3″),3.42 (1H,d,J=10.9 Hz,H-5″β)；13C-NMR (150 MHz,CD3OD)δ:176.1 (C-4),163.9 (C-7),160.9 (C-5),157.1(C-9),146.1 (C-2),146.1 (C-3′),146.1 (C-5′),137.2 (C-4′),136.1 (C-3),122.1 (C-1′),109.9 (C-6′),109.0 (C-2′),102.8 (C-1″),103.7(C-10),98.1 (C-6),93.1 (C-8),75.9 (C-3″),72.9 (C-2″),68.9 (C-4″),66.0 (C-5″)。以上數(shù)據(jù)與文獻(xiàn)［23］ 報(bào)道基本一致,故鑒定為楊梅素-3′-O-β-D-木糖苷。

化合物13:黃色粉末。ESI-MSm/z:584.3［M-H］-。1H-NMR (600 MHz,CD3OD)δ:7.83(1H,d,J=1.8 Hz,H-2′),7.63 (2H,d,J=8.6 Hz,H-2?and H-6?),7.62 (1H,dd,J=8.4,1.8 Hz,H-6′),6.91 (1H,d,J=8.5 Hz,H-5′),6.72(2H,d,J=8.6 Hz,H-3?and H-5?),6.43 (1H,d,J=1.5 Hz,H-8),6.25 (1H,d,J=1.6 Hz,H-6),5.27 (1H,d,J=7.8 Hz,H-1″),4.41 (1H,dd,J=11.3,8.2 Hz,H-6″α),4.43 (1H,dd,J=11.3,4.5 Hz,H-6″β),3.93 (3H,ddd,J=12.5,10.7,4.1 Hz,H-2″,H-3″ and H-5″),3.71 (1H,dd,J=9.5,3.4 Hz,H-4″)；13C-NMR (150 MHz,CD3OD)δ:177.9 (C-4),165.8 (C-7?),165.1(C-7),161.8 (C-4?),161.1 (C-5),156.9 (C-2),157.1 (C-9),147.8 (C-4′),145.1 (C-3′),133.8 (C-3),130.9 (C-2?and C-6?),120.9 (C-6′),120.9 (C-1′),120.9 (C-1?),115.9 (C-2′),115.1 (C-5′),115.1 (C-3?and C-5?),103.9 (C-10),103.9 (C-1″),99.0 (C-6),92.3 (C-8),72.9 (C-3″/C-5″),72.9 (C-5″/C-3″),72.0 (C-2″),69.0 (C-4″),63.0 (C-6″)。以上數(shù)據(jù)與文獻(xiàn)［24］ 報(bào)道基本一致,故鑒定為異槲皮素-6″-O-p-對(duì)羥基苯甲酸丁酯。

化合物14:淡黃色粉末。ESI-MSm/z:213.1［M+H］+。1H-NMR (600 MHz,CD3OD)δ:8.25(1H,d,J=5.4 Hz,H-4),8.21 (1H,d,J=8.0 Hz,H-5),7.95 (1H,d,J=5.4 Hz,H-3),7.71(1H,dd,J=8.2,0.9 Hz,H-8),7.62 (1H,ddd,J=8.2,8.0,0.9 Hz,H-7),7.31 (1H,t,J=8.0 Hz,H-6),5.39 (1H,q,J=6.6 Hz,H-1′),1.68(3H,d,J=6.6 Hz,CH3-2′)；13C-NMR (150 MHz,CD3OD)δ:143.5 (C-13),138.6 (C-4),137.5(C-10),136.9 (C-1),133.3 (C-11),130.3 (C-7),122.7 (C-5),121.7 (C-12),121.6 (C-6),120.1 (C-3),113.5 (C-8),70.5 (C-1′),19.4(C-2′)。以上數(shù)據(jù)與文獻(xiàn) ［25］ 報(bào)道基本一致,故鑒定為cordysinin C。

化合物15:黃色粉末。ESI-MSm/z:213.1［M+H］+。1H-NMR (600 MHz,DMSO-d6)δ:11.21(1H,s,H-9),8.53 (1H,d,J=5.4,H-3),8.55(1H,d,J=5.4,H-4),8.42 (1H,d,J=8.1,H-5),7.93 (1H,d,J=8.1,H-8),7.72 (1H,d,J=8.1,H-7),7.42 (1H,d,J=8.1,H-6)；13CNMR (150 MHz,DMSO-d6)δ:142.9 (C-1),135.6(C-3),118.5 (C-4),122.3 (C-5),129.5 (C-6),120.3 (C-7),113.3 (C-8),131.9 (C-10),119.9(C-11),131.6 (C-12),135.6 (C-13),165.9 (1-COOH)。以上數(shù)據(jù)與文獻(xiàn)［26］ 報(bào)道基本一致,故鑒定為β-carboline-1-carboxylic acid。

4 討論

研究表明茶藨子葉的主要活性成分為黃酮類,杜偉偉、鄒維娜［11,27］分別對(duì)茶藨屬不同種茶藨子活性成分和藥效做了總結(jié),并以香茶藨子葉為研究對(duì)象,針對(duì)其黃酮類化學(xué)成分建立了相應(yīng)的富集分離工藝,并驗(yàn)證黃酮的藥理活性。本實(shí)驗(yàn)對(duì)五裂茶藨子中黃酮類成分進(jìn)行探究,結(jié)果表明五裂茶藨子的黃酮類化學(xué)成分既與茶藨屬的其他種的化學(xué)成分有相似之處,又有其化學(xué)成分的獨(dú)特性。