周媛媛,劉寧雨,馬丹娜,高蕙蕊,于海龍,劉 艷*
(1.黑龍江中醫(yī)藥大學(xué)藥學(xué)院,黑龍江 哈爾濱 150040;2.哈爾濱市食品藥品檢驗(yàn)檢測(cè)中心,黑龍江 哈爾濱 150525)
核桃青皮為胡桃科胡桃屬植物胡桃楸JuglansmandshuricaMaxim.的未成熟青果皮,別名青胡桃皮、青龍衣,主要分布在我國(guó)的東北地區(qū),在內(nèi)蒙古、河北、山東等地區(qū)也有少許分布,資源豐富,為我國(guó)民間常用中藥,其味苦、澀,性平,具有清熱解毒、止痢、療癬等功效,可用于治療皮膚瘙癢、發(fā)熱咳嗽、水痢等[1-2]。目前,從核桃青皮中主要分離得到三萜類、黃酮類、萘醌類等成分。現(xiàn)代藥理研究表明核桃青皮具有抗腫瘤、抗氧化、抗菌等作用[3]。近年來(lái)胡桃屬的藥用價(jià)值引起了人們的關(guān)注,為了進(jìn)一步研究其藥效物質(zhì)基礎(chǔ),本實(shí)驗(yàn)對(duì)其化學(xué)成分進(jìn)行系統(tǒng)研究,共分離得到14個(gè)化合物,其中3個(gè)黃酮類、7個(gè)黃酮苷類、4個(gè)萘苷類。
Bruker-400型超導(dǎo)核磁共振光譜儀(瑞士Bruker公司);Finnigan LCQ質(zhì)譜儀;Heidolph-Laborota 4000型旋轉(zhuǎn)蒸發(fā)儀(德國(guó)海道夫公司);柱層析硅膠(青島海洋化工廠);柱色譜ODS (日本YMC公司);其他試劑均為分析純。
藥材于2019年7月采自吉林省長(zhǎng)白山地區(qū)。經(jīng)黑龍江中醫(yī)藥大學(xué)藥學(xué)院楊炳友教授鑒定為胡桃科植物核桃楸JulansmandshuricaMaxim未成熟外果皮,標(biāo)本(20190813)保存于黑龍江中醫(yī)藥大學(xué)中藥化學(xué)教研室。
取干燥的核桃青皮粉末10.0 kg,用甲醇浸泡1 d,回流提取3次,減壓濃縮得到提取物。以適量蒸餾水添加并分散所得提取物,依次用二氯甲烷、乙酸乙酯及水飽和正丁醇進(jìn)行多次萃取,萃取液經(jīng)減壓濃縮得各部位浸膏。
取正丁醇浸膏(208 g),經(jīng)硅膠柱,以二氯甲烷-甲醇系統(tǒng)梯度洗脫,得到8個(gè)組分Fr.1~Fr.8。Fr.1經(jīng)硅膠柱,以二氯甲烷-甲醇(15∶1~0∶1)梯度洗脫,得化合物12(6.8 mg)、13(5.4 mg)、14(7.2 mg);Fr.6經(jīng)硅膠柱以二氯甲烷-甲醇(15∶1~0∶1)梯度洗脫后,得7個(gè)組分Fr.6.1~Fr.6.7,F(xiàn)r.6.4經(jīng)硅膠柱以二氯甲烷-甲醇(20∶1~0∶1)系統(tǒng)梯度洗脫后,得到化合物2(3.6 mg)、5(6.3 mg)、7(4.7 mg);Fr.6.6經(jīng)硅膠柱以二氯甲烷-甲醇(10∶1~0∶1)系統(tǒng)梯度洗脫后,得化合物4(5.8 mg)、6(5.6 mg)、8(7.2 mg)、10(5.7 mg);Fr.6.7經(jīng)硅膠柱以二氯甲烷-甲醇(15∶1~0∶1)系統(tǒng)梯度洗脫后,得到化合物1(4.2 mg)、11(4.8 mg);Fr.8經(jīng)硅膠柱色譜二氯甲烷-甲醇(10∶1~0∶1)與ODS硅膠柱甲醇-水(40∶60~100∶0)反復(fù)梯度洗脫得化合物3(3.3 mg)、9(5.1 mg)。
化合物1:棕褐色粉末,分子式C25H26O12。ESI-MSm/z:541.1[M+Na]+。1H-NMR (400 MHz,acetone-d6)δ:9.26 (1H,s,8-OH),8.87 (1H,s,4-OH),8.10 (1H,s,4″-OH),7.71 (1H,dd,J=8.2,0.8 Hz,H-5),7.42 (2H,s,H-2″,6″),7.28 (1H,t,J=8.2 Hz,H-6),7.21 (1H,d,J=8.2 Hz,H-3),6.80 (1H,dd,J=8.2,0.8 Hz,H-7),6.53 (1H,d,J=8.2 Hz,H-2),5.10 (1H,d,J=7.2 Hz,H-1′),4.76 (1H,dd,J=10.6,2.8 Hz,H-6′),4.47 (1H,dd,J=10.6,6.2 Hz,H-6′),3.95 (1H,m,H-5′),3.84 (3H,s,3″,5″-OCH3),3.66 (1H,m,H-3′),3.64 (1H,m,H-2′),3.58 (1H,m,H-4′);13C-NMR (125 MHz,acetone-d6)δ:148.6 (C-1),107.6 (C-2),112.6 (C-3),149.6 (C-4),113.7 (C-5),127.2 (C-6),112.2 (C-7),154.7 (C-8),117.3 (C-9),128.4 (C-10),104.4 (C-1′),77.6 (C-2′),74.6 (C-3′),71.4 (C-4′),75.5 (C-5′),64.6 (C-6′),121.3 (C-1″),108.2 (C-2″,C-6″),148.4 (C-3″,C-5″),141.5 (C-4″),166.3 (C-7″),56.5 (C-3″,5″-OCH3)。以上數(shù)據(jù)與文獻(xiàn)[4]基本一致,故鑒定為1,4,8-三羥基萘-1-O-β-D-[6″-O-(4″-羥基-3″,5″-二甲氧基苯甲酰)]-葡萄糖苷。
化合物2:白色粉末,分子式C18H20O10。ESI-MSm/z:397.1[M+H]+。1H-NMR (400 MHz,CD3OD)δ:7.72 (1H,dd,J=0.8,8.0 Hz,H-5),7.56 (1H,s,H-2),7.28 (1H,t,J=8.0 Hz,H-6),6.86 (1H,dd,J=0.8,8.0 Hz,H-7),4.86 (1H,d,J=8.0 Hz,H-1′),3.84 (3H,s,11-COOCH3);13C-NMR (125 MHz,CD3OD)δ:147.6 (C-1),109.2 (C-2),105.8 (C-3),155.3 (C-4),116.2 (C-5),128.6 (C-6),116.4 (C-7),157.7 (C-8),120.3 (C-9),128.8 (C-10),172.3 (C-11),105.6 (C-1′),80.1 (C-2′),75.4 (C-3′),71.8 (C-4′),78.4 (C-5′),62.8 (C-6′),53.1 (C-11-COOCH3)。以上數(shù)據(jù)與文獻(xiàn)[5]基本一致,故鑒定為1,4,8-三羥基-3-萘甲酸-1-O-β-D-葡萄糖苷甲酯。
化合物3:棕色粉末,分子式C21H26O12。ESI-MSm/z:493.1[M+Na]+。1H-NMR (400 MHz,CD3OD)δ:7.64 (1H,dd,J=0.8,7.7 Hz,H-5),7.26 (1H,t,J=7.7 Hz,H-6),7.20 (1H,d,J=8.0 Hz,H-3),6.81 (1H,dd,J=0.8,7.7 Hz,H-7),6.69 (1H,d,J=8.0 Hz,H-2),4.97 (1H,d,J=0.8 Hz,H-1″),4.95 (1H,d,J=7.8 Hz,H-1′);13C-NMR (125 MHz,CD3OD)δ:148.8 (C-1),108.5 (C-2),113.3 (C-3),150.7 (C-4),114.8 (C-5),127.3 (C-6),112.3 (C-7),154.8 (C-8),117.9 (C-9),129.1 (C-10),105.5 (C-1′),75.4 (C-2′),78.3 (C-3′),71.6 (C-4′),77.1 (C-5′),68.1 (C-6′),110.3 (C-1″),85.4 (C-2″),83.1 (C-3″),78.5 (C-4″),62.6 (C-5″)。以上數(shù)據(jù)與文獻(xiàn)[6]基本一致,故鑒定為1,4,8-三羥基萘-1-O-β-D-葡糖醛基-(1→6)-α-L-阿拉伯糖苷。
化合物4:白色粉末,分子式C25H28O13。ESI-MSm/z:559.2[M+Na]+。1H-NMR (400 MHz,DMSO-d6)δ:12.02 (1H,s,8-OH),8.10 (1H,s,4-OH),7.48 (1H,d,J=8.6 Hz,H-6),7.36 (2H,s,H-2″,6″),6.56 (1H,d,J=8.6 Hz,H-7),4.74 (1H,d,J=7.8 Hz,H-1′),4.61 (1H,brd,J=4.2 Hz,H-4),3.02 (1H,m,H-2),2.20 (1H,m,H-3);13C-NMR (125 MHz,DMSO-d6)δ:206.6 (C-1),33.7 (C-2),30.9 (C-3),64.9 (C-4),149.3 (C-5),128.9 (C-6),118.3 (C-7),158.7 (C-8),115.6 (C-9),135.6 (C-10),105.0 (C-1′),75.6 (C-2′),78.0 (C-3′),71.9 (C-4′),75.8 (C-5′),60.9 (C-6′),121.4 (C-1″),108.5 (C-2″,6″),148.8 (C-3″,5″),142.3 (C-4″),166.7 (C-7″),57.1 (3″,5″-OCH3)。以上數(shù)據(jù)與文獻(xiàn)[7]基本一致,故鑒定為4,5,8-三羥基-α-四氫萘酮-5-O-β-D-[6′-O-(4″-羥基-3″,5″-二甲氧基苯甲酰)]葡萄糖苷。
化合物5:無(wú)色簇狀結(jié)晶,分子式C21H20O9,ESI-MSm/z:417.1[M+H]+。1H-NMR (400 MHz,DMSO-d6)δ:9.59 (1H,s,4′-OH),8.43 (1H,s,H-2),8.07 (1H,d,J=8.8 Hz,H-5),7.44 (2H,d,J=6.2 Hz,H-2′,6′),7.26 (1H,d,J=1.6 Hz,H-8),7.17 (1H,dd,J=1.6,8.8 Hz,H-6),6.82 (2H,d,J=6.2 Hz,H-3′,5′),5.49 (1H,d,J=7.8 Hz,H-1″);13C-NMR (125 MHz,DMSO-d6)δ:153.2 (C-2),123.4 (C-3),174.1 (C-4),126.3 (C-5),115.0 (C-6),160.7 (C-7),99.4 (C-8),156.2 (C-9),117.6 (C-10),121.6 (C-1′),129.3 (C-2′,6′),114.2 (C-3′,5′),156.5 (C-4′),102.6 (C-1″),71.1 (C-2″),72.4 (C-3″),69.2 (C-4″),76.3 (C-5″),61.7 (C-6″)。以上數(shù)據(jù)與文獻(xiàn)[8]基本一致,故鑒定為4′-羥基異黃酮-7-O-β-D-半乳糖苷。
化合物6:淺黃色粉末,分子式C21H20O10,ESI-MSm/z:433.1[M+H]+。1H-NMR (400 MHz,DMSO-d6)δ:7.72 (2H,d,J=8.6 Hz,H-2′,6′),6.88 (2H,d,J=8.6 Hz,H-3′,5′),6.34 (1H,d,J=1.2 Hz,H-8),6.13 (1H,d,J=1.2 Hz,H-6),5.29 (1H,d,J=2.0 Hz,H-1″)0.96 (3H,d,J=5.8 Hz,-CH3);13C-NMR (125 MHz,DMSO-d6)δ:157.3 (C-2),134.9 (C-3),177.7 (C-4),161.5 (C-5),98.8 (C-6),164.5 (C-7),93.8 (C-8),156.0 (C-9),103.7 (C-10),120.1 (C-1′),130.3 (C-2′,6′),115.1 (C-3′,5′),160.4 (C-4′),101.4 (C-1″),70.3 (C-2″),70.1 (C-3″),71.2 (C-4″),70.3 (C-5″),17.8 (C-6″)。以上數(shù)據(jù)與文獻(xiàn)[9]基本一致,故鑒定為山柰酚-3-O-α-L-鼠李糖苷。
化合物7:淺黃色粉末,分子式C21H20O12,ESI-MSm/z:487.1[M+Na]+。1H-NMR (400 MHz,DMSO-d6)δ:7.74 (1H,d,J=1.8 Hz,H-2′),7.59 (1H,dd,J=1.8,8.4 Hz,H-6′),6.91 (1H,d,J=8.4 Hz,H-5′),6.74 (1H,d,J=1.8 Hz,H-8),6.38 (1H,d,J=1.8 Hz,H-6),5.11 (1H,d,J=7.8 Hz,H-1″);13C-NMR (125 MHz,DMSO-d6)δ:148.0 (C-2),136.7 (C-3),176.8 (C-4),161.0 (C-5),99.3 (C-6),163.4 (C-7),94.5 (C-8),156.5 (C-9),105.4 (C-10),122.3 (C-1′),115.5 (C-2′),145.4 (C-3′),148.3 (C-4′),116.4 (C-5′),120.1 (C-6′),100.0 (C-1″),73.4 (C-2″),76.6 (C-3″),70.1 (C-4″),77.3 (C-5″),60.9 (C-6″)。以上數(shù)據(jù)與文獻(xiàn)[10]基本一致,故鑒定為槲皮素-7-O-β-D-吡喃葡萄糖苷。
化合物8:淺黃色針狀結(jié)晶,分子式C21H20O11。ESI-MSm/z:449.1[M+H]+。1H-NMR (400 MHz,DMSO-d6)δ:8.15 (2H,d,J=8.8 Hz,H-2′,6′),7.02 (2H,d,J=8.8 Hz,H-3′,5′),6.84 (1H,d,J=2.2 Hz,H-8),6.46 (1H,d,J=2.2 Hz,H-6),5.14 (1H,d,J=7.8 Hz,H-1″);13C-NMR (125 MHz,DMSO-d6)δ:147.7 (C-2),136.2 (C-3),176.3 (C-4),160.4 (C-5),98.8 (C-6),162.5 (C-7),94.2 (C-8),155.4 (C-9),104.4 (C-10),121.7 (C-1′),129.8 (C-2′,6′),115.6 (C-3′,5′),159.6 (C-4′),99.6 (C-1″),73.3 (C-2″),76.9 (C-3″),69.7 (C-4″),77.0 (C-5″),60.8 (C-6″)。以上數(shù)據(jù)與文獻(xiàn)[11]基本一致,故鑒定為山柰酚-7-O-β-D-吡喃葡萄糖苷。
化合物9:黃色粉末,分子式C28H24O15,ESI-MSm/z:623.1[M+Na]+。1H-NMR (400 MHz,CD3OD)δ:7.33 (1H,d,J=2.0 Hz,H-2′),7.31 (1H,dd,J=2.0,8.2 Hz,H-6′),7.06 (2H,s,H-2?,6?),6.90 (1H,d,J=8.2 Hz,H-5′),6.31 (1H,d,J=2.0 Hz,H-8),6.13 (1H,d,J=2.0 Hz,H-6),5.61 (1H,m,H-2″),5.49 (1H,d,J=1.5 Hz,H-1″),0.98 (3H,d,J=5.8 Hz,-CH3);13C-NMR (125 MHz,CD3OD)δ:159.2 (C-2),136.3(C-3),180.1 (C-4),163.8 (C-5),101.3 (C-6),168.1 (C-7),95.5 (C-8),159.9 (C-9),106.5 (C-10),123.6 (C-1′),117.1 (C-2′),147.1 (C-3′),150.5 (C-4′),117.6 (C-5′),123.8 (C-6′),100.0 (C-1″),74.1(C-2″),71.0 (C-3″),74.1 (C-4″),72.9 (C-5″),18.5 (C-6″),121.9 (C-1?),110.7 (C-2?,6?),149.5 (C-3?,5?),140.6 (C-4?),166.1 (C-7?)。以上數(shù)據(jù)與文獻(xiàn)[12]基本一致,故鑒定為槲皮素-3-O-α-L-(2″-沒(méi)食子?;?-鼠李糖苷。
化合物10:黃色粉末,分子式C23H22O12,ESI-MSm/z:491.1[M+H]+。1H-NMR(400 MHz,DMSO-d6)δ:7.48 (1H,dd,J=8.4,2.2 Hz,H-6′),7.45 (1H,d,J=2.2 Hz,H-2′),6.94 (1H,d,J=8.4 Hz,H-5′),6.85 (1H,d,J=2.2 Hz,H-8),6.79 (1H,s,H-3),6.51 (1H,d,J=2.2 Hz,H-6),5.34(1H,d,J=7.8 Hz,H-1″),4.14 (2H,overlapped,-CH2),1.22 (3H,t,J=7.0 Hz,-CH3);13C-NMR (125 MHz,DMSO-d6)δ:164.3 (C-2),103.1 (C-3),182.2 (C-4),157.3 (C-5),99.1 (C-6),162.4 (C-7),94.9 (C-8),161.0 (C-9),105.3 (C-10),119.5 (C-1′),113.6 (C-2′),145.7 (C-3′),150.3 (C-4′),116.3 (C-5′),121.8 (C-6′),99.2 (C-1″),72.7 (C-2″),75.4 (C-3″),71.5 (C-4″),75.2(C-5″),168.9 (C-6″),60.7 (-CH2-),14.3(-CH3)。以上數(shù)據(jù)與文獻(xiàn)[13]基本一致,故鑒定為木犀草素-7-O-β-D-葡萄糖醛酸乙酯苷。
化合物11:淺黃色粉末,分子式C21H20O11,ESI-MSm/z:449.1[M+H]+。1H-NMR (400 MHz,DMSO-d6)δ:12.96 (1H,s,5-OH),7.47 (1H,dd,J=2.2,8.4 Hz,H-6′),7.44 (1H,d,J=2.2 Hz,H-2′),6.92 (1H,d,J=8.4 Hz,H-5′),6.76 (1H,d,J=2.2 Hz,H-8),6.71 (1H,s,H-3),6.42 (1H,d,J=2.2 Hz,H-6),5.10 (1H,d,J=7.8 Hz,H-1″);13C-NMR (125 MHz,DMSO-d6)δ:164.7 (C-2),103.3 (C-3),182.1 (C-4),161.4 (C-5),99.7 (C-6),163.5 (C-7),95.4 (C-8),157.1 (C-9),105.5 (C-10),121.7 (C-1′),114.2 (C-2′),146.1 (C-3′),150.5 (C-4′),116.2 (C-5′),119.4 (C-6′),100.1 (C-1″),73.4 (C-2″),76.7 (C-3″),70.1 (C-4″),77.9 (C-5″),61.3 (C-6″)。以上數(shù)據(jù)與文獻(xiàn)[14]基本一致,故鑒定為木犀草苷。
化合物12:淺黃色針狀結(jié)晶,分子式C18H16O8,ESI-MSm/z:383.1[M+Na]+。1H-NMR (400 MHz,DMSO-d6)δ:12.68 (1H,s,5-OH),7.46 (1H,dd,J=2.2,8.4 Hz,H-6′),7.40 (1H,d,J=2.2 Hz,H-2′),7.02 (1H,d,J=8.4 Hz,H-5′),6.59 (1H,s,H-3),3.97 (3H,s,-OCH3),3.96 (3H,s,-OCH3),3.94 (3H,s,-OCH3);13C-NMR (125 MHz,DMSO-d6)δ:163.1 (C-2),102.7 (C-3),182.5 (C-4),148.2 (C-5),131.1 (C-6),150.6 (C-7),128.1 (C-8),145.1 (C-9),102.6 (C-10),121.5 (C-1′),109.7 (C-2′),148.6 (C-3′),150.6 (C-4′),115.7 (C-5′),120.2 (C-6′),60.4 (6-OCH3),61.4 (8-OCH3),56.0 (3′-OCH3)。以上數(shù)據(jù)與文獻(xiàn)[15]基本一致,故鑒定為4′,5,7-三羥基-3′,6,8-三甲氧基黃酮。
化合物13:黃色針狀結(jié)晶,分子式C15H12O5,ESI-MSm/z:295.1[M+Na]+。1H-NMR (400 MHz,DMSO-d6)δ:11.92 (1H,s,5-OH),10.90 (1H,s,7-OH),7.53 (2H,m,H-2′,H-6′),7.42 (2H,m,H-3′,H-5′),7.41 (1H,m,H-4′),5.95 (1H,d,J=2.2 Hz,H-8),5.91 (1H,d,J=2.2 Hz,H-6),5.94 (1H,d,J=6.4 Hz,3-OH),5.16 (1H,d,J=11.2 Hz,H-2),4.60 (1H,dd,J=11.2,6.4 Hz,H-3);13C-NMR (125 MHz,DMSO-d6)δ:82.7 (C-2),71.3 (C-3),197.4 (C-4),163.2 (C-5),96.0 (C-6),166.5 (C-7),95.3 (C-8),162.1 (C-9),100.2 (C-10),137.5 (C-1′),128.3 (C-2′,C-6′),128.5 (C-3′,C-5′),128.9 (C-4′)。以上數(shù)據(jù)與文獻(xiàn)[16]基本一致,故鑒定為短葉松素。
化合物14:白色粉末,分子式C21H22O7,ESI-MSm/z:409.1[M+Na]+。1H-NMR (400 MHz,CD3OD)δ:6.92 (1H,s,H-6′),6.34 (1H,s,H-3′),5.94 (1H,s,H-6),5.62 (1H,dd,J=12.4,3.6 Hz,H-2),5.16 (1H,t,J=6.6 Hz,H-2″),3.77 (1H,s,5′-OCH3),3.16 (2H,brd,J=6.6 Hz,H-1″),2.86 (1H,dd,J=17.0,12.4 Hz,H-3a),2.78 (1H,dd,J=17.0,3.6 Hz,H-3b),1.63 (3H,s,H-5″),1.62 (3H,s,H-4″);13C-NMR (125 MHz,CD3OD)δ:74.5 (C-2),42.2 (C-3),197.4 (C-4),161.6 (C-5),95.9 (C-6),164.6 (C-7),108.2 (C-8),160.1 (C-9),102.2 (C-10),116.7 (C-1′),148.3 (C-2′),103.3 (C-3′),146.5 (C-4′),140.4 (C-5′),110.2 (C-6′),21.4 (C-1″),122.8 (C-2″),131.7 (C-3″),17.4 (C-4″),25.4 (C-5″)。以上數(shù)據(jù)與文獻(xiàn)[17]基本一致,故鑒定為苦參醇 W。
核桃青皮是我國(guó)傳統(tǒng)中藥材,有著悠久的藥用歷史,近年來(lái),隨著現(xiàn)代科學(xué)技術(shù)的發(fā)展,對(duì)于核桃青皮化學(xué)成分的研究日益深入,但是就目前而言,對(duì)其正丁醇部位的研究報(bào)道較少。本實(shí)驗(yàn)通過(guò)現(xiàn)代提取分離技術(shù),對(duì)核桃青皮正丁醇部位進(jìn)行系統(tǒng)研究,共分離得到14個(gè)化合物,包括萘醌苷類、黃酮及其苷類,其中化合物1~5、7~11、13為首次從該植物中分離得到,化合物5、9為首次從胡桃屬中分離得到。本研究在一定程度上豐富了核桃青皮的化學(xué)成分庫(kù),完善了核桃青皮正丁醇部位化學(xué)成分的研究,以期為核桃青皮正丁醇部位的藥理活性研究提供物質(zhì)基礎(chǔ),對(duì)今后核桃青皮的合理開發(fā)與利用具有指導(dǎo)意義。