青錢柳葉的化學(xué)成分研究△

李龍宇，羅榮華，徐明杰，伍一煒，趙迪，潘巧玲，金佳怡，趙田甜，徐暾海*，鄭曉杰，姜程曦*，朱寅荻

1.北京中醫(yī)藥大學(xué) 中藥學(xué)院，北京 102488；2.溫州大學(xué) 生命科學(xué)研究院，浙江 溫州 325035；3.溫州醫(yī)科大學(xué) 藥學(xué)院，浙江 溫州 325035；4.溫州科技職業(yè)學(xué)院，浙江 溫州 325000

青錢柳Cyclocarya paliurus（Batal）Iljinsk 又名山化樹或山麻柳，為胡桃科（Juglandaceae）青錢柳屬（Cyclocarya）植物，屬于高大喬木，生長(zhǎng)于我國(guó)安徽、江蘇等省。其小葉數(shù)量、外形差異極大[1]，樹葉廣泛用于制作保健茶。目前，圍繞青錢柳栽培、繁殖、采摘、化學(xué)成分、藥理作用、產(chǎn)業(yè)發(fā)展等已有大量的研究?，F(xiàn)代研究發(fā)現(xiàn)，青錢柳葉的主要化學(xué)成分為三萜[2]、多糖[3]、黃酮及其對(duì)應(yīng)的糖苷、酚酸[4]等類，具有降血糖、調(diào)血脂、降血壓和抗氧化等藥理作用[5]。為了更全面地認(rèn)識(shí)青錢柳葉的化學(xué)成分，本實(shí)驗(yàn)對(duì)其乙醇提取物的正丁醇萃取部位進(jìn)行研究，采用多種柱色譜方法分離得到10 個(gè)化合物，并用核磁共振法（NMR）鑒定其分別為山柰酚（kaempferol，1）、杜仲樹脂酚（medioresinol，2）、（+）-松脂醇[（+）-pinoresinol，3]、（+）-表松脂醇[（+）-epipinoresinol，4]、積雪草酸（asiatic acid，5）、山楂酸（maslinic acid，6）、熊果酸（ursolic acid，7）、1α，3β-二羥基-12-齊墩果烯-28-酸（1α，3β-dihydroxy-12-oleanen-28-oic acid，8）、石榴萜酮（punicaone，9）、青錢柳苷Ⅰ（cyclocarioside Ⅰ，10）。其中化合物2～4、8 均為首次從青錢柳屬植物中分離得到。各化合物結(jié)構(gòu)見圖1。

圖1 青錢柳葉中分離得到的化合物1～10結(jié)構(gòu)式

1 材料

青錢柳葉由文成縣圣山食品開發(fā)有限公司提供，經(jīng)溫州醫(yī)科大學(xué)藥學(xué)院汪洪副教授鑒定為胡桃科青錢柳屬植物青錢柳Cyclocarya paliurus（Batal.）Iljinsk 的葉。樣品標(biāo)本現(xiàn)存于溫州醫(yī)科大學(xué)藥學(xué)院，編號(hào)：20180613。

1100 型高效液相色譜儀（美國(guó)Agilent 公司）；Bruker Avance Ⅲ600 型核磁共振儀（德國(guó)Bruker 公司）；GF254薄層硅膠板和柱色譜硅膠（100～200 目）均來(lái)自青島海洋化工廠；Pack ODS-A 型半制備色譜柱（250 mm×10 mm，5 μm，日本YMC 公司）；D101 大孔吸附樹脂（南開大學(xué)）；Sephadex LH-20（瑞典Amersham Biosciences 公司）；C18反相填料（日本YMC 公司）；色譜級(jí)甲醇（美國(guó)Tedia 公司）；其余化學(xué)試劑均為分析純（西隴科學(xué)股份有限公司）；超純水為實(shí)驗(yàn)室自制。

2 提取與分離

取干燥的青錢柳葉（35 kg）切碎，用70%乙醇加熱回流提取2 次，濾過(guò)后減壓回收乙醇，得浸膏3.7 kg，用水分散，分別用石油醚、乙酸乙酯、正丁醇各萃取3 次，濃縮后得到石油醚萃取物840 g、乙酸乙酯萃取物789 g、正丁醇萃取物1.1 kg。取正丁醇萃取物，溶解后用D101 型大孔吸附樹脂分離，以乙醇（30%～95%）梯度洗脫，將各洗脫部位濃縮、干燥。取75%乙醇部位約100 g，乙醇溶解后與硅膠208 g 拌勻，烘干后上硅膠柱色譜分離，以二氯甲烷-甲醇（100∶0～100∶50）洗脫，得到Fr.Z1～Z12 組分。其中，F(xiàn)r.Z6 經(jīng)C18反相柱色譜（30%～100%甲醇）分離，得到的各組分分別反復(fù)經(jīng)Sephadex LH-20和HPLC分離純化，得到化合物1～5。Fr.Z3 經(jīng)過(guò)C18反相柱色譜（30%～100%甲醇），將70%甲醇洗脫部位合并，再用硅膠柱色譜分離，以二氯甲烷-甲醇（100∶40～100∶100）洗脫，將二氯甲烷-甲醇（100∶40～100∶45）部位合并，再用Sephadex LH-20分離得12個(gè)組分，將4～8號(hào)組分用HPLC進(jìn)行制備，得到化合物6～9。Fr.Z5經(jīng)C18反相柱色譜，以甲醇（30%～100%）梯度洗脫，將60%～80%甲醇洗脫部位合并，上C18反相柱色譜，以同樣溶劑洗脫，每250 mL 收集1 瓶，在80%甲醇洗脫部位第六、七瓶析出固體，抽濾，保留固體成分即為化合物10。

3 結(jié)構(gòu)鑒定

化合物1：黃色粉末，分子式為C15H10O6。核磁共振氫譜（1H-NMR）(600 MHz,CD3OD)δ:6.21(1H,d,J=2.1 Hz,H-6),6.42 (1H,d,J=2.4 Hz,H-8),6.93(2H,d,J=8.4 Hz,H-3′,5′),8.11 (2H,d,J=8.4 Hz,H-2′,6′)。其氫譜數(shù)據(jù)與文獻(xiàn)報(bào)道基本一致[6]，故鑒定化合物1為山柰酚。

化合物2：白色粉末；分子式為C21H24O7。1HNMR (600 MHz,CD3OD)δ:6.68 (2H,s,H-2′,6′),6.79 (1H,d,J=8.4 Hz,H-5),6.84 (1H,dd,J=1.8,8.4 Hz,H-6),6.98 (1H,d,J=1.8 Hz,H-2),4.74 (2H,d,J=4.2 Hz,H-7,7′),3.88 (3H,s,3-OMe),3.87 (6H,s,3′,5′-OMe),3.87 (2H,m,H-9a,9′a),3.15 (2H,m,H-8,8′),4.25(2H,m,H-9b,9′b)；13C-NMR(150 MHz,CD3OD)δ:55.5 (C-3′-OMe),55.7 (C-5′-OMe),56.9(C-8,8′),56.5 (C-3-OMe),72.9 (C-9′),72.8 (C-9),87.7 (C-7),87.9 (C-7′),104.6 (C-2′,6′),111.1 (C-2),116.2 (C-5),120.2 (C-6),130.1 (C-1′),133.3 (C-1),133.9 (C-4′),147.5 (C-4),149.5 (C-3′,5′),149.2 (C-3)。其波譜數(shù)據(jù)與文獻(xiàn)報(bào)道基本一致[7-8]，故鑒定化合物2為杜仲樹脂酚。

化合物3：白色粉末，分子式為C20H22O6。1HNMR(600 MHz,CD3OD)δ:6.97(2H,d,J=1.8 Hz,H-2,2′),3.83 (2H,m,H-8,8′),6.80 (2H,dd,J=1.8,8.4 Hz,H-6,6′),3.85 (6H,s,3,3′-OCH3),6.76 (2H,d,J=8.4 Hz,H-5,5′),4.23 (2H,m,H-9a,9′a),4.70(2H,d,J=4.2 Hz,H-7,7′)；13C-NMR (150 MHz,CD3OD)δ:133.9 (C-1,1′),111.1 (C-2,2′),149.3 (C-3,3′),56.5 (3,3′-OCH3),147.5 (C-4,4′),116.2 (C-5,5′),120.2 (C-6,6′),87.7 (C-7,7′),55.6 (C-8,8′),72.8(C-9,9′)。其波譜數(shù)據(jù)與文獻(xiàn)報(bào)道基本一致[8-9]，故鑒定化合物3為（+）-松脂醇。

化合物4：白色粉末，分子式為C20H22O6。1HNMR(600 MHz,CD3OD)δ:6.94(1H,d,J=1.8 Hz,H-2),6.96(1H,d,J=1.8 Hz,H-2′),6.76(1H,d,J=2.4 Hz,H-5),6.79 (1H,d,J=1.2 Hz,H-5′),6.80 (1H,d,J=1.8 Hz,H-6′),6.81 (1H,d,J=1.8 Hz,H-6),4.41 (1H,d,J=7.2 Hz,H-7),4.85 (1H,d,J=6.0 Hz,H-7′),2.93(1H,m,H-8) 3.38 (1H,m,H-8′),3.78 (1H,m,H-9′a),4.10 (1H,d,J=9.6 Hz,H-9a),3.84 (3H,s,3′-OCH3),3.85 (3H,s,3-OCH3),3.83 (1H,m,H-9b),3.18 (1H,m,H-9′b)；13C-NMR(150 MHz,CD3OD)δ:134.0(C-1),131.5 (C-1′),111.0 (C-2),110.7 (C-2′),149.3 (C-3),149.0 (C-3′),147.6 (C-4),146.8 (C-4′),116.2 (C-5),116.1 (C-5′),120.4 (C-6),119.5 (C-6′),89.7 (C-7),83.7 (C-7′),55.9 (C-8),51.5 (C-8′),72.2 (C-9),70.8(C-9′),56.5 (3,3-OCH3)。其波譜數(shù)據(jù)與文獻(xiàn)報(bào)道基本一致[9-10]，故鑒定化合物4為（+）-表松脂醇。

化合物5：白色粉末，分子式為C30H48O5。1HNMR (600 MHz,CD3OD)δ:0.78 (3H,s,H-27),0.84(3H,s,H-26),0.88 (3H,d,J=6.6 Hz,H-30),0.96(3H,d,J=4.8 Hz,H-29),1.02 (6H,s,H-24),1.13(3H,s,H-25),2.20 (1H,d,J=11.4 Hz,H-18),3.52(1H,d,J=10.8 Hz,H-3),3.87 (3H,m,H-2,3),5.24(1H,t,J=3.6 Hz,H-12)；13C-NMR(150 MHz,CD3OD)δ:17.6(C-24),17.8(C-25),17.9(C-30),18.0(C-29),19.1 (C-6),181.9 (C-28),21.8 (C-26),24.4 (C-27),24.6 (C-11),25.5 (C-21),29.3 (C-16),32.0 (C-15),33.8(C-7),38.3(C-22),39.3(C-10),40.6(C-19,20),41.0 (C-8),42.5 (C-4),42.7 (C-14),43.6 (C-17),44.4 (C-5),49.8 (C-9),54.5 (C-18),67.4 (C-2),71.5(C-23),78.9(C-3),126.9(C-12),139.9(C-13)。其波譜數(shù)據(jù)與文獻(xiàn)報(bào)道基本一致[10]，故鑒定化合物5為積雪草酸。

化合物6：白色粉末，分子式為C30H48O4。1HNMR (600 MHz,C5D5N)δ:1.02(3H,s,H-30),0.96(3H,s,H-29),1.28 (3H,s,H-27),1.04 (3H,s,H-26),1.00(3H,s,H-25),1.10(3H,s,H-24),1.39(3H,s,H-23),2.28(2H,dd,J=12.6,4.2 Hz,H-1),4.13(2H,m,H-2),3.42 (2H,d,J=9.6 Hz,H-3),1.06 (1H,s,H-5),5.45 (1H,t,J=4.2 Hz,H-12),3.32 (1H,dd,J=14.4,4.2 Hz,H-18)；13C-NMR (150 MHz,C5D5N)δ:180.7(C-28),145.3 (C-13),122.9 (C-12),84.2 (C-3),69.0(C-2),56.3 (C-5),48.6 (C-9),48.2 (C-1),47.1 (C-17),46.8 (C-19),42.6 (C-14),42.4 (C-18),40.3 (C-4),40.2 (C-8),38.9 (C-10),34.6 (C-21),33.7 (C-29),33.6(C-7,22),31.4(C-20),29.8(C-23),28.7(C-15),26.6 (C-27),24.3 (C-16),24.2 (C-30),24.1 (C-11),19.3 (C-6),18.1 (C-24),17.9 (C-26),17.3 (C-25)。其波譜數(shù)據(jù)與文獻(xiàn)報(bào)道基本一致[11]，故鑒定化合物6為山楂酸。

化合物7：白色粉末，分子式為C30H48O3。1HNMR (600 MHz,C5D5N)δ:1.27 (1H,s,H-27),1.25(1H,s,H-23),1.08 (1H,s,H-25),1.05 (1H,s,H-30),1.02 (1H,d,J=6.0 Hz,H-24),0.97 (1H,d,J=6.0 Hz,H-29),0.91 (1H,s,H-26),5.52 (1H,t,J=3.6 Hz,H-12),3.49 (1H,dd,J=10.8,5.4 Hz,H-3),2.67 (1H,d,J=11.4 Hz,H-18)；13C-NMR (150 MHz,C5D5N)δ:180.4(C-28),139.7(C-13),126.0(C-12),78.5(C-3),56.2 (C-5),54.0 (C-18),48.5 (C-9),48.4 (C-17),42.9 (C-14),40.4 (C-8),39.9 (C-20),39.8 (C-19),39.8 (C-4),39.5 (C-1),37.9 (C-22),37.7 (C-10),34.0 (C-7),31.5 (C-21),29.2 (C-15),29.1 (C-23),28.6 (C-2),25.3 (C-16),24.3 (C-27),24.0 (C-11),21.8 (C-30),19.2 (C-6),18.0 (C-29),17.9 (C-26),17.0(C-24),16.1(C-25)。其譜學(xué)數(shù)據(jù)與文獻(xiàn)報(bào)道一致[12-13]，故鑒定化合物7為熊果酸。

化合物8：白色粉末，分子式為C30H48O4。1HNMR (600 MHz,C5D5N)δ:5.58 (1H,t,J=3.6 Hz,H-12),4.41(1H,dd,J=12.0,4.8 Hz,H-3),3.35(dd,J=13.8,4.8 Hz,H-1),2.97 (1H,dd,J=11.4,6.6 Hz,H-19);1.02(3H,s,H-30),0.95 (3H,s,H-29),1.36 (3H,s,H-27),1.13 (3H,s,H-26),1.02 (3H,s,H-25),1.13(3H,s,H-24),1.37(3H,s,H-23)；13C-NMR(150 MHz,C5D5N)δ:180.7 (C-28),145.1 (C-13),123.3 (C-12),73.1 (C-3),72.1 (C-1),49.0 (C-5),47.1 (C-19),47.0(C-17),43.1 (C-14),42.5 (C-18),41.9 (C-10),40.1(C-8),40.0 (C-4),38.8 (C-9),36.2 (C-2),34.6 (C-21),33.7 (C-28),33.6 (C-22),33.5 (C-7),31.4 (C-20),29.3 (C-23),28.9 (C-15),26.7 (C-27),24.2 (C-16),24.2(C-30),24.0(C-11),19.2(C-6),18.0(C-26),16.9(C-24,25)。其波譜數(shù)據(jù)與文獻(xiàn)報(bào)道基本一致[14]，故鑒定化合物8為1α，3β-二羥基-12-齊墩果烯-28-酸。

化合物9：淡黃色粉末，分子式為C30H48O4。1HNMR(600 MHz,C5D5N)δ:6.63(1H,d,J=10.2 Hz,H-1),6.05 (1H,d,J=10.2 Hz,H-2),5.59 (1H,dd,J=4.5,3.0 Hz,H-12),3.70 (1H,d,J=10.8 Hz,H-25),3.62 (1H,d,J=10.8 Hz,H-25),2.55 (1H,dd,J=11.4,5.4 Hz,H-9),1.30 (3H,s,H-24),1.20 (3H,s,H-23),1.13(3H,s,H-26),1.06(3H,s,H-27),1.00(3H,s,H-29),0.94 (3H,s,H-30)；13C-NMR (150 MHz，C5D5N)δ:206.8 (C-3),180.7 (C-28),154.0 (C-1),144.1(C-13),127.1(C-2),124.3(C-12),69.9(C-25),48.4 (C-10),47.3 (C-17),46.4 (C-19),45.5 (C-5),42.9 (C-4),42.9 (C-18),42.6 (C-14),40.5 (C-8),39.2 (C-9),34.7 (C-21),33.7 (C-29),33.5 (C-7),32.9 (C-22),31.3 (C-20),28.7 (C-15),27.1 (C-11),26.1 (C-23),24.2 (C-27),24.1 (C-16),19.5 (C-6),18.8(C-30),17.9(C-26),16.8(C-24)。其波譜數(shù)據(jù)與文獻(xiàn)報(bào)道基本一致[15-16]，故鑒定化合物9為石榴萜酮。

化合物10：白色粉末，分子式為C40H70O12，1HNMR (600 MHz,C5D5N)δ:5.53 (1H,d,J=1.8 Hz,H-1′ of arabinos),5.05 (1H,d,J=7.8 Hz,H-1′′ of quinovos),4.18 (1H,t,J=9.0 Hz,H-3′′ of quinovos),3.03 (1H,m,Ha-1),2.92 (1H,dt,J=12.6,4.2 Hz,Ha-11),1.61 (3H,d,J=6.0 Hz,H-6′′),1.08 (3H,s,H-18),1.42 (3H,s,H-19),1.18 (3H,s,H-21),1.45(3H,s,H-26),1.37 (3H,s,H-27),0.95 (3H,s,H-28),1.28 (3H,s,H-29),0.72 (3H,s,H-30)；13C-NMR(150 MHz,C5D5N)δ:87.0 (C-20),84.7 (C-24),79.7(C-3),77.3 (C-12),71.7 (C-25),54.5 (C-9),51.4 (C-5),50.6 (C-14),49.6 (C-17),42.0 (C-8),41.5 (C-13),40.5 (C-10),38.4 (C-4),36.8 (C-7),36.1 (C-1),35.0(C-11),34.4 (C-22),32.1 (C-15),30.5 (C-29),28.3(C-27),27.3(C-2),26.8(C-23),26.3(C-26),25.0(C-21),23.4 (C-28),21.8 (C-16),18.8 (C-6),17.5 (C-18),17.3 (C-30),17.2 (C-19),3-O-arabinos 糖基的數(shù)據(jù) 為δ：106.8 (C-1′),84.5 (C-2′),79.8 (C-3′),86.1(C-4′),63.4 (C-5′)；12-O-quinovos 糖基的數(shù)據(jù)為δ：101.9 (C-1′′),76.0 (C-2′′),78.8 (C-3′′),79.8 (C-4′′),73.2(C-5′′),19.1(C-6′′)。其波譜數(shù)據(jù)與文獻(xiàn)報(bào)道基本一致[17]，故鑒定化合物10為青錢柳苷Ⅰ。

4 討論

對(duì)青錢柳活性成分的報(bào)道多集中在多糖、三萜上，本實(shí)驗(yàn)從青錢柳葉乙醇提取物的正丁醇萃取部位分離鑒定了10 個(gè)化合物，其中有6 個(gè)三萜類和3個(gè)木脂素類化合物，其中1個(gè)三萜類和3個(gè)木脂素類化合物為首次從青錢柳屬植物中分離得到，擴(kuò)大了對(duì)青錢柳化學(xué)成分多樣性的認(rèn)識(shí)，為青錢柳活性成分的研究和進(jìn)一步開發(fā)利用提供了參考。