黄独、光皮木瓜的化学成分及抗癌促发的活性研究
摘要
为有效利用天然的可食用植物资源，从中发现新的抗癌药物，本文通过体外活性筛选体系—对抗化学促癌剂TPA(12-O-tetradecannoylphorbol-13-acetate)诱导两种肿瘤克隆原细胞(clonogenic cell)在0.33％软琼脂糖培养基上集落形成作用，以平均抑制率为评价指标，从100多种可食用植物中筛选出了两种具有明显抗癌促发活性的植物--黄独(Dioscorea bulbifera L.)和光皮木瓜(Chaenomeles sinensis (Thouin) Koehne)，并对它们进行了系统的化学成分研究，同时，对有代表性的单体化合物进行了抗癌促发活性测试，分析总结了相应的构效关系。
实验结果表明：黄独块茎75％的乙醇提取物(DB75-E)与光皮木瓜枝条部分的甲醇提取物(CSM)具有明显的抑制癌促发活性。对JB-6两种肿瘤克隆原细胞集落形成的平均抑制率分别为76.5％、96.8％。DB75-E经溶媒CHCl_3(DB-C)、EtOAc(DB-A)、BuOH(DB-B)萃取后，回收溶剂得3种萃取物，对每种萃取物及残留水层组分DB-W进行活性检测，在各自最大测试浓度下，其平均抑制率分别为38.6％、100％、92.6％、66.4％。则乙酸乙酯萃取部分的活性最强，而正丁醇层也显出很强的抑制活性，通过反复硅胶柱色谱、制备薄层色谱及反相HPLC等分离手段从DB-C，DB-A，DB-B中共分得28个化合物，利用理化性质和谱学分析手段(UV、IR、~1H-NMR、~(13)C-NMR、MS、2D-NMR)鉴定了其中26个化合物的结构。从CHCl_3层中分得四种单体成分，为黄独素-B(diosbulbin-B，1)、胡萝卜苷(daucosterol，2)、β-谷甾醇(β-sitosterol，3)、棕榈酸(palmatic acid，4)，从乙酸乙酯层中，除分得化合物1、2、3、4外，还分离得到了琥珀酸(succinic，5)、莽草酸(shikimic acid，6)、3,5-二甲氧基山奈酚(3,5-dimethoxykaempferol，7)、 3,5,3′-三甲氧基槲皮素(3,5,3′-trimethoxyquercetin，8)、山核桃素(caryatin，9)、(+)儿茶素((+)catechin，10)、杨梅树皮素(myricetin，11)、杨梅树皮素-3-O-β-D-吡喃半乳糖苷(myricetin-3-O-β-D-galactopyranoside，12)、杨梅树皮素-3-O-β-D-吡喃葡萄糖苷(myricetin-3-O-β-D-glucopyranoside，13)、金丝桃苷(hyperoside，14)、山奈酚-3-O-β-D-吡喃半乳糖苷(kaempferol-3-O-β-D-galactopyranoside，15)、原儿茶酸(protocatechuic acid，16)、香草酸(vanillic acid，17)、异香草酸(isovanillic acid，18)等十四个单体成分，从正丁醇萃取物中分得了化合物10、11及(+)表儿茶素((+)epicatechin，19)、甲基-O-α-D-呋喃果糖苷(methyl-O-α-D-fructofuranoside，20)、丁基-O-α-D-呋喃果糖苷(butyl-O-α-D-fructofuranoside，21)、乙基-O-α-D-吡喃果糖苷(ethyl-O-α-D-fructopyranoside，22)、丁基-O-α-D-吡喃果糖苷(butyl-O-α-D-fructopyranoside，23)、苄基-O-β-D-吡喃葡萄糖苷(benzyl-O-β-D-glucopyranoside，24)、2-(4-甲氧基苯基)乙基-O-β-D-吡喃葡萄糖苷(2-(4-methoxyphenyl)ethyl-O-β-D-
日匕EB亡s乃丰一一白卜和尽e白慎t日b二t 摘要
glucoPyranoside，25）、3－苯基－2－烯丙醇基－O－60－＊g南葡萄糖昔（3－Phenyl－2－ProPenol－
O－卜D－glucopyranoside，26），其中化合物7、8． 9、11、12、13、14、15、16、20、
ZI、22、23、25、26等 15个化合物为首次从该属植物中分离得到的己知化合物，化
合物5、6、17、18、24为首次从该种植物中分离得到的已知化合物。
光皮木瓜枝条甲醇提取物CSM依次用溶媒EtOAC（CSMA卜BuOH（CSMB）革取
后回收溶剂得两种苹取物，对上述革取物和水层oSMW）进行活性测试，在各自最
大测试浓度下，对 JB＊两种肿瘤克隆原细胞集落形成的平均抑制率分别为 gi石％、
76．3％、44．9％。对活性最强的乙酸乙酯层进行化学分离，共分得27个化合物，利用
理化性质和波谱分析鉴定了23个的结构。其中被命名为木瓜酮（chaenomon）的化合
物27与化合物34诩扇－20（29）－烯－3 p，24，28－三醇，flip－20（29）－en－3 p，24，28－triol）、
48染料木素－5－O－p－D－回哺半辛糖昔，genistein－5－O－B－D－galactopyranoside）、49染料
木素－7－O－6－D－毗哺半孪糖苦，genistein－7－O B－D－gal砒top一side）为4个新化合物。
其余 19 个已知化合物分另为＊谷笛醇usitosterol，28）、古柯二醇（etythdio，29）、
马斯里酸（maslinic acid，30）、白晔酸（betulinic acid，31）、2 a－羟基白烨酸（2 a－
h叫r＊X加咖＊＊ic砒i＊，3二）、白烨脂醇O＊恤ifi，3＊）、3（ 个香豆酞基白掸月醇（－仰－p
coumaroylbetulin，35）、3－（斗卜香显酚基白烨脂醇（3－①．－coumaro＊lbetUlin，36）、3－闪－
阿魏酚基白烨月醇（3－（切－feruloylbetulin，37）、3仰－咖叫酚基白烨月醇（3－阅－
caffeoylbetulin，38）、2 a－羟基乌索酸（2 a－hydroxyursolic acid，39）、2 a，3 a，19 a－
三羟基乌索－20（29）－烯－28－酸（euscaphic砒id，40）、2 a，3 p，19 a－三羟基乌索－20（29－
烯－28－酸（tormellti Old，41）、lyonirysinolg’－O卜D－＊回葡?
In order to take an advantage of the edible plants and find most promising chemopreventers from the natural sources, by means of the assay- anti the promotion of JB-6 tumorigenic cell lines Cl 22, Cl 41 induced by TPA (Ing/ml, as a promoter) in vitro, the inhibitory effects of more than 100 kinds of plants were evaluated. Among them, 75% ethanol extract of the rhizomes of Dioscorea bulbifera L. (DB75-E) and methanol extract (CSM) from twigs of Chaenomeles sinensis (Thouin) Koehne showed potent the inhibitory effects on the tumor promotion. Further investigation on the chemical constituents of two kinds of plants were carried out, at same time, the activity of some typical compounds were examined, and relationship between the activity and structures were discussed.
DB75-E and CSM showed the potent activity on anti tumor promotion. The average of inhibitory rate were 76.5% and 96.8% respectively. The rhizome of Dioscorea bulbifera L.(Dioscoreaceae), as a traditional medicine was usually used to treat sore throat and struma in ancient times of China. Chaenomeles sinensis (Thouin) Koehne (Rosaceae) is a special woody plant of eastern Asia, and distributes widely in China and Japan. The fruit, called "Mu Gua" is a traditional medicine showed the activity against the pneumonia, pulmonary tuberculosis, cholera and so on.
DB75-E was suspended in water and partitioned successively with CHC13(DB-C), EtOAc(DB-A), BuOH(DB-B). The activity of every layer including the residues of aqueous (DB-W)was evaluated by the same method, and the average of inhibitory rates were 38.6%, 100%, 92.6%, 66.4% respectively at their maximal concentration. The fact was shown that the activity of DB-A was the most potent. Further investigation on the chemical constituents of the extract of Dioscorea bulbifera L. by the chromatographs and HPLC techniques, 28 compounds were isolated and the structures of 26 compounds were identified by the physical-chemical properties and spectral methods. They were elucidated as diosbulbin-B (1), daucosterol(2), P -sitosterol(3), palmatic acid(4), succinic acid(5), shikimic acid(6) 3,5-dimethoxykaempferol(7), 3,5,3 ' -trimethoxyquercetin(8), caryatin(9), (+)catechin(10), myricetin(11), myricetin-3-O- β -D-galactopyranoside(12), myricetin-3-O-β -D-glucopyranoside(13), hyperoside(14), kaempferol-3-O-β -D-galactopyranosid
e(15), protocatechuic acid (16), vanillic acid(17), isovanillic acid(18), (+)epicatechin(19), methyl-O- a - D-fructofuranoside (20), butyl-O- a - D-fructofuranoside, (21), ethyl-O- a -D-
fructopyranoside(22), butyl-O- a -D-fructopyranoside(23), benzyl-O-β -D-glucopyranoside(24), 2-(4-methoxyphenyl)ethyl-O-β -D-glucopyranoside(25), 3-phenyl-2-propenol-3-O-β -D-glucopyranoside(26). Among them compounds 7, 8, 9, 11, 12, 13, 14,15,16, 20, 21, 22, 23, 25, 26 were obtained from Dioscorea genus for the first time, and 5,6,17,18,24 were isolated from this plant for the first time.
One part of the extract of twigs of Chaenomeles sinensis (Thouin) Koehne was suspended in water and partitioned successively with EtOAc(CSMA),n-BuOH(CSMB). The bioactivity of each layer was tested by the same way. The EtOAc soluble fraction exhibited the most potent inhibitory effects on the tumorigenic cells' promotion. By the chromatographs and HPLC techniques, 27 compounds were isolated and the structures of 23 were elucidiated by the physical-chemical properties and spectral methods. Compounds 27(chaenomone), 34(lup-20(29)-en-3 P , 24, 28-triol), 48(genistein-5-O- β -D-galactopyranoside) and 49(genistein-7-O- β -D-galactopyranoside) were four novel constituents, while the other known components were identified as β -sitosterol(28), erythodiol(29), maslinic acid (30),betulinic acid(31), 2 a -hydroxybetulinic acid(32), betulin(33), 3-(E)-p-coumaroylbetulin(35), 3-(Z)-p-coumaroylbetulin(36), 3-(E)-
feruloylbetulin(37), 3- (E)-caffeoylbetulin(38), 2 a -hydroxyursolic acid (39), euscaphic acid(40), tormentic acid(41), lyoniresinol -9 ' -O- β -D- glucopyranoside(42), lyoni
实验结果表明：黄独块茎75％的乙醇提取物(DB75-E)与光皮木瓜枝条部分的甲醇提取物(CSM)具有明显的抑制癌促发活性。对JB-6两种肿瘤克隆原细胞集落形成的平均抑制率分别为76.5％、96.8％。DB75-E经溶媒CHCl_3(DB-C)、EtOAc(DB-A)、BuOH(DB-B)萃取后，回收溶剂得3种萃取物，对每种萃取物及残留水层组分DB-W进行活性检测，在各自最大测试浓度下，其平均抑制率分别为38.6％、100％、92.6％、66.4％。则乙酸乙酯萃取部分的活性最强，而正丁醇层也显出很强的抑制活性，通过反复硅胶柱色谱、制备薄层色谱及反相HPLC等分离手段从DB-C，DB-A，DB-B中共分得28个化合物，利用理化性质和谱学分析手段(UV、IR、~1H-NMR、~(13)C-NMR、MS、2D-NMR)鉴定了其中26个化合物的结构。从CHCl_3层中分得四种单体成分，为黄独素-B(diosbulbin-B，1)、胡萝卜苷(daucosterol，2)、β-谷甾醇(β-sitosterol，3)、棕榈酸(palmatic acid，4)，从乙酸乙酯层中，除分得化合物1、2、3、4外，还分离得到了琥珀酸(succinic，5)、莽草酸(shikimic acid，6)、3,5-二甲氧基山奈酚(3,5-dimethoxykaempferol，7)、 3,5,3′-三甲氧基槲皮素(3,5,3′-trimethoxyquercetin，8)、山核桃素(caryatin，9)、(+)儿茶素((+)catechin，10)、杨梅树皮素(myricetin，11)、杨梅树皮素-3-O-β-D-吡喃半乳糖苷(myricetin-3-O-β-D-galactopyranoside，12)、杨梅树皮素-3-O-β-D-吡喃葡萄糖苷(myricetin-3-O-β-D-glucopyranoside，13)、金丝桃苷(hyperoside，14)、山奈酚-3-O-β-D-吡喃半乳糖苷(kaempferol-3-O-β-D-galactopyranoside，15)、原儿茶酸(protocatechuic acid，16)、香草酸(vanillic acid，17)、异香草酸(isovanillic acid，18)等十四个单体成分，从正丁醇萃取物中分得了化合物10、11及(+)表儿茶素((+)epicatechin，19)、甲基-O-α-D-呋喃果糖苷(methyl-O-α-D-fructofuranoside，20)、丁基-O-α-D-呋喃果糖苷(butyl-O-α-D-fructofuranoside，21)、乙基-O-α-D-吡喃果糖苷(ethyl-O-α-D-fructopyranoside，22)、丁基-O-α-D-吡喃果糖苷(butyl-O-α-D-fructopyranoside，23)、苄基-O-β-D-吡喃葡萄糖苷(benzyl-O-β-D-glucopyranoside，24)、2-(4-甲氧基苯基)乙基-O-β-D-吡喃葡萄糖苷(2-(4-methoxyphenyl)ethyl-O-β-D-
日匕EB亡s乃丰一一白卜和尽e白慎t日b二t 摘要
glucoPyranoside，25）、3－苯基－2－烯丙醇基－O－60－＊g南葡萄糖昔（3－Phenyl－2－ProPenol－
O－卜D－glucopyranoside，26），其中化合物7、8． 9、11、12、13、14、15、16、20、
ZI、22、23、25、26等 15个化合物为首次从该属植物中分离得到的己知化合物，化
合物5、6、17、18、24为首次从该种植物中分离得到的已知化合物。
光皮木瓜枝条甲醇提取物CSM依次用溶媒EtOAC（CSMA卜BuOH（CSMB）革取
后回收溶剂得两种苹取物，对上述革取物和水层oSMW）进行活性测试，在各自最
大测试浓度下，对 JB＊两种肿瘤克隆原细胞集落形成的平均抑制率分别为 gi石％、
76．3％、44．9％。对活性最强的乙酸乙酯层进行化学分离，共分得27个化合物，利用
理化性质和波谱分析鉴定了23个的结构。其中被命名为木瓜酮（chaenomon）的化合
物27与化合物34诩扇－20（29）－烯－3 p，24，28－三醇，flip－20（29）－en－3 p，24，28－triol）、
48染料木素－5－O－p－D－回哺半辛糖昔，genistein－5－O－B－D－galactopyranoside）、49染料
木素－7－O－6－D－毗哺半孪糖苦，genistein－7－O B－D－gal砒top一side）为4个新化合物。
其余 19 个已知化合物分另为＊谷笛醇usitosterol，28）、古柯二醇（etythdio，29）、
马斯里酸（maslinic acid，30）、白晔酸（betulinic acid，31）、2 a－羟基白烨酸（2 a－
h叫r＊X加咖＊＊ic砒i＊，3二）、白烨脂醇O＊恤ifi，3＊）、3（ 个香豆酞基白掸月醇（－仰－p
coumaroylbetulin，35）、3－（斗卜香显酚基白烨脂醇（3－①．－coumaro＊lbetUlin，36）、3－闪－
阿魏酚基白烨月醇（3－（切－feruloylbetulin，37）、3仰－咖叫酚基白烨月醇（3－阅－
caffeoylbetulin，38）、2 a－羟基乌索酸（2 a－hydroxyursolic acid，39）、2 a，3 a，19 a－
三羟基乌索－20（29）－烯－28－酸（euscaphic砒id，40）、2 a，3 p，19 a－三羟基乌索－20（29－
烯－28－酸（tormellti Old，41）、lyonirysinolg’－O卜D－＊回葡?
In order to take an advantage of the edible plants and find most promising chemopreventers from the natural sources, by means of the assay- anti the promotion of JB-6 tumorigenic cell lines Cl 22, Cl 41 induced by TPA (Ing/ml, as a promoter) in vitro, the inhibitory effects of more than 100 kinds of plants were evaluated. Among them, 75% ethanol extract of the rhizomes of Dioscorea bulbifera L. (DB75-E) and methanol extract (CSM) from twigs of Chaenomeles sinensis (Thouin) Koehne showed potent the inhibitory effects on the tumor promotion. Further investigation on the chemical constituents of two kinds of plants were carried out, at same time, the activity of some typical compounds were examined, and relationship between the activity and structures were discussed.
DB75-E and CSM showed the potent activity on anti tumor promotion. The average of inhibitory rate were 76.5% and 96.8% respectively. The rhizome of Dioscorea bulbifera L.(Dioscoreaceae), as a traditional medicine was usually used to treat sore throat and struma in ancient times of China. Chaenomeles sinensis (Thouin) Koehne (Rosaceae) is a special woody plant of eastern Asia, and distributes widely in China and Japan. The fruit, called "Mu Gua" is a traditional medicine showed the activity against the pneumonia, pulmonary tuberculosis, cholera and so on.
DB75-E was suspended in water and partitioned successively with CHC13(DB-C), EtOAc(DB-A), BuOH(DB-B). The activity of every layer including the residues of aqueous (DB-W)was evaluated by the same method, and the average of inhibitory rates were 38.6%, 100%, 92.6%, 66.4% respectively at their maximal concentration. The fact was shown that the activity of DB-A was the most potent. Further investigation on the chemical constituents of the extract of Dioscorea bulbifera L. by the chromatographs and HPLC techniques, 28 compounds were isolated and the structures of 26 compounds were identified by the physical-chemical properties and spectral methods. They were elucidated as diosbulbin-B (1), daucosterol(2), P -sitosterol(3), palmatic acid(4), succinic acid(5), shikimic acid(6) 3,5-dimethoxykaempferol(7), 3,5,3 ' -trimethoxyquercetin(8), caryatin(9), (+)catechin(10), myricetin(11), myricetin-3-O- β -D-galactopyranoside(12), myricetin-3-O-β -D-glucopyranoside(13), hyperoside(14), kaempferol-3-O-β -D-galactopyranosid
e(15), protocatechuic acid (16), vanillic acid(17), isovanillic acid(18), (+)epicatechin(19), methyl-O- a - D-fructofuranoside (20), butyl-O- a - D-fructofuranoside, (21), ethyl-O- a -D-
fructopyranoside(22), butyl-O- a -D-fructopyranoside(23), benzyl-O-β -D-glucopyranoside(24), 2-(4-methoxyphenyl)ethyl-O-β -D-glucopyranoside(25), 3-phenyl-2-propenol-3-O-β -D-glucopyranoside(26). Among them compounds 7, 8, 9, 11, 12, 13, 14,15,16, 20, 21, 22, 23, 25, 26 were obtained from Dioscorea genus for the first time, and 5,6,17,18,24 were isolated from this plant for the first time.
One part of the extract of twigs of Chaenomeles sinensis (Thouin) Koehne was suspended in water and partitioned successively with EtOAc(CSMA),n-BuOH(CSMB). The bioactivity of each layer was tested by the same way. The EtOAc soluble fraction exhibited the most potent inhibitory effects on the tumorigenic cells' promotion. By the chromatographs and HPLC techniques, 27 compounds were isolated and the structures of 23 were elucidiated by the physical-chemical properties and spectral methods. Compounds 27(chaenomone), 34(lup-20(29)-en-3 P , 24, 28-triol), 48(genistein-5-O- β -D-galactopyranoside) and 49(genistein-7-O- β -D-galactopyranoside) were four novel constituents, while the other known components were identified as β -sitosterol(28), erythodiol(29), maslinic acid (30),betulinic acid(31), 2 a -hydroxybetulinic acid(32), betulin(33), 3-(E)-p-coumaroylbetulin(35), 3-(Z)-p-coumaroylbetulin(36), 3-(E)-
feruloylbetulin(37), 3- (E)-caffeoylbetulin(38), 2 a -hydroxyursolic acid (39), euscaphic acid(40), tormentic acid(41), lyoniresinol -9 ' -O- β -D- glucopyranoside(42), lyoni
引文
1．肿瘤化学预防及药物治疗 韩锐 北京医科大学中国协和医科大学联合出版社1991 P 3．
2．《抗癌药物研究与实验技术》中国协和医科大学北京医科大学联合出版社韩锐 1997 P 3．
3. Genes and signal transduction in tumor promotion, Thomas D.G., Yoshiyuki Nakamura, Linda A. et al, Carcinogenesis ,8, 1985, 341～367.
4．天然食物中药用成分的研究进展 陆阳，赵霞，陈聪颖等，国外医药植物药分册，1999，14(12)；52～57．
5．中华本草 上海科学技术出版社 1999(8卷) p7278～7280．
6．黄药子古今临床应用研究．唐迎雪，中国中药杂志 1995，20(7)：435～438．
7．黄药子的研究概况．徐增莱，丁志尊；中草药，1998，29(2)：125～128．
8．中国薯蓣属根状茎组系统分类的初步研究．裴鉴 丁志遵 秦慧贞等，植物分类学报，1979，17(3)：61～71．
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