王不留行环肽研究

王不留行环肽研究 3 王不留行环肽研究 12 张荣平邹澄谭宁华周俊 ( ) 中国科学院昆明植物研究所植物化学开放实验室 , 昆明 650204 ( ) 摘要 从中药王不留行 V acca ri a seget alis种子中分离并鉴定了 4 个环肽化合物 , 分别命名为王不 ( ) 留行环肽 A , B , C , D vaccarins A , B , C , D, 其中王不留行环肽 A 为新环肽化合物 。其结构通过光 1 2 ) ( 谱和化学方法分别确定为 : vaccarin A — —cyclo - Trp - Ala- Gly - Val - Ala, vaccarin B ———cy2 1 2 1 2( ) ( clo - Pro - Gly - Leu - Ser - Phe- Ala - Phe, vaccarin C ———cyclo - Pro- Gly - Tyr - Val - Pro 2 1 ) ) ( . Gly - Val- Leu - Trp, vaccarin D ———cyclo - Pro - Val- Trp - Ala - 关键词 王不留行 , 石竹科 , 王不留行环肽 A , 王不留行环肽 B , 王不留行环肽 C , 王不留行环肽 D分类号 Q 946 Cyclopept ides f rom Vaccar ia segetal is 21ZHAN G Ro ng - Ping ZOU Cheng TAN Ning - Hua ZHOU J un ( )L aboratory of Phytochem ist ry , Ku n m i ng I nst it ute of B ot any , T he Chi nese A cadem y of S ciences , Kunming 650204 Abstract Fro m t he seeds of V acca ri a seget al is , a well - known t raditio nal Chinese medicine named “wang bu liu xing”, four cyclopep tides vaccarins A - D were isolated in which o nly vaccarin A is a new cyclopep tide. Their st ruct ures were elucidated by spect ral and chemical met hods as vaccarin A ———cyclo 1 2 1 ( ) ( - Trp - Ala- Gly - Val - Ala, vaccarin B ———cyclo - Pro - Gly - L eu - Ser - Phe- Ala - 2 1 2 ) ( ) ( Phe, vaccarin C ———cyclo - Pro- Gly - Tyr - Val - Pro- L eu - Trp, vaccarin D ———cyclo - Pro 1 2 ) - Val- Trp - Ala - Gly - Val. Key words V acca ri a seget al is , Caryop hyllaceae , vaccarin A , vaccarin B , vaccarin C , vaccarin D ( ) ( ) V acca ri a seget alis Neck. Garcke Caryop hyllaceae, dist ributed all over China except Sout h China , is used as a well - known t raditional Chinese medicine t reated in amenorrhea , milk secretion blockin g , dystocia , carbuncle , and () blooding J iangsu College of New Medicine , 1995. As part s of our investigations on cyclopeptides in Caryop hyllaceae ( ) ( plant s Tan et al , 1993 ; Zhao et al , 1995 ; Zhang et al , 1997 ; Wang et al , 1998,in t he communication Zhang ) et al , 1995we reported one new cyclopeptide vaccarin A f rom V acca ri a seget alis which is t he first cyclopeptide found in t he genus of V acca ri a. In t his paper we describe t he isolation and st ruct ure determination of vaccarin A and ot her t hree cyclopeptides vaccarins B , C , D f rom t he plant based on spect ral and chemical met hods. 3 This work was supported by grant s f ro m Nat ural Science Foundatio n of China and Laboratory of Phytochemist ry , Kunming Instit ute of Botany , The Chinese Academy of Sciences. 1 Aut her for correspo ndence. 2 Address now : Faculty of Pharmacy , Kunming Medical College , Kunming 650031 1997 - 11 - 20 收稿 RES UL TS A ND D ISCUSSIO N ext racted wit h The EtO H ext ract of t he dried seeds of V acca ri a seget alis was suspended in HO and 2 () pet roleum , EtOAc and n - BuO H respectively. Removal of solvent f urnished an EtOAc f raction 147 g. The E2 ( tOAc f raction was repeatedly chromatograp hed on a silical gel and a RP - 18 column and afforded vaccarin A 538 ) () () () mg, vaccarin B 205 mg, vaccarin C 317 mgand vaccarin D 316 mg, respectively. 25 . 5 α() Vaccarin A ( 1) disc crystals , ]- 104 . 23?c , 0 . 662 , DMSO,gave a negative ninhydrin reaction ,but D positive when hydrolyzed wit h 6 mol/ L HCl. It s moleclular formula was deduced as CHONby means of D EP T 24 32 5 6 + ( ) spect ral analysis and FAB - MS M + 1at m/ z 485 . The IR s pect rum in KBr disc showed intense amide C = O - 1 - 1 ( ) at 1666 , 1650 cm and amide N - H at 3336 cm . In t he D EP Tspect rum DMSO, a total of five amide CO signals could be seen between 169 . 5 and 172 . 1 pp m. Meanwhile , t he middle and high field signals of five met hines , two met hylenes , four met hyls , and seven low field signals between 109 . 9 and 136 . 0 pp m were identified. The low field signals showed t he p resence of one indole group . From t hese data , vaccarin A appeared to be a pentapeptide. By means of 2D NMR techniques , t he amino acid composition could be identified to be one t r yptop han , two ala2 nines , one glycine , one valine , which were in correspondence wit h t hat of amino acid analysis af ter complete acidic () hydrolysis 6 mol/ L HCl , 110 ?, 24 h. For t he compound gave a negative response to ninhydrin test , it must be a cyclic pentapeptide. The amino acid sequence could be determined p reliminarily by positive FAB - MS which showed t he f ragment s of I to V I as following : + I m/ z 228 Ala - Gl y - Val + H + II m/ z 299 Ala - Gl y - Val - Ala + H + III m/ z 258 Ala - Tr p + H + IV m/ z 329 Ala - Tr p - Ala + H + V m/ z 386 Ala - Tr p - Ala - Gly + H + V I m/ z 485 Ala - Tr p - Ala - Gly - Val + H ( ) These f ragment s could give t he gross st ruct ure as cyclo - Trp - Ala - Gly - Val - Ala. 1 1 1 13 Furt her evidences were p rovided by H - H COS Y , H - C COS Y , TOSC Y and COLOC spect ra. At first we ( assigned p roton and carbon singals of every amino acid residues wit h t hose 2D NMR experiment s The data are shown ) in Table 1, and t hen determined t he sequence of amino acid residues based on t he correlations between amide CO and ( ) N H in COLOC spect ra as following Tan et al , 1993: Therefore , t he st ruct ure of t he compound named vaccarin A , a new pentacyclopeptide , was elucidated as cyclo - ( ) t ryptop hyl - alanyl - glycyl - valyl - alanyl. 20 α() Vaccarin B ( 2) crystals , ]+ 5 . 58?c , 0 . 448 , CHCl. It s moleclular formula was deduced as CHO D3 37 49 8 + ( ) Nby means of D EP T spect ral analysis and FAB - MS M + 1 at m/ z 720 . The IR s pect rum in KBr disc 7 - 1 - 1 () showed intense amide C = O at 1650 , 1616 cm and amide N - H at 3310 cm . In t he D EP T spect rum DMSO, a total of seven amide CO signals could be seen between 168 . 6 and 171 . 3 pp m. Meanwhile , t he middle and high field signals of seven met hines , eight met hylenes , t hree met hyls , and t he low field signals of two p henyls were identified. Applications of 2D NMR techniques and amino acid analysis af ter hydrolysis , t he amino acid composition could be i2 dentified to be one p roline , one glycine , one leucine , one serine , two p henylalanines , one alanine. These data indicat 2 ed t hat vaccarin B appeared to be a cyclic heptapeptide. The amino acid sequence could be determined p reliminarily by positive FAB - MS which showed t he f ragment s of I to V I as following : + I m/ z 155 Gl y + H Pro - + 268 II m/ z Gl y - Leu + H Pro - + III m/ z 355 Pro - Gl y - Leu - Ser + H + IV m/ z 502 Pro - Gl y - Leu - Ser - Phe + H + V m/ z 573 Pro - Gl y - Leu - Ser - Phe - Ala + H + V I m/ z 720 Pro - Gl y - Leu - Ser - Phe - Ala - Phe + H ( Therefore , t he gross st ruct ure of t he compound could be deducde as cyclo - Pro - Gly - Leu - Ser - Phe - Ala - ) Phe. Wit h t he same met hods mentioned above , we could determine t he se quence of amino acid residues as t he follow2 ing peptide based on t he correlations between amide CO and N H in COLOC spect ra : (Then t he st ruct ure of t he compound named vaccarin B , a heptacyclopeptide , was elucidated as cyclo - p rolyl - ) glycyl - leucyl - seryl - p henylalanyl - alanyl - p henylalanyl. 19 α() Vaccarin C ( 3) amorp hous powder , ]- 47 . 85?c , 0 . 768 , CHO H. It s moleclular formula was deduced D 3 + ( ) as CHONby means of D EP T spect ral analysis and FAB - MS M + 1 at m/ z 813 . The IR s pect rum in 43 56 8 8 - 1 - 1 KBr disc showed intense amide C = O at 1660,1611 cm and amide N - H at 3346,3240 cm . In t he D EP T () spect rum DMSO, a total of seven amide CO signals could be seen between 171 . 1 and 174 . 6 pp m. Meanwhile , t he middle and high field signals of eight met hines , ten met hylenes , four met hyls , one indole group and one p - hydrox2 yp henyl were identified. Applications of 2D NMR techniques and amino acid analysis af ter hydrolysis , t he amino acid composition could be identified to be two p rolines , one glycine , one t yrosine , one valanine , one leucine , one t r ypto2 p han. These data indicated t hat vaccarin C appeared to be a cyclic heptapeptide too . The amino acid sequence could be determined p reliminarily by positive FAB - MS which showed t he f ragment s of I to V as following : + I m/ z 318 Pro - Gly - Tyr + H + 417 II m/ z - Gly - Tyr - Val + H Pro + III m/ z 514 Pro - Gly - Tyr - Val - Pro + H + IV m/ z 627 Pro - Gly - Tyr - Val - Pro - Leu + H + V m/ z 813 Pro - Gly - Tyr - Val - Pro - Leu - Trp + H ( So t he gross st ruct ure of t he compound could be deduced as cyclo - - Gly - Pro ) Tyr - Val - Pro - Leu - Trp. Wit h t he same met hods mentioned above , we could determined t he partial sequence of amino acid residues as t he () () following peptides 1and 2based on t he correlations between amide CO and N H in COLOC spect ra : Therefore , t he st ruct ure of t he compound named vaccarin C , a heptacyclopeptide , was elucidated as cyclo - () p rolyl - glycyl - t yrosyl - valyl - p rolyl - leucyl - t ryptop hyl. 24 α() Vaccarin D ( 4) amorp hous powder , ]- 95 . 26?c , 0 . 454 , CHCl. It s moleclular formula was deduced D3 + ( ) as CHONby means of D EP T spect ral analysis and FAB - MS M + 1at m/ z 610 . The IR s pect rum in 31 43 6 7 - 1 - 1 KBr disc showed intense amide C = O at 1658 , 1630 cm and amide N - H at 3330 , 3234 cm . In t he D EP T () spect rum DMSO, a total of six amide CO signals could be seen between 169 . 2 and 172 . 8 pp m. Meanwhile , t he middle and high field signals of seven met hines , five met hylenes , five met hyls and one indole group were identified. Applications of 2D NMR techniques and amino acid analysis af ter hydrolysis , t he amino acid composition could be i2 dentified to be one p roline , two valanines , one t ryptop han , one alanine , one glycine. These data indicated t hat vac2 carin D appeared to be a cyclic hexapeptide. The amino acid sequence could be determined p reliminarily by positive FAB - MS which showed t he f ragment s of I to V as following : + ,I m/ z 258 Tr p - Ala + H + II m/ z 315 Gly + H Tr p - Ala - + III m/ z 414 Tr p - Ala - Gly - Val + H + IV m/ z 511 Gly - Val - Pro + H Tr p - Ala - + V m/ z 610 Gly - Val - Pro - Val + H Tr p - Ala - ( ) So t he gross st ruct ure of t he compound could be deduced as cyclo - Trp - Ala - Gly - Val - Pro - Val. Wit h t he same met hods mentioned above , we could determined t he partial sequence of amino acid residues as t he () () following peptides 1and 2based on t he correlations between amide CO and N H in COLOC spect ra : (Therefore , t he st ruct ure of t he compound named vaccarin D , a hexacyclopeptide , was elucidated as cyclo - p ro2 ) lyl - valyl - t ryptop hyl - alanyl - glycyl - valyl. EXPERIMENTAL General Mp s. uncorr . Optical rotations were recorded on SEPA - 300 wit h 2 cm cell. IR were taken for KBr disc. NMR were measured wit h AMX - 400 spect rometer using TMS as t he internal standard. 1 13 ( ) ( ) Table 1 H and C NMR spect ral data of vaccarins A 1and B 2in DMSO , 400 HMz for δδ, 100 M Hz for, TMS H C ( ) ( ) vaccari n A 1vaccari n B 2 Amino acid Amino acid H C H C residues residues Trp Pro α α ( )( )4 . 21 1 H ,m 4 . 12 1 H ,m 56 . 1 61 . 0 β β ( )( ) ( )3 . 20 2 H ,dd ,14 . 4 ,6 . 4 2 . 04 1 H ,m,1 . 72 1 H ,m 28 . 9 26 . 3 γ indole ( )( )10 . 86 1 H ,d ,2 . 0 1 . 82 2 H ,m 24 . 4 1 δ ( )( ) ( )7 . 12 1 H ,d ,2 . 0 3 . 48 1 H ,m,2 . 92 1 H ,m 47 . 4 2 123 . 5 CO 3 109 . 9 171 . 1 Gly 4 127 . 4 α ( )( )5 118 . 2 42 . 5 7 . 54 1 H ,d ,7 . 6 4 . 13 1 H ,m ( )( )6 7 . 06 1 H ,dd ,7 . 6 ,6 . 8 120 . 9 3 . 33 1 H ,dd ,16 . 8 ,4 . 3 ( )7 ( )6 . 97 1 H ,dd ,8 . 0 ,7 . 2 118 . 2 N H 8 . 84 1 H ,m 8 ( )111 . 3 CO 7 . 33 1 H ,d ,8 . 4 168 . 6 136 . 0 9 Leu α ( )( )7 . 93 1 H ,m N H 4 . 53 1 H ,d ,10 . 0 52 . 4 β ( )CO 171 . 0 1 . 55 2 H ,m 42 . 9 γ 1 ( )1 . 56 1 H ,m 24 . 0 Alaδ α (() )( )0. 7 73 H ,d ,5. 7,0. 73 H2 ,d ,5. 7 22 . 6 ,21 . 6 4 . 07 1 H ,m 49 . 7 N H β ( )( )8 . 06 1 H ,d ,10 . 3 1 . 22 3 H ,d ,7 . 2 17 . 0 CO N H ( )7 . 98 1 H ,d ,8 . 8 171 . 1 Ser CO α 171 . 3 Gly β ( )4 . 41 1 H ,d ,6 . 1 54 . 4 αN H ((( ) ( )) )43 . 4 4 . 10 1 H ,m,3 . 28 1 H ,m 4. 1 21 H ,m,3. 715 H ,d ,22. 062 . 2 N H CO ( )( )8 . 44 1 H ,t ,5 . 6 8 . 43 1 H ,d ,6 . 4 CO 1 169 . 5 170 . 3 PheVal α α β ( )( )3 . 90 1 H ,t ,7 . 6 59 . 4 4 . 19 1 H ,m 56 . 6 β φ ( )( )γ 1 . 96 1 H ,m 29 . 6 3 . 02 2 H ,d ,6 . 4 35 . 7 (() )( ) 0. 8 73 H ,d ,5. 6,0. 83 H5 ,d ,6. 4 19 . 0 ,18 . 0 7 . 16,7 . 32 5 H ,m126 . 4 N H ( )128 . 2 7 . 75 1 H ,d ,8 . 0 CO 2 170 . 3 128 . 9 Alaα 137 . 3 β ( )( )8 . 51 1 H ,d ,4 . 5 4 . 19 1 H ,m 48 . 4 N H N H ( )CO 170 . 3 1 . 19 3 H ,d ,6 . 8 16 . 9 CO ( )7 . 95 1 H ,m Ala α ( )172 . 1 4 . 11 1 H ,m 49 . 0 β ( )1 . 04 3 H ,d ,7 . 3 17 . 8 N H ( )7 . 73 1 H ,d ,8 . 6 CO 171 . 3 2 Pheα ( )4 . 72 1 H ,dd ,14 . 0 ,8 . 0 51 . 8 β ( )2 . 99 1 H ,m 38 . 5 ( )2 . 65 1 H ,dd ,13 . 2 ,5 . 8 ( ) φ126 . 4 7 . 16,7 . 32 5 H ,m 128 . 2 129 . 3 136 . 7 ( )N H 7 . 22 1 H ,m CO 169 . 0 1 13 δδ( ) ( ) Table 2 H and C NMR spect ral data of vaccarins C 3and D 4in DMSO , 400 M Hz for , 100 HMz for, H C TMS ( ) ( ) vaccari n C 3vaccari n D 4 Amino acid Amino acid H C H C residues residues 1 Pro Proα α ( )( )4 . 15 1 H ,m 64 . 0 4 . 33 1 H ,d ,8 . 0 60 . 3 β β ( ) ( )( ) ( )2 . 23 1 H ,m,1 . 83 1 H ,m 29 . 8 2 . 09 1 H ,m,1 . 90 1 H ,m 31 . 3 γ γ ( ) ( )( ) ( )1 . 92 1 H ,m,1 . 52 1 H ,m 22 . 6 1 . 90 1 H ,m,1 . 70 1 H ,m 21 . 4 δ δ ( ) ( )( )3 . 51 1 H ,m,3 . 28 1 H ,m 47 . 5 3 . 56 2 H ,dd ,8 . 8 ,4 . 8 46 . 7 CO CO 1174 . 6 171 . 5 Val Gly α α ( )( )43 . 6 3 . 69 2 H ,m 4 . 11 1 H ,d ,10 . 0 59 . 8 βN H ( )( )2 . 02 1 H ,m 30 . 0 7 . 97 1 H ,m γCO (() )171 . 1 0. 7 53 H ,d ,3. 6,0. 73 H4 ,d ,3. 6 19 . 1 ,18 . 4 N H Tyr ( )α 7 . 34 1 H ,d ,8 . 4 CO β ( )172 . 5 4 . 61 1 H ,m 55 . 8 Trp α ( )2 . 70 1 H ,t ,12 . 0 36 . 8 β ( )( )2 . 60 1 H ,dd ,18 . 0 ,4 . 0 4 . 25 1 H ,d ,6 . 4 55 . 6 indole 1 (() ) ( )6. 6 32 H ,d ,6. 0,6. 92 H4 ,d ,8. 4 ψ116 . 0 3 . 10 2 H ,m 25 . 9 2 ( )10 . 9 1 H ,s 130 . 2 3 ( )131 . 4 7 . 13 1 H ,d ,1 . 6 123 . 3 4 156 . 4 109 . 2 5 ( )127 . 0 N H 8 . 31 1 H ,t ,6 . 5 6 ( )CO 118 . 3 173 . 1 7 . 54 1 H ,d ,7 . 6 7 ( )Val 7 . 06 1 H ,t ,7 . 2 121 . 0 α 8 ( )( )118 . 3 6 . 96 1 H ,t ,7 . 2 3 . 92 1 H ,d ,7 . 4 60 . 1 β 9 ( )( )111 . 3 1 . 95 1 H ,m 30 . 9 7 . 33 1 H ,m γN H (() )1. 0 13 H ,d ,6. 7,0. 93 H3 ,d ,7. 0 136 . 1 19 . 5 ,19 . 1 N H CO ( )( )8 . 39 1 H ,d ,4 . 0 8 . 51 1 H ,d ,6 . 0 CO Ala 173 . 2 172 . 8 2 α Proβ α ( )( )β 4 . 59 1 H ,m 62 . 5 3 . 65 1 H ,m 49 . 4 N H ( )( )2 . 41 1 H ,dd ,16 . 0 ,10 . 0 1 . 12 3 H ,d ,6 . 8 32 . 0 15 . 4 CO ( )( )1 . 89 1 H ,m 8 . 98 1 H ,d ,6 . 8 Gly ( ) ( ) γ2 . 06 1 H ,m,1 . 84 1 H ,m 26 . 7 171 . 2 α ( ) ( )δ48 . 4 3 . 68 1 H ,m,3 . 50 1 H ,m N H ( ) ( )CO 173 . 2 3 . 65 1 H ,m,3 . 35 1 H ,m 42 . 8 CO ( )2 7 . 44 1 H ,t ,5 . 2 Leu Val( )α 169 . 2 4 . 17 1 H ,m 56 . 5 α β ( ) ( )β1 . 58 1 H ,m,1 . 26 1 H ,m 41 . 3 γ ( )( )γ1 . 43 1 H ,m 26 . 3 4 . 51 1 H ,m 55 . 3 (() )( )δ0. 8 13 H ,d ,6. 5,0. 73 H7 ,d ,6. 5 23 . 3 ,21 . 3 2 . 17 1 H ,m 29 . 7 ( )( )8 . 39 1 H ,d ,4 . 0 0 . 92 3 H ,d ,10 . 4 19 . 4 ,17 . 4 N H ( )CO 174 . 6 0 . 80 3 H ,d ,6 . 8 ( )Trp 7 . 41 1 H ,d ,9 . 2 N H α ( )CO 170 . 7 5 . 00 1 H ,m 54 . 1 β ( )3 . 53 1 H ,m 26 . 7 ( )3 . 23 1 H ,dd ,13 . 4 ,5 . 4 indole 1 ( )2 7 . 26 1 H ,d ,9 . 2 125 . 7 3 109 . 4 4 129 . 4 ( )5 120 . 1 7 . 02 1 H ,m ( )6 7 . 02 1 H ,m 122 . 5 ( )7 119 . 3 7 . 63 1 H ,d ,7 . 2 8 ( )112 . 6 7 . 26 1 H ,d ,9 . 2 9 137 . 5 ( )7 . 74 1 H ,d ,7 . 2 N H CO 172 . 7 FAB - MS were determined wit h V G Autospec - 3000 mass spect rometer . ( ) Extraction and isolation The powdered seeds of V acca ri a seget alis 15 kgwere ext racted wit h 95 % EtO H t hree times at t he refluex condition. Removal of t he solvent under reduced p ressure , t he EtO H syrup was suspended in HO and ext racted wit h pet roleum , EtOAc and n - BuO H , respectively. Then t he EtOAc solutions were evaporat2 2 () ed and t he residues 147 gwere subjected to a silica gel column eluting wit h CHCl- MeO H. Then by combination 3 () () ()of a silical gel and RP - 18 column , we obtained vaccarin A 538 mg, vaccarin B 205 mg, vaccarin C 317 mg () and vaccarin D 316 mg, respectively. 25 . 5 α( ( ) ?, ]- 104 . 23?c , 0 . 662 , Vaccarin A ( 1) CHON, disc crystals MeO H, mp 268 . 0 ,273 . 5 24 32 5 6 D KBr- 1 + ) ν( ) ( ) ( ) DMSO. cm : 3336 , 1666 , 1650 . Pos. FAB - MS m/ z : 485 M + 1, 40 , 386 14 , 329 4 , 299max 1 13 () () () ( ) ( ) 17, 258 18, 228 27. H and C NMR : see Table 1 . Amino acid anal ysis standard met hod: Trp 1eq, () () () Ala 2eq, Gly 1eq, and Val 1eq. Amino acid analysis of Vaccarin A ( 1) : The hydrolysate of vaccarin A af ter hydrolysis wit h 6 mol/ L HCl at 110 ?for 24 h was analysed for amino acids using t he standard met hod. 20 α( ) Vaccarin B ( 2) CHON, cubic crystals , mp 167 . 0 ,170 . 0 ?, ]+ 5 . 58?c , 0 . 448 , CHCl3 . 37 49 8 7 D - 1 + KBr ν( ) ( ) ( ) ( ) cm: 3310 , 1650 , 1616 . Pos. FAB - MS m/ z : 720 M + 1, 76 , 573 2 . 5, 502 1 . 8, 355 4 . 5, max 1 13 () () ( ) ( ) 268 6 . 5, 155 15 . 0. H and C NMR : see Table 1 . Amino acid analysis standard met hod: Ser 1eq, Leu () () () () () 1eq, Ala 1eq, Pro 1eq, Phe 2eq, and Gly 1eq. 19 Vaccarin C ( 3) α(CHON, amorp hous powder , mp 187 . 0,189 . 5 ?, ]- 47 . 85?c , 0 . 768 , CH 43 56 8 8 D3KBr - 1 + ) ν( ) ( ) ( ) O H. cm : 3346 ,3240 , 1660 ,1611 . Pos. FAB - MS m/ z : 813 M + 1, 100 , 627 3, 514 3 ,max 1 13 () ( ) ( ) ( ) 417 8, 318 16 . H and C NMR : see Table 2 . Amino acid anal ysis standard met hod: Tyr 1eq, Trp () () () () () 1eq, Val 1eq, Leu 1eq, Pro 2eq, and Gly 1eq. 24 Vaccarin D ( 4) α( CHON, amorp hous powder , mp 184 . 0 , 185 . 5 ?, ]- 95 . 26?c , 0 . 454 , 31 43 6 7 D KBr - 1 + ) ν( ) () (CHCl. cm : 3330 , 3234 , 1658 , 1630 . Pos. FAB - MS m/ z : 610 M + 1, 77 , 511 4, 414 1 . max 3 1 13 ) () () ( ) () 5, 315 4, 258 2 . 5. H and C NMR : see Table 2 . Amino acid analysis standard met hod: Trp 1eq, Ala () () () () 1eq, Pro 1eq, Gly 1eq, and Val 2eq. Ref erences J iangsu College of New Medicine , 1995 . Zho ngyao Dacidian , Vol . 1 . Shanghai : The Shanghai Scientific and Technological Press , 311,312 Tan N H , Zhou J , Chen C X , et al , 1993 . Cyclopeptides f ro m t he root s of Pseu dostell ari a heterophyll a. Phytochem ist ry , 32 ( ) 5: 1327,1330 Tan N H , Wang D Z , Zhang H J , et al , 1993 . Determinatio n of amino acid sequences of t wo new cyclopeptides by 2D NMR. ( ) Chi nese Jou r nal of M agnet ic Resonance , 10 1: 69,74 Wang Y C , Tan N H , Zhou J , et al , 1998 . Cyclopeptides f ro m Di ant h us s uperbus . Phytochem ist ry , in p ress ( ) Zhang R P , Zou C , He Y N , et al , 1997 . Three new cyclopeptides f ro m S ilene szech uensis . A ct a B ot anica Y u n nanica , 19 3: 304,310 ( ) Zhang R P , Zou C , Chai Y K , et al , 1995 . A new cyclopeptide f ro m V accari a seget alis . Chi nese Chem ical L et ters , 6 8: 681 ,682 ( ) Zhao Y R , Zhou J , Wang X K , et al , 1995 . Cyclopeptides f ro m S tell ari a y u n nanensis . Phytochem ist ry , 40 5: 1453,1456