• Nghien CIFU - Ky thuat
Ban lu|in
Nghien CCPU nay Cpng dung ma vach ADN (ITS) trong phan tich da dang di truyen cde lodi thugc chi Stephania Lour, d- Viet Nam. Cac ket qua nghien c y u trude day tren the giai khang djnh vung gen ITS ed the giup nhan dien lodi vd d y d i lodi nhy mdt ma vach phdn t y , ddng thdi edng nghg nay cd the d y g c dung cho cdc nghien euu tiep theo ve tien hda phan t y va nghien cuu bao t6n ngudn gen eua lodi d y g c lieu quy.
Ket luan
Nghien c y u da xac djnh d u g c 12 doan trinh t y ITS1-5.8S-ITS2 eCia 12 mdu Stephania Lour cd chieu ddi t y 628 bp den 646 bp.
Thdnh phan GC cua doan trinh t y ITS1-5.8S- ITS2 cac mdu nghien cdu cd ty le dao dgng ti>
50,4% den 62,6%
Miie t y a n g ddng trinh t y nueleotid cua doan trinh t y ITS1-5.8S-ITS2 cua 12 mau nghien edu dao dgng t y 79% den 99%.
Dya vdo s y sai khdc trinh t y nueleotid doan gen ITS1-5.8S-ITS2 ed the ehia cdc mdu Stephania Lour, nghien cuu thanh 2 nhdm tdch bi$t dl truyen.
Tai lieu tham khao 1 V6 Van Chi (1997), Tu dien cay thuoc Vi$t Nam,
NXB Y hpc Ha Npi. tr. 95-97, 326-327, 385-386, 388- 389,895-896,1439.
2. Vo Van Chi (1994), TiFdien thuc vat thong dung.
NXB Khoa hpc va Ky thuat Ha Noi, Tap II, tr 2334-2340 3. Nguyin Chieu (1982), "Nghien ci/u xac djnh ten cay cu dom". Tap chi Du^ hoc. s6 1, tr 15-17.
4. Nguyin Chieu, Ngo Van Trai (1986), "Nghien CLFU cay binh voi a Viet Nam", Tap chi Duac hoc, so 4. tr. 10-12
5. H6 Huynh Thuy Duang (2005), Sinh hoc phan tu. NXB Giao dye, tr 200 - 205
6. Nguyin Quoc Huy (2010), "Nghien cCru v l thyc vat, thanh phin hoa hoc, mot s6 tac dung sinh hpc cua mpt s6 loai Ihupc chi Stephania Lour d Viet Nam", Luan an Tien sT Duac hoc. Trifdng Dai hpc Duac Ha Noi
7. Khuat HOu Thanh (2006), "Ky thuat gen nguyen ly va iJng dung", NXB Khoa hpc va ky thuat Ha Noi, tr.
173-179.
8 Vijayan Deepu, C P Archana, S.P Geetha, Balachandran Indira (2013), "Developing RAPD mark- ers for identification of three source plants of Ayurvedic raw drug 'Patha'", Medicinal Plants - International Jour- nal of Phytomedicines and Related Industries. Volume, 5, Issue. 1, First page ( 55) Last page. ( 58),
9. Zuo Yan, Huang Ning Zhen, Tang Shao Qing (2006), Genetic Diversity of Stephania Kwangsiensis Lo Germplasm Resources by ISSR and RAPD Mark- ers. Master's thesis. Course' Conservation and Utiliza- tion of Wild Fauna and Flora, Guangxi Normal Univer- sity.
10 http.//www.ncbi,nim nih.gov/genbank (Ngay nhan bai: 08/08/2014 - Ngay duyet dang: 08/09/2014)
Phan lap chrysophanol, physcion va emodin trong cay chiit chit nhan
(Rumex crispus L., Polygonaceae)
Dinh Thi Kim Phirong', Pham Dong Phiro"ng^*
' Bo mon Duac lieu - Khoa Dirm. Dai hac Y Duac TP. Ho Chi Minh ' Trung ldm Dao tgo va Nghien cihi phai trien ihuoc nguon goc nr nliien - Dgi hoc YDifOC TP H6 Chi Minh
* E-mail phuongpcl56(a^yahoo.com vn
Summary
The phytochemistry of the plant Chut chit nhan (Rumex crispus L). a well-known herbal remedy in folk medicine for constipation, liver congestion and skin scrofularia. . grown in Datat. Lam Dong
T^P C H i D U Q C H Q C - 9/2014 (SO 461 NAM 54)
• Nghien cu'u - Ky thuat
province was studied. The dried root powders (5 kg) were extracted with ethanol 96%, evaporated in vacuum to condensed extracts. The obtained extracts were partitioned successively with chloroform and ethyl acetate. From the chloroform fractions, 3 compounds were isolated, and structurally elucidated as Chrysophanol (chrysophanic acid), Physcion and Emodin by interpreting their UV, MS, NMR spectra. It is for the first time, these 3 anthraquinones were isolated from this native species of Vietnam.
Keywords: Rumex crispus L, Anthraquinones, Chrysophanol, Physcion, Emodin.
Dat van de
Rumex la mgt chi Idn trong hp Polygonaceae vdi khoang 200 lodi phdn bd rgng rai ^ong t y nhien '^-^l Rd cay chut ehit nhan (Rumex crispus L.) ed tdc dung nhudn trdng (do cde ddn chdt eua anthraqulnon). Ngodi ra, edn ed tde dung bdi bd ea the, Igc mau...'''^i, bao ve gan, khang viem, khang khuan, khang ndm, khang s6t ret f^"^'. Thanh phdn hda hgc chinh eua eac loai trong chi Rumex Id ddn xudt anthraqumon vd tannin, ngoai ra cdn cd eac ddn xudt naphtalen, flavonoid, triterpenoid, anthocyanidol vd coumarin i^'°'.
Viet Nam cd 7 lodi thuge ehi Rumex t'l nhung ehi ed 2 loai da d y g c nghien c y u . Vien Nghien euu Hda hge edng nghiep Viet Nam da bdo eao eong trinh nghien eiiu cao chiet ehut chit nhan (R. crispus) de lam thuoc chong ndm tren cay trdng i'*i. Cae nghien cdu ve tdc dyng d y g e ly eua cao Idng va dieh ham rd ehijt chit {Rumex wallichii Meisner.,) thi nghiem tren rugt thd ed lap lam tang t r y a n g lyc, bidn do co bdp va tdn sd co bdp eua ruot '^l Nghien edu ve hda hgc ehl mdi thye hien tren sae ky Idp mdng khi chdm so sanh vdi chdt chuan eho thdy dich ehidt chut chit (Rumex wallichii) cd chrysophanol vd emodin '^^
Trong bai bdo nay, cde tde gia tidn hdnh khao sat thdnh phan hda hge, phan lap vd xac dinh cau true 3 hgp chdt thude nhdm anthraqulnon la chrysophanol (acid chrysophanic), physcion vd emodin trong rd chut chit nhan a thanh phd Da Lat, tinh Lam Ddng.
Ddi tu-pTig va phu'cng phap nghien
CU'U
D o i t y g n g n g h i e n cLru
Nguyen lieu: Rd chut ehit nhdn (Rumex crispus L.) d y g e thu hdi tai p h y d n g 8, thdnh phd Da Lat, tinh Lam Ddng, vdo thang 4 nam 2011,
da d y g c TS. Vd Van Chi djnh danh. Re dugc n>a sach, bd re eon, eat Idt, phai khd trong didu kien kho mat trdnh dnh sang, xay vd rdy thanh bpt.
D u n g m o i hoa chdt, t h i e t bj nghien ciPu Cae dung mdi hda chat nghien cuu: EtOH, MeOH, CHCI3, EtOAe, n-hexan, aceton, benzen, toluen, CH3CI2, diethylamin (DEA), ether ddu hda (PE) dat tieu chuan tinh khidt phan tich (PA). Cdc thuoc t h y (TT): TT KOH 5%/MeOH, TT vanilin-sulfuric (VS). Sdc ky Idp mdng (SKLM) pha thugn:
dung ban silica gel F254 trdng s i n tren nen nhom (Merck,Art. 1,05554). Sdc ky cgt (SKC):
silleagel c d hat 15-40 pm dimg eho sdc ky cpt (Merck),
Pho khoi (MS) d y g c t h y c hien tren may LC- MS/MS Alliance 2695 XE-Trung tam Dao tao va Nghien c d u phdt then thudc ngudn goc t y nhien - Dai hpc D H . Y D y g e TP. Hd Chi Minh.
May do ph6 cgng h y d n g t y hat nhdn (NMR) BRUKER - AV - 500, tai Vien Hda hge thugc Vien Han Idm Khoa hgc Cdng nghe Viet Nam.
P h u ' c n g phdp n g h i e n cu<u
Phan tich so'bg thanh phan hoa thuv vat Tidn hdnh phan tieh sa bg thanh phdn hda hge eua re ehut chit bang p h y a n g phap I. Ciulei eai tidn.
Phan tich bing sic ky lop mong Kiem tra anthraqulnon toan phdn trdn SKLM vdl he dung moi: ether ddu hda - EtOAe - acid formic (85:15:1), Sau khi khai trien, soi UV a cac b y d c sdng 254, 365 nm, phun TT KOH 5%/
MeOH va ehup hinh sdc ky do. So sdnh cdc v4t anthraqumon d y a tren gid trj R,, mau sac vet vd dien tieh eac vet t y a n g d y a n g .
Chiet xuat
Chiet bdng p h u a n g phap ngam kigt vdi c6n 96%, thu hoi con de d y a c eao Idng.
60 TAP CHi DUQC HQC - 9/2014 (SO 461 NAM 54)
• Nghien ciPU - Ky thuat
Phan tach cac phan doan
Cao long d y g c phan b6 long-long vdi cdc dung moi cd do phdn cyc khdc nhau (CHCI3, EtOAC), thu hdi dung moi de thu d y g c cac cao tyang y n g .
Phan lap
Thyc hidn viee phan lap cae hgp ehdt thugc nhdm anthraqulnon tren sdc ky cgt nhanh va sac ky cgt CO dien, pha tmh Id silica gel c d hat 15-40 pm (Merck).
Dung moi dong: khai trien vdi he dung mdi thich hgp cho cac mdu t h y (se d u a c trinh bay rd dphan ket qua).
Kiem tra cac phan doan va kit tinh Hyng cdc phdn doan, kiem tra tren SKLM, phat hien bdng UV-254, 365 vd TT KOH 5%/
MeOH. Cac phdn doan gidng nhau d y g e ggp chung, thu hdi dung mdi d y d i dp suat giam.
Ket tinh hay ket tinh phdn doan trong dung mdi thich hgp. Kiem tra chdt ket tinh tren SKLM vdi 3 hd dung mdi khde nhau Id PE - EtOAc - add formic (85:15.1), benzen - EtOAe - acid formic (80:20'1) va CHCI3 - MeOH (9:1), phat hien bang quan sdt dudi dnh sdng thudng, soi UV, phun TT KOH 10%/MeOH.
Xac djnh cau tnic
Sy dung kdt qud eua ede phuang phap pho nghigm gdm pho khoi (MS), pho cdng hudng t y hgt nhan (NMR) de xac dinh cdu true, so sanh ddi chieu vdi eae d y lieu vd pho NMR, MS cua ede ch^t tyang t y da edng bo trong tai lieu
Ketqua va banluan
Ket qua phan tich so" bo thanh phan hoa hoc Rd chut chit nhan cd chu'a ede nhdm hgp chat anthraqulnon, flavonoid vd tannin.
Chiet xuat
Bgt rd chut chit nhan (5 kg) dyac lam am va chiet ngam kiet vdl cdn 96% cho den khi djch ehiet g^n nhy khong mau (90 tit), cd dudi dp sudt giam thu dygc cao Idng (3 lit). Tien hanh ldc phdn bo cao I6ng vdl CHCI3 den khi djch CHCI^ khdng cdn eho mdu hong vdi TT KOH 10% trong MeOH, gop eac djch CHCl3(19 lit), cd thu hoi dung moi thu dugc cao CHCI3 (RCc, 100 g). Djch edn lai (sau khi chidt bdng CHCI3) duvc ldc phan bo vdi ethyl acetat, ed thu hoi dung mdi dygc cao ethylaeetat (RCe, 115 g).
Phdn lap cac anthraquinon Phan lap bing sac ky cgt nhanh Cao RCc (10 g) dugc phdn lap bang sde ky cgt nhanh (cot sic ky 4,8 x 40 em), silica gel Merk cd hat 0,015-0,040 mm, khoi lygng 100 g, nhdi cot khd, Khai trien vdi he dung mdi ether ddu hda (PE) - CHCI3 - acid formic vdi do phan eye tang dan theo ty le t y (98'2:1) ^ (80:20:1) sau dd Id PE - EtOAe - acid fonnic (95'5:1),
Kiem tra cdc phan doan bang SKLM vdi he dung mdi PE - EtOAc - acid formic (85:15:1), phat hien bdng UV 254 nm, UV 365 nm, TT KOH 10%/MeOH, Kdt qua thu d y g c 11 phdn doan'
- Cac phan doan 2, 3, 4: tren sde ky dd ddu cd 2 Viet hien mau dd vdi TT KOH 10%/ MeOH, ggp ehung, thu hdi dung mdi thu d y g c 1,50 g, dat ten Id RCx vd d y g e tidn hanh phan lap tiep.
- Phan doan 5 (0,23 g): Tien hdnh kdt tinh phdn doan nay trong hon hgp CHCI3 - MeOH (8:2), thu dugc tinh the hinh kim mau vang, ddt ten Id RC2(34 mg),
- Phan doan 11 (0,44 g): Ket tinh trong hdn hgp CHCI3- MeOH (8:2), Igc, thu d y g c mot tinh the hinh khdi, mau cam, tan trong CHCI3, tan it trong MeOH, Kiem tra tren SKLM vdi 3 he dung mdi nhy tren cho mdt vet tren sac ky dd, dat ten la RC3 (48 mg).
Phan lap bang sic ky cgt co dien Sac ky cdt co dien dugc thyc hien tren silleagel 60, cd hat 0,015-0,042 mm, dung moi khai tnen:
ether dau hda - EtOAe (98:2), Mau phdn lap la hon hgp RCx (1,5 g phdn doan 2, 3, 4 cCia cot nhanh) Kiem tra cac phan doan tren sdc ky Idp mdng, thu d y g c 8 phan doan, Kdt tinh phdn doan RCx, va RCx^ trong CHCi3-MeOH (8:2), thu dugc tmh the dang hat, mau vdng cam, dat ten Id RC, (92 mg).
Xac djnh cdu true cac chat phan lap Xac dmh ciu true cua hgp chit RCI RC, la chdt tmh the dang hat eat, mau vdng cam, It tan trong MeOH, tan trong CHCI3, eho mdu hong tim vdi dung djch kidm (NaOH 10%, KOH 10%).
Pho L/V(MeOH) >.^^, 225 (0,38); 256 (0.16);
286 (0,09); 428 (0,08)™'
Pho khdi ESI-MS (positive) eua RC, eho ede
TAP CHi DUQC HQC-9/2014 (SO 461 N.\M 54)
• Nghien CUKU - Ky thuat
ion vdi m/z ( c y d n g dp t y a n g ddi % ) : 393 (100):
309 (18); 277 (78,5) [M+Na^; 255 (16) [M+H]^;
253 (7) [M-Hr; 236 (19), N h y vay, RC, cd khdi l y g n g phdn t y (M) bdng 254,
Pho "C-WMR eua RC, cho tin hieu eua 15 C, trong dd ed 9 carbon bac IV, 5 carbon bdc 111 vd 1 carbon bde 1 Trong s6 cde tin hieu edng h y d n g carbon bde IV, co 2 tin hieu cgng h y d n g d h^ 181,9; 192,5 ppm dae tryng cua carbon eeton (quinon) va 2 tin hieu d 5^ 162,4 vd 162,7 ppm dac tryng cua carbon tren nhan tham mang nhdm th§ oxy. Nam tin hieu con lai h^ 113,7; 5^
149,3; 5^ 133,3; 5^ 133,6 va 5^ 115,8 ppm ddu !a cua cae carbon vdng tham Nam carbon bdc 111 ddu eho 5 tin hieu eua carbon tham khdng nhdm thd cdng hydng d 124,3, 121,3; 136,9; 119,9 va 124,5 ppm Nhdm methyl cgng h y d n g d vung trydng cao 5^, 22,2; 5^ 2,45 ppm dac tryng eua nhdm methyl the tren nhdn tham.
Vdi edng t h y c nguyen C,5H,^0^(M=254), cd 5 H cua vdng tham, 3 H cua nhdm methyl va 2 nhdm earbonyl, 2 nhdm chdc cdn lai cd 5^ 11,96 va 12,07 ppm cua RC, phai la nhdm OH, Cac dae diem pho nhy tren chCpng to rdng RC,, ed edu true ciJa anthraquinon ed mot nhom thd methyl (C3) vd 2 nhdm OH ( 0 1 , C8) thudc nhom nhudn tay (oxymethylantraquinon).
Tren pho ''H-NMR, cac tin hieu cua 3 proton nhdn tham xudt hien vdi 2 proton la dinh ddi (d) (dd) va 1 proton la dinh gia 3 (pseudo. t), dae
t a m g cua 3 proton d y n g kd can cd [5^ 7,26 ( I H , m) ( I H , m), 7,64 ( I H , pseu.-t.; 8,5 Hz) va 7,78 ( I H , dd, 12,5; 7,5 Hz) ppm]. N h y vay 3 nguyen ti>
H phai d 3 carbon eanh nhau tren vdng benzen vd phd COSY cua RC, eung cho thdy rd tuang tdc cua 3 proton nay. Trong sd 3 proton tren, proton ed tin hieu [5„ 7,64 ( I H , pseu.-f.; 8,5 Hz) ppm] edn eho thdy cd t y a n g tae vdi 1 carbon ed 5;; 181,9 ppm Dieu nay da giup cho viec xac dinh nhdm OH (5„ 12,07 ppm) a eung phia vdi 3 proton tren vdng tham vd se d gdn nhdm earbonyl quinon cd 5^ 192,5 ppm. Nhu vay, nhdm OH (5^ 11,96 ppm) se d eung phia vdi 2 proton nhan t h a m edn lai vd proton nhdm methyl.
Tren pho HMfiC, nhdm OH (5„ 11,96 ppm) cd s y t y a n g tae vdi C l a , va C2 nen phai d vi t r I C I . 3 proton eua nhdm methyl S„ 2,45 ppm cho thay t u a n g tac vdi 1 carbon mang nhdm methyl ed S^, 149,3; 1 carbon cd 5^. 124,3 va mgt carbon eo
§^ 121,3 ppm Trong khi dd 2 proton cdn lai (5^
7,06 va 7,61 ppm khong d gdn nhau vi khong ed t y a n g tdc tren pho COSY, Nhy vdy, nhom methyl phai lien ket vdi C3 cd 6^149,3 ppm nhy edu true t h y d n g gap trong nhdm 1,8-dihydroxy antraquinon.
Vl tri eija ede carbon vd eac nhdm thd cua RC, d y g c xac dmh chdc chdn bdi eae tyang tae quan sat d y g c trong pho HMBC nhy trinh bay trong Bang 1. Cdu true eua RC, d y g c xac djnh la 3-methyl-1,8-dihydroxy-9,10-anthraquinon va 36 chinh la chrysophanol (aeid chrysophanic).
Bang 1 : DO' iieu NMR cua RCI so
Vi tri 6 j 1 162,7 2 124,3 3 149,3 4 121,3 5 119,9 6 136,9 7 124,6 8 162,4 9 192,5 10 181,9
sanh vcyi RC, fCDCy 500 MH2 6„ ppm (J, Hz)
7,06 (1H,S)
7,61 (IH, d, 1,0) 7,78(1H, drt 2,5, 7,5) 7,64 (IH, pseiido.fiS.O) 7,26 (1H,m)
COSY
C5, C7
C6 C6
zhrysopfianoi i^°i
HMBC
C9a, C4; Cl
C9a, C2;C10 C8a; C7. CIO C10a, C8;C10 C8a; C5, 08
Chrysophanol (CDCIJ 400 MHz ""
5c 162,7 124,4 149,3 121,4 119.,9 136,9 124,5 162,4 192,5 181,9
S^ ppm (J, Hz)
7,09 (1H.S)
7,64 (1H,S) 7.81 (IH, dd, 2,7;8,0) 7.67 (1H,m) 7,52(1H, dd, 2,0;8.8)
TAP CHI Diroc H p c - 9/2014 (SO 461 NAM 54)
• Nghien CLFU - Ky thuat
9a 113,7 4a 133.3 10a 133.6 8a 115.8 Me-1 22.2 0H-C1 0H-C8
2.45 (3H. s) 12.07 11.96
C4; C2; C3 C9a:C1;C2 C8a; 08; 07
113.7 133.3 133.6 115,9
22.3 2.47 (3H, s) 12,0 12,1 Xac dmh cau true cua hgp chat RC^
RC; Id tinh the hinh kim, mdu vdng, it tan trong MeOH, lan trong CHCl^, cho mau hong vdi dung djch kidm (NaOH 10%, KOH 10%),
Ph6 UV/(MeOH) X^^^ 224 (0,39); 250 (0,15);
267 (0,15); 272 (0,15); 285 (0,16); 433 (0,10), Pho khoi ESI-MS (positive) cua RC^ cho eae Ion vdi m/z (cudng do t y a n g ddi %): 307 (100) [M+Nar; 339 (16); 285 (13) [M+H]*, Pho khdi ESI-MS (negative) cua RC^eho cdc ion vdi m/z (cydng dg t y a n g doi %): 283 (100); 325 (24); 311 (8,5); 255 (61) [ M + H f ; 227 (18); 212 (28). Nhy v^y, RCj ed khoi lygng phan t y (M) bdng 284,
Pho "C-NMR cua RC^cho tin hieu cua 16 C, trong dd cd 10 carbon bgc IV, 4 carbon bac 111 vS 2 carbon bde I, Trong so eae tin hieu edng hudng carbon bde IV, cd 2 tin hieu cdng hudng d 5p 181,9; 190,8 ppm ddc tryng cua carbon eeton (quinon) va 3 tin higu d 5^ 162,5; 166,5 va 165,2 ppm dae tryng eua carbon tren nhan tham mang nhom the oxy. Nam tin hieu cdn lai S^ 113,7;
148,4; 133,2; 135,3 vd 110,3 ppm deu la eua c^c carbon vdng tham. Bon carbon bac III deu cho 4 tin hieu cua carbon tham khdng nhdm thd
edng hydng d 8^ 124,5; 121,3; 108,2 va 106,8 ppm. Nhdm methyl cgng hyang d vung trydng eao 6^ 22,1; 5„ 2,45 ppm dac tryng cua nhdm methyl the tren nhan tham. Khdc vdi hgp chat chrysophanol d tren, RC^ cdn cd tin hidu vdi 5^
56,1; 5„ 3,93 ppm ddc trung eua nhdm methoxy thd tren nhdn tham.
Vdi cdng thyc nguyen C^gHj^O^ (M=284), cd 4 H cua vdng tham, 6 H cCia nhdm methyl vd 2 nhdm earbonyl, 2 nhdm ehde edn lai cd 5„ 11,96 va 12,07 ppm cua RC^ phai Id nhdm OH. Cac dac diem pho nhy tren chdng td rdng RC^ ed eau true cua anthraquinon cd 1 nhdm the methyl (C3), 1 nhdm methoxy vd 2 nhdm OH C l , C8) thude nhdm nhudn tay (oxymethylantraquinon),
Vi tri cua eae carbon va cdc nhdm the cua RC^
d y g c xac dinh chdc chdn bdi cac tuang tae quan sat d y g c trong pho HMBC nhy trinh bay trong bang 2,
Nhy vdy, cau true cua RC^ dyge xac djnh la 3 - m e t h y l - 1 , 8 - d i h y d r o x y - 6 - m e t h o x y - 9 , 1 0 - anthraquinon va chinh la physcion - mgt chat da d y g c bao eao trude ddy.
Bang 2: DQ' lieu phd NMR cua RC^ so sanh vai physcion "
RC, (CDCiJ 500 MHz Physcion (CDCI) 400 MHz
Vj tri 6^ ppm (J, Hz) COSY 5„ ppm (J. Hz)
2 3 4
124,5 148,4 121,3
7.06 ( I H . d ; 0,5)
7.61 ( I H . d ; 1,0)
Ola, 0 1 . 0 4
Ola, 02.CIO
121.2 148,4 124.4
7.08 (1H. brs)
7,63 (IH, brs)
7.35 (IH.d); 2,5 7.37 ( I H . d , 2.4)
106,8 6.67(1H. d. 2,5) 05; C8a, 06; 08 6,69(1H, d, 2 4)
T^P C H i D U q C HQC-9/2014 (SO 461 NAM 54)
• Nghien ciru - Ky thuat
8 9 10 9a 4a 10a 8a Me-1 O0H3 OH-01 OH-08
165,2 190,8 181,9 113.7 133.2 135.3 110.3 22.1 56,1
2,45 (3H, s) 3,93 (3H 12.09 12.29
l.s)
C4;
C9£
07;
02;
i:C2;
08a:
03
08 01
166.5 190.7 181.9 113,7.
133.2 135,2 110.2 22.2.
56.1
2.46 (3H. s) 3.94 (3H, s).
12,31 (IH, s) 12,11 (IH. s) Xac djnh ciu true cua hgp chat RC^
RCj la ehdt tmh the dang hat khoi, mdu vdng cam, It tan trong MeOH, tan trong CHCI3, cho mau hdng vdi dung dieh kidm (NaOH 10%, KOH 10%).
Pho i ; V ( M e O H ) X^^^ 224 (0,39); 250 (0,15);
272 (0,15); 285 (0,16); 433 (0,10).
Pho khoi ESI-MS (positive) eua RC3 eho eac ion vdi m/z (eudng do t y a n g ddi % ) : 393 (100);
420 (12); 309 (16) [M+K]*; 293 (28) [ M + N a f ; 271 (13) [M+H]-; 236 (10), Pho khoi ESI-MS (negative) eua RC3 eho cac ion vdl m/z (eudng do t u a n g ddi
%): 269 (100) [M-Hr; 270 (23); 271 (4). Nhy vay, RC3 cd khoi lygng phdn tip (M) bdng 270.
Pho "C-NMR cua RC3eho tin hieu cua 15 C, trong do cd 10 carbon bde IV, 4 carbon bdc 111 va 1 carbon bde 1. Trong so cac tin hieu cpng hydng carbon bde IV, cd 2 tin hieu cgng h y d n g d 6^ 189,6; 181,2 ppm ddc tryng eua carbon eeton (quinon) va 3 tin hieu d 6^; 161,3; 165,5 va 164,4 ppm dac trung eua carbon tren nhdn tham mang nhdm the oxy. Nam tin hieu edn lai 5^ 113,2; 5^
148,1; S^ 132,7; 5^ 135,0 va 5^ 108,9 ppm ddu la cua eac carbon vong tham. Bdn carbon bac 111 deu cho 4 tin hieu cua carbon tham khdng nhom the eong hudng d 124,0; 120,4; 108,7 va 107,8 ppm. Nhdm methyl edng h y d n g d vung tradng eao 5^ 21,4, 5^ 2,39 ppm dae tryng eua nhdm methyl the tren nhan tham,
Vdi edng thdc nguyen C,5H,(,O5(M=270), cd 4 H eua vdng tham, 3 H cua nhdm methyl va 2 nhdm earbonyl, 3 nhdm ehyc cdn lai (mgt nhom khdng ed tin hieu proton), 2 nhdm cd 5„ 11,96 va 12,07 ppm eua RC3 phai la 3 nhdm OH. Cae dac
diem pho nhy tren ehyng to rdng RC3 cd cau true cua anthraquinon cd 1 nhdm thd methyl (C3) va 3 nhdm OH ( C l , C6, C8) n h y cau tao thdng thydng cua nhdm nhuan tay (oxymethylantraquinon).
Tren pho ^H-NMR, cae tin hieu cua 4 proton nhdn tham xudt hien vdi 4 dinh dan ehiing to rdng khdng eo proton nao d canh nhau. Tren pho HMBC, trong sd 2 proton cd 5„ 11,96 vd 12,04 ppm th) proton 5„ 11,96 cd s y t y a n g tdc vdi eac carbon ed 6f. 113,2; 124,0 vd 161,3 ppm. Trong khi do 3 proton cua nhdm methyl cd 5„ 2,39 ppm cd s y t y a n g tac vdi eae carbon ed 6^, 124,0; 148,1 vd 120,4 ppm. Proton 5^ 7,43 ppm cd s y tyang tae vdi cac carbon cd 5^ 113,2; 124,0 va 181,2 ppm.
N h y vay cae nhom OH, nhdm methyl va 2 proton cd 5^ 7,43 vd 7,12 ppm phai d cung mgt ben va theo cdu tgo t h y d n g cd cua nhdm anthraquinon thi nhdm OH (proton cd 5^ 11,96 ppm) gdn vao C l ; proton S^ 7,12 ppm gan vdo vdng tham d C2 (do C2 cd t u a n g tac vdi proton eua nhdm OH).
Cac vi tri cdn lai id methyl d C3 vd proton 6„ 7,43 ppm a C4. Proton cd 6„ 7,08 ppm tyang tac vdi carbon cd 5^ 166,5; 107,8 va 182 ppm ndn se phdi gan vdo carbon gdn vdi 0 4 (vi 2 carbon nay ddu t y a n g tac vdi CIO ed 6^ 182 ppm) nen proton ed 5„ 7,08 ppm se d gan vdi C5. Proton cdn lai se chi ed the gdn vdo 0 7 . Proton cd 8^ 12,04 ppm (nhdm OH) cd s y t y a n g tae vdi carbon cd 5^, 108,9, 164,4 va 120,4 ppm nen nhdm OH nay se gan vao 0 8 va nhu vay nhdm OH cdn lai sd d C6.
Vj tri eua ede carbon vd cdc nhdm the cua RC, d y a c xde djnh ehdc chdn bdi eac t u a n g tdc quan sat d y g c trong pho HMBC nhu trinh bdy trong bang 3,
TAP CHI DUQC HQC - 9/2014 (SO 461 NAM 54)
• Nghien CIFU - Ky thuat Nhy vgy, cau tnJc cua RC3 d y g c xac i^nh la 3-methyl-1,6,8-lrihydroxy-9,10-anthraquinon vd
dd ehinh la emodin, mdt chdt da d y g e bdo cao va hien didn trong hau het cde loai thudc chi Rumex Bang 3: DO- lieu NMR cua RC^ so sanh vai emodin
Vi tri 1 2 3 4 5 6 7 8 9 10 9a 4a 10a 8a Me-1 0H-C1 0H-C8 OH
5r 161,3 124.0 148.1 120.4 108,7 165,5 107,8 164,4 189.6 181.2 113,2 132,7 135,0 108,9 21,4 q
RC, (DMSO) 6„ ppm (J, Hz)
7.12 (IN, s)
7,43(1 H.S) 7.08 (IH.S)
6.66(1 H.S)
2.39 (3H, s) 11,96 12,04
c o s y HMBC
09a; 04; 01
09a; 02, CIO 07; 06:010
C5, 08
02; 03; 04 09a; 02, 01 08a; 08
Emodin (CDCI,J 8.
164,1 (0-1) 120,2(0-2), 148,0(0-3) 123,9(0-4) 108,7 (C-5) 161,1 (C-6) 107,7 (C-7) 165,2 (C-8) 189,4 (0-9) 181.0(0-10) 113,1 (C-9a) 132.6 (C-4a) 134.9 (C-lOa) 108.5 (C-8a) 21,4 (-CH3)
1,0,
S^ppm (J, Hz)
7.15(1H. d. 1,2)
7,48(1H, d. 1,2) 7,11 (IH, d, 2,0)
6,59(1H. d, 2.0)
2.38 (3H.S) 12 00 (IH.S) 12 07 (IH.S) 11 35(1H, brs)
Ket luan
Ty eao rd chut chit nhdn ( R crispus I.) thu miu tai thanh pho Da Lat, tinh Lam Ddng da ehiet xu^t va phan lap d y g c chat tinh khidt RC,, RC^
vd RC3, Bdng cac d d lieu ve pho UV, MS, NMR vd so sanh vdi d y lieu pho ^H-NMR, '^C-NMR cua ede nghien c d u trydc day da xae dinh dygc 3 chat tren lan lu'at Id chrysophanol (acid chrysophanic), physcion vd emodin. Chut chit (R.
wallichii) da d y g c nghien c y u ve hda hgc d trong nude nhyng ehi mdi d m y e dp djnh tinh nhdm chit vd sdc ky ldp mdng, Ddy la lan ddu tien eae chJit ndy d y g c phdn lap trong chut chit nhan de c6 t h i s y dpng ldm chat chuan ddi chidu. Vide phdn l^p d y g e cdc chat nay se hd trg cho viee xfly dyng p h y a n g phap kiem nghidm va tieu
chuan hda d y g e lieu, eao chiet va danh gia chinh xac ham lygng hoat chat trong thanh pham.
L&i cam an: Nhom cac tac gia cam an Ong Dang VO Luang, Phong Cong huang tu Hat nhan.
Vien Hoa hoc, Vien Han lam Khoa hoc Cong nghe Viet Nam da giup da do pho NMR; ThS. Ma Chi Thanh, Labo Hoa hap chat tu nhien, Trung tam Dao tao va Nghien cuu phat tnin thuoc nguon gdc tu nhien, Dai hoc YDuac TP. Hd Chi Mmh da giup dd do phd MS
Tai iieu tliam khao 1 Vo Van Chi, (2012), Tu'diSn cay thuoc Viet Nam.
NXB Khoa hpc va ky thugt, tap 1, tr 477-478 2 D6 Tat Lot (2000), Nhung cay thuoc va vi thudc Vi0t Nam, NXB Y hoc, tr 221-222
TAPCHi DlTQC HQC - 9/2014 (S6 461 N \\I 54) 65
• Nghien CIFU - Ky thuat
3. Ngo Van Thu (2011), Dupc//eu hoc, NXB Y hpc.
tap 1,fr, 343-344.
4. Lyu Hoang Ngoc, Nguyin Thanh Loan, Nguyin Nggc Thanh, Nguyen Thi Hoai Anh, Nguyin Thj Thu Hyang, Nguyin Mai Cycng, Le Ngpc Thyc (2009), Bao cao tdng k4t de tai "Nghien cyu cong nghe san xuat cao djch chiet cua r i cay chut chit Rumex crispus djnh hydng lam thuoc try nam thao mpc than thien moi trydng", Vien Hoa hoc cong nghiep Viet Nam
5. Heng-Xing Liang, Huan-Qin Dai, Hai-An Fu, Xiao-Ping Dong, Abiodun Humphrey Adebayo, Li- Xin Zhang, Yong-Xian Cheng (2010), "Bioactive compounds from Rumex plants", Phytochemistry Letters.pp. 181-184.
6. Keyong Ho Lee, Ki-Hyeong Rhee (2013),
"Antimalanal activity of nepodin isolated from Rumex crispus". Arch. Pharm. Res., 36, pp 430-435.
7. Yildirim A., Mavi A., Kara A. A. (2001),
"Determination of antoxidant and antimicrobial activities of Rumex crispus L. extracts", J. Agric. Food Chem., 49 (8), pp, 4083-4089.
8. L. Omur Demirezer, Ayse Kuruuziim (1997), "A comperative chemotaxonomlc study on eleven Rumex species growing in Turkey", FABAD J. Pharm. Sci., 22(4), pp. 153-158,
9. Fan Ji-ping, Zhang Zhen-liang (2009), "Studies on the chemical constituents from Rumex chspus L.', Joumal of Guangdong College of Pharmacy. Issue 6, pp, 585-587,
10. Selda Balkan, Ay§e Daut-Ozdemir, Kenman Gunaydin, F Bedia Enm (2007), "Analysis of anthraquinones in Rumex cnspus by micellar electrokinetic chromatography", Talanta, 71, pp. 747- 750.
[Ngay nhan bai: 03/06/2014 - Ngay duyet dang: 08/09/2014)
Nghien cmi djnh lirong luteolin, apigenin trong dirffc lieu va bot cao kho ciic hoa vang
(Chrysanthemum indicum L.)
bang phirong phap sac ky long hieu nang cao
Nguyin Trong Diep', Chir Van M^n', Vu Binh Duffng'"
Nguyen Vu MinhS Nguyen Thanh Hai^
' Hoc Vien Qudn Y
^ Vien Kiem nghiem nghien cim Duac Quan dot
^Khoa Y Diroc - Dgi hoc Quoc gia Ha Noi
* E-mail [email protected]
Summary
Luteolin and apigeninin in the Flos Chrysanthemi Indici and dried extracts of Chrysanthemum indicum were quantitatively analyzed by HPLC-UV/VIS (338 nm) with a reversed phase column C18 (5 pm; 4.6 mm X 150 mm), gradient elution by solvent A (formic acid 0.01% in water), followed by solvents (acetonitrile): flow rate - 0.45ml/min. The limit of detection (LOD) was 3.1 and 15.2 ng/mL and the limit of quantification (LOQ) was 8 and 38ng/mL for Luteolin and Apigenin, respectively. The method was validated for the suitability precision and accuracy Applying to quantitation of luteolin and apigenin in the Flos materials and dried extracts of Flos Chrysanthemi Indici, the method revealed no loss of the active compounds through preparing the dried extracts.
Keywords: Chrysanthemum indicum L, HPLC, luteolin, apigenin.
TAP CHI DUQC HQC - 9/2014 (SO 461 NAM 54)
