HQl NGHI KHOA HQC T O A N Q U O C V £ SINH T H A I V A TAI N G U Y E N SINH VAT L A N THl> 5
CAC HOP CHAT FLAVONOID PHAN LAP T l / GO CAY C A M L A I {Dalbergia oliveri)
PHAM THANH LOAN Dgi hgc Hirng Virang, Phii ThQ HOANG LE TUAN ANH, BUI HlTU TAI, PHAM HAI YEN,
DAN THI THUY H A N G , NGUYEN THI CUC, DirONG THI HAI YEN, DU'ONG THI DUNG, NGUYEN XUAN NHIEM, CHAU VAN MINH, PHAN VAN KIEM Vien Hda sinh bien.
Vien Hdn ldm Khoa hgc vd Cong nghf Vi^l Nam TRAN HUY THAI, NGUYEN THI HIJ&N Viin Sinh thdi vd Tdi nguyen sinh vdt, Vien Hdn ldm Khoa hgc vd Cong ngh? Viet Nam Cam iai (hay con co ten la Cim Iai vu. Cam Iai ba rla) (ten khoa hpc: Dalbergia oliveri Gamble ex Prain; syn: Dalbergia bariensis Pierre, Dalbergia duperreana Pierre, Dalbergia dongnaiensis Pierre), thupc chl Trac {Dalbergia), hp Dau (Fabaceae). Ciy phan bo chu yeu a cac nuac Dong Nam A nhu Myanmar, Thai Lan, Lao, Campuehia va Viet Nam. 0 Viet Nam, cay mgc rai ric trong rimg, nai im, dit bing phing hay dat co dp doc nho, a dp cao den 800m a mpt so tinh phia Nam nhu: Gia Lai, Kon Turn, Dak Lak, Lam Dong, Dong Nai^ Tay Ninh... (Sich Do Vi?t Nam, 2007). Cay Cam Iai xua nay dupe sir dung nhu mot Ioai cay liy go qu^, ben chic, co van va mau sic dep. La loai bj san liing khai thic cgn kiet, den nay kho tim thay ca the truang thinh nhu truac trong lu nhien Loii dugc ghi trong Sich Do Viet Nam 2007 vai muc nguy cap EN AIa,c,d. Nhu'ng nghien ciru ve thanh phan hoa hpc cua loii nay a trong va ngoai nuac con it.
Bii bio niy thong bao kit qui phin lip vi xic dinh cau triic cua bon hop chat flavonoid:
formononetin (1), pratensein (2), violanone (3) vi isollquyritigenin (4) ti> djch chiet metanol cua gfl cay Cim Iai vai viec sir dung cac phuong phip pho cpng huang tir hat nhan mpt chieu va hai chieu (ID va2D-NMR).
I. VAT LIEU VA PHU'ONG PHAP NGHIEN CUtJ
Miu go Cam Iai dupe thu tgi Vuon Quoc gia Yok Don (Dik Llk) vao thing 4/2012. Mau tieu bin d\xgc luu tai Phong Tai nguyen thuc vat cua Vien Sinh thai va Tal nguyen sinh vat.
Sic ky lap mong (TLC): Thuc hien tren ban mong trang sin DC-AIufoIien 60 F2sj (Merck 1,05715), RP-18 F254s (Merck); cac v?t chit dugc phit hien bang den tir ngogi 0 hai buoc song 254nm va 365nm hoac dimg thu6c thCr la dung dich H2SO4 10% phun diu len bin mong, say kho roi ha nong tir tir den khi hien mau.
Sac ky cgt (CC): Dupe tien hanh voi chat hip phu Ii silica gel pha thuang va pha dao.
Silica gel pha thuang co c5 hat la 0,040-0,063mm (240-430 mesh). Silica gel pha dio YMC (30-50 p,m, Fujisillsa Chemical Ltd.).
Ph6 cgng huang tix hat nhan (NMR): Do tren miy Bruker AVANCE500 tal Vl?n Hoa hpc, Vi?n Hin lam Khoa hpc va Cong ngh? Viet Nam.
Go cay Cim lai dupe phai kho, nghiln nho thu dupe 8kg bpt kho. Bpt nay dupe ngam chiet*' vol metanol (3 lan x 5 lit) tren thilt bj chilt sieu am (a 50°C, m6l Ian I gio). Cac djch chiet dupe gom Iai, ipc qua giay Ipc va cit thu h6i dung moi dual ap suat giim thu dugc 700g cjn chiet metanol. Can chilt nay dugc lie diu tao huyin phu vai 2 lit nuac cat sau do tiln hanh
HQl NGH! KHOA HQC T O A N Q U O C V £ SINH T H A I V A T A I NGUYEN SINH VAT L A N THU' 5
^iet phan bo lan lugt vai clorofoc, etyl axetat thu dupe cac can dich tuang ung clorofoc (DOI, I30g), etyl axetat (D02,270g) v i lap nu6c (D03).
Can chiet etyl axetat (270g) dugc hoa tan bang metanol, tim silica gel (ty le 1: 1,5, w/w), cat Ioai dung moi din kho, nghiln m|n. H6n hgp niy dugc phan tich thanh nim phin doan D02A-D02E tren cpt sac ky silica gel pha thuang vol he dung moi rua giai gradient clorofoc/metanol CO dgphin cue tang din (50/1->l/I,v/v). Phan doan D02B (2g) tiep tuc dugc
phan tich thanh 4 phan dogn nho ban D04A-D04D tren cpt sic ky silica gel pha thuang vai he dung moi rira giii clorofoc/axeton (10/1, v/v). Hgp chit 1 (6mg) vi 3 (8mg) thu dugc sau khi tinh che phan doan D04A tren cpt sac ky silica gel pha thuang sii dung he dung moi rira giai clorofoc/etyl axetat (7/1, v/v).
Phan doan D04D dugc phan tach thanh ba phan doan nho D05A-D05C bang sic ky cpt silica gel pha dio vol he dung moi rua giii axeton/nuac (1/1, v/v). Hgp chit 2 (8mg) va 4 (lOmg) thu dugc sau khi tinh che phan doan D05B bang cpt sac ky pha dao sir dung he dung (nfli r&a giii metanol/nuac (1,5/1, v/v) kit hgp vol sic ky cot silica gel pha thuong siJr dung he
^ung moi rira giai clorofoc/metanol (10/1, v/v).
I Thong tin ve 4 chat flavonoid phan lap dupe nhu sau:
Formononetin (1): Bgt vo djnh hinh, miu vang; cong thuc phan tir Ci6H\iOA (M = 270);
'H-NMR (500 MHz, DMSO-rf,) vi '^C-NMR (125 MHz, DMSO-d,), xem bang 1.
Pratensein (1): Bpt vo djnh hinh, mau vang; cong thirc phin tir CiaHiiOe (M = 300);
'H-NMR (500 MHz, DMSO-i^s) va "C-NMR (125 MHz, DMSO-do), xem bing 1.
Violanone (3): Tinh the hinh kim, mau trang; cong thuc phan tir C17H14O6 (M = 316);
'H-NMR (500 MHz, CDCI3) vi '^C-NMR (125 MHz, CDCI3), xem bang 2.
Isoliquyritigenin (4): Bpt vo djnh hinh, miu tring; cong thirc phin tOr C1SH12O4 (M = 256);
'H-NMR (500 MHz, DMSO-rf^) va '^C-NMR (125 MHz, DMSO-t/^), xem bang 2.
Hinh 1. Cdu triic hod hgc ciia cdc chdt 1-4
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II. KET QUA NGHIEN ClTU
Hgp chat 1 thu dugc dual dgng bgt vo djnh hinh mau vang, hien mau vang dgm hen ban TLC ggi y day Ii mgt hgp chit dgng flavonoid. Pho 'H-NMR ciia 1 cho nhgn thay mgt tin hieu cpng huang ciia cac proton tham 6H 8,33 (IH, s), mpt cgp tin hi?u thugc h? tuang tic spin AA'BB' 5H 6,98 (2H, d, J = 9,0 Hz) va 7,50 (2H, d, / = 9,0 Hz) va ba tin hi?u thugc he tuang tic spin ABX 5H 7,97 {\H,d,J = 8,5 Hz), 6,94 (1H, dd, 7 = 2,5; 8,5 Hz) va 6,87 (IH, d,J= 2,5 Hz). Ngoai ra, tin hl?u cgng hucmg tgi 5H 3.78 (3H, s) cho thay s\rc6 mat ciia mgt nhom metoxi trong phin tix 1. Phin tich ph6 '•'C-NMR va DEPT cho thay tin hieu cua 16 nguyen tir carbon bao g6m bay nguyen tOr carbon bic 4, tim nguyen tir carbon bac 3 va mpt nguyen tir carbon bge 1 (nhom metoxi). Tir cac dir kien tren cimg vdi tuong tic tr\rc tilp nhan dugc tren HSQC giira H-2 (5H 8,33) va C-2 (5c 153.09) v i cic tucmg tac HMBC giira H-2 vai C-3 (8c 123.16), C-4 (5c 174.61), C-9 (5c 157.44) cho thiy hgp chatl la mpt isolavon. Tiep d6, vj tri cua cac nhom the trong 1 mpt Ian nita dugc xic djnh dua tren phin tich phi 2 chieu HMBC (hinh 2). Tuong tic HMBC gitra hydro cua nhom metoxi (5(1 3.78) vai C-4' (5c 158.95) cho phep khang djnh nhom metoxi the para tai vong B. Cac tuang tic HMBC giua H-6 (5H 6 , 9 4 ) / C - 1 0 (5C 116.60), H-8 (5n 6,87)/C-10 va H-5 (5H 7,97)/C-4 cho phep khang dinh nhom hydroxy the tal vj tri C-7 cua v6ng A. Dieu n^y cung phii hgp vai hing s6 tuang tic J^ea = 2.5 Hz nhgn dugc giiia H-6 va H-8 tren pho 'H- NMR. Hon nira, su tuang dong khi so sanh pho "C-NMR ciia 1 vcri formononetin (Almeida J.G. vd nnk., 2008) (bing 1) cho phep xac dinh cau triic hoa hpc cua hgp chat 1 la formononetin (hinh I).
Hgp chit 2 thu dupe duai dang bpt vo djnh hinh miu vang. Gilng nhu 1, cac tin hi?u cgng huang nhin dugc tren phd 'H-, '^C-NMR cho bilt 2 cung la mot Isoflavon (5H 8.30,S, H-2 va 5c 154.14, C-2). Cic cap tin hieu 5H 6.22 (d, 2.0) 5c 98.99 va 5H 6.38 (d, 2.0) 6c 93,66 die trung cho hydro vi carbon tgi vj tri C-6 vi C-8 ciia flavonoid da bi thi a vj tri C-5 va C-7.
Nh6m hydroxy the tai vj tri C-5 con dupe die trung boi sy: xuat hien tin hieu c^ng hircmg ciia OH tren phi 'H-NMR nim a viing trucmg thap 5H 12.96 do anh hudng cua viec hinh thinh lien kit hydro npi phan tir vai nhom cacbonyl a C-4. Ba tin hieu cpng hucmg cua hydro tham con lgi 5H 7.04 {d,J=\.5 Hz), 6.95 (d, J = 8.5 Hz) va 6.94 (dd, y = 8.5,1.5 Hz) cho thay cic hydro cua vong B thupc he tuang tic spin ABX. Han nira, cic tin hi?u tuong tic nhan dugc tren ph6 2 chilu HMBC (hinh 2) giira H-2' (5H 7.04) va C-3 (5H 123.36), H- 6' (5H 6.94) vi C-3 chung to vong B bj thi tgi hai vi tri 3',4'. Ngoai ra, cgp tin hi?u 5H3.79 (3H, s), 6c 55.66 cho thiy sir co mat cua mpt nhom metoxi trong 2. Va cac tuang tac HMBC gitra H-MeO (5H 3.79)/C-4' (5c 147.72), H-2VC-4' v i H-6'/C-4' da chi ra ring nhom metoxi thi tgi vj tri C-4'. Nhu vay, ciu true hoa hpc cua hgp chit 2 dugc xic djnh la 5,7,3'- trihydroxyl-4'-metoxlisoflavon hay pratensein (hinh 1). Cic kit qua phan tich pho "C- NMR cua 2 cung hoan toin phii hgp vai cic dir ki^n ph6 tuang ung cua pratensein nhudi dugc cong bo trong tai lieu [1] (bing 1).
HQI NGHI KHOA HOC T O A N Q U O C V S SINH THAI VA TAl N G U Y S N SINH VAT L A N THO' 5 Bang 1 D i r kien pho N M R ciia 1,2 va cac ch^t t h a m khao
No.
2 3 4 5 6 7
e
9 10 1 2 3 4 5 6 4-OMe
50H
FomiononetJn 5c" [1]
153.4 123.5 174,9 127,6 115.5 162.8 102.4 167.8 117.0 124.6 130,4 113,9 159.3 113.9 130.4 55.4
1 5 c "
153,09 123,16 174,61 127.27 115,23 162,62 102,11 157.44 116.60 124.23 130,05 113.59 158,95 113,59 130,05 55.12
S H " (mult, J in Hz) 8,33 (s)
7.97 (d. 8.5) 6,94 (dd, 2.5, 8,5)
6,87 (d, 2,6)
7.50 (d, 9,0) 6,98 (d, 9,0)
6,98 (d. 9.0) 7,60 (d. 9,0) 3,78 (s)
Pratensein Sc-Pl
154,0 126.2 181.5 164.1 100,5 166,3 95,0 158,0 106.0 122.7 118.1 148.5 149.3 112,8 120,8 66,3
2 5 c "
154,14 122,16 180,11 162,01 98,99 164,28 93,66 157.53 104.47 123.36 116,40 146,12 147,72 112,01 119,80 55,66
6H'''^ (mulL, J In Hz) 8,03 (s)
-
6,22 (d. 2,0)
6.37 (d, 2,0)
7,03 (d, 1,6)
6,96 (d, 8.5) 6.94 (dd, 1,5; 8,5.)
3.79 (s) 12.96 (s) Do trong"DMSO-rftf, "'CsDjN-c/i, '"125 MHz. "^'SOO MHz.
Hmh 2. Cdc tirang tac HMBC (H->C) chinh ciia I vd2
Hgp chat 3 thu dugc duai dgng tinh the hinh kim mau tring. Pho 'H-NMR ciia 3 nhan thay [ tin hi^u ciia hai nhom metoxi 5H 3,77; 3,75 (moi tin hieu 3H, s), tin hi?u ciia nam hydro thom, I tin hi?u ciJa mgt nhom oxymetylen 5H 4,48 va 4,37 va mgt nhom metin 5H 4.08. Cac ket qua phan tich tren pho "C-NMR, DEPT, kit hgp vol cac tuang tic tr^c tilp H-C nhan dugc tren phi HSQC cho thay trong phan tir hgp chat 3 co 17 nguyen tir carbon. Hai tin bleu a 6c 60.16 va . 56.0 thugc ve hai nhom metoxi. 15 tin hi|u con lai thupc vl aglycon, gim tin hieu ciia mot nhdm oxymetylen 5c 71.24, mot nhom metin 6c 47.69, mgt nhom cacbonyl 6c 192.44 vi 12 tin hi|u khac nim trong viing cpng huong ciia nguyen tir carbon olefin. Qua do cho thiy cau triic
HQI NGHI KHOA HQC T O A N Q U O C V £ SINH T H A I V A TAI NGUYgN SINH VAT L A N THLf 5 hgp chit 3 co dang mpt dihydroIsoflavon. Cung gilng nhu chat 1, cac tuang tac HMBC (hinh 3) giffa H-5 (5H 7'.75)/C-4 (6c 192.44), H-6 (6H 6.44)/C-10 (5c 114.48), H-8 (5H 6.30)/C-lO, h?
tuang tic spin ABX vai tin hieu H-8 co Jmwa = 2.0 Hz chumg to vong A bj the tai vj tri C-7. H?
tuongticspin AB cua cac hydro vong B ( 5 H 6,53, d , J = 8,5 Hz va 6,50, d, 7 = 8,5 Hz) cung vdi tucmg tic hai chilu nhan dupe tren HMBC giiTa H-6' (5H 6.50)/C-3 (6C 47.69) cho phep khJng dinh vong B cua 3 bi the tgi ba vj tri 2',3',4'. Tiep do, vj tri cac nhom metoxi ciing dugc xic djnh dua tren cac tuong tie tren ph6 2 chilu HMBC. Tuang tac HMBC gitra H-3 (5H 4.08)/C-2' (5C 145.62) vi H-OMe (6H 3.75)/C-2' cho phep khing djnh nhom metoxi thir nhit the tgi vj tri C-2'. Tuang tie HMBC gliJa H-6' (5H 6.50)/C-4' (5C 147.81) va H-OMe (6H 3.77)/C-4* cho phep khing dinh nhom metoxi thu hai the tgi vj tri C-4'. Nhu vgy ciu true cua hgp chat 3 dirpc xic djnh la 7,3'-dihydroxyI-2',4'-dimetoxl-2,3-dihydrolsofiavon hay violanone (hmh I). Hon nira cac du kien pho '^C-NMR cua 3 cung phii hgp vai cac dQ kien pho ' C-NMR cua hgp chat violanone da dupe cong bl trong tai lieu [2] (bing 2).
No.
2 3 4 6 6 7 8 9 10 1 2 3 4 5 6 4- OMe 2'-0Me
DO- kicn pho N M R 3
6 c "
71,24 47,69 192.44 129,43 110.67 164.39 102.67 163,92 114.48 121.58 145.62 138.82 147.81 106,49 119,63 56.00 60.16
BH''" (mult, J in Hz) 4,48(1.11.5) 4.37 (dd. 5.5; 11.5) 4.08 (dd, 5,0; 11.5)
7.75 (d, 8,6) 6,44 (dd, 2,0; 8.5)
6,30 (d, 2,0)
6,63 (d, 8,5) 6.50 (d, 8.6) 3.77 (s) 3.75 (s)
ciJa 3 , ' va chat t h a m khao 4 Sc"
144.14 117,41 192,41 165.17 102,58 165,78 108.17 132.76 112,88 126,72 131,13 115,82 160.25 115,82 131,13
B H " ' ( m u l t , J i n Hz) 7.75' 7.75'
-
6,27 (d, 2.5)
6.40 (dd, 2,5; 8,0) 8.15 (d, 8.0)
-
7,75' 6,84 (d, 8,5)
6,84 (d. 8,5) 7.76'
6 H " (mult, J i n HZ) 7 80 (d, 15,5) 7.62 (d. 15.5)
6,31 (d, 2,5)
6,44 (dd, 2,5; 8,5) 7,98 (d, 8,5)
7,63 (d, 8,5) 6,87 (d, 8,5)
-
6,87 (d, 8,6) 7,63 (d, 8,5)
Bo trong "CDCIj-CDjOD, "DMSO, "MeOD, "l25MHz, "500MHz, '^In hi|u bi chdng chap.
HQl NGHj KHOA HQC T O A N Q U 6 C V E SINH T H A I V A TAI NGUYEN SINH VAT L A N THCf 5
Hinh 3. Cdc tirang tde HMBC (H-K:) chinh cua 3vd4
Chat 4 phin lap dupe duai dgng hot vo dinh hinh mau tring. Tin bleu ciia 15 nguyen tOr carbon a vung truang thip tren p h i ' C-NMR va cic tin hieu xuat hien a vung hydro tham quan sat dugc tren pho 'H-NMR cho thiy chat 4 ciing thupc lap chat flavonoid. Cac cap tin hieu C-2 (5H 7,75; 5c 144,14), C-3 (5H 7,75; 6c 117,41), C-4 (6c 192.41) ggi y chit 4 la mgt chalcon.
Tirong tic HMBC (hinh 3) gi&a H-9 (5H 8 . 1 5 ) / C ^ (6c 192.41), C-5 (6c 165.17), C-7 (Sc 165.78), he tuang tic spin ABX quan sat dugc tren pho 'H-NMR (5H 8.15, d, 7 = 8.0 Hz;
6,40. dd, J = 2,5; 8,0 Hz, va 6,27 d, ^ = 2,5) cho thiy vong A cua chalcon 4 bj thi tai hai vj tri C-5 va C-7. Dilu nay phii hgp vai tin hieu ciia 5-OH tren pho ' H - N M R xuit hien tal 5H 13.62 do co lien ket hydro npi phan tu" voi nhom cacbonyl tgi C-4. Ngoii ra cic tin hieu cpng huang thupc h? AA'BB' quan sit dugc tren pho ' H - va 'X-NMR da khang djnh vong B trong ciu true cua 4 bj the para. Cau hinh ciia nli doi C-2/C-3 dugc xic dinh dua tren hing si tuang tic spin H-2/H- 3 nhan dugc tren phi ' H - N M R . Khi do trong DMSO cac tin bleu H-2, H-3, H-2' va H-6' bj chong chgp Ian nhau do do pho ' H - N M R dupe do Iai trong CD3OD. Trong trucmg hpp niy dg
!6n ciia hing si tuang tic spin yH-2,H-3 = 15.5 Hz (bang 2) cho phep khang dinh noi doi C-2/C-3 c6 cau hinh trans. Cull cung, sir tuang dong giita phi '^C-NMR ciia 4 va isoliquyritigenin (Deesanier S. va nnk., 2007) (bang 2) cho phep xic djnh cau triic hoa hpc ciia 4 Ii Isoliquyritigenin (hinh I).
m . KET LUAN
Bing cic phuang phap sic ky, formononetin (1), pratensein (2), violanone (3) va isoliquyritigenin (4) da dugc phan lip tir go cay Cam Iai {Dalbergia oliveri). Cau triic hoa hgc ciia chung dugc xic djnh dua tren phan tich pho cpng huong tir hat nhan mot chilu va hai chieu.
L&i cant an: Cdng trinh dirge hoan thdnh nh& su ho trg kinh phi ciia de ldi cdp Vi^n Hdn lam Khoa hgc vd Cdng nghe Vi^t Nam. theo hu&ng da dgng vd cdc chdt cd hogt linh sinh hgc.
TAI UEU THAM KHAO
1. Nguyen Tien Ban (chu bien), 2005. Danh lyc cic loai thuc vit Vi?t Nam. NXB. Nong nghi§p Tjip 2.
779-786.
2 Bg Khoa hpc vi Cong ngh?, 2007. Sich Do Vi?t Nam. Phin 2. Thirc vat. NXB. KHTN & CN.
Tr: 194-195.
3. Almeida J.G., Silveira E.R., Pessoa O.D.L., 2008. Magn. Reson. Chem., 46: 103-106.
1. Deesamcr S., Kokpol U., Chavasiri W., Douillard S., Peyrot V., Vidal N., Combers S., Finet J.P.,2007. Tetrahedron, (>y. 12986-12993.
5. Han T., Li H., Zhang Q., Zheng H., Qin L., 2008. Chcm. Nat. Comp , 42- 567-570.
6. Rayanil K.O., Bunchornmaspan P., Tuntiwachwuttikul P., 2011 Arch. Pharm Res., 34: 881-886.
HQI NGHI KHOA HQC T O A N Q U O C V E SINH THAi VA TAl NGUYEN SINH VAT tAU THLf 5 ISOLATION OF FLAVONOIDS FROM STEM WOOD OF Dalbergia oliveri
PHAM THANH LOAN. HOANG LE TUAN ANH, BUI HUU TAl PHAM HAI YEN, DAN THI THUY HANG. NGUYEN THI CUC, DUONG THI HAI YEN. DUONG THI DUNG. NGUYEN XUAN NHIEM CHAU VAN MINH, PHAN VAN KIEM, TRAN HUY THAI, NGUYEN THI HIEN
SUMMARY
Four flavonoids: formononetin (1), pratensein (2), violanone (3), and isoliquyritigenin (4) were isolated from the methanol extract of Dalbergia otiven by various chromatographic experiments. The structure elucidation of isolated compounds was done by spectroscopic methods including 1D- and 2D-NMR as well as in comparison with the literatures.
