Nghien CIFU - Ky thuat
(H-4) va 6,07 (H-6) (tuong irng moi tin higu I H , t, J = 2,0 Hz) dge trung cho s y xuat hien cua mpt vdng thom bj the ba vj tri khdng lien tiep v d ddi xirng. S y cd mgt cua mdt don vj d u d n g ^ D - glueopyranose cung d u p c dac trung bdi tfn hieu eOa mgt proton anome tai 5H 4,86 ( I H , d, J = 7,5 Hz, H-1') vd sdu carbon tgi 8c 102,18 (CH, C-1'), 74,82 (CH, C-2'), 77,95 (CH, C-3'), 71,34 (CH, C-4'), 78,08 (CH, C-5') va 62,48 (CHs, C-6').
Ngodi ra, mdt nhdm methoxy cung d u p c xac djnh bdi cac tin higu cdng h u d n g tgi 8c 55,68 va 5H 3,73 (3H, s). So sanh sd lieu phd ^^C-NMR cua 4 vdi cdc sd ligu dd d u p e edng bd cue picraquassioside D"^ nhdn d u p c s y phii hp'p hoan toan tai tdt ca cac vj tri t u o n g irng (bdng 2). Tir ede dCr kign thu dwac, kdt hpp vdi cac ket qua phd HMBC va ESl-MS cho phep khang djnh hpp chat 4 chfnh la picraquassiosid D.
Ket luan
Sir dyng cac phuong phdp sac ky kdt hpp, bdn hpp chat trao ddi thir cap, bao gdm mdt ddn xudt mdi eiia acid glutaric, tetraglabrifolioside (1), vd ba hpp chat phenol giyeosid, syringin (2), saikolignanosid A (3) va picraquassiosid D (4), da d u p c tinh sach tir djch ehiet methanol cua la cay Tetradium glabrifolium. Cau trOe hda hpc ciia ehiing d u p c xac djnh bang cde p h u o n g
phap phd cgng h u d n g ttr hat nhdn mpt chidu vd hai chidu ( I D - and 2D-NMR) va phd khdi lupng (ESI-MS), kdt hpp so sanh vdi cdc sd ligu phd da d u p c cdng bd. Cde hpp chat 2-4 d u p e phdn Igp lan dau tien tir cay T. Glabrifolium (Benth.) Hartl.
Tai lieu tham l<hao
1. Pham Hoang Hg (2003), "Cgy co Vigt Nam", Nha xuat ban Tre, tgp II, tr 413.2. Dictionary of Natural Products on DVD, version 18.1, Copyright® 1982-2009 CRC Pressr
3. Song D.; Chou G. X.; Zhong G. Y ; Wang Z. T.
(2008), "Two new phenylpropanoid derivatives from Codonopsis tangshen Oliv.". Helvetica. Chimta.
Acta., 91, 1984-1988.
4 Kim S. K; Park J. H.; Kim T B.; Lee H. H.; Lee K.
Y.; Kim Y. C; Sung S. H. (2010), Inhibiton of antigen- induced degranuiation by aryl compounds isdated from the bark of Betula platyphylla in RBL-2H3 cells", Bioorganic and Medicinal Chemisiiy Letters 20,2824-2827.
5. Sugiyama M., Nagayama E., Kikuchi M. (1993),
"Lignan and phenylpropanoid glycosides from Osmanthus asiatbu^'. Phytochemistry, 33,1215-1219.
6. Tan L . Wang B., Zhao Y. Y. (2004), "A lignan glucoside from Bupleurum scorzonerlfolium", Chinese Chemical LeUers. 15, 1053-1056.
7. Yoshikawa K., Sugawara S.. Arihara S. (1995).
"Phenylpropanoids and other secondary metabolites from fresh fruits of Picrasma quasbkles".
Phytochemistry, 40, 253-256.
Nghien ciru xay dyng phtfong phap xac dinh ham Iu'O'ng mot so hop chit co trong vo than
cay gao (Bombax malabaricum DC.)
Pham Le Minh, Ho Thi Thanh Huyen NguySn Thai An, Thai Nguyin Hung Thu*
Truang Bgi hgc Duac Hd Ndi 'E-mail: [email protected]
Summary
An HPLC method was developed for quantitative determination of the 6 compounds isolated from the stem barks of Bombax malabaricum DC... The method was validated In terms of specificity, linearity, precision and accuracy; and thereby, proved practically suitable for indentification and assay of epicatechin, catechln, lupeol, friedelin, stigmasterol and daueosten)!.
Keywords: Bombax malabaricum DC, epicatechin, catechln, lupeol, friedelin, stigmasterol, daucosterol, HPLC.
TAP CHi DlTQfC HQC - 07/2013 (S6 447 NAM 53)
0 Nghien CLFU - Ky thuat
Dat van 6k
Cdy ggo {Bombax malabaricum D C . ) la eay gd eao ddn 15 m, cd hoa mau do t u o i , d u p c bidt ddn nhu Id mpt cdy thude dan gian tgi cac nude Pakistan, An Dd, Trung Qude, Viet Nam vd chau Phi.
Nghidn ciru vdi vd than cdy gao thu hai tgi Hd Npi, cdc tde gia da phan lap d u p c mot sd thanh phan nhu epicatechin'^', eateehin. momor- cerebroside l'^', lupeol, stigmasterol'^, friedelin va daucosterol. Trong khudn khd bdi bdo ndy, chung tdi tidp tye md td qua trinh nghien cuu phuong phdp xdc dinh hdm lupng ede hpp chat da phdn lap d u p c d trdn trong vd thdn eay gao.
N g u y e n l i e u , p h u ' a n g p h a p n g h i e n CLTU
Hoa chat va thiet bj
- Mdy sdc ki Idng khdi phd Shimadzu LC/MS 8030 ed kdt ndi them detector SPD-20A (cd kha ndng phdn tich UV-VIS trong viing 190-700 nm) ciia Trung tdm Kidm nghigm Dupe pham va My pham Hd Ngi.
- Cpt sac kf Zorbax C18 (250 mm x 4,6 mm;
5 \im) kdt hpp cgt bdo vd Phenomenex C18 (ODS, Octadecyi; cartridge 15'<30 mm ID).
- Cdn phdn tich Mettler Toledo dg ehinh xac 0,01 mg.
- Hda chdt tinh khidt phdn tich: acid fomiic.
- Dung mdi loai diing cho HPLC: methanol, acetonitril.
- Cdc hp'p chat da phan lgp d u p e tir vd thdn cdy ggo Id epicatechin. eateehin. lupeol, stigmasterol, friedelin vd daucosterol da d u p c nhgn dgng cdu triic bang cdc phuong phdp phd lyiS vd NMR.
M l u nghien c i r u
Mdu nghidn cO-u Id phdn v d thdn cua cay ggo, d u p c thu hdi tai H u o n g Son (My Dire, Ha Ngi) vd da dupe TS. Tran Huy Thai (Vign Sinh thdi vd Tdi nguydn sinh hpe) giam ^ n h ten khoa hpc Id: Bombax malabaricum D C , ho Ggo (Bombacaceae).
PhLTong phdp n g h i d n c u u
- Ddnh gid tdng lupng tap chat cd tnDng ede mdu hpp ehdt phdn lgp vd tinh chd d u p c bang phuong phdp chudn hda dign tfch.
TAP rui nifrtr unr - 07/201.1 « 6 447 N A M 53)
- Khao sat cac dieu kidn sdc ky vdi HPLC de ed the tdeh hodn todn ede hpp chat trong hdn hp'p 6 hpp chdt nghidn euu vdi nhau va vdi cdc hp'p chdt khac trong cao Idng vd than cdy gao.
- Oinh tinh cac pie tren sde ky dd d y a vdo thdi gian luu kdt hpp vdi MS kdt ndi.
- Tham djnh eac dieu kien djnh lupng mdt sd hpp chat tren vdi chdt ddi chidu Id cdc chat phan lap d u p c da qua nhgn dgng eau true vd xae djnh tdng iupng tgp chdt bang phuong phap chuan hda dien tich.
- Ap dyng de so bp xac djnh ham lupng 6 hpp chat trdn cd trong mdu vd thdn cdy gao da thu hai d\rcfc-
K e t q u a v a b a n l u a n Chuan bj mau t h u
Qua thir nghidm vdi nhieu bidn phap xir 1^
m l u khdc nhau, ehung tdi ehpn phuong phdp xir ty mdu tuong ddi phu hpp nhu sau: can 500,08 g d u p c lidu vd cay da dupc lam nhd, ngdm Ignh 3 lan vdi methanol trong 24 gid, 48 gid va 72 gid. Thu dich chiet, Ipc, ggp djch Ipe cdt thu hdi dung mdi dudi dp suat giam, thu d u p e 25,12 g can. Can d u p c bdo qudn d 4''C.
Hieu suat chldt la 5,4%. Lay khodng 0,50 g cdn, pha vdo btnh d|nh mire 25 mL, Ipc qua bdng thiiy tinh, rdi qua mang Ipe 0,45 pm trudc khi tidm m l u vdo hd thdng HPLC.
Chon dieu kien sac ky
Cdt sac ky sir dung la cdt Zorbax C18 (250 mm X 4,6 mm; 5 pm) kdt hpp cdt bdo vg Phenomenex C18 (ODS. Octadecyi; cartridge 15x30 mm ID) d nhiet dg 25°C.
Pha ddng: Sii" dung chd dg gradient dung mdi vdi kenh A Id acid fonnic 0 . 1 % trong acetonitril va kdnh B Id acid fonmic 0 , 1 % trong methanol (B). Ty Id kenh A-B ban dau Id (30:70), thay ddi tuyen tfnh dgt den A - B (40:60) d phut thir 6, rdi thay ddi tuydn tinh dgt den A - B (50:50) d phut t h u 23. Thay ddi tuydn tfnh ve Igi A - B (40:60) d phut thir 27 vd eudi eiing pha ddng d u p c thay ddi tuydn tfnh ve ty Ig A - B (30:70) ban dau d phut thir 30.
Tde dp ddng ia 0.7 mL/phut. The tich tiem mau Id 10 pL. Budc sdng phdt hign Id 280 nm (detector SPD-20A).
49
• Nghien CIFU - Ky thuat
a) b)
Hinh 1: Sic ky dd cua hon hgrp cic chat nghien cOv (a) vi miu thw chuin bj tO v6 ciy gao (b) (hinh la). Tren sac ky do, mlu thir cd thdi gian luu triing vdi thdi gian luu ciia pic epicatechin, eateehin, daucosterol, lupeol, stigmasterol va friedelin Idn luot la 4,370; 5,401; 7.890; 11,380;
Vdi dieu kien sae k]? va phuong phdp xu- ly mau da lya ehpn, sde ky do thu duoc cho cdc pic taeh rd rdng, nhidu ndn thdp the hien qua sac kif db ciia mlu thu- ehuan bj tir vd than edy
ggo (hinh lb) va hdn hop cde chdt nghien ciru 17,910 va 21,350 phut (hinh 2)
Hinh 2: Sie ky dd ctia cic chdt nghiin euv epicatechin (a), catechln (b), daucosterol (c),
lupeol (d), stigmasterol (e) vi friedelin (f)Xac djnh do tinh khiet cua cdc hap chat nghien cu>u
Cac chdt nghidn euu dup'c cdn chinh xde va hda tan thdnh eae dung dich trong methanol va tiem vdo hg thdng sdc ky.
Hdm lupng ede hpp chat nghidn ciru dup'c ddnh gid qua tdng lupng tgp chdt cd trong
ehiing sau khi duo'c phan lgp va tinh ehe dd xae djnh edu true. Tdng lupng tgp ehdt ciia eae hpp ehat dupc danh gia theo phuong phap ehuan hda dign tfch vdi cdt Zorbax C18 (250 mm; 4,6 mm, 5 pm). Pha dgng su- dyng che dp gradient nhu da xdc di\nh dupc d trdn. Tde dg ddng: 0,7 mL/phut. Thd tich tiem mau: 10 ^JL.
T4iP CHi DU'OC HQC - 07/2013 (S6 447 NAM 53)
• Nghien ciPu - Ky thugt
Nhgn dgng ede pic bang phd khdi lupng.
Chd dp do: ESI+ vdi tde dp khf phun Id 3 L/phut, tde dg khi ldm khd la 15 L/phut, nhiet dg CDL Id 250°C, nhidt dg eua heat block Id 400°C, viing qudt m/z la 100-1000. The pha manh +4,5 kV.
Cac sac ky dd d hinh 3 cho thay ed 6 hpp chdt deu khdng xudt hign pie tgp chat (trong thdi
Tham dmh p h u v n g phap
gian s i c ky la 30 phiit) nen cd thd sir dyng ede hpp chdt ndy Idm nguyen ligu de thidt Igp chat ddi chidu. Tuy nhidn do l u p n g ede hpp chat phdn lap d u p e cdn ft vd thdi gian ehua eho phep, do vgy ehiing tdi tgm dung chiing nhu chdt ddi chidu dd khao sat phuong phdp djnh lupng chiing trong vd thdn cdy gao.
Hinh 3: Phd khdi luK?ng Crng vol cic pic cda epicatechin (a), catechln (b), daucosterol (c), lupeol (d), stigmasterol (e) vi friedelin (f)trin sic k^ do eua miu thd' va hdn hgp chat ddi ehieu Djnh tinh bdng so sdnh phd khdi lupng ciia
cdc pie eiing thdi gian luu cua mau thir vd hdn hpp chat ddi chidu. Kdt qua hodn toan phii hpp.
Ngodi pic cOa 6 hpp chat nghien c u u , tren sac k;^ dd eua mdu thir ehudn bj tir vd than eay ggo cdn xuat hign thdm 2 pie khdc d 14,003 phiit vd
Bang 1: Ket qui khao sat tinh thich hgp
25,431 phut (hinh l b ) .
Kiim tra tinh thich hgp cua he thong Tinh thieh hpp cOa he thdng sac ky d u p c ddnh gia qua kdt qua tidm 6 lan m l u chuan vao hg thdng HPLC, tidn hdnh sac ky vdi didu kign da chpn. Ket qua thu dupe nhu d bang 1.
Hoat chilt Epicatechin Catechin Daucosteroi
Lupeol Stigmasterol
Friedelin
RSD cua thai gian Itm
0,11 0,13 0,12 0,14 0,16 0,15
CM
RSD cOa di^n tich pic (%}
0,12 0,14 0,15 0,21 0,20 0,14
S6 3ia ly thuyet trung binh
22320 23230 24550 34660 35140 32340
H# s6 bit (l6i trung binh
1,11 1,05 1,10 1,07 1,04 1,12
' NAM 53)
• Nghien CLFU — Ky thuat
Cac (ffleu kien sac ky da lya ehpn va he t h ^ g HPLC sii' dyng phO hpp va dam bdo dn djnh eiia phep phan tfch djnh lupng cdc hpp ehdt trdn.
Bang 2: Ket qua khio sit khoang tuydn tinh
Khio sat khoang nong dg tuyen tinh Ket qua khao sat khoang ndng dg tuydn tfnh cOa eac h p p chat nhu d bang 2.
Hgrp chat Epicatectiin Catectiin
Lupeol Friedelin Stigmasterol Daucosterol
N6ng do C (tig/mL) 4,6-45,9 3,8-38,0 4,5^4,8 5,3^2,7 2,4-24,0 1,7-34,0
Phuvng trinh hoi quy (Dien tich pic S: mAU.s)
S=1603,26.C + 0,57 S = 4226,32.C+280.58 S = 2126,83.C + 27,08 S = 1375,75.0 281,07 S = 1456,32.0- 3,05 S = 2103,73.0-231,97
H$ so tu/cmg quan r 1,0000 0,9999 1,0000 0,9999 1,0000 0,9999 Gioi hiin phit hi^n va gidi h^n dinh twong
Gidi hign phdt hien va gidi han dinh l u p n g cua cdc h p p chat xac dinh d u p e Id epicatechin (0,50 va 1,25 Mg/ml-), catechin (0,50 va 1,25 pg/mL), lupeol (0,61 va 1,43 pg/mL), friedelin (0,80 vd 1,60 pg/mL), stigmasterol (0,32 va 0,91 Mg/mL) vd daucosterol (0,34 va 0,80 pg/mL).
Khao sit do dung cua phwong phip Dg dung ciia p h u o n g phap d u p c kiem tra bang phuong phdp them chuan eiia tO-ng hpp chat nghidn ciru vao m l u thir. Tidn hanh vdi 6 m l u Idy gid trj trung binh. Kdt qua thu d u p c n h u d bang 3.
Bang 3: Ket qua kidm tra do dung
Hop chat Nong do
thim vao miu Trung (pg/mL) "'""
TylSthuh6i(%)
Epicatechin Catechin
Lupeol Friedelin Stigmasterol Daucosterol
7,34 3,04 7,17 4,27 1,92 1,36
99,20 99,21 100,88 99,77 100,61 101,42
97,44 98,58 - 99,82.
95,33 - 99,07 - 99,97 •
- 99,84 101,26 101,74 102,68 102,43
•102,19 Cde kdt qua trdn eho phep ed the tidn hdnh dinh tinh, djnh l u p n g 6 h p p ehdt ndy trong v d than edy ggo bdng HPLC.
Kdt qua xdc djnh ham l u o n g mot so hop chdt trong m l u vd than cay gao
Tir mau v o than cay gao dung nghien ciru chiing tdi xac djnh d u p e hdm l u p n g cua 6 chat nghidn ciru ed trong 100 g d u p c ligu nhu sau:
epicatechin Id 12,3 m g ; catechin Id 6,7 mg;
daucosterol la 1,8 m g ; lupeol Id 9.4 mg;
stigmasterol la 1,9 mg va friedelin la 7,6 mg.
Can lam thdm tren sd l u p n g mdu du Idn dd cd sd ligu chfnh xac hon v d ham l u p n g 6 chat tren cd trong v d thdn cay gao.
Ket luan
Mdt p h u o n g phap HPLC da d u p c xay dyng vd thdm i^nh cho phep cd the xac dinh ddng thdi d u p e ham l u p n g cua 6 h p p ehdt cd trong vd than cay gao la catechin, epicatechin, lupeol, friedelin. stigmasterol va daucosterol.
Tai lieu tham khao
1. Hd Thi Thanh Huyen, Thdi Nguyin Hung Ttiu, Nguygn Thai An, Phan VSn Kigm (2011), "Phgn Igp v^ nhan dang epicatechin tir vd t h i n cay gao Bombax malabaricum D C , hp Gao Bombacaceae", T$p chi Dwac hQC. 6/2011 (422), 19-20, 44,57.2. Hd Thi Thanh Huydn, Thdi Nguydn Hung Thu, Nguyen Thai An (2012), "PhSn lgp catechin va momor-cerebrosid 1 tir vd thfin cay gao (Bombax malabaricum DC.)', Tap chi Duoc hoc 12/2012 (440), 49-52
3. Hd Thi Thanh Huydn, Thdi Nguygn Hung Thu, Nguyin Thai An (2013). "Phin lgp jupeol vd stigmasterol tCr v6 than cdy ggo (Bombax malabaricum DC.)", Tgp chl DWQC hoc. 3/2013 (443)
TAP CHi DlTQC HpC-07/2013 (SO 447Niiu s
