Ba dgng sinh kgc vi cde ckit cd kogt tink sink kgc
PHifaNG PHAP O|NH LisgtiG HOP CHAT O-METHYL FURODYSININ
LACTONE TLf CAO CHI^T LOAl HAI MI^N DYSIDEA FRAGILIS
(MONTAGU, 1818) BANG HPLC-DAD
v a Anh Td, Chin Ngflc Digp, NgnySn Th| Cue, Dan TM Thiy Hing, Hoing LS Tufin Anh, Nguyin van Thanh*, Nguyin Xuin Ciring, Nguygn Hoii Nam, Chiu Vfin Minh Vign Hoa sinh bien (IMBC), Vifn Hdn ldm Khoa hpc vd Cong nghf Vigt Nam (VAST)
Abstract
High performance liquid chromatography with diode array detector (HPLC-DAD) was developed for flu quantitative determination of O-methyl furodysinin lactone (1) m methanol extract of the sponge Dysidea fivgilit (Montagu, 1818). The separations were effected by usmg a mobile phase of acetonitrile and water. The gradient elution of the mobile phase was 5-20% ACN in 0-12 min, 20-60% ACN m 12-50 min, 60-75% ACN m 50-65 min, 75-95% ACN in 65-95 min. The flow rate was 0.5 ml/min and the detector wavelength was set at 210 nm. This method was validated in terms of compatibility, specificity, linearity, precision, accuracy, limit of detection and limit of quantitation.
Keywords. 0-methyI fiirodysinin lactone, Dysidea fivgilis, HPLC, validation.
1. DJ5LT V A N Dfi
Furodysmin lactone li nhung hpp chSt sesquiterpene rit d$c tnmg cho cic loii hii mi8n chl Dysidea. Nhflng nghidn cuu trSn Hik gidi trudc day
^ tim thiy ldp chat niy trong cic loii hii mien D.
herbacea, D. sp., D. fragilis, D. arenaria [1] [2] [3]
[4] [5]. d n diy, trong chuong trinh nghiSn ci3ru tim kiem cic bo^t chit sinh hpc tit cic loii hii mi6n vimg biln Ddng t ^ Vi^t nam, chung tdi di phin l|p dupe 7 hpp chat fiinxfysinin lactone tfr loii hii miSn Dysideafragilis thu thjp t^i vdng bifin Quing Ninh, trong d6 h?p chit O-methyl furodysioin lactone cd him lupng vupt tr§i so vdi cic chdt cdn Ud. Trong bii bio niy chung tdi bio cio phuong phip dpih lupng hpp chit O-methyl fiuodysinin lactone td cao chiet MeOH mSu hii mien D. fragilis bhig phuong phip HPLC-DAD.
H 9 OMe
H ^'^
11 10
Hmh 1: Cku true hpp chat O-methyl fiirodysmin lactone
2.THU'CNGHieM 2.1. Thi^t b{ vi hda chit
H? thong sic ky ldng tii^u n ^ g cao (HPLC) Agilent 1200,4 kenh dung mdi, bom mSu tg dgng, detector DAD, cpt s5c k^ Zorbax EcUpse XDB-C18 (4.6 X150 mm, 5 jim).
Cin phin tich Ohaus-USA do cUnh xic 0,0001 g, miy siSu im, cic dyng cu thdy tinh, blnh djnh mdc, micropipetie cd dd chinh xic phd hpp.
Dung mdi Methanol (MeOH), Acetonitril (ACN) lo^ dung cho HPLC (Merok).
Sic k^ cdt (CC) sd dyng silica gel (Kieselgel 60, 70-230 mesh and 230-400 mesh, Merck) vi YMC RP-18 (30-50 ^m, Fuji Silysia Chemical Ltd.). sSc \$ l(5p mdng (TLC) sd dyng bin tring sin siUca gel 60 Fi54 (1.05554.0001, Merck) vi RP-18 F254S plates (1.15685.0001, Merck). V§t chit duffc phit h i ^ blag each phun deu thudc thi^ axit HzSO^
10% vi ha ndng tu tft den khi hifn miu.
2.2. Ngnyln U^u
M^u hii mien Dysidea fragilis (MontagUi 1818) dupe tiiu th§p t^i V9n D^n, Quing Ninh, vao thing 8 nim 2014 vi dupe PGS. TS. D 5 Cdng Thung vi§n Tii nguySn Mdi trudng bilu djnh loii' MSU tiSu bin dupe luu gid tgi Vifn Hda sinh bi^
vi Vifn Tii nguySn vi Mdi tiiidng biln.
23. Phin % hpp chdt (1)
M5U hii mifin Dysideafragilis tuoi, lanh, duP»=
lim khd vi xay nhd (2 kg), sau dd chilt trong MeOH
Hgi thdo kkoa kgc fty ni^m 40 ndm thanh l^p Vifn Hin ldm KH&CN Vift Nam ddng ba lln. Sau khi clt Iogi dung mdi, C$Q chiSt
(120 g) tilp tyc dupe chilt phan ldp vdi dung mdi CH2C!2.
C$n chilt CH2CI2 (50 g) dupe phan tach bing bf tiling sic k^ long trung ip tiSn silica gel, rda giai gradien bing hSn hpp n-hexan/aceton (40/1 - 0/1) tbu dupe cic phin do^a Phin dogn chda hpp chat (1) tilp tyc dupe chgy sic ky cpt pha dio vdi hf dung mdi acetone/HzO thu dupe 65 mg chat (1).
Hpp chit niy dupe tinh chl thSm bing hf tiiong HPLC dieu che, rda giii bing hSn hpp ACN/H2O fim dupe 50 mg O-methyl fiirodysinin lactone tinh khilt, sd dyng lim cbit chuin dl dinh lupng.
ciu trie hpp chit O-methyl fiirodysinin lactone dupe xic djuh bing cich so sinh dd lifu phd cfng hudi^ td hgt nhin (NMR) va cic b^ng sd v$t 1^ vdi cic tii lifu da cdng b l [1].
2A. Chudn h\ man phin tich
CSn Idioing 100 mg cao chiet MeOH n^u hii miSn Dysideafragilis, chuyin vio blnh djnh mdc
10 ml, them MeOH tdi vgch mdc vi lie dlu. Dung dich tiiu dupe dupe Ipc qua ming Ipc 0.45 pm trudc khi phin tich bing HPLC
2.5. Chuin b | mSu chuin
Cic dung dich chuan dupe pha tii O-methyl furodysinin lactone tioh Idiilt trong MeOH d cac nong dp 10, 20, 50, 100, 250, 500 pg/ml vi dupe bio quin trinh inh sing d 4°C cho din khi phin ti'ch.
2.6. Dieu kifn sic k^ (*)
Cft sic k^ Zorbax Eclipse XDB-C18 (4.6 x 150 mm, 5 Jim), detector DAD, budc sdng phit hifn 210 nm, tic df dong 0.5 ml/ph, thl t£ch tiSm mSu 5 pi, pha dOng ACN - H2O vdi chuong trinh dung mdi: 5-20% ACN td 0-12 phut, 20-60% ACN tii 12- 50 phut. 60-75% ACN tir 50-65 phut, 75-95% ACN td 65-95 phdt
Hmh 2: Slc k^ dS miu tring (a), chk chuin (b) ning dp 500 (tg/ml vi djch chilt MeOH cda bii mign Dysideafragilis ning df 10 mg/ml (c)
Ba dgng sinh hgc vi cdc ckat co kogt tinh sink hgc
3. K ^ QUA VA T H A O L U ^ N 3.1. Xiy d^mg phinmg phip phan tich
Hinh 3: Kit qui khio sit budc sdng hap thy Qua tiln hanh Idiio sit budc sdng cho df hip thy eye dgi, ty If thinh phin pha dpng, thl tich tiSm miu, toe df ddng, chdng tdi di xiy dyng dupe dieu kifn sic Ity thich hpp de dinh lupng hpp chit (1) li diiu kifn (*). Vdi dieu kifn sic 1^ nay chit (I) cd thdi gian luu la 54.06 phdt vi dupe tich khdi cic pic khic trong miu phin tich (hlnh 2). Him lupng cbit phin tich dupe xic dinh dya vio difn tich pic thu dupe vi dudng chuia
3.2. Thim djnh phmmg phip
Tham dinh phuong phip phin tich dupe tiln hinh theo cic chl tieu: df tuong thfch cda hf thing sic ky, df chpn Ipc - <^c hifu, khoing ning df tuyin tinh, dp chinh xic, df dung, gidi Iign phdt hifn vi gidi hgn ti^nh lupng [6], [7].
3.2.1. Bd tucmg thich cda hf thong sdc ky Bdng 1: Ket qui khio sit tinh tuong thfch cda hf thong
TT 1 2 3 4 5 6 RSD(V.)
TbM gUn luu (phdt) 54.189 54.014 53.928 53.86 54.055 54.028 0.21
Difn tich pic chdt (1) (mAu)
642.20 628.92 644.57 639.04 629.75 637.21 1.00
Chgy sic ky 6 lin mSu chuin nong dp 20 Mg/nd tiieo dilu kifn sic k^ di chpn. Kit qui cho thiy RSD cua tiidi gian luu vi difn ti'ch pic deu nhd hon 2% (bing 1) chdng td hf th6ng sic k^ hiong tiiich cho qui tiirdi phin tich miu.
3.2.2. Dg chgn lgc - B$c hifu
Tien hAnh phin tich n^u tring, nuu chuin ^l^
miu phin tich theo phuang phip di xiy d\mg. KJI qui cho thiy (hlnh 2) tren sic 1^ d l cda miu trii^
tgi V) tri thdi gian luu cda (1) khdng xuit h i f n ^ nhu d miu c h i ^ vi miu phin tich. Pic cua (1) tnjiw^
miu phan tich dupe tich bift Ididi cac pic khic. Ntd v|y phuong phip cd tfnh chpn Ipc tit vi df d$c hifaj 3.2.3. Khodng nong dg tt^in tinh
TiSn hanh sic 1^ cic mau chuan cd nong df &, 10 - 500 (ig/ml theo phuong p b ^ 6h chpn vi du^
Ifp moi quan hf gitta difn tich pic vdi nong df eh^
chuin. Ket qui cho thiy trong khoing nong df trea cd sy tuong quan tuyin tfnh gida difn tich pfc vi ning dp (1) vdi hf so tuong quan r = 0.99949.
Bdng 2: Ket qua khio sit khoing ning dp tuyin tioh Ning df chit
chain O^g/ml)
Difn tich pic (1) (mAU)
Phuong trinh hii quy: y = 27.6x + 137.26 Hf so tuong quan: r=0.99949
IRnh 4: £>udng chuin bilu di&i mdi tuong qui"
gida difn tich pic vdi ndng df chit (1)
Bpi thdo khoa hpcky ni§m 40 nam thdnh ldp yi^n Hdn ldm KH&CN Vi^t Nam
1.2.4.
\
Bp chinh xac3; K$t qui khao sit dO chinh xic ci^ ptmong
TT 1 l 2 i 3
" 4
| 5
u
Ningdf mSn ptadn tich (mg/ml)
9.6 10.2 10 9.8 10.6 10.4
Di|n tfch pic (l)(mAU)
2382.42 2561.51 2459.2 2435.37 2630.02 2597.74 Trung binh
RSD (%)
Him lugng
(•/.)
0.847 0.861 0.841 0.850 0.852 0.857 0.85 0.83
Tien hinh djnh lupng 6 miu phin tich khac nhau fthipc chuin bj theo quy trinh trong myc 2.4) vdi dilu kifn sic k;? di lya chpn. Kit qui (bing 3) xac djnh dupe him lupng (1) trong cao chilt mau hii mien Dysidea fragilis li 0.85 %, vdi dp Ifch chuin tuong dli li 0.83 %. Nhu vfy phuong p h ^ cd df chfnh xic tot, dip ung yeu clu phin tich 3.2.5. Dg dung
ThSm chfnh xac dung djch chuin vio cic mau phan ti'ch (ty If 1:1 vl thl ti'ch) dl thu dupc cic dung djch cd nong df O-methyl fiirodysinin lactone tuong dng Idioing 80%, 100% vi 120% so vdi ning df dinh lupng. Tiln hinh sic k^ theo phuong phip da xiy dyng dl xic djnh lupng chit chuin tiiu hii.
Kit qui cho thiy (bang 4) phuong phip cd df dung dip dng dupc yeu clu cua phuong pMp phin tich HPLC vdi t j If tiiu hii tiung bmh la 100.55% vi RSD =1.34%.
3ing 4: Ket qui khio sit df dung cda phuong phip
TT 1 2 3 4 5 6 7 8 9
Nongd^
mau (mg/ml)
9.8 10 10.2 9.8 10 10.2 9.8
10 10.2
None <$ chit chuan thSm vio (fig/mi)
50 50 50 85 85 85 120 120 120
Di|n tich pic chit (1) (mAU)
1964.79 1995.41 2027.14 2450.72 2488.03 2532.51 2948.76 2997.51 3050.87
Luffng chit chuan tim i ^ (Hg/ml)
49.16 50.52 49.11 84.38 86.22 85.73 120.47 123.14 123.30
TJ-llthu h4i(%) 98.33 101.04 9823 99.27 101.43 100.86 100.39 102.61 102.75 p Trung binh
2.6 Vng
Gicd hpn ph (LO0
it hifn (LOD) vd gi&l hpn dinh qui thim din tuong thich c
ii cho thiy ] Aa h$ thSng
Trung blnh 99.20
100.52
101.92 100.55 huong phip sic k;^, d$ c
RSD(%)
1.61
1.12
1.30 1J4 ihoa min dp ion IQC - d ^ Tiln hioh chgy sic 1^ miu chuin theo dilu
d ^ (*) (lip Igi 4 lln) d ning df thip sao cho chilu
^ pic ch^ chuin ^ p 3 lan df nhilu dudng nIn.
^ qui cho tiiiy gidi hgn phit hifn cda (1) li 0.04 ig/ml vi ^di hgn dinh lupng li 0.13 pg/ml.
' K ^ L U A N
Dfi xiy dyng dupc phuong phip dinh lupng O- lyl furodysinin lactone trong cao chilt miu hii piSu Dysidea fr-agilis bing phuang phip HPLC,
•ctector DAD vdi cic dilu k i ^ sic Iq^ nhu tr&L Kit
hifu, khoang ning dp taykn tfoh, df chfnh xic, df ddng, gidi hgn phit hifn vi gidi hgn djnh lupng.
Ldi cim on: Cong trinh ndy dugc hodn thdnh vai s}f tdi tn; kinh phi cda Vign Hdn ldm Khoa kgc vd Cong nghf Vift Nam, de tdi md so:
VAST.mMB.06/13-lS.
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Liin hf: NguySn Vfin Thanh
Vifn Hda sinh biln, Vifn Hin lim Khoa hpc vi Cdng nghf Vift Nai s l 18, Hoing QuIc Vift, Clu Giiy, Hi Nfi, Vift Nam
