Nghien ciru - Ky thuat
Xay dyng phirtmg phap xac dinh ham Iirong
r r
mot so hop chat trong co seo ga (Pteris multifida Poir.) bang sac ky long
hieu nang cao
N g u y i n Duy Chi", N g u y i n Hai D u * n g ' N g u y i n Thai A n ' , Thai N g u y i n H u n g Thn^' ' nin KN-NC Duf/c vd TTBYT Quan itqi
^ Truang Dai liQc Dirac Hd Npi 'E-mail, [email protected]
Summary
An HPLC method was developed for quantitative determination of 5 compounds Isolated from Pteris multifida Poir The method was validated in terms of specificity, linearity, precision and accuracy, showing fit for indentification and assay of maltol-3-O-p-D-glucopyranoside (PM3); kaempferitrin (PM 9); 3,4-dihydroxybenzandehyde (PIvl 12); kaempferol -3-0-a-L -rhamnopyranoside - 7 - 0 -P-D- glucopyranoside (PM15J and kaempferol 3-0-a-L- rhamnopyranoside - 7 - 0 - [a • D-apiofuranosyl-(1-2) -p-D -glucopyranoside] (PI\A18).
Keywords: Pteris multifida, apiofuranosyi, glucopyranoside, kaempferitrin, kaempferol, rhamnopyranoside.
D^t van de
Co seo ga (Pteris multifida Poir.), thuoc hg Co Iu6ng (Pteridaceae)'" la loai cay thao cao l<hoang 20 - 40 cm, cay moc ph6 bign a mi§n Bgc va mien Trung nLroc ta, ngoai ra con co ca o Trung Quoc, Nhat Ban. Theo y hpc co truygn, co seo ga CO tac dung thanh nhiet, glai doc, ioi th§p, giam dau... thu'ong du-oc dung 6e ch&a benh ly, viem tu" cung, vi^m da day, ruot, viem du-ong tigt nieu, viem hong, viem tuyen nuoc bot, sung vu, mun nhot, lo ngira, benh ngoai da, ung thu'...'^'^!. Nam 2013, tCr miu co seo ga thu hai tai Ba Vi, Ha Nol, chung tSi da phan Igp, tinh ehe du-oc mot s6 hap chat. Cac chit phan lap dugc da du'ge xac dinh va khing djnh cau true bang pho MS va NMR. De danh gia tiem nang va gop phan tieu ehuan hoa du'ge IISu chung toi da tien hanh nghien cuu xay dung phuang phap xac dinh ham lu-gng mot so hgp chit phan lap duge tip eo seo ga bang sac ky long hieu nang eao.
Nguyen lieu va Phu'O'ng phap nghien c i r u
Dung mdi, hoa chat
- Cac hoa chat dung mol loai tinh khiit phSn tich hoac diing cho HPLC.
Cac hgp chat da dugc phan Up, tinh Che va xae djnh cau true \i maltol-3-O-p-D- glucopyranosid (PM3), kaempferol 3,7-di-O- a-L-rhamnopyranosId (Kaempferitrin -PM9), 3,4-dihydroxybenzandehyd(PM12), kaempferol- 3-0-a-L- rhamnopyranosid - 7 - 0 - p - D - glucopyranosid (PIVI16) vS kaempferol - 3 - 0-a-L-rhamnopyranosld - 7 - O - [a - D - apiofuranosyl-(1-2)-3-D-glueopyranosldJ(PM18).
Thiet bj
May sac ky long Shimadzu vcS'i detector UV - VIS, may sic k j long hl^u nang cao HEWLETT - PACKARD 1100 vai detector DAD da du'ge hl$u ehuan va dap Crng y§u eau theo GLR
TAP CHi Dl/giC HQC - 7/2014 (S6 459 NAM 54)
Nghien ciru - Ky thuat
P h m y n g phap n g h i e n CIFU Chuin bj mau thu- va chit doi chiiu Dung dieh miu thi>: Qua t h u nghiem vui nhieu bien phap x u ly m l u khac nhau. chiing tdi chon phuang phap x u ly m i u tuong doi phu hgp nhu sau: can khoang 50 g d u g c lieu toan cay co seo ga da d u g c lam nho, c h i l t si6u am vai methanol den kiet, thu dich chiet, loc, boc hai each thuy dung m6i, thu d u g c dn (khoang 2 g). CSn d u g c bao quan a 4 ^ . Lay khoang 0,3 g can, hda tan hoan toan dn b^ng methanol va dinh muc thanh 50 mL, loc qua mang lgc 0,45 |im t r u ^ c khi sSc ky.
Dung djch doi chiiu: Can chinh xac cac ch^t phan lap va tinh chi d u g c pha thanh cac dung djch d6i chieu trong methanol cd n6ng dp nhu sau: PM3 la 102 ng/mL, PM12 \i 92 pg/mL, PM18 la 196 ng/mL, PM15 la 202 ng/mLva PM9 la 240
|jg/mL. Loc qua mang Ipc 0,45 pm de d u g c dung djch tiem s i c ky.
Xay duvig phuvng phip phin tfch Diiu kien sic ky
Khao sat vi loai va kich t h u a c cot sic ky, pha dpng va cac yeu to khac d ^ chpn d u g c dieu ki#n sac ky cho phep tach cac chat nghien cuu trong m i u t h u .
Tham djnh phuung phap phan tieh Quy trinh phan tich d u g c tham ^ n h vai cac tieu chl c a ban gom: dp thich hgp he th6ng,
khoang tuyen tinh, gid'i han phat hien, giai hgn djnh lugng, dp dung, dp lap lai cua phuang ph^p.
Ket qua va banluan
Xay di^png p h i r c n g phdp p h a n t i c h Tign hanh phan tich bang HPLC vai detector UV-VIS, s u dung cac cot tach la c6t C18. Thanh phSn pha dpng va chuang trinh dung mSi d u g c khao sat va lua chpn d u g c digu kien sic ky phu hgp n h u sau:- Cdt sic kyf s u dung la cot Zorbax Eclipse XBD - C I 8 (250 mm x 4,6 mm, 5 Mm) k6t hgp cpt bao ve Phenomenex C I 8 (cartridge 30 x 4 mm ID), nhiet do 25''C.
- Pha ddng: S u dung hp pha dpng acetonitril (kenh A) va n u a c co 0 , 1 % acid formic (kenh B) vd'i ehe dd gradient dung moi. Ty le A:B s u dung ban ddu la (5:95), thay do! tuyen ti'nh den (9:91) a phut thu 4, roi thay doi tuyen tinh dat (16:84) a phut thO- 5, duy tri den phut t h u 12, sau do t i l p bjc thay doi t u y i n tinh tdi (28:72) a phut t h u 22, thay doi tuyen tfnh tra ve ty I? ban dau (5:95) a phut 29, duy tri d i n phiit 30.
- Tic dd ddng la 1,0 mL/phiit.
- The tich tiem miu la 20 |iL.
Quet pho DAD cua cac pic t u a n g ung vai cac c h i t nghien c u u thu d u g c dac tinh pho nhu a bang 1.
Bang 1 : Mgt so gii th phd DAD cua pic ung vai cie chat nghien cuii
X (nm)
190 198 210 228 258 264
PUS A (mAU)
15,9 12,8 20,7 9,1 24,0 209
A (nm) 190 206 230 250 264 280
298 312
PM12 A (mAU)
15,3 34,6 31,0 5,1 14.8 25,3
19,2 21,1
P«f»8
X(nm) 198 218 226 264
286 342
A (mAU)
105,5 46,7 47,4 67.3
28,1 45,2
PM15 A (nm)
200 238 264
286 344
A (mAU) 38.6 16,3 25^
10,6 17.4
pm
A (nm) 198 226 264
286 342
A (mAU) 80,1 37,0
SU.
22,3 34,7
TAP CHi Dl/gfC H p c - 7/2014 (SO 459 NAM 54)
Nghien CIFU - Ky thuat
PM12
Hinh 1 ; Pho DAD cija PM3 vi PM12
Pho UV - VIS cua ci 3 chat PM18, PM15 va PM9 deu c6 h i p thu cue dai tgi bw&c s6ng 265 nm. Cac c h i t edn Igi la PM12 va PIVI3 cd dg hap thu tai b u a c sdng nay khdng khac bipt qui n h i i u so vai b u a c sdng cy-c dai (bang 1, hinh 1). Do vay chung tdi ehgn b u a c song phat hi?n la 265nm.
V a i d i l u kien sae ky nay, h6n hgp cac chat nghien c u u da d u g e tich khdi nhau va tach v6i cac thanh phan khac trong mau t h u (vai R £ 1,5). Sac ky do cua hdn hgp chat doi chiiu v^
m l u thi> thu dwgc n h u 6' hinh 2.
Thue hien c h u a n g trinh sac ky da chpn v6i cac hgp c h i t phSn Igp d u g c d l xac djnh thd'i gian luu t u a n g ung vd'i tOng chat nghien cuu vi kiim tra them dp tinh k h i l t ciia eac hgp c h i t phsin l|p dugc. Trong dieu kien sac ky tren, thai gian luu cua PM3, PM12, PIW18, PM15, PM9 lan lugt 1^
khoang 6,1; 10,0; 10,5; 10,9 va 19,2phCit.
Cae s i c ky d l thu d u g e eho thay ea 5 hgp chat phan lap dwgc deu khdng xuat hien pic tgp chat trong thd'i gian sae ky la 30 phiit. Tuy nhten do thd'i gian khdng cho phep va lugng cac hg'p c h i t ph§n lap dwgc cdn it chua du de thilt l$p chat chuan, do vay chung tdi tgm dung chiing n h u c h i t ddi chieu d l khao sat phuang ph^p x^c djnh ham lugng cac hgp c h i t phan lap dugc ti>
cd seo ga.
- ^ i l i U i - - —
il
Hinh 2: Sie k^ do cCia hdn hop 5 chit nghiin cuu (tren) va cua dich ehiit toin phin eo seo ga (du&i)
TA.P CHI DlTgfC HQC - 7/2014 (SO 459 NAM 54)
• Nghien ci>u - Ky thuat
Tham djnh phiromg phap Tinh d$c hiiu
s i c ky lan l u g t dung mdi pha m l u , dung djeh t h u va dung djch ddi e h i l u vd'i dieu kien HPLC da chpn. K i t qua eho t h i y s i c ky dd cua dung mdi pha mau khdng ed pic cua cac c h i t nghien CLFU tgi thai gian luu t u a n g u n g . T i l n hanh kiem tra va chong pho UV-VlS cua cac pic t u a n g ung tren s i c ky dd cua m i u thCp v^ m l u c h i t doi c h i i u thu dwgc eac hp sd match eiia cac hgp c h i t : PM 3 la 0,988; PM 12 la 0,999; PIVI 18 la 0,998; PM 15 la 0,990 va PM 9 la 0,999. Cac k i t qua cho thay cac pic cd dp tinh k h i l t cao va khang dinh cac c h i t nghien CLPU da d u g e taeh khdi eac c h i t khac cd trong cac hon hgp phan tich.
D p thich hgp
Tien hanh s i c ky lap lai 6 lan dung djch d i i c h l l u da c h u i n bj d tren, ghi Igi cac gia trj v l thdi gian luu, dien tich pic. K i t qua d u g c trinh bay a bang 2 cho t h i y he thong s i c ky cd dp lap lai tot vdi thdi gian luu va dien tich pie ciia eae e h i t (RSD < 2 % , bang 2).
B a n g 2: Kit qua kiem tra do thich hgp Hpp
chit PM3 PM12 PM18 PM15 PM9
Thai gian lipu (phut) Dien tich pic (mAU.s) Trung binh
6,04 9,97 10,53 10,88 19,18
RSD(%) 0,18 0,16 0,14 0,14 0,07
Tmng binh 154852 246030 642688 254480 461200
RSD (%) 1,11 0,86 0,70 0,65 0,85 Khoang nong do tuyin tinh T u cae dung djeh gdc c h i t d i i c h i i u , pha cac day dung djch c h u i n gdm 6 m l u cd nong dp tang dan trong khoang thich hgp, t i l n hanh s i c ky de khao sat khoang nong dp t u y i n tinh. Ket qua thu d u g c n h u sau:
- P h u a n g trinh hdi quy vdi PM3 xac djnh d u g c la S = 19976.C - 7 7 7 1 , trong khoang n l n g dd 2,0 - 20,4 Mg/mL vd'i r = 0,9996.
- P h u a n g trinh hdi quy vdi PM12 xac djnh d u g c la S = 59403.C - 21783, trong khoang nong dO 1,0 - 13,8 pglmL vai r = 0,9997.
- P h u a n g trinh hoi quy vdi PM18 xae dinh d u g e la 8 = 37253.C - 64445, trong khoang nong do 4,9 - 49,0 MQ/mL vai r = 0.9990.
- Phuang trinh hdi quy vdi PM15 xac djnh d u g c la S = 35286.C - 20621, trong khoang nong do 2,0-20,2 lig/mL vdi r = 0,9992.
- Phuang trinh hdi quy vdi PM9 xac dmh d u g e la S= 53034.C - 36684, trong khoang ndng do 2,4 - 24,0 ng/mL vdi r = 0,9994
Gioi han phit hien va giai han dmh Iwgmg Gidi han phat hien va gidi hgn djnh lugng cua cac hgp chat xac dinh d u g c nhu d bang 3.
Bang 3: Kit qua khao sit LOD. LOQ(pg/mL)
LOD LOQ
PM3 0,18 0,59
PM12 0,18 0,61
PMie 0.18 0,59
PM15 0.16 0,52
PM9 0.13 0,43 Do dOng
Them vao mau t h u mdt lugng chinh xac c h i t doi chieu sao cho tong lugng hoat c h i t ed trong mau nam trong khoang tuyen tinh, Mdi m l u them 3 muc nong do, moi nong dp t i l n hanh 3 m l u . K i t qua khao sat do dung d u g c tong hgp trong bang 4.
Bang 4 : Kit qui do dung eua eae ehit Ty le thu hoi (%)
PM3 PM12 PM18 PM15 PM9
Min - max 97,58-102,23 98,86-104,76 95,39 - 97,77 96.55 - 99,71 99,63-103,49
Trung binh 100.64 102,61 96,27 98.01 101,30
1,48 2,17 0,83 1,21 1.28 Do lap lai
S l e ky 6 m i u t h u d u g c c h u i n bj vd'i cac khdi lugng can la 0,2717; 0,2990; 0,2651; 0,2994;
0,2776 va 0,2605 g. Xac dinh gia tn F la ty le giua diSn tich pic thu d u g c tren s i c ky dd vol khdi lugng e i n d u g c l l y de chuan bi m l u (tinh rieng theo tung pic ung vdi cac hgp c h i t nghien CLFU).
Bang 5: Kit qua khao sit do lap lai HOP
chat PM3 PM12 PM18 PM15 PM9
Khoang dien tich pic S (MAU.S) 141394-166267 249448 - 287688 661434 - 767336 256447-301582 455519-522532
Gia trip trung binh
553256 956788 2540269 983129 1747785
RSD
(%)
1,32 1,58 1,81 1,43 0,50
TAP CHi DU<fC HOC - 7/2014 (SO 459 NAM 54)
Nghien ciru - Ky thuat
STT 1 2 3 4 5 6 Trung binh
Ham Iwomg (mg/100 g dinyc ii^u) PM3
7,42 7,29 7.52 7.37 7.35 7,26 7,37
PM12 4,50 4,39 4,39 4,41 4,46 4.44 4,43
PM18 19,30 18,33 18,87 18,89 18.88 18.94 18,87
PM15 PM9 7,56 9.06 7,83 8,90 7,62 8,96 7,50 6,92 7.47 8,99 7,63 9,02 7,60 8,98
So' bo xac dinh ham luwng cac chat co ph«p co the xae dinh dftng thM hSm luang olia trong co seo ga 5 hap chit co trong eo seo ga va so bo xac 1 J >, I.- uoi r~ ,»« ™ , rfi.nn dinh du'ae h4m luang cua chCing trong 100 g Ap dung phuong phap HPLC da xay dung ^^^^ ^..^ ^^^ |,. ma|,o|.3.0-|3-D-glucopyranosid duoc de xac djnh ham luang mot so hap chat ^p^^,^, -^^ ^^ kaempferitrin (PM9): 9,0 mg;
trong mau dugc lieu CO seo ga thu hai tai Ba Vl, 3 4.dihydroxybenzandehyd (PM12): 4,4 mg;
Ha NOI (bang 6). l<aempferol-3-0-a-L- rhamnopyranosld-7- Bing 6: Ket qua xac dinh ham lu^ng cac chit 0-p-D-glucopyranosid (PM16): 7,6 mg v4 nghien ciru tmng CO seo ga kaempferol - 3 - 0-a-L-rhamnopyranosid - 7 - 0
- [a - D - apiofuranosyl-(1-2)-3-D-glucopyranosid]
(PM18):18,9mg.
Tai lieu tham V\\ho
1 D 5 Huy Bich va cong su (2004), Cay thudc va dpng vaf lam thuoc a Vi§t Nam, t$p 2, NXB Khoa hpc v i ky tiiuat, 729 - 730.2. JIangsu New Medical College (1985), Dictionary of Traditional Chinese Drug, Vol 1, Shanghai Science and Technology Press, Shanghai, 487.
3. Lee H., Lin J. Y. (1988), "Antimutagenic activity of K e t i u a n extracts from antipancer drugs in Chlnense medicine",
' . , j „ , „ . . , . Mutat Res,, 2 0 4 , 2 2 9 - 2 3 4 .
Phuang phap HPLC xay dung dugc cho
(Ngay nhan bai: 05/06/2014 - Ngay duyet dSng: 10/07/2014)
Phan tich ciu triic va xay dwng tieu chuan
r r
CO" SO" cho phospholipid chiet xuat fluffc lam nguyen lieu tao dang thuoc tiem liposom
Nguyen Thi Lap'*, Le Dang Quang^
' Tnrong Dai hoc Dupe Ha Noi
^ Vien Hoa hoc Cong nghiep Viet Nam 'E-mail: [email protected]
Summary
Natural phospholipids were extracted, purified from fowl eggs and hydrogenated with activated NiekelRaney. Their chemical structures were determined by LC-MS, GC-MS. The principal phospholipid ingredient was palmitoyi oleoyl phosphatidyleholin. Home speci^cations of the obtained phospholipids were introduced based on LJSP34-NF 29. Analytical results showed the obtained products fit for use as materials of injectable liposome formulations.
Keywords: Phospholipid, supercritical earbondioxide extraction, liposome.
