Tai lifu tham khao
1. V6 van Chi (2012), Tif diin cdy thuoc Viet Nam, NXB Y hgc, t$p 1, u. 853-854. 2. Hgi dong y tinh Ninh thujn (2008), Nhimg cdy thuoc. bai thudc dong bdo Chdm. 3. Ngo VSn Thu (1998), Bdi gidng du^c lieu, Tnrcmg D?i hgc Dupc H4 NOi, t|p 1. tr. 324-338. 4. NguySn Thi Thircmg (2012), Nghien diu ddc diem thuc vgt vd thdnh phdn hoa hgc cdy da tir bien (Limnocitrus littoralis (Miq.) Sv/.). hg cam (Rutaceae) a Ninh Thu4n, Lu?n van T h ^ sJ Dugc hpc, Djii hpc Dugc Ha Npi 5 Silverstcin RM et al. (1998), Spectrometric identification of organic compounds. Sixth edition. John Wiley &Sons, Inc.
USA. 6. Zhang XQ, Hu TT, Wang L, Wang GC (2012), A new coumarin from Citrus grandis 'Shatianyu', Biochemical Systemalics and Ecology, 42, 124-127. 7. Zhao J. Zhou M. Liu Y (2011), Chromones and coumarins from the dried fhictus of Cnidium monnieri, Fitoterapia, 82, 767-771. 8 Zhou P. Takaishi Y, Duan H, Chen B (2000), Coumarins and bicoumarin from Ferula sumbul: anti-HIV activity and inhibition of cytokine release. Phytochemistry, 53, 689-697. 9. Chebrolu KK, Jayaprakasha GK, Jifon J, Patil PS (2013), Purification of coumarins, including meranzin and pranferin, from grapefniitby solvent partitioning and a hyphenated chromatography. Separation and Purification Technology, 116, 137-144. 10. Huy LD.
Caple R. Kamperdick C, Diep NT, Karim R (2004), Isomeranzin against Herpes simplex virus in vitro from Clausena heptaphytla {RoKb)VJ.& Am.: isolation, structure and biological assay, Vowrna/o/cAemis/o', 42(1), 115-120.11. Bank BR.
Dey AK, Das PC, Chatterjee ., Shoolery JN (1983), Coumarins of Murraya exotica - absolute configuration of auraptenol, Phytochemistry, 22(3), 792-794. 12. Gray AI, Waterman PG (1978), Coumarins in the Rutaceae, Phytochemistry, 17(5), 845- 864. 13. Bandara BMR et al. (1990), Acridone alkaloids and coumarins from Pleiospermium alalum, Phytochemistry, 29(1), 297-301.
Tap chi Dugc li$u, tap 19, so 4/2014 (Trang 224 - 231)
DANH GIA Sir THAY DOI NONG D p CUA HESPERIDIN TRONG HUYET TU'ONG CHUOT SAU KHI DUNG THUOC DU^OfNG UONG
Nguyen Thj Hd Ly, Khong Trpng Qudn, Do Thi Phuang, Nguyen Thf Pkiegng, Nguyen Minh Khdi, Phuang Thien Thuang*
Vien Dugc lieu 'E-mail: phuongthienthuong@yahoo.com
(Nhan bdi ngay 05 thang 7 nSm 2014) Tdm tat
Trong c6ng trinh n^y, chiing toi da xay dimg dugc phucmg phap dmh lugng hesperidin trong huyet thanh va huySl tuong chugt bang phuong ph^p sac ky long hi?u nang cao (HPLC). Dung moi pha dgng su dung 1^ acetonilri! va nucrc, chucmg trinh rira giai gradient v6i t6c dp dong 0,5 ml/phiit, quan sat tgi bu6c s6ng 280 nm. Gidi han phat hi^n (LOD) v^ gidi hgn dinh lugng (LOQ) trong phan tich hesperidin lan lugt la 0,44 va 1.46 pg/ml. Dg thu hoi cua hesperidin tir mlu huySt tuong la 94,4
± 2.4 % va dg l?ch chu3n tucmg doi ciia di?n tich pic la 2,0 %. Nhom nghien cihi da sur dyng phuong phap chi6t long - long viri dung moi chgn Igc ethyl acetat de loai tgp va lam giau mhi phan tich. SiJt dyng phucmg phap xay dimg dugc, nhom nghien ciiu da dinh lugng nong d$ hesperidin trong huyet tuong chugt tai nhieu thai diem khac nhau sau uong thu6c, qua d6 xay dyng duong cong nong dg - thd gian va tinh loan cac thong s6 dugc dgng hgc ciia hesperidin.
Tir kh6a: Hesperidin. Huyet tuang chugt, Dugc dgng hgc. Sdc ky long hi^u ndng cao.
Summary
Evaluation of Hesperidin Concentration Changes in Mice Plasma Following Oral Administration We developed in this study a quantitative analysis for determination of hesperidin, an active principle of Citrus species, in murine serum and plasma by using a RP-HPLC-UV-VIS system. Mobile phase was a stepwise gradient of acetonitril and water, flow rate of 0.5 ml/min. detector UV-VIS at 280 nm The limit of detection (LOD) and limit of quantitation (LOQ) for the analysis of hesperidin were 0.44 and 1.46 pg/ml, respectively. The recovery was 94.4 ± 2 4 % and the relative standard deviation percentage (RSD %) of the peak areas was 2.0 %. A liquid-liquid extraction method using ethyl acetate as the specific solvent was applied to remove impurities and enrich the free-hesperidin concentration in analytical samples. By means of these methods, we quantified the concentrations of hesperidin in plasma of mice after oral dosing at varied times and subsequently built the dose-response curve as well as evaluated pharmacokinetic parameters of oral hesperidin in mice.
Keywords: Hesperidin. Pharmacokinetic. Murine plasma. HPLC.
224 Tgp chi Dugc lieu, tap 19, so 4/2014
1. Bit van d l
Hesperidin la flavonoid chinh trong mot s6 loai thuOc chi Citrus du<jc sit dung nhieu trong y hpc CO truyen nhu cam {Citrus sinensis), cam chua (C. aurantium), chanh (C. lemonosa), va quat (C japonica), quyt (C reticulata), thuQC ho cam (Rutaceae) [1], [2]. Hesperidin c6 mpt s6 tdc dyng dupc ly ddng quan tdm nhu ha mS mau, bao V? tim m^ch, ha huy^t dp, di6u trj tieu duong, lien v6t lo^t, chong stress oxy hoa, chong trdm cam nho ca che chong oxy hoa va urc ch6 tao thanh cac tien chat gay viem [2], [3]. Nhieu nghien cihi, k6 cd nghien curu Idm sang da chung minh nhChig tac dung quy ndy cua hesperidin [4- 7] nen ban thdn no da dupc su dung rpng rai trong y hpc hi?n dai [2], [4]. Da co mpt s6 nghien cuu tren the gioi cho biSt tinh chat dupc dpng hpc ciia hesperidin [8], [9] dS co cdc thong tin can thiet cho vipc nghien ctjru bdo chS, hutjng ddn phdn liSu trong sir dung thuoc vd cdc thanh phdm c6 cht^a ho^t chat nay. Tuy nhien, chua co nghien cihi nao dupc tien hdnh de ddnh gid dupc dpng hpc ciia hesperidin noi rieng hay cac chat ttr dupc li^u noi chung cr Vi$t Nam. Do do nghien cthi nay dugc thirc hi?n nhdm ti'nh todn cac thong so dupc dpng hpc cua hesperidin, dong thoi xdy d\mg dupc mpt mo hinh de nghien ciiu dupc dpng hpc cua mpt don chat c\i th6, hucmg tai vipc danh gid dupc dupc dpng hpc ciia cua dupc li?u, cac thu6c vd cdc bai thuoc tiir dupe li?u.
2. s d i t i r ^ g va phiromg phap nghien cuu 2.1. Nguyen li^u
Nguyen li?u diing cho nghien curu Id bpt hesperidin phan lap dupc tii vo qud quyt (Periearpium Citri iieticulatae) co ham lupng 50,0 %.
2.2. Dung moi, hod chdt
Chat chuan Hesperidin co dp tinh khiet 94,5 % dupc cung cap boi Vi^n Kiem nghifm thuoc Trung uang. Cdc dung moi, hod chat su- diing deu d?it tieu chuan tinh khi^t phdn tfch (P.A.), nudc cat hai lan. Ngay truac khi sii dyng, dung moi ch^y HPLC dupc Ipc qua mdng Ipc cellulose acetat 0,45 pm vd sieu am duoi khi.
2.3. Dgng vgt thi nghi?m
Dpng vdt thi nghiem la chupt nhdt trdng truang thdnh, chung Swiss - Albino trpng lirpng 20-22 g dupc mua cua Vien V? sinh Dich tl trung uang. Chupt dupc nuoi trong phong nuoi dpng vdt t^i Khoa Dupc ly Smh hoa (Vi^n Dupc Heu).
2.4. Mdy mdc, trang thiet hi H^ thong phdn tich:
Phan tich dupe thuc hi?n tren he thong HPLC (Shimadzu, Nhdt Bdn), gdm: bom LC-20AD, detector PDA, phdn m6m Labsolution d6 truy xuat hinh dnh va s6 li?u Iren may HPLC. Phan mim Excel (Microsoft Office) va Minitab 14 dupc sii dung d6 tinh todn kit qua vd cdc so lieu th6ng ke.
Thiet bi chudn bi vd bdo qudn mdu sinh hgc:
Mdy ly tdm, tu lanh am sau (Khoa Dupc ly - Sinh hoa, Vien Dupc lieu).
2.5. Phuong phdp nghien ciru
2.5.1. Chuan bj thudc va cho chupt uong thu6c:
Chupt dupc diing thuoc theo duang uong [10].
Bpt hesperidin dupc nghien mjn sau do phdn tan trong nuac co chtia 0,5 % CMC de t^o thanh h6n djch cho chupt uong. O 16 thir chupt dupc cho uong bdng bam tiem co ddu tu de dua thuoc vao da ddy chupt mpt cdch nh$ nhdng. Moi chupt uong 0,4 ml hon djch hesperidin. Mdu trdng Id Id chupt uong nudc cd phdn tan CMC vdi nong dp 0,5 %.
2.5.2. Chuan bj mdu:
Chudn bi huyit tuang: mau dupe Idy tir dpng mgch cd cua chupt dupc cho ngay vao dng chua chat chong ddng EDTA. Mdi chupt Idy tii 1,0 den 1,5 ml mdu. Mau sau dd dupc dem ly tam vdi toe dp 3000 vdng/phiit trong ID phiit d6 loai huyet cau. Ldy Idp huyet tuang ldng a phia tren cho vdo dng eppendorf sau dd bao qudn d tii l^nh dm sau nhipt dp - 80°C cho tdi khi sir dyng.
Chudn bf huyit thanh: tiln hdnh tucrng ty nhu chudn bi huylt tuang nhung khong sii dyng chat chdng ddng EDTA.
Dung dich chudn hesperidin: Cdn chinh xdc khoang 5,0 mg chat chudn hesperidin, hda tan trong 5 ml methanol : nudc (70 : 30) thu dupc dung djch chudn hesperidin cd nong dp chinh xae khoang 1 mg/ml. Tir dung djch ndy, tiln hanh pha loang bdng hdn hqs dung mdi tren theo cdc Tgp chi Dugc liiu, tdp 19, so 4/2014 225
ty le khdc nhau thu dupc cac dung dich hesperidin chuan co ndng dp nho hon. Lay chinh xae 200 pi cdc dung djch nay them vac 200 pi huylt tuang (mdu trdng), them tiep 400 pi ethyl acetat, lac trong 10 phut, de yen 10 phut de phan Idp dung moi. Diing micropipet hiit ldy phan lap ethyl acetat d phia tren. Lam lap lai 3 lan, gpp djch ethyl acetat, d l bay hai het dung moi tdi cdn. Hda tan cdn trong 200 pi methanol, Ipc qua mang cellulose acetat 0,45 pm thu duoc dich phan tich HPLC.
Mdu thu- huyet tuang dimg cho phdn tich HPLC: Ldy chinh xdc 200 pi mdu huyet tuang chupt, them chinh xae 200 pi dung mdi ethyl acetat, lac trong 10 phiit, de yen 10 phiit de phdn
Idp dung moi. Dung micropipet hiit lay phdn Idp ethyl acetat a phi'a tren. Lam Idp l^i 3 lan, gpp dich ethyl acetat, de bay hoi hit dung mdi thu tai can. Hoa tan can trong 200 pJ methanol, lpe qua mang cellulose acetat 0,45 pm thu dupc dich phan tich HPLC.
2.5.3. Dinh lupng hesperidin trong huyet tuong chupt bdng phuong phap HPLC:
Dieu kien tiln hanh phdn tich: cdt pha dao Zorbax ciia hang Agilent (150 mm x 4,6 mm, 5 pm), detector UV-VIS (budc sdng 280 nm); pha ddng la nudc(B) va acetonitril (A) vdi chi dp riia gidi gradient; tdc dp nia giai la 0,5 ml/phdt;
the tich mlu ti6m vao cpt la 10 pi. Detector UV- VIS quan sdt tai budc sdng 280 nm.
Bang 1. Chirong trinh nia giai hesperidin Thjii gian (phijt)
0 - 3 5 - 1 0 1 0 - 2 0 2 0 - 3 0 3 0 - 4 0
A (%, tt/tt) 0 - 1 5 1 5 - 2 5 2 5 - 6 0 6 0 - 9 0 Stop
B (%. tt/lt) 100-85
8 5 - 7 5 7 5 - 4 0 4 0 - 1 0
Kilu rtra giili Gradient Gradient Gradient Gradient
2.5.4. Xdy dtptg dirang cong nong dd - thai gian vd tinh todn cdc chi so dicQc dong hoc cita hesperidin du&ng uong tren chuQt nhdt trdng
So bp tmh todn cac chi sd vl Dupc dpng hpc ctia thudc hesperidin nhu hp sd hap thy (Kg), ndng dp toi da trong mau (Cmax), thai gian de ndng dp d^t tdi da (Tnax), thdi gian ban thai (ti,^), he so thanh thdi (Ke), dien tich duoi dudng cong (AUQ^o)...
theo cdc tdi lieu tham khao [10], [11]. Cdc thdng sd Dupc ddng hpc dupc bieu diln dudi dang chi sd trung binh ± SD ciia 3 ldn thi nghifm ddc lap [ 12].
3. Ket qua
i. /. Toi uu hoa dieu /ci^n phdn tich HPLC Nhdm nghien ciiru da tien hanh khdo sat mdt so dieu kipn phan tich HPLC khac nhau vl toe dp
nia gidi, he dung moi va chuang trinh dung mSi riia gidi. Cac kit qud duac danh gia dya tren cac thong so ddc trung cho phan tich sdc k;y' nhir: tin hieu dudng nin, thdi gian luu (tR), hp sd ddi xung pic (As) va dp phan giai (R). Tii cdc ket qua do, nhdm nghien ciiu da lua chpn dupc dilu ki?n toi uu, phii hop dl phdn tich djnh tinh va djnh lupng hesperidin trong huylt tuang chu^t nhu myc 2.5.3.
3.2. Xay dimg ducmg chudn hesperidin Cac ndng dp hesperidin diing de xdy dyng ducmg chudn dupc trinh bay d Bdng 3. Cac dung djch nay deu dupc Ipc qua mang cellulose acetat 0,45 pm trudc khi dem phan tich HPLC. Kit qua dupc tinh todn tren phdn mem Excel va trinh bay ti-en Bang 2 va Hinh 1.
Bang 2. Sy phii thuge gi&a gid trj di?n tich pic vio nong AQ hesperidin Nong d9 (Mg/ml)
Difn tich pic (mAu.miii)
500 9197044
250 4776381
50 I2I4896
25 480065
5 178655
2.5 43659
226 Tgp chi Duac Ueu. lap 19, so4l20U
10000000 • 9000000 \ 8000000 -^
- ^ 7000000 ^ ' I 6000000 :
^ 5000000 i J ^ 4000000 -i
^ 3000000 ^ 2000000 J 1000000 -I 0 # *
0 100
y = 2.10'.l +11012.-^
R3 = 0.999...-"
200 300 400 C(Mg/ml)
500 600
Hinh 1. Dudng chuan hesperidin Phuong trinh dudng chuan (y = a + b.x) ciia
hesperidin Ihu dupc Id y = 2.10'.x + 11012, trong do: y Id gia trj dipn tich pic, x la ndng dp hesperidin trong mau phan tich (pg/ml). H? sd tuang quan R^ = 0,999 cho thay giira nong dp hesperidin va gid trj dipn tich pic co tuang quan tuyen tinh tot, vi v|iy phuang phap ndy cd the dp dyng de phan tich djnh lupng hesperidin. Phuang trinh duong chuan dd dupc danh gid ve sg sai khdc g i ^ gid trj a va gid trj 0, kit qua cho thdy sy sai khdc nay khong co y nghTa thong ke hay phuang phap xdy dvmg dupc khong mdc sai so hp thong.
3.3. Khdo sdt phuang phdp xir ly mdu 3.3.1. Khdo sat n6ng dp hesperidin frong huylt thanh chupt vd huylt tuang chupt:
De danh gid dupc dOng hpc ciia hesperidin, chung tdi tien hanh khdo sat de lya chpn doi tupng phdn tich giOra huyet thanh (da lo^i cdc yeu to ddng mdu) va huyet tuong (cd them chdt chong ddng). Kit qua thu dugc cho thdy ung vdi moi thi nghipm diu cho kit qud ham lupng hesperidin trong mdu huyet tuang cao han trong mau huyet thanh (khodng 2 lan). Dieu nay cd thi dupc gidi thich do cac flavonoid trong mau lien kit vdi cac protein huyet tucmg d cdc miic dp khdc nhau [13]. Huyet thanh la huylt tucmg da lofti di cdc ylu to ddng mdu vd fibrinogen, trong qud trinh nay cd thi lam giam ham lupng
hesperidin dudi dang lien ket. Vi vdy, chung toi lya chpn huyet tucmg de tien hdnh cdc khdo sdt tilp theo.
3.3.2. Khao sat dung mdi chiit:
De lya chpn dung mdi chiet doi vdi hesperidin tir mdu huyet tuang chupt, chiing tdi khdo sdt vdi hai lo^i dung mdi chiet Id ethyl acetat va n-butanol, cd so'sanh vdi trudng hi;^ khdng sir dyng dung mdi chiit. Qud trinh khdo sdt dupc thyc hifn tuang ty nhu myc 2.5.2 tren huyet tuang chupt sau khi uong hesperidin mirc lieu 125 mg/kg. Moi thi nghiem dupc ldm Idp l^i 3 ldn va tinh ket qud trung binh. Ket qud cho thdy vi?e sir dyng dung mdi hiiu co de chiet hesperidin ra khdi nen mau huylt tuong cho ket qud phdn tich tot ban vipc tiem mdu huylt tuang true tilp vao h^ thong HPLC. Dilu nay dupc giai thich do trong nen mdu huylt tuang, hesperidin cd the t^io Hen ket vdi cac protein nen khong thay xudt hi?n pic tin hi?u ciia hesperidin ty do. Tuy nhien, khi sii dyng n-butanol ldm dung mdi chiit ciing khdng thdy cd mat thanh phdn hesperidin dang ty do, khac vdi trudng hpp sii dyng ethyl acetat. Ket qud ndy phu hpp vdi nghien eiiu ciia Fang T. va cpng sy khi cdc tac gid cung sii dyng ethyl acetat la dung mdi chiet xuat [14]. Vi vdy, chiing tdi lya chpn ethyl acetat lam dung mdi chiit hesperidin ra khdi nen mau huylt tuang chupt.
r«> chi Dugc liiu, tdp 19, s^ 4/2014 227
3.4. Ddnh gid phuong phdp phdn tick 3.4.1. Ddnh gia tinh thich hpp cua he thdng:
Ddi voi he thdng mdy sdc ky, khi da lua chpn dupc dieu kien tdi uu cho qua trinh tach, thi mpt yeu td quan trpng quyet djnh hieu qua phdn tich dd la tinh thich hop ciia he thdng sdc ky dd. Dai lupng nay dupc danh gia thong qua dp lap \^i ciia thiet bj, bao gdm cd dp lap dien tich pic vd thdi
gian luu. Thi nghipm dupc tiln hanh bdng each tiem lap lai 6 ldn dung djch hesperidin chudn co ndng dp la 50 pg/ml. Kit qua thu dupc la cdc gia trj dp Ipeh chudn tuang ddi (RSD %) ciia gia trj thdi gian lim va dien tich pic deu nhd hon 3,0 % chung td dieu kien vd he thong sdc ky HPLC da lya chpn la dn dinh, phii hpp cho phep phan tich djnh tinh vd dinh lupng hesperidin.
SKD chuan hesperidin SKB HPLC mdu trang SKD HPLC m&ulh&
Phd VV chudn hesperidin Phd UV cua pic cd thdi gian luu triing vdi pic cUa Hesperidin chuhn Hinh 2. S3c ky do HPLC phan tich hesperidin trong mhi huyet tircmg chugt
3.4.2. Danh gia dp lap lai ciia phuang phap phdn tich:
De xdc nhdn gid tri sir dyng ciia phuang phap phdn tich, chung tdi tien hanh danh gia dp lap \^\
ciia phuang phap phdn tich. Vi?c danh gid dya tren ket qua phdn tich lap lai 6 lan ndng dp hesperidin trong huyet tuang chupt d miic lilu udng 125 mg/ kg the trpng chupt tai thdi dilm 3h.
Ket qud thu dupc dp lech chudn tuang doi RSD {%), nhd hem 3,0 %, chiing td phuang phdp xdy dyng dupc cd dp lap l^i tdt.
3.4.3. Ddnh gia hipu sudt thu hdi cua phucmg phap phan tich:
De danh gia hipu sudt thu hdi cua phuang phdp, chiing tdi sir dyng phuang phdp them chudn. Chdt chuan hesperidin dupc them vao nen mau thyc la mdu huylt tuong chupt dupc ldy sau khi cho chupt udng 3h, lilu uong la 125 mg/kg.
Cac mdu sau khi them chuan dupc Idc trong 10 phiit, tdc dp Idc Id 1400 vdng/phiit. Sau do tiln hdnh xu Iy mau nhu myc 2.5.2 vd phan tich tren h?
thdng HPLC. Ket qua thu dupc cho thdy hi?u suat thu hdi ciia phuang phap dat 94,44 ± 2,40 (%).
3.4.4. Gidi han phdt hien (LOD) vd gidi han dinh lupng (LOQ) cua phuang phap:
Gidi han phat hi^n (LOD) va gidi h^n djnh lupng (LOQ) Id cac thdng sd ddc trung cho dg nh^y ciia mpt phuang phap phdn tich. Hai gia trj nay cd the dupc tinh todn dya vao dp l?ch chuan cua dudng nen (SB). Tuy nhien, khi tin hi?u nen qud thap ta khdng thi tinh toan gid trj LOD dsra vao dp l?ch chudn ciia dudng nen SB dupc, khi d6 vi?c tinh todn hai gia trj nay cd the dya vdo dp Ipch chudn ciia gid trj y trong phuang trinh hoi qui tuyen tinh (y = a + b.x) Sy, tire la xem SB = Sy va tin hieu nen khi phan tich mdu nin y^ = a. Khi
228 Tgp chi Dugc lieu, tap 19, so 4/2014
dd tin hipu thu dugc iimg vdi nong dp phdt hi^n YLOD = a + 3.Sy. Sau dd diing phuang trinh h6i qui cd thi tim dupc LOD [12].
XLOD= —T- ( l ) v d L O Q = 3,33xLOD(2) Trong dd:
Sy Id dp Ipch chu4n ciia gia trj y trong phucmg
Bing 3. G i « hgai phat hi|n (LOD) v^ gidri han djnh hi^ng (LOQ) cua phirong phip
trinh hoi qui tuyen tinh y = a + b.x dupc tinh toan bang phan mem thdng ke Origin 6.1.
b la d§ ddc cua phucmg trinh hdi qui Dua tren cac cdng thiic (1), (2) vd cac gia trj thu dupe tir phucmg trinh dudng chudn (Sy = 2954, b = 2.10'), chung tdi tinh toan vd thu dupc cdc gia trj LOD va LOQ nhu Bang 3.
Th&ng &h Hesperidin
D$ l^ch chuSn ciia gid trj y trong phinmg trinh hdi qui
tuyen tinh S, 2954
{>$ doc cQa phirong trinh Yihi qui b
2.10'
Gidl han phjt hi^n (LOD) 0,44 (Mg/ml)
Gi6i han djnh lirgng (LOQ) l,46(pg/ml) 5.5. Djnh luxmg hesperidin trong cdc mdu
huyet tucmg chuqt
Qua trinh dinh lupng hesperidin trong huylt tuong chupt dupc thyc hi$n vdi hai lilu thudc cho chupt uong khac nhau, gom: lilu 50 mg/kg va lieu 125 mg/kg. Ung vdi mdi lieu nay, chung tdi ddnh gid sy thay doi nong dp hesperidin trong
huyet tucmg chupt sau nhihig thai dilm udng khdc nhau. Mdi id gdm 5 mau. Cdc mlu diu dupc xu ly nhu muc 2.5.2 va tiln hdnh phdn tich tren he thong HPLC nhu muc 3.1. Kit qud dupc tinh toan dya theo phuang trinh dudng chuan da xay dyng dupc d myc 3.2. Ky hipu Id va kit qud dupc trinh bay d Bdng 4.
B&ng 4. Ky hi^u cSc mlu huyet tuong v& k^t qua djnh lupng hesperidin Liiu 125 mg/kg
Klhifu IS
1 2 3 4 5 6 7 8 9 10
ThiVi dilm l^y huyit tmmg sau khi cho chu$t
ujng thuic 15 phot
30 philt i g i a 2gi6 3 gid 4gi&
6gi&
8gi»
10 gid 24gi(r
Hiim l u ^ g hesperidin trung
binh (fig/mi) 0.9 ±0,2 (<LOQ)- 2,0 ± 0,2 6,0 ± 0.4 21.0 ±0.5 31.0 ±0.4 10.5 ± 0.4 3,5 ± 0.3 1.8 ±0.2 1.1 ±0,1 (<LOQ)
<LOD
Liiu SO mg/kg KI hifu
16 11 12 13 14 15 16 17 18
Thdi diim lay huyit tutmg sau khi cho chu9t uongthuoc
15 phut 30 phut Igid 2 gid 3 gid 4 gid 6 gid 8 gid
Him \\itjn%
hesperidin trung binh (pg/ml)
<LOD 1,2 ±0,3 (<LOQ) 2,4 ±0,4 5,0 ±0,3 12,0 ±0,2 6,1 ±0,5 2,1 ±0,3 0,9 ± 0.2 (< LOQ)
i. 6. Xay dyng duang cong ndng df - thai gian vi dinh gia cac Ihong so duac dong hoc
3.6.1. Xay dimg duimg cong nong d? - thM gian:
Dira tien ket qua * Bang 4, chung toi xay
d^mg duang cong bieu dien s\r bien doi nong dp hesperidin trong huy8t tucmg chupt theo thai gian tai hai lieu diing khac nhau (50 mg/kg va 125 mg/kg). Ket qua dugc bieu dien tren Hinh 3.
Tfp chi Duac lieu, tdp 19. so 412014 229
Hinh 3. Dirdng cong n6ng dg - thiri gian Dudng 1 ung vdi lieu cho chupt udng la 125 'Irdn phat hipn (LOD).
mg/kg, dudng 2 ung vdi lieu 50 mg/kg. Nhdn xet thdy, umg vdi thdi diem 3h sau khi cho chuOt udng hesperidin thi ndng dp hesperidin trong mau d^t cue dai va gidm dan den lOh (ung vdi lilu 125 mg/kg) va 8h (iing vdi lieu 50 mg/kg) thi ndng dp thuoc giam dudi gidi
3.6.2. Tinh todn cdc thdng so dupc dpng hpc;
Dua tren dudng cong nong dp - thdi gian da xdy dyng dupc, kit hpp vdi cdc tai li§u tham khdo [1], [10] (myc 2.5.4), chung tdi xdc dinh dupc cdc thdng sd dupc dpng hpc cua hesperidin trong Bdng 5.
STT 1 2 3 4 5 6
Bing 5. Cac thong s6 dupc d9ng hoc cua hesperidin Thdng so DiTsfc d^ng h9C
C™. (mg/ml) T „ , ( h ) AUCts (mg.h/L) AUCoa (mg.h/L)
K,(l/h) T,n(h)
cm trj Liiu 125 mg/kg
30,0 ±0.4 3h 76.6 81.7 0.69 1,00
Liiu 50 mg/kg 11.0 ±0.2
3h 33,3 36,3 0.67 1,03 Cdc thdng sd dupc dpng hpc dupc xdc djnh tii
2 miic lieu 50 mg/kg va 125 mg/kg cd gia trj khd tuang ddng, cac gid tr} nay cd su khac nhau chut it vdi nghien cuu cua Tong L. vd cpng sy [8] (ti/2 (h) = 3,17; t^ax (h) = 0,25; K^ (1/h) = 0,245 ). Sy khac biet nay cd the dupc giai thich do nghien cuu dupc dpng hpc ciia cdc tdc gid tren dupc tien hanh tren doi tupng khac (chupt cong) va nguyen li^u nghien cihi !a nhiing bai thudc ma thanh phdn cd nhieu chdt khdc, se cd nhung tuong tdc cd the anh hudng den dpng hpc ciia hesperidin (tdng toe dp hdp thu, giam thdi trir).
4. Ket luan
Ldn ddu tien d Vipt Nam da cd nghien ciiu ve dupc ddng hpc ciia mpt dan chat dupc chiet xuat ti!r dupc li$u tren md hinh in vivo. Trong cdng trinh nay, chung tdi dd xdy dyng dupc phuang phap dinh lupng hesperidin trong huylt thanh va huyet tucmg chupt, sir dyng phuang phap chiet Idng-ldng vdi dung mdi ethyl acetat dl lo^i t^p va lam giau mdu phdn tich. Phuang phap xdy dyng dd dupc thdm djnh vl dp Idp l^i, dp thu hoi, c6 gidi h£in phdt hipn vd gidi h ^ djnh lupng thap.
Nhdm nghien ciiu dd djnh lugng dupc nong d?
230 Tgp chi Dugc lieu, tap 19, so 4/2014
hesperidin trong huylt tuang chupt sau khi diing ndy cho cac nghien ciiu tilp theo v l dupc dpng thuoc dudng udng tgi cac thai diem khac nhau, hpc ciia thudc, dupc lieu hay cdc bdi thuoc y hpc dong thdi qua dd xay dyng dudng cong nong dp- co truyen nhdm ddng gdp cac thdng tin quan thdi gian vd ddnh gia dupc cdc thdng s6 dupc trpng cho viee nghien ciiu ve phdn lilu, bao ehi dpng hpc ciia hesperidin dudng udng tren ddi vd cdch su dpng dupc lieu hay thudc y hpc ed tupng chupt nhdt trdng trudmg thanh. Nghien ciiu truyin an toan, hpp ly va hieu qud.
Tk\ li|u tham khdo
1. BO Y tl (2010), Duipc diin Vi^t Nam IV. NXB Y hpc. 2. Phirong Thi|n Thuong (2012). Nghien cuu qui trinh cong nghe chiet xudt citrojlavonoid ttr vo qud ciia mpt so lodi thupc chi Citrus, hq Rutaceae. BSo cao t6ng k^t d i Iii cip So Y Khoa hgc vi Cflng nghe H i Ngi. 3. Rizza S et al (2011), Citrus polyphenol hesperidin stimulates production of nitric oxide in endothelial cells while improving endothelial fiinction and reducing inflammatory markers in patients with metabolic syndrome. Clinical Endocrinol Metabolism Journal 96(5), 782-792. 4. Anand AZ et al (2014), Polyphenols in Human Health and Disease: Chapter 76 - Cardiovascular Effects of hesperidin: A Flavanone Glycoside. Academic Press 2, 989 - 992. 5.
Leandro CS et al (2013), Evidence for the involvement of the serotonergic 5-HTIA receptors in the antidepressant-like effect caused by hesperidin in mice. Progress in Neuro-Psychopharmacology and Biological Psychiatry 40, 103 - 109. 6.
Milenkovic D et al (2011), Hesperidin displays relevant role in the nutrigcnomic effect of orange juice on blood leukocytes in human volunteers: a randomized controlled cross-over study. /'LoSOA'£6(ll), e2666. 7, Morand C ef fl/(2011), Hesperidin contributes to the vascular protective effects of orange juice: a randomized crossover study in healthy volunteers. American Journal of Clinical Nutrition 93, 73 - 80. 8. Tong L. et al (2012), Simultaneous determination of naringin, hesperidin, neohesperidin, naringenin and hesperetin of Fruclus auranlii extract in rat plasma by liquid chromatography tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis 58, 58 - 64. 9. Sirlak M et al (2010), Micronized purified flavonoid fraction in pretreating CABG patients. Texas Heart Institute Journal 2il{2), 1 7 2 - 177. 10. Sampath Cera/(2012), Pharmacokinetics of valerenic acid in rats after intravenous and oral administrations. Planta Medica 78, 575-581.11. BQ Y tfi (2011), Duvc ldm sdng. NXB Y hpc, trang 2 4 - 3 5 . 12. T? Thj Thao (2010), Gido trinh mon hoc thong ke trong hda phdn tich. Tru&ng D^i hgc Khoa hgc Ty nhien - D^i hgc QGHN H^ NOi. 13. Xiao J et al (2011), Molecular propeny-binding affinity relationship of flavonoids for common rat plasma proteins in vitro Biochimie 93(2), 134 - 140. 14. Fang T et al (2006), A rapid LC/MS/MS quantitation assay for naringin and its two metabolites in rats plasma. Journal of Pharmaceutical and Biomedical Analysis 40, 454 - 459.
Tgp chi Dirge li^u, tap 19, s6 4/2014 (Trdng 231 - 234)
KHAO SAT THANH PHAN HOA HOC TREN CAO CHIET CLOROFORM -
C A Y BORRERIA ALATA (AUBL.) DC.
To Cdm Loan', Phgm Nguyin Kim Tuyin\ Nguyin Kim Phi Phun^\ Tir Biic Dun^' ' Dgi hQc An Giang, ^Dgi hgc Sdi Gon.
^ Dgi hpc Khoa hpc tu nhien, Dgi hgc Qudc gia TP HCM
*Email: kimphiphung@yahoo.fr hodc koibito_tdd@yahoo.com (Nhan bdi ngdy 20 thdng 5 nam 2014)
T6m ik
Borreria alata (Aubl.) DC. (hay Spermacoce alata Aubt., Borreria latifolia K. Schum.) la cay pho bien d viing nhi?t ddi v4 cfin nhi$I Chau MJ, ChSu Phi, Chau Au v^ Chau A. CJ Vi^t Nam, cay Borreria alata chi dir<?c diing lim cay phu dat \i chira c6 nghien ciiii nSo v^ thinh ph4n hda hgc ciJng nhu ho^t tinh sinh hgc. Tit dich chiel cloroform ciia cSy Borreria alata, chiing tdi dfl ph9n ISp dupc 3 h(Fp ch^t: Acid ursolic (1), acid olcanolic (2) va kaempfcrol (3). Cau injc cua cic hgp ch4t dugc xdc dinh thdng qua phi 'H-, '^C-NMR vft so s ^ h vdri cic tii Ii?u tham khao.
Til' khfla: Rubiaceac. Borreria alata. Acid ursolic, Acid oleanolic. Kaempferol.
Tgp chi Dugc liiu. tdp 19, sd'4/2014 231
