Nghien
CLFU- Ky thuat
Ket luan
Chiing tdi da xay dung dupe quy trinh ehidt xuit stigmasterol toan phin (dang t u do va kdt hgp) bing ehidt Soxhlet sau 5h vdi ehloroform.
hieu xuit chidt duge kidm seat bing phat hien vdt stigmasterol trong dieh ehidt thuy phan tren TLC vdi he dung mdi then khai la n-hexan/ethyl acetat (8:2), phat hien bing thudc thir Liebermann-Burchard, Quy trinh djnh lugng stigmasterel trong dich chidt thuy phan duge thuc hien bing HPLC tren cot C8, nhiet do cot 30 ± 1°C, pha ddng ACN/dem phosphat 0,025M (93:7), pH=4,8, tde dp ddng Iml/phiit, the tich tiem 20pl, Amax=195nm, Phuang phap phan tieh cho tinh tuang thieh he thdng eao (RSD% <
2%) va duge danh gia t h i m djnh tdt (tinh dac hieu, dp tuydn tinh (10-300ppm), do diing. dp chinh xae dat yeu c i u . LOQ = lOppm), Ham lugng stigmasterol trong bpt duge lieu ray khd la 0.0089%, Do dd, phuang phap nay ed thd ap dung dd danh gia chit lugng eiia eay ray ndi heng va cae dugc lieu chira phytosterol ndi chung.
Summary
Total stigmasterols from the Alocasia odora (Roxb.) C Koch powders was extracted on Soxhlet apparatus for 5h by ehloroform being followed by TLC (with n-hexane-ethyl axetate - 8:2. detected by Liebermann-Burchard reagent).
The content of stigmasterols was determined by
a sensitive and time-saving HPLC technique: C8 column (30 ± 1°C): mobile phase: acetonitente- phosphate buffer pH 4.8 (93:7 v/v), Iml/min, injection volume: 20pl: det. UV 195nm. The method was validated: RSD < 2% (for ehromatogaphic parameters): linearity range: 10 to 300ppm (r = 0.9995). The method proved to be accurate and precise with the mean percent recovery of stigmasterols from the powders:
95.2% and LOQ: lOppm.
Tai lieu tham khao
1. D 5 Tat Lgi. NhCrng cay thuoc va vi thu6c Viet Nam. NXB Yhgc va TW, 2007. tr.l40.
2. Bo Y t l , Quy che danh gia tinh an toan va hieu luc thuSc c6 truyin. Tap chi dwgc hgc, 1996. s6 5 , tr,3,
3. Le Ngpc Kinh. Nghien cuu thanh phin hoa hgc ciia cil ray d thanh pho Hue. NXB Y hgc thwc banh, Bg Yte. 2005, s6 521.tr, 24,
4. Le Nggc Kinh, Tac dung ha cholesterol trong huyet thanh tho ciia dich chiet tir than r l cay ray. Tap cbi Dwgc hoc, 2007, so 374, tr. 11.
5. Ellen Odeen Thesis, Investigation of phytosterols in Alocasia Odora Roxb, by reversed phase HPLC, A study made in Hue City, Vietnam, 2009, p.23.
6. World Health Organisation, General guidelines for Methodologies on Research and Evaluation of Traditional Medicine, 1998.
7. Ludw/ing huber. Validatio n of Analytical Methods, Review and Strategy, 2001. p i 3,
Nghien ciru dmh liro'ng apigenin trong ciic hoa vang (Flos Chrysanthemi indici)
bang phuo'ng phap HPLC
Nguyen Trong Diep', Dao Van Don', Hoang Viet Dung' Nguyen Van Long', Nguyen Minh Chinh', Doan Cao So'n^
Dat van de
Ciic hoa vang (Flos Chrysanthemi indici) la dugc lieu duge sir dung kha phd bidn trong cac bai thude y hpc ed truyen, Dugc lieu nay ed tac dung tan phong thip, thanh d i u mue, giang
Hgc vien Qudn y. ' Vien Kiem nghiem tliuoc TW
hda, giai ddc nen dugc dimg dd chQ'a phong ma sinh hoa m i t , nhirc d i u , dau m i t . cao huydt ap.
sdt'^', Theo cac cdng trinh khoa hge da edng bd, nhdm heat ehit chinh trong eiie hoa vang la flavonoid vdi mpt sd hpp chit nhu: apigenin.
TAP CHi DUQC HQC - 3/2011 (SO 419 NAM 51)
45Nghien CLFU - Ky thuat
luteolin, luteolin - 7 - (3 - D - glucopyranosid, acacetin - 7 - (3 - D - glucopyranosid, ehrysanthemin'^'^',,. Trong dd, apigenin la heat chit thu hilt duge nhidu su quan tam nhit bdi boat ehit nay ed mdt sd tac dung sinh hpc nhu: ngan ngu'a ung thu, chdng oxy hda, dpn gde tu do,,,
De dam bao hieu qua chira benh cho eae thang thude ed thanh phin ciie hoa vang thi ehit lugng dugc lieu nay ddng vai rit quan trpng bdi ham luang boat ehit trong dugc lieu cd su thay ddi rit Idn giira cae viing trdng nguyen lieu, giii'a eac thdi diem thu hai va dieu kien ehe bidn, bao quan, Han nira. qua tim hidu tai lieu duge didn cac nudc nhu: Viet Nam, Trung Qudc, Nhat, My,,,, chiing tdi chua thiy cd tai lieu nao edng bd phuang phap djnh lugng apigenin trong dugc lieu, Vi vay, nhu c i u c i n phai cd mpt phuang phap djnh lugng apigenin treng ciic hoa vang la c i p thidt. cd y nghTa khoa hpe va thue tidn,
Nguyen lieu va phu'O'ng phap nghien
CLFUNguyen lieu
- Duge lieu: Ciic hoa vang dugc mua d phd Lan Ong - Ha Ndi vao thang 10/2010, Dugc lieu dugc xae djnh dat eae tieu ehuin djnh tinh quy dinh trong Duge didn VN III,
- Hda ehit:
+ Chit ehuin apigenin (Shanghai Tauto Biotech Co,, Ltd, -China).
+ cdn 96°, methanol... dat tieu chuan tinh khidt phan tieh.
+ Acetonitril, aeid formic: dat tieu ehuin phan tieh HPLC-Merek.
- Thidt bi. dung eu:
+ Binh ehidt sieu am Sonielean.
+ May HPLC Alliance Water 2695D; Detector PDA,
+ Can phan tieh Sartehus dp chinh xac 0,1mg.
+ Bd Ipc hilt chan khdng Alltech - My.
+ Mang Ipe nylen Sartprius Minisart kieh thudc Id Ipc 0,45 pm.
Phwang phap nghien ciru
- Xay dung phuang phap djnh lugng apigenin trong duge lieu dua tren danh gia eae thdng sd: tinh tuang thieh he thdng, tinh dac hieu, khoang tuydn tinh, dp lap lai, dp diing, gidi ban phat hien va gidi ban dinh lupng dudi.
- Qua tham khao tai lieu '^"^' va thue nghiem chiing tdi da xay dung dugc didu kien phan tich HPLC nbusau:
+ He thdng HPLC (nhu tren).
+ Detector PDA, djnh lugng d budc sdng 338nm.
+ Chuang trinh rii'a giai isocratic vdi he pha dpng aeetonitril/dung djeh aeid formic 0 , 1 % ty le 40/60.
+ Tdc do ddng: 1 ml/phiit.
+ Thd tich tiem: 20 pi.
- Dung dich thir: Can chinh xae khoang 5g bdt dugc lieu (dugc nghidn tdi kich thudc thich hgp) eho vao dng nhua dung tieh 50ml. Them khoang SOmI methanol, ehidt sieu am trong thdi gian 30 phut d 50°C (chidt 3 lin). Gdp eae djch chidt vao binh djnh mirc dung tich 100ml, them methanol vii'a dii den vach. Dung djeh trong binh dugc Ipc qua mang 0,45mcm sau dd dem phan tich s i c ky.
- Dung djeh chuan: Can ehinh xae kheang 50 mg c h i t chuan apigenin va hda tan vae binh dinh mirc 100ml vdi methanol dd dugc dung dich chuin gdc cd ndng dp khpang 0,5 mg/ml.
K § t q u a n g h i e n CLFU Tinh tu'O'ng thich he thdng
Dupe danh gia b i n g each tiem lap lai 6 l i n mpt dung dich chuan vao he thdng s i e ky vdi didu kien neu tren. Kdt qua dugc trinh bay d hinh vS bang dudi dciy:
BCD B!D W
Hinh 1 : SSc ky do dung dich chuin apigenin
Hinh 2: Sic ky dd dich chidt dwqe lieu
46 TAP CHl DUQC HQC - 3/2011 (SO 419 NAM 51)
Nghien CLFU - Ky thuat
Bang 1: Ket qua khao sat tinh thich hqp cua he thdng sic ky
Noi dung Dien tich pic Thoi gian luu He so bat doi xirng
So dTa ly thuyet
Ket qua
S,b= 866312; RSD = 0,67%
tR tb = 8.69 phiit; RSD = 0.52%
1,27 3200
Ket qua eiia bang 1 eho thay be thdng sac ky tuang thich vdi m i u phan tich vdi RSD ciia dien tich pic va thdi gian luu ddu nhd ban 1 %.
Tinh dac hieu
Sir dung detector PDA dd danh gia tinh dac hieu ciia phuang phap: tai thdi gian luu eiia pie tren s i c ky dd m i u ehuin va m i u thir, xae dinh phd UV-VIS, Kdt qua cho thiy phd tai cae diem tren hai pic la gidng nhau ve hinh dang va sd dinh hip thu cue dai ehirng td phuang phap ed tinh dae hieu cao.
Khoang tuyen tinh
De khae sat kheang tuydn tinh eua phuang phap, chiing tdi tidn hanh pha mdt day dung djch ehuin ed ndng dp: 2,40; 4,80; 9,60; 19,20;
38.40 pg/ml sau dd xac djnh tuang quan dien tich peak va ndng do cac dung dieh ehuin. Kdt qua duge trinh bay d bang va hinh sau:
Bang 2: Sw phu thude cita dien tich peak vao ndng do apigenin chuin
Nong do (pg/ml) Dien tich
2.40 210542 4.80 426674 9.60 870312 19.20 1707954 38,40 3527603 y = 91967 X -19853 vai R^ = 0,9997
3500000 - ^ -, , V - 9 " " « - 1 9 8 5 3
" ' ' ™ ' ' ° R' - 0,9997 ^ " ^
0 10 20 30 40 50
Hinh 3: Dd thj twang quan giira dien tich peak va ndng do apigenin
Kdt qua bang tren cho thiy trong khoang ndng do khae sat, dien tieh peak va ndng dp
apigenin ed su tuang quan tuyen tinh ehat ehe vdi R^ = 0,9997,
Do lap lai
Op lap lai cua phuang phap dugc tidn hanh bing each dinh lugng apigenin vdi 6 thi nghiem rieng biet bing phuang phap da ehpn, Kdt qua dugc trinh bay d bang dudi day:
Bang 3: Kdt qua khao sat dp lap lai
^^ Lug'ng can Ham Iwtyng jhong ke
(mg) (%)
1 2 3 4 5
5,0003 4,9998 5.0023 5,0012 5,0016
0,0186 0,0170 0,0175 0,0181 0,0183
HL,b=0.0178%
RSD= 3.43 %
5.0021 0.0174
Ket qua eua bang 3 eho thay phupng phap djnh lupng ed dp lap lai tdt vdi RSD = 3.43%,
Dp dung
Xac dinh dp diing ciia phuang phap bing phuang phap them ehuan.
- Dung dich ehuin gdc: hiit ehinh xac 1ml dung djeh ehuin. bda loang vao binh dinh mire 50ml bing methanol, l i c ky,
- Dung dich thir gdc: tidn hanh chuin bi nhu phin dung dieh thir,
- Dung djeh hdn hgp: hiit ehinh xac 5ml dung dieh thir gde eho vao binh djnh mirc 10ml, them dung djch ehuin gde vii'a du tdi vach, lae deu.
Dung djch dugc Ipc qua mang 0,45pm sau dd dem phan tich s i c ky.
Tidn hanh do HPLC vdi didu kien da neu tren, lap lai thue nghiem vdi 5 dung djch thir khae nhau, Kdt qua dugc trinh bay d bang sau:
Bang 4: Kdt qua khao sat dd diing
TT
1 2 3 4 5
Lwo'ng them vao (pg) 48.00 48,00 48,00 48,00 48,00 Thong ke
Lucmg tim lai .. ^. , . , , %tim lai (pg)
45,96 97,78 46.23 98.36 46.59 99.12 47.69 101.47 45,43 96.65
%tim lai ,b = 98,68%
RSD = 1,83%
Ket qua bang 4 cho thay phuang phap djnh lugng cd dp diing eae vdi % tim lai tir 96,65 - 101,47%; RSD= 1.83%,
TAP CHi DUQC HQC-3/2011 (SO 419 NAM 51) 47
Nghien CLFU - Ky thuat
Giai han phat hien va glal han dmh Iwg'ng dwai
Da khae sat va xae djnh duge:
+ Ndng dp m i u thir khpang 0,2 pg/ml la gidi ban phat hien, tai ndng do nay dap irng pic m i u thir ed dien tieh g i p han 3 l i n dien tich dap irng ciia m i u tring,
+ Ndng dp m i u thir khoang 0,4 pg/ml la gidi ban dinh lugng dudi, tai ndng dp nay dap irng pie m i u thir ed dien tieh g i p ban 10 l i n dien tieh dap irng eiia m i u tring vdi dp diing va do lap lai d muc cd thd c h i p nhan khi tidn banh phan tich d ndng dp t h i p nay,
K § t l u a n
Da xay dung dupc phuang phap djnh lugng apigenin trong ciic hoa vang bing phuang phap HPLC vdi didu kien s i e ky la: edt Phenomenex Genimi RP-C18; pha ddng:
acetonitnl : aeid formic 0 , 1 % (40:60); detector UV: 338nm, Phuang phap djnh lupng nay ed su tuang quan tuyen tinh chat ehe g'lwa dien tieh pic va ndng dp apigenin trong khoang ndng do khao sat vdi R^ = 0,9997, dp lap lai (RSD = 3,43%) va dp diing (tir 96,65 - 101,47%, RSD = 1,83%) cao, Thdi gian phan tich mdt mau b i n g HPLC la phil hgp (khoang 8,7 phiit).
Summary
An HPLC method was developed for quantifying the main active compound apigenin
of Flos Chrysanthemi indici: RP-C18 column:
detector UV: 338nm: mobile phase: acetonitrile- aqueous 0.1% formic acid (40 : 60): flow rate:
Iml/min: injection volume: 20pl. The method was validated to be adequately time-saving (taking ca. 8.7 minutes) and of good linearity (R^=0.9999), high repeatibllity (RSD = 3.43%) and high accuracy (96.65 - 101.47%,, RSD:
1.83%>).
Tai lieu tham khao
1. D6 Tit Lgi. Nhung cay thuoc va vi thuoc Viet Nam. NXB Y bgc. Ha Npi - 2004, trang 604 - 605.
2. Liang-Yu Wu, Hong-Zhou Gao. Xun-Lei Wang, Jian-Hui Ye, Jian-Liang Lu, Yue-Rong Liang. Analysis of chemical composition of Chrysanthemum indlcum flowers by GC/MS and HPLC. Joumal of Medicinal Plants Research Vol. 4(5), pp. 421-426, 4 March, 2010.
3. Qing-Lei Sun, Shu Hua, Jian-Hui Ye, Xin-Qiang Zheng, Yue-Rong Liang. Flavonoids and volatiles in Chrysanthemum morifolium Ramat flower from Tongxiang County in China. African Joumal of Biotechnology yo\. 9(25), pp. 3817-3821, 21 June, 2010.
4. Long-Ze Lin, James M. Harnly. Identification of the phenolic components of chrysanthemum flower (Chrysanthemum morifolium Ramat). Food Chemistry 120(2010)319-326.
5. Mustafa Bener, Mustafa Ozyurek, Kubilay Guclii, Resat Apak. Polyphenolie contents of natural dyes produced from industrial plants assayed by HPLC and novel spectrophotomethc methods.
Industnal Cropsand Products 32 (2010) 499-506.
N g h i e n C L F U . . . (Tidp theo trang 1 7) Anh hu'dng ciia dung djch HL den chiHC nang than
Than la ca quan tidt nieu. ed vai trd quan trpng bae nhit dd dam bao su hing djnh ndi mdi. Khi dua thude vao ea the ed the gay tdn thuang than, anh hudng ddn ehirc nang than. Dd danh gia anh hudng ciia thudc ddn ehire nang than, ngudi ta djnh lugng ndng dp creatinin treng huydt thanh'"'.
Creatinin la chat chuydn hea eudi ciing, san phim phan buy eiia ereatin phosphat, ndng do creatinin khdng phu thupe vao ehd dp an. Khi cd tdn thuang than, creatinin tang sdm han va tin eay ban ure nen hien nay day la mdt chi sd thudng diing dd danh gia va theo ddi chirc nang than''*'.
Kdt qua nghien ciru eua chiing tdi cho thiy sau 2 tuin, 4 tuin udng thude thii' lien tuc va sau ngii'ng thudc 2 tuin. ndng dp creatinin trpng huydt thanh thd d 2 Id trj khdng thay ddi SP vdi trudc khi udng thude va se vdi Id chirng. Didu nay ehirng td thudc
thir dung djch HL lidu 0,08ml va 0,4ml/kg/ngay udng lien tuc trong 4 tuin khdng anh hudng ddn chire nang Ipe eiia e i u than.
Ban luan vd kdt qua mo benh hoc
Giai p h i u me benh hpc la xet nghiem quan trpng b i t bupc phai tidn hanh khi nghien eiru danh gia dpc tinh ban trudng didn eiia thudc thu tren thuc nghiem'^'. Cac kdt qua nghien ciru cho thdy, khdng cd tdn thuang dac hieu tren giai phiu benh d gan va than thd sau udng dung djeh HL, didu nay phii hgp vdi cac kdt qua xet nghiem sinh hda.
K§t luan
Thude thii' dung djeh HL lidu 0,08ml va 0,4ml/kg/ngay (tuang duang 0,3 va 1,52g/kg/ngay) udng lien tuc trong 4 t u i n khdng anh hudng ddn chirc nang gan va than tren thd thuc nghiem.
(Xem tiep trang 56)
48 TAP CHi DUQC HQC-3/2011 (SO 419 NAM 51)
