Nghiên cứu định lượng apigenin trong cúc hoa vàng (Flos Chrysanthemi indici) bằng phương pháp HPLC

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