• Nghien CIFU - Ky thuat
3. Ngo Van Thu (2011), Dupc//eu hoc, NXB Y hpc.
tap 1,fr, 343-344.
4. Lyu Hoang Ngoc, Nguyin Thanh Loan, Nguyin Nggc Thanh, Nguyen Thi Hoai Anh, Nguyin Thj Thu Hyang, Nguyin Mai Cycng, Le Ngpc Thyc (2009), Bao cao tdng k4t de tai "Nghien cyu cong nghe san xuat cao djch chiet cua r i cay chut chit Rumex crispus djnh hydng lam thuoc try nam thao mpc than thien moi trydng", Vien Hoa hoc cong nghiep Viet Nam
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6. Keyong Ho Lee, Ki-Hyeong Rhee (2013),
"Antimalanal activity of nepodin isolated from Rumex crispus". Arch. Pharm. Res., 36, pp 430-435.
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"Determination of antoxidant and antimicrobial activities of Rumex crispus L. extracts", J. Agric. Food Chem., 49 (8), pp, 4083-4089.
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[Ngay nhan bai: 03/06/2014 - Ngay duyet dang: 08/09/2014)
Nghien cmi djnh lirong luteolin, apigenin trong dirffc lieu va bot cao kho ciic hoa vang
(Chrysanthemum indicum L.)
bang phirong phap sac ky long hieu nang cao
Nguyin Trong Diep', Chir Van M^n', Vu Binh Duffng'"
Nguyen Vu MinhS Nguyen Thanh Hai^
' Hoc Vien Qudn Y
^ Vien Kiem nghiem nghien cim Duac Quan dot
^Khoa Y Diroc - Dgi hoc Quoc gia Ha Noi
* E-mail [email protected]
Summary
Luteolin and apigeninin in the Flos Chrysanthemi Indici and dried extracts of Chrysanthemum indicum were quantitatively analyzed by HPLC-UV/VIS (338 nm) with a reversed phase column C18 (5 pm; 4.6 mm X 150 mm), gradient elution by solvent A (formic acid 0.01% in water), followed by solvents (acetonitrile): flow rate - 0.45ml/min. The limit of detection (LOD) was 3.1 and 15.2 ng/mL and the limit of quantification (LOQ) was 8 and 38ng/mL for Luteolin and Apigenin, respectively. The method was validated for the suitability precision and accuracy Applying to quantitation of luteolin and apigenin in the Flos materials and dried extracts of Flos Chrysanthemi Indici, the method revealed no loss of the active compounds through preparing the dried extracts.
Keywords: Chrysanthemum indicum L, HPLC, luteolin, apigenin.
TAP CHI DUQC HQC - 9/2014 (SO 461 NAM 54)
• Nghien ciru - Ky thuat
Dat van de
Cue hoa vang {Flos Chrysanthemi indici) dygc SLF dung lam pho bien trong cdc thudc co tnjyen tO hang ngan nam nay. Nhieu nghien cuu cho thay, cue hoa cd tac dung tot eho sue khde nhy chong oxy hda, bao ve tim mach, bao ve gan, tang c y d n g midn dich, khang khuan, khang nim, ye ehe s u nhdn len eua HIV '^'. Nhdm hoat chdt lien quan ddn cac tac dung sinh hgc chinh trong cue hoa vdng Id flavonoid, trong dd luteolin (3, 4',5,7-tetrahydroxyflavon) va apigenin (4',5,7-trihydroxyflavon' ['"'^L. d y g e eoi la ehat danh ddu khi xdy dyng tieu chuan doi vdi cue hoa vang i^i. Tuy nhien, hien nay trong DDVN IV chya cd chi tieu djnh lygng flavonoid nen khd kiem soat chat lugng nguyen lieu trong qua trinh bao chd iit d u g c lieu ndy.
Ham lygng flavonoid trong cue hoa vdng phy thupc ldn vdo vung trdng, thdi diem thu hdi t^^ ndn 6k dam bao chat lugng cua d y g c lieu va cac san pham tu' cue hoa vdng, chung tdi tien hanh nghien CLPU nay nhdm xdy dung phyang phap dinh lygng dong thdi luteolin va apigenin trong dyge lieu vd bgt eao khd cue hoa vang bdng phyang phap sdc ky long hieu ndng eao.
Nguyen vat lieu va phu'cng phap nghien cu'u
Hoa chat va thiet bj
- Mdy sde ky long hieu ndng eao Waters 2487 vdi detector PDA cua Phdng D y g c hge va cae hgp chat t y nhien, Hgc vien Qudn y, Thidt bi da flifgc hieu ehuan vd dap yng yeu eau theo GLP v^ ISO/lEC 17025.Cgt sde ky la Symmetry- C18 (150 mm X 4,6 mm, 5 pm) d y g c bao ve bdi cdt C18.
- Cac hda chat dung mdi loai tinh khiet phdn tich hogc dung eho HPLC, Chdt ehuan luteolin, apigenin cua Hang Sigma Aldnch (Han Qudc).
M l u nghien cii'u
Cym hoa eua eay cue hoa vang {Chrysanthemum indicum L.) dat tieu ehuan DDVN IV. Bgt eao khd cue hoa vang d y g e ehiet Ku^t sau dd lam khd bdng phyang phap phun sly tgi Trung tdm Dao tgo - Nghien cdu d y g c . Hoc vi^n Qudn y.
PhiFO'ng phap nghien c i r u Phuxmg phap xu- ly mau
Qua t h y nghiem vdi nhidu bien phdp XLF ly mau khdc nhau, chung tdi chgn phyang phap xi>
ly mau nhy sau;
Dung djch mau thu cue hoa vang: Can chfnh xac khoang 1,0 g bdt d y g e lieu cue hoa vang cho vao binh djnh mdc 50 ml, them khoang 40 ml methanol, chidt sieu am 60 phut. Dd ngugi, them methanol v y a du den vach, Ide ddu, Igc djch chidt qua mang Ipe 0,45 pm, d u a c dung djch mdu thy cue hoa vang.
Dung dich miu thu hot cao kho cue hoa vang Can chinh xac khoang 200 mg bdt cho vdo binh dinh mye 50 ml, them khoang 40 ml methanol, chidt sieu am 60 phut. De ngupi, thdm methanol vua du den vach, lae deu, loc djch chiet qua mang loc 0,45 pm, duac dung djch mdu thy bgt cao khd cue hoa vang
Dung dich doi chiiu
Cdn chinh xac cae ehat chuan pha thanh cac dung djeh doi ehieu trong methanol cd nong luteolinva apigenin khoang 250 pg/mL, Lgc qua mdng Ige 0,45 pm de d y g c dung djch tiem sdc ky,
Xay dyng phuang phap phan tich Diiu kien sic ky: Khao sat ve loai va kich thude cdt sdc ky, pha ddng va cae ydu to khae de chon d y g c dieu kien sac ky eho phep taeh eae chat nghien cdu trong mdu thy,
Thim dmh phuang phap phan tich. Quy trinh phan tieh duac tham dmh theo Thdng t y 22'^' vdi cac tieu chi ca ban gom, dp thieh hgp he thong, tinh dac hieu, khoang tuydn tinh, dp dung, do chinh xae.
Kit qua va banluan
Xay d y n g p h y a n g phap phan tich Tidn hdnh phan tieh bang HPLC vdi detector UV-VIS, sy dung cgt C18, Thanh phan pha dgng va chyong trinh dung mdi d y g e khao sdt vd lya chon dieu kien sde ky phu hgp nhy sau'
- Pha ddng: Su' dung he pha ddng acetonitril (kenh A) va acid formid trong n y d c 0 , 1 % (kenh B) vdl chd dp gradient sau:
TAP CHi DUQC HQC - 9/2014 (SO 461 \.\M 54)
• Nghien ciru - Ky thuat
Thin gian (phut)
0 20 30 40 50 55 60 70
T6C do dong (ml/phut)
0,45 0,45 0,45 0,45 0.45 0.45 0.45 0.45
Kenh A 19 20 32 36 38 100 19 19
KSnhB
81 80 68 64 62 0 81 81
B y d c sdng: 338 nm.Tdc dO ddng: 0,45 mil phut.The tich tiem mau:10 pL.
V d i dieu kien sdc ky nay, h6n hgp cac chat nghien euu da d y g c tach khoi nhau va tach vdi cac thanh phdn khac trong mdu t h i i (vdi R^ > 1,5).
T h y c hien c h y a n g trinh sae ky da chgn vdi cae hgp chdt de xac djnh thai gian luu tyang yng vdi t y n g ehdt nghien c u u . Trong dieu kien sac ky trdn, thdi gian luu cua luteolin, apigenin lan lygt la khoang 24,15 vd 34,03 phut (hinh 1 a, b).
Hinh 1 ; Sac ky dd cua miu chuin luteolin (a) va apigenin (b)
Dua tren ket qua quet pho UV-Vis thdy pie eua Cae sde ky do thu d y g e cua dung djch ddi chieu luteolin va apigenin cd cue dai hdp thu d b y d c
sdng khoang 337 -347 nm, nen chung tdi lua chgn b y d c sdng 338 nm Idm b y d c sdng dmh l y g n g .
vd dung dieh mdu t h y cho thay pie cua luteolin va apigenin taeh hdn nhau vd tach rd so vdi cae pic eua ede chdt co trong mdu thCp (hinh 3).
Hinh 2: Sic ky dd mau hdn hap chuin luteolin, apigenin (a) va dung mdi (b)
Hinh 3: Sic ky do mau thu cue hoa vang (a) va bot cao kho cue hoa vdng (b)
TAP CHI DUQC HQC - 9/2014 (SO 461 NAM 54)
Nghien CIFU - Ky thuat
Tham djnh phipong phap Tinh d^c hi^u
Sdc ky lan l y g t dung moi pha mau, dung djch doi chieu, dung djch mdu thy cue hoa vdng va m i u thy bgt cao khd cue hoa vang vdi didu kien HPLC dd chgn. Kdt qua cho thay sdc ky 66 cua dung mdi pha m l u khdng co pic cua cac ehdt nghien c y u tai thdi gian iuu tuang yng (hinh 2).
Sac ky do cua hai chat luteolin vd apigenin trong Bang 2; Ket qua kiim tra do thich hap
mdu ddi chidu va cac mdu thi> tdch nhau ro, vdi dg phdn gidi R ^ 1,5 (hinh 3).
£)o thich hgp
Tidn hanh sac ky lap lai 6 ldn dung djeh doi chidu da ehuan bi d tren, ghi lai cdc gid tri ve thdi gian l y u , dien tieh pie. Kdt qua d u g c trinh bay d bang 2 cho thay he thdng sdc ky ed do lap lai tot vdi thdi gian lyu vd dien tfch pic cua cac chdt (RSD <2%, bang 2).
Hgp chat Luteolin Apigenin
Thai gian luu (phut) Trung binh RSD(%)
24,155 1,78 34,035 1,29
Dien tich pic (mAU.s) Trung binh RSD(%)
3957523 0,94 4118484 0,84 Khoang nong dg tuyen tinh
T y cac dung djeh gdc ehdt doi chidu, pha cac day dung djch chuan gdm 5 mau cd nong do tang ddn trong khoang thich hgp, tien hdnh sdc ky de khao sat khoang ndng dd tuyen tinh. Ket qua thu dyge nhy sau:
- Phyang trinh hoi quy vdi luteolin xae djnh dyge la y = 91,942,68x - 409.744,70, trong khoang nong dd 5 ddn 250 pg/mL vdi r = 0,9993 (hinh 4a), Phuang trinh hdi quy vdi apigenin xac djnh d y g c la y = 86 654,92x -103.103,38, trong khoang nong do 5 den 250 pg/mL vdi r = 0,9997 (hinh 4b).
0 50 100 150 20D 250 300 0 100 200 31
Hinh 4: Phuang trinh hoi qui tuyin tinh cua luteolin (a) va apigenin (b)
Gidi han phat hien va gioi han djnh Iwong Gidi hgn phdt hien vd gidi han djnh luang cua cac hgp chat xac djnh d y g c nhy d bang 3.
Bang 3,- Kit qua khao sat LOD. LOO (yg/mL) Chi tieu
LOD LOQ
Luteolin 0,3093x10'
0.0080
Apigenin 1,517x10-5
0.038 Dg dung
Thdm vdo mau t h y mot lugng chinh xac chat doi chi^u sao eho tong lygng hoat chat cd trong m§u ndm trong khoang tuyen tinh Ket qua khao sdt dO dung d y g c trinh bay trong bang 4,
Bang 4: Ket qua do dung cua cac chat Ty te thu hoi (%)
Apigenin
RSD
(%)
Dg lap lai:
Sdc ky 6 mdu thy d y g e chuan bj vdi cac khoi luang cdn la 1.0823, 1.0546, 1,0411; 1,0386, 1,0732 vd 1,0261 g. Ket qua do lap lai nhy sau
Ti^P CHi DUQC HQC - 9/2014 (SO 461 N.\M 54)
• Nghien cifu - Ky thuat
Bang 5: Ket qua khao sat do lap lai Chit phan tich Khoang dien tich pic S
(^U.s)
RSD
(%)
Ket qua xac dinh ham Iwang luteolin va apigenin trong duw: lieu va bgt cao kho c6c hoa vang
Ap dung phyang phap HPLC da xay diing dyge de xac djnh ham lygng luteolin, apigenin trong dygc lieu va bgt cao kho cue hoa vang. Ket qua dygc the hien d bang 6.
Bang 6; Kit qua xac dinh ham luang luteolin, apigenin trong duvc lieu va bot cao kho cue hoa vang
Ham Iwgng (mg/g)Apigenin
Cue hoa vang
1 2 3
X±SD
1 2 3
1.4054 1.3881 1.4024 1,3986± 0.0092
4.2596 4.4487 4.2751
0.2277 0,2248 0,2296 0.2274 ± 0,0024
0.7415 0.7563 0.7389 Bpt cao kho cue hoa vang
X ± S D 4,3278 ±0,1050 0,7456 ± 0,0094 Tien hdnh phdn tieh vdi eae dieu kien sac ky
da xay dung cho thay, ham lugng luteolin va apigenin trong cue hoa vdng lan lugt la 1,3986 mg/g, 0,2274 mg/g va trong bdt cao khd ciic hoa vang lan luat la 4,3278 mg/g va 0,7456 mg/g.
Ket luan
Phyang phdp HPLC xdy d y n g d y g e cho phep xde djnh dong thdi luteolin, apigenin ed trong d y g c lieu vd bdt cao khd cue hoa vdng vdi didu kien sde ky. cdt pha dao C18 (5 pm, 4,6 mm x 150 mm), vdi chuang trinh gradient s y dung dung mdi A (acetonitril) va dung mdi B (acid formic 0,1 % trong nude), b y d c sdng 338 nm, tde do ddng 0,45 ml/
phut. Gidi han phat hien cua luteolin vd apigenin t y a n g yng la 3,1 vd 15,2 ng/mL. Gidi han dmh lygng cua luteolin vd apigenin tuang yng la 8 vd 38 ng/ml. Phyang phap nay ed dO dac hieu, do tuyen tinh, do ehinh xac, do dung cao. Kdt qua dinh lygng luteolin vd apigenin trong d y a c lieu la 1,3986 vd 0,2274 mg/g, trong bdt cao khd cue hoa vang la 4,3278 vd 0,7456 mg/g.
Tai lieu tham khao 1. Tran Van Hien va cong sy (2008), "Inhibitory effect of extracts from Chrysanthemum indicum in Nitrite Formation in LPS and HiV-1/MULV- induced Macrophage", Tap chi Duac lieu, s6 4, ir 175-180.
2. Trdn Van Hi§n va cong sy (2009), "Phan lap va nhan dang mpt so flavonoid tLF cue hoa vang {Chrysanthemum indicum L.) trdng d Viet Nam", T$p chi Duac lieu, so 4. tr. 195-207.
3. Bd Y ti (2009), Huang dan cua Asean ve thim dmh quy trinh phan tich. Phu luc 7 - Thong tu 22/2009/rT-BYT Quy djnh vi dang ky thuoc.
4. Liang-Yu Wu, Hong-Zhou Gao, Xun-Lei Wang (2010), "Analysis of chemical composition of Chrysanthemum indicum flowers by GC/MS and HPLC" Journal of Medicinal Plants Research, Vol 4(5), pp 421-426.
5. Zhongjian Chen, SIsi Kong, Feifeng Song, Liping Li, Huidi Jiang (2012), "Pharmacokinetic study of luteolin, apigenin, chrysoenol and diosmetin after oral administration of Flos Chrysanthemi extract in rats", Fitoterapia. 83, 1616-1622
6 Hai-ling Fang, QIao-sheng Guo, Hal-jin Shen, Yu-chuan Li (2012), "Genetic diversity evaluation of Chrysanthemum indicum L. by medicinal compounds and molecular biology tools". Biochemical Systematics and Ecology 41, 26-34
(Ngay nhan bai: 16/07/2014 - Ngay duyet d§ng: 08/09/2014)
