Khao sat sy thay doi thanh phan flavonoid
r '^
cua vi thuoc hoang cam (Radix Scutellariae) qua cac phvong phap che bien
cua Y hoc CO truyen
Dat van de
Hodng cim Id vj thuoc dugc diing nhilu trong Y hoc eo truyin, cd cdng nang thanh nhiet ta hda, tdo thip. Chu trj ede chirng tire nguc, budn ndn, ndn, diy b7, kilt ly, tieu chay, hodng dan, phi nhiet ho, sdt cao, huylt nhiet, tho huylt nuc huylt, ung thung sang dde, ddng thai ramdu.,,'^'^'^1,
Tiiy thude vdo benh chirng khdc nhau md hodng cim dugc ehl biln theo cac phuang phdp khdc nhau khi sir dung. Qua qua trinh ehl biln, dudi su tde ddng cua cac ylu td nhu nhiet dp, dp am, phu lieu, thdnh phin hda hpc cua hodng c i m thay doi nhu ndo? £)h tra Idi cdu hdi ndy, ehiing tdi tiln hdnh khao sdt su thay doi thdnh phin hda hpc chinh flavonoid cua hodng c i m qua mpt sd phuang phdp ehl biln khdc nhau.
Nguyen lieu va phu'ang phap
Nguyen lieu nghien CLFU
- Vj thudc hodng c i m , dat tieu chuan Dugc dien Viet Nam III, do Vien Y hpc co truyin Qudn dpi eung cip.
- Cde mau thir:
+ Hodng c i m philn (HCP): R l hodng earn dugc Idm mem bdng cdch do trong 60 phiit, u ndng, thdi philn mdng, kich thudc ddi 3-5cm, day 1-3 mm siy khd d nhiet dp 60-70°C, be ngodi philn cd mdu vdng ndu.
+ Hodng c i m sao vdng (HCV): Dun chao ndng gid, cho HCP vdo, dao deu din khi mat ngodi phien ed mdu vdng dam, ben trong ed mdu cua philn song.
Phiam Xuan Sinh
Nguyen Manli Tuyen, Nguyen Phu'O'ng Nhj Truang dai hoc Duac Ha Noi
+ Hodng c i m sao den (HCD): Dun chao ndng gid, cho HCP vao, dao diu, sao din khi mat ngodi philn mdu den, ben trong ed mdu vdng ndu.
+ Hodng cam chich girng (HCG): HCP 200g, girng tuai 40g, Dem girng tuai rua sach, gid ndt, them nude, vdt lay djch cho du 30ml. Trdn deu djch nudc girng vdo HCP. 0 60 phiit cho ngim hit nudc girng. Sao nhd lua din khi hodng cam khd, cd mdu vdng.
+ Hodng edm chich mat ong (HCM): HCP 200g, mat ong 80g. Lay mat ong dem dun din khi cd bpt, cho hodng earn philn vdo dao deu sao din khi cd mdu hai vdng, sd khdng dinh tay.
+ Hodng cam chich rugu (HCR): HCP 200g, rugu trdng (40°) 30ml. Dem HCP tam vdi rugu trdng. G 10 phut cho ngim hit rugu. Dem sao din khi mat ngodi ed mdu vdng ddm.
Phu'ang phap nghien ciru
- Chiet xuit, phdn lap, nhdn dang mdt so thdnh phdn flavonoid chinh trong hodng cim '^'.
- Ojnh tinh thdnh phan flavonoid trong hodng cim bdng cac phan irng thudng quy trong ong nghiem vd bdng sdc ky Idp mdng '^'.
- Djnh lugng flavonoid trong hodng cim bing phuang phdp do dp hdp thu quang d budc sdng 279 ± 1 nm theo Dugc dien Vi$t Nam '^l
K§t qua va ban luan
Chilt xuit, phan lap, nhan dang mot s6 chit trong hoang c3m philn
- Chiet xuat chat Ll bang etfianol
Nghien
CLPU- Ky thuat
Cdn khoang 500 g hodng c i m philn, s i y khd, tdn nhd 2-5 mm, them 1000 ml ethanol 70%, ngdm trong 48 h, riit djeh ehilt, L i n 2 vd lan 3, them 500 ml ethanol 70%, ngdm trong 24 h mdi lan, riit djch chiet, Gdp djeh chilt, C i t thu hoi dung mdi dudi dp suat giam, d i n edn khoang 200 ml, thu dugc djch ehilt dam ddc.
Them 300 ml ethanol 96%, hda tan. Lpc, thu duge dieh ehilt ethanol. Cd tren edeh thuy d i n thu duge e i n .
Hda tan c i n trong 100 ml aceton, lpc. De djeh lpc d nhiet dp phdng trong 24 h, x u i t hien tua T l , Lpc l i y tiia, RCra tua bang aceton 3 l i n , mdi l i n 10 ml. Bay hai aceton. Hda tan tua thu duge b i n g ethanol 70%. Lpc, d l yen dung djch ethanol 48 h trong tu lanh, x u i t hien tinh t h i hinh kim mdu vdng chanh, Gan l i y tiia, hda tan vd k i t tinh lai trong 50 ml ethanol 25 % (ldp lai 3 lin), thu duge tinh the L1, K i e m tra do t i n h khiet cua L l : D i i n g S K L M 2 chieu v a i ban m o n g silicagel GF254 trang san dugc khai trien tren 2 he dung m o i : He 1: toluen: c h l o r o f o r m : aceton (8:5:7) va He 2: toluen: ethyl acetat: acid f o r m i c (5:6:1).
Hien mau bang soi den tir ngoai d buac song 254 nm va h a i NH3 dac.
K i t qua trdn s i c ky do d i u eho 1 v i t (hinh 1).
vdng, d l yen 24 h d nhiet do phdng, Loc l i y tua, RCra tua nhilu ldn b i n g nudc eat. S i y tua d nhiet dp 70-80°C, T i l p tuc ru-a nhilu l i n bdng chloroform d i n khi djch rira khdng cdn mdu nau, thu duge tua mdu vdng. De bay h i t hai dung mdi, hda tan tiia trong ethanol tuyet doi ndng. Loc, thu duge dung dieh ethanol, Oe bay hai tu nhien d nhiet dp phdng, Sau 48h t h i y x u i t hien tua mau vdng tuai, gan l i y tua. Hda tan vd k i t tinh lai trong ethanol 3 l i n , mdi l i n 100ml thu duge tua T2. RCra tua bdng 50ml methanol, thu dugc mdt c h i t mdu vdng tuai L2.
K i l m tra do tinh khilt cua L2: Sir dung SKLM vdi he dung mdi khai trien: He 1: Toluen : EtOAc: Acid formic (10:15:11) (chilu 1); He 2:
n-BuOH : Acid acetic : H2O (4:1:5)-pha tren (chilu 2).
K i t qua tren sdc ky do d i u eho 1 v i t (hinh 2).
•IS r~
:S
Hinh 1: Sde ky do 2 c h i l u c h i t L1(quan sat dudi UV 254nm)
Nhdn xdt: tren SKLM hai ehilu d dnh sang thudng vd dudi dnh sdng UV254 nm d i u eho mdt vit, Vi vdy sa bd k i t ludn L l Id mpt chat tinh khilt,
- Chiet xuat chat L2 bang nuKrc
Cdn khoang 500 g hodng c i m philn, thdm nudc c i t vira du, s i c 3 l i n . L i n 1 sde trong I h , l i n 2 vd 3 sdc 30 phiit, Gdp djch chilt, ed trong cdch thuy sdi d i n edn khoang 200ml. Cho djch chilt vdo coc thuy tinh cd md vd d i l u ehinh d i n pH d i n 1-2 b i n g HCl ddc (TT), x u i t hien tua
Hinh 2: S i c ky do 2 chieu chat L2 quan sdt dudi UV 254nm
Nhdn xet: tren SKLM hai chilu quan sdt d dnh sdng thudng vd dudi dnh sang UV 254nm d i u eho mpt v i t . Vi vdy sa bp k i t ludn L2 Id mdt c h i t tinh khilt,
- Nhan dang chat Ll:
+ Tinh chdt: C h i t L1 thu duge dudi dang tinh t h i hinh kim mdu vdng chanh, tan trong MeOH, EtOH, Aceton, hdu nhu khdng tan trong nude, Cho mdu vdng vdi dd NaOH 10% (TT), mau hdng vdi phan irng cyanidin vd'mdu xanh den vdi dd FeCI 5% (TT).
+ Pho UV (MeOH) >.max (nm): 209,0; 247,0;
277,3 va 345,0, Pho UV mang ddc d i l m h i p thu tCr ngoai ddc trung cua khung flavon.
+ Pho IR: dugc do dudi dang vidn ndn KBr, cho cac bdng h i p thu cue dai ddc trung eua eae nhdm ehire: OH-phenol (3274,5 cm''), carbonyl (C=0) eua vdng y-pyron (1656,8 cm" )
vd bdng hap thu cua hgp chat nhan tham (1660 cm"').
+ Pho MS: pho khoi do dugc theo ky thudt EI-MS cho thiy ion phan tir [M]* irng vdi peak 284 m/z phan tir lugng 284 tuang irng vdi cdng thirc C16H12O5 Cdc peak khdc the hien cho su phdn manh cua L1 khi bj pha vd.
Doi chieu vdi ngdn hang dir lieu pho khoi cho thiy L1 phii hgp vdi pho khdi cua wogonin (chinh xdc 93%). Do dd chiing tdi nhdn dang L1 Id wogonin.
- Nhan dang chat L2:
+ Tinh chit: Chit L2 Id chit bdt mdu vdng tuai, tan trong methanol, ethanol, hdu nhu khdng tan trong nudc. Cho mdu vdng vdi dd NaOH 10% (TT), mdu hong vdi phan irng cyanidin vd mdu xanh den vdi dd FeCI 5%(TT).
+ Pho UV (MeOH) X^^^ (nm): 215,0; 246,1;
277,7 vd 315,3. Pho UV mang dac diem hip thu tip ngoai ddc trung cua khung flavon.
+ Pho IR: duge do dudi dang vien nen KBr, cho cdc bdng hip thu cue dai dac trung eua cdc nhdm chirc: OH-phenol (3391.1 cm"'), carbonyl (C=0) cua vdng y-pyron (1660.1 cm"') vd bdng hip thu cua hgp chit nhdn tham (1490.0 em"').
+ Pho MS: pho khoi do dugc theo ky thuat EI-MS cho thay ion phdn tir [Mf irng vdi peak 270 m/z phdn tir lugng 270 tuang irng vdi cdng thirc C15H10O5 Cdc peak khdc the hien cho su phdn manh ciia L2 khi bj phd vd.
Doi chieu voi ngan hang du lieu pho khoi cho thay L2 phu hgp vai pho khoi cua baicalein (chinh xac 96%). Do do chung toi nhan dang L2 la baicalein.
OCH3
OH O
Wogonin
Qua tinh chit vd phdn tich pho, chiing tdi kit lu$n chat Ll Id wogonin vd chat L2 Id baicalein, ddy Id hai thdnh phin hda chinh cd trong vi thuoc hodng cam dd duge phdn ldp vd nhdn dang.
Djnh tinh flavonoid trong hoang c i m phiin vd hoang c i m c h i
Chiet xuat: Cdn 5.g bdt thd hoang cam (phien hoac che) cho vdo binh ndn dung tich 100 ml, them 20 ml ethanol 80%, dun sdi each thuy 10 phut, loc ndng, bdc hai cdch thuy thu dugc cdn, dem hod cdn vdi 20ml nudc cat.
Cho djch vdo binh gan 150 ml Idc vdi ethyl acetat (3 lan, mdi ldn 10ml) gdp djch chiet, boc hai ethyl acetat lay cdn, hod tan cdn bdng 10 ml ethanol 80% de lam phan irng trong ong nghiem va cham sic ky.
-Phan irng trong dng nghiem: cdc phan Crng cyanidin, phan irng vdi kiem, vdi dd NaOH, vdi dd FeCIa 5%, vdi Pb(CH3COO)2 cua miu thir d i u cho phan irng duang tinh. Chirng td thdnh phan hda hpc chinh flavonoid van ton tai trong hodng c i m sau e h l biln.
-Djnh tinh bing sic ky Idp mdng:
Dimg chit doi chilu la Ll vd L2 dugc phdn lap tir hoang cam philn. Trien khai SKLM djch ehilt flavonoid trong hodng cam bdng hd dung mdi toluen: ethylacetat: acidformic (5: 6: 1) (He I) vdi chat doi chieu la L l ; dimg he dung mdi khai trien n-BuOH: acid acetic: H2O (4:1:5) (pha tren) (He II) vdi chat doi chieu Id L2. Hidn mdu bdng hai amoniae va soi UV 254nm.
Tren sic ky do quan sdt d dnh sdng thudng vd UV 254nm, djch chilt flavonoid hodng cim philn vd che deu cho 6 vit, trong dd vet thir 6 ed mau sic vd Rf tuang irng vdi Ll khi then khai vdi he dung mdi I (Hinh 3). Chirng td sau khi ehe bien thdnh phan wogonin trong cdc miu van ton tai. Sic ky dd quan sdt d dnh sdng thudng vd UV 254nm, djch chiet hodng cim philn vd ehe deu cho 5 vit, trong dd cd vit thir 5 cd mdu sdc vd Rf tuang irng vdi L2 khi triln khai bdng he dung mdi II (Hinh 5). Dilu ndy chirng td sau khi che biln thdnh phan baicalein
OH _ , ^ - \
// V H
Baicalein ' trong cdc miu vdn ton tai.
Nhu vdy qua cdc phuang phdp che bien c6 truyen ndi tren, v i n giQ- dugc thdnh phin flavonoid chinh cd tde dung quan trgng ciia vj thuoc.
Nghien
CLPU- Ky thuat
1 2 5 6 7
Hinh 3: Sic ky dd dich chiit flavonoid trong djeh chiit hoing cim (hiin miu bing NH3) -
Hi dung mdi I Ghi ehii: 1:L1;2:HCP
3: HCV; 4: HCD 5: HCR; 6: HCG 7: HCM
sassi^erx 1 2 3 4 5 6 7 Hinh 4: Sic ky dd dich chiit flavonoid trong dieh chiit hoing cim (hiin miu bing
NH3) -Hi dung mdi II
Ghi chii: ^:L2 2: HCP 3: HCV; 4: HCD
5: HCR; 6: HCG 7: HCM
Djnh lu>9>ng flavonoid
T i l n hdnh djnh lugng flavonoid trong cde mdu hodng c i m philn vd e h l bdng phuang phdp do dd h i p thu d bude sdng 279 ±1 nm theo Dugc d i l n Vi$t Nam III, ehuydn ludn hodng c i m '''. Hdm lugng flavonoid trong edc m i u thir duge tinh theo baicalein. Mdi m i u t i l n hdnh 3 l i n , lay gid tri trung binh, k i t qua dugc trinh bdy trong bang 1.
Bang 1 : Him Iwgng flavonoid cOa hoing cim phiin vi hoing cim chi
1 2 3 TB
Him Iwgfig flavonoid tinh theo baicalin (%) HCP HCV HCD
5,09 5,87 2,94 5,39 6,27 3,13 5,22 5,88 3,35 5,23 6,08 3,14
HCR 8,20 7,73 8,07 8,00
HCG HCM 8,26 8,91 8,58 7,39 7,08 8,03 7,97 8,10
Cy edc mdu hodng cam philn, sao vdng, chich rugu, chich girng vd hodng c i m chich mdt ong, d i u cd hdm lugng baicalein > 4%, phii hgp vdi tieu c h u i n DDVN III (>4%). Cde m i u ndy d i u eho ham lugng flavonoid cao han m i u philn, cd the do e h l bien Idm thay doi v l mdt e i u triic ciia vj thuoc, giam dp b i n ea hpe, nhd dd ede thdnh p h i n flavonoid duge ehilt tdch r l ddng han, Hoang cam sao den cho k i t qua hdm lugng flavonoid todn p h i n t h i p nhit (3,14%) ed t h i Id do tde ddng cua nhiet do cao, thdi gian ddi dd phdn huy cde hoat c h i t ed trong vj thuoc, khiln cho hdm lugng flavonoid giam di.
K § t l u a n
Dd chilt x u i t vd phdn lap duge hai thdnh phin hda hpe ehinh Id baicalein vd wogonin trong hodng cam philn d l Idm c h i t doi chilu.
Kit qua nghien ciru cho thay hodng edm philn vd hodng c i m che d i u cd flavonoid vd thdnh phin ndy khdng bj thay doi nhilu sau khi ehl biln. Tren sdc ky do vdi chit ddi chilu Id baicalein vd wogonin cho thiy cde phuang phdp c h i biln khdng lam mit di hai thdnh phdn hda hpe ehinh ndy.
Kit qua djnh lugng flavonoid tinh theo baicalein theo phuang phdp ghi trong Dugc dien Viet Nam III, cdc mdu diu cd ham lugng dat tieu chuan cua Dugc diln, trir miu hodng cam sao den ed ham lugng thap han 4%
(3,14%).
Summary
Radix Scutellariae (Hoang earn - in Vietnamese or Hoangsin - in Chinese), mainly its processed items, were tested for antibaeteral and antifungal activities. Antibacterially, they showed inhibitive effects on the following 7 bacteria: Proteus mirabilis, Shigella flexneri, Sarcina lutea. Bacillus cereus. Bacillus subtilis.
Bacillus pumilus, and Staphylococcus aureus.
The black-roasted Huangqin had inhibitive effects exclusively on 3 types of experimental bacteria, e.g. Bacillus pumilus. Shigella flexneri, Proteus mirabilis. On antifungal tests, the experimented samples showed inhibitive effects on 4 bacterial types: Candida albicans, Aspergillus ni^er, Penieillium notatum, Botrytis cineca.
Tai lieu tham khao
1. Bp Y fe, Dugc dien Viet Nam ldn xuat ban thip ba(2002), A/Xey/7(?c
2. Bo mon Dugc hpc co truyen - Trudng dgi hpc Dugc Ha Npi (2006), Dugc hpc co truyen, NXB Y
hQC
3. NguySn Van Dan, Nguyen Viet Tuu, Phuong phap nghien ciru hoa hpc cay thuoc, NXB Y /7pc(1985)
4. Pham Xuan Sinh, Phuang phap che bien thuoc CO truyen, NXB Y hpc (2006).
5. Hoang Tri^u Thing, Trung Dugc hpc, NXB V$
sinh nhan dan, (2002) 357-358 (Tieng Trung)
r r w
Nghien cuu chiet xuat, phan lap hop chat andrographolid tir la cay xuyen tam lien (Andrographis paniculata fBurm.f.) Nees)
r •> r r •?
dung lam chat chuan doi chieu trong kiem nghiem
Hoang Viet Dung, Nguyen Minh Chinh, Nguyen Van Long Hoc vien Qudn y
Dat van de
Xuyen tdm lien (XTL) la dugc lieu dugc sir dung tir lau d nhilu nudc tren t h i gidi de didu tri cde trudng hgp: viem dudng hd hip, viem dudng tieu hda, cam ciim, sit, nhidt mieng,., '^' Tuy nhien, viee sir dung cdc ehl pham bdo ehl tir duge lieu nay d Viet Nam hien nay cdn han ehl md nguyen nhdn chu ylu vi cdng tac quan ly chit lugng gdp nhilu khd khan do thilu phuang tien ky thudt vd chit ddi chilu hda hpc.
Vi vay chiing tdi tiln hanh thue hien d l tdi vdi muc tieu ehilt xuit, phdn lap chit chuan' andrographolid tir Id cay XTL nhdm cung cip chit chuan doi chilu phuc vu cdng tac kiem
nghiem dugc lieu vd cdc ehe pham bdo ehl tir duge lieu ndy.
N g u y e n lieu va phu'O'ng phap n g h i e n CLPU
Nguyen lieu - Hda chat:
+ Chit chuan andrographolid (hdng Sigma - Aldrieh).
+ Methanol (MeOH), Cloroform (CHCI3), cin tuyet doi, ejn 95°, ethylacetat (EtAc), ether: dat tieu ehuan PA.
+ Bpt Silicagel GF254 (Merck - Dire).
-Thilt bj, dung eu:
