• Nghien ci>u - Ky thuat
of 3 ml/kg/day (equivalent to the human dose) and another a( the triple dose (9 ml/kg/day, 3 times tiie normal human dose), both for 4 consecutive weeks.
At these experimented doses, the extracts exhibited no perceivable effects on the t^ood counts, including let^ocytes, lymphocytes, polynuclear neutrophils, f^atelets.
Keywords: "Khing man thdng ty", white blood cells, lymphocytes, neutrophils, platelets.
Tai lieu tliam l(tiao
1. Nguyin Nang /\n (1998), "Viem mui dj ung", Pf ling miin djch lam sang, tr. 2 - 5 .2. Nguyen Nang An (1967), "Nghidn ci>u tdc dyng chong dj ung cua kim ngan hoa", T^p chi Y hoc Vi$t Nam, So 3,77-84.
3. D6 Tat Lgi (1991), Cay thuSc vi vi thuoc Viet Nam. NXB Khoa hoc Ky thugt, tr. 88-93, 328-330, 403-409, 571-572, 601-606, 670-672, 879-882.
Xac dinli cac ginsenosid Rgl, Re, Ro, Rf, Rbl, Re, Rb2 va Rd trong thyc pham
chifc nang co chifa cao nhan sam
Trinh Ho^ng Dirong, Thi^u Thj Thu Lilu, Ha Di@u Ly
Vien Kiem nghiem Thuoc Thanh pho Ho Chi MinhDat van de
Rh hay cu nhdn sdm Id mflt trong nhu'ng du'g'c lidu qwjf vd ph6 bien nhidu nhat trong chi Aratiaceae, da du'p'c dCing rdt nhidu trong cdc bdi thu6c y hoc co truyen. Cac nghien c d u d a a c lye hpc da chiing minh nhdn sdm co rdt nhleu tac dung sinh hpc nhu- keo ddi tuoi thp, cai thidn tinh trgng tinh thdn va the chdt, tang cu-ang he midn djch '''. Cac hogt chmh cua nhan sdm la cac saponin ginsenosid cd khung Oammaran- 20-S-protopanadiol vd 20-S-protopanatriol. Cdc saponin ginsenosid gom co saponin trung tinh nha R g l , Re, Rf, R b l , Rb2, Re va Rd, vd 01 saponin acid co khung olean la Ro. Ngodi ra, cdc d i n xuat malonyl saponin acid trong nhdn sdm thu'd'ng khong dn djnh, dd ddng bj thuy phdn trong qua trinh bdo qudn, Idm khd, hay chidt xuSt de cho cdc gensinosid tu'ong Cpng nhu' R b l , Rb2, Re vd Rd. Theo ede nghien ciru dd edng b6, saponin acid chiem khoang 35-60
% tong s6 ham l u v n g saponin; do do vide xac djnh ddng thd'i cdc saponin ginsenosid trung tfnh v d acid Id cdn thiet cho vide ddnh gid chat lopng nhdn sdm '^ ^.
Cdc phaang phdp xde djnh nhdn sdm da d a p c de cdp trong Dop^e didn Trung Qudc (PRC 2005) va NF29 (2011). Tuy nhien, vipc xac djnh cdc thdnh phdn saponin cua nhdn sdm trong sdn p h i m thyc phdm chCrc ndng v i n chu-a eo quy trinh, trong khi dd mdt luxp'ng Id'n cdc logi thyc phdm chO-c nang co ehipa nhdn sdm dang du'p'c quang cdo nhu' Id tdc nhdn tang cu'6'ng si>c khde, keo dai tudi thp du'pc nguxri tieu dCing rat aa ehupng. Mat khdc, khi dp dgng dieu kidn s i c ky theo du-ac dien cho sdn phdm thyc pham chCpc nang thi cae ginsenosid acid bj mdt hode tnling vd'i ginsenosid trung tfnh do ndn mau rat da dang vd phao tap.
Vi vdy, vipc xay dyng quy trinh xac djnh cdc saponin ginsenosid (hinh 1) ed trong nhdn sdm cOa thye phdm chiic ndng giup cdc nhd quan ly ngdn chdn cdc san pham ehi>a "rde nhdn sdm"
lu'u hdnh trdn thj tru'd'ng dd bdo v d siic khoe ngu'ai tieu dung Id nhu cdu cap thiet.
Tren ca sa cac dieu kipn tham khdo '^'^' vd ket hp'p vai nghien ciiu tren thye nghipm, trong bai bao ndy chung tdi giai thidu phu'ong phdp HPLC dan gidn de djnh tinh cdc ginsenosid trong mpt sd dgng t h y c pham chiic nang e6 ehiia nhdn sam.
T^M* CHl Dlf0C HQC - 04/2013 (S6 444 NAM 53)
> Nghien cipu - Ky thuat
OR, 20(S)-protopanaxatriol
R2O .;
Ginsenosid Rg1 Ginsenosid Re Ginsenosid Rf
R1 Glc Glc'^-Rha Glc''-Glc
R2 Glc Glc H
Ginsenosid Rb1 Ginsenosid Rb2 Ginsenosid Re Ginsenosid Rd Malonyl Ginsenosid Rb1 Malonyl Ginsenosid Rb2 Maionyl Ginsenosid Re Malonyl Ginsenosid Rd
R1 Gic'-Glc Glc'-GIc Gic'-Gic Gic'-GIc Glc^-Gic'-rrai Glc'-Glc'-mal Gic'-Glc"-mai Glo'-Glc'-mal
JU
Glc°-Glo Glc'-Ara(p) Glc°-Ara(f)
Glc Gic'-Gio Glc'-AraCp) Glo''-Ara(f)
Glc
1 '^^
J p-D-Giucose POT Gl a-L-Arabinopyra nose
a-L-Arabinofurose a-L-Rhanose P-D-Giucuronic acid
Gic Ara(p) Ara(f) Rha GicUA Ginsenosid Ro
Hinh 1 : Cac ginsenosid chinh trong cao nhan sam D o i tifCFng v a p h u ' O ' n g p h a p n g h i d n
CU'U
M l u n g h i d n ci>u
M$u chuin: B6t Asean Ginseng extract CRS, Cao chudn USP, Sd 16: G0F321.
Mau thw 1: Nu'd'c uong Korea red Ginseng extract, Han Quoc, so 16: G29672, 2014.
Miu th^ 2: Vidn nang mem Koligin D-5240, Hdn Qudc, sd 16: 12-T06-ROD, 2015.
Mau thO- 3: Keo hdng sam Sugar free, Han Quoc. s6 16: TS-912, 2014.
D u n g m o i va hoa c h a t
Acetonitril (HPLC), methanol (HPLC), kali dihydro phosphat (PA), hgt nhya Daion.
Thiet bj
Thidt bj va dgng ey da duwe hidu chuan theo ISO/IEC 17025 v d G L P .
24
May sac ky long Shimadzu CBM 20A bao gdm bg phan dudi khi DGU-20A5, hd thdng b a m LC-20AD, bg phgn tiem m a u t y dgng SIL 20-AC, budng dn nhipt CTO-10AS vp, dau do UV. He thong d i m e kdt ndi v d i may tfnh bang phdn mem LC solutions.
P h i p o n g p h a p n g h i e n ci>u Dieu kien sac ky'
Cot sac k^: Dai 150 m m , du-ang kinh trong 4 m m , ehCra ehat nhdi cOt silica gel RP18 Pyramid, 3 pm. Hang san xudt: Nueleodur.
Toe dO d o n g : 1,5 ml/ phOt Nhipt dO cdt : 30 °C Detector UV : 203 nm The tich tidm: 20 jil
Pha dOngA: Dung djch kali dihydro phosphat 0,272 g/L. Chinh pH 6,8 bdng dung djch natri hydroxyd loang.
T ^ CHI DirgfC H p c - 04/2013 (S6 444 NAM 53)
• Nghien ci>u - Ky thuat
Pha dgng B: Acetonitril Chaang trinh sdc ky nhu- sau:
PhUt 0-20 20-50 50-60 60-61 70
Pha dgng A 80 8 0 - 6 5 6 5 - 2 0 20-80 80
Pha tfpng S 20 2 0 - 3 5
35-80 80-20 20 Quy trinh xCr ly mau
Mdu chuan: Hda tan 100 mg cao nhdn sdm chuan trong 10 ml ethanol 30 %. Lpe qua Ipe 0,45pm.
Miu thir dang vien nang mem
Hoa tan mpt lapng che pham t a a n g ling vd'i 100 mg cao nhdn sdm trong 10 ml ethanol 30
%. Chidt 3 Idn mdi lan 20 ml n-butanol bao hoa vai nu'd'c. Ggp djch chidt vd ed quay ddn cdn.
Hoa tan c i n trong 10 ml ethanol 30 %. Lpe qua Ipc 0,45 pm.
Miu thw d^ng k?o mem
Hoa tan mdu tuang ling vd'i 100 mg cao nhdn sdm trang nu'dc dun nhe vd sidu am den khi m§u tan hodn todn, tidn hdnh chidt vd'i 3 Idn mdi lan 20 ml n-butanol bao hda vd'i nu'd'c. Gdp djeh chiet vd CO quay den can. Hoa tan can trong 10 ml methanol, rdi sac ky egt (hgt nhya Daion) bdng each ngp mau vdo cpt, nia egt v6i nuxi'c ddn khi nudc nra khong con phdn ling vdi dung djeh Fehling, sau do nia gidi vdi methanol (3 x20 ml), hiing djch methanol, co dudi dung m6i, can hoa trong 10 mi ettianol 30 %. Lpe qua Igc 0,45 pm.
M§u thi> dang dung djch uong
Lay mgt the tich mau toang ling vdi 100 mg
cao nhdn sdm, tidn hdnh ehiet vdi 3 lan m6i Idn 20 ml n-butanoi bdo hda vdi nude. Gpp djch chidt va CO quay den cdn. Hoa tan can trong 10 ml methanol, rdi sde ky cpt (hgt nhya Daion) bdng each ngp mau vao cpt, nia cOt vdi node den khi nude n i a khong cdn phdn ling vdi dung djch Fehling, sau do nia giai vdi methanol (3 x 20 mi), hiing djch methanol, c6 dudi dung moi, can hoa trong 10 ml ethanol 30 %. Lpe qua Igc 0,45 pm.
Tien hanh
Lan lu-at tiem mau chuan va mdu t h i i vao hd thdng sdc ky. Ghi iai sac ky dd. Phep t h a chf ed gid trj khi dO phdn giai giCra ginsenosid R g l vd ginsenosid Re Idn han 1,5.
K e t q u d v a b d n l u a n Khao sdt pH cua pha d d n g
Anh hodng cua pH ddn hd sd dung lu'gng cua cae ginsenosid. Thdi gian lu'u cCia ede ginsenosid c6 the thay ddi theo pH t i i 4,5-7 eua node trong pha dpng, neu sir dgng pha ddng id aeetonltrii- nu-dc. Khdo sat trdn cdc malonyl ginsenosid cho thdy ede pie cd thdi gian lau khdng dn djnh hode pie b i ^ mdt khi tidm lap iai. Ngodi ra, pic cda saponin acid Ro vd cdc malonyl gisenosid co thd chdng pie Idn cae saponin trung tinh. S y thay ddi pH cua pha dgng c6 thd Idm ion hda cae malonyl ginsenosid vd saponin acid d i n den gidm tdng sd saponin trong mdu. Dieu nay gdy khd khan trong vide xac djnh ede ginsenosid dae biet trong nen mau phiic tap nhu" kpo. Vi vgy chung t6i da thay ddi nudc bang dung dich dpm. Tham khao tdi lidu ehung toi chpn hd ddm phosphat pH 6,8 vdi ndng dp kali dihydrophosphat Id 0,272 g/L trong pha dgng.
Hinh 2: Sic k'jr dd cao nhin sim chuin USP vd? pha dgng acetonltril-nwac
tf^ CHi DirgfC HQC - 04/2013 (S6 444 NAM 53)
• Nghien C I J U - Ky thuat
Hinh 3: Sic k^ dd cao nhin sim chuan USP Khao sdt n6ng dp acetonitrii
Thdi gian lu-u cua cac ginsenosid rat nhgy ddi vdi dung mdi hO'u ea e6 trong pha dgng. Sy thay ddi ti s6 pha dpng du rdt It cung co thd cho ket qud thay ddi quan trgng vd thdi gian lu'u IR trong sac kj dd. Khao sat ti Id dung mdi hu-u ca dd taeh 2 hap chat Rgl vd Re (co cung cau trile 20-S-protopanaxatriol), eho thay sy thay ddi ndng dO acetonitril theo ehu-ang trinh gradient d khoang 18-20 % cho sy riia giai co do phdn giai R>1,5.
Khao sdt quy trinh chiet mau Dung moi hoa tan ethanol-nud'c (30:70) dya vdo dgc diem hoa tan eua cdc ginsenosid trong
pha d^ng acetonitril-dim phosphat pH 6,8 bgt nhdn sam ehuin USP. •
Dg phuc hoi cOa quy trinh chiet
Can ehinh xac mot lu'p'ng mdu thu- tuwng dng vdi 100 mg cao nhan sdm, them vao mau mpt the tich mdu ehudn eo ndng dp tu-ang dng 10 mg eao nhan sdm. Tien hanh chidt theo quy trinh tren mau keo mem. Ti id hdi phyc trung binh cua 3 mdu chiet td 95-102%, do do quy trinh chiet eo do Igp lai chdp nhgn.
Trien khai phan tich mlu vdi eac dieu kipn neu tren. Cdc ginsenosid trong che phim du-pc nhgn danh thdng qua vide so sanh thdi gian luu vdi eac pic trong sdc ky dd eua mau chuan. Kit qua phan tfch duxyc trinh bdy trong bang 1.
Bang 1: Tinh twang thich hi thdng
Ginsenosid He so phan giai H^s6d6ixung Ginsenosid H^ so phan giai H^sodSixuug Rgl 1,093 Rbl 16.274 0,941
Re 1,847 1,068 Re 3,372 1,244 Ro 21.037 1,447 Rb2 3,407 0,941
Rf 11,685
0,975
Rdeua mdu ehuan. Dp sai Ideh RSD cua dp Igp lai cda cdc pic co thdi gian lu'u khdng qua 2%.
Tinh dac hi$u
Thdi gian luu cda cdc pic cua mau thd' eo thdi gian lu'u taang dng vdi eac pie tren sde do
Bang 2: B0 lip lai thdi gian Iwu cOa cic ginsenosid cua miu chuan vi miu thw
Mau
Mau ctiuan
M l u nu'dc uong
Koiigin D-5240
Rg1 15,985 16,002 16,120 RSD=0.46
15,763 15,936 16,214 RSD=1,42
16,638 16,210 16,325 RSD=1,15
Re 17.872 17,852 17,961 RSD=1,25
17,451 17,825 17,562 RSD=1,09
18,128 17,998 17,854 RSD=0,64
Ro 31,126 31,235 31,268 RSD=0,21
31,258 31,110 31,351 RSD=0,34
31,315 30,998 31,045 RSD=0,24
Thiri gian Itru (ti^
Rf 37,119 37,216 36,995 RSD=0,30
37,015 37,259 37,317 RSD=0,37
37,120 37,159 37,016 RSD=0,24
Rb1 44,695 44,584 44,751 RSD=0,18
44,592 44,625 44,752 RSD=0,19
44,767 44,565 44,660 RSD=0,21
Re 46,405 46,125 46,335 RSD=0,26
46,314 46,258 46,401 RSD=0,16
46,524 46,446 46,440 RSD=0,10
Rt)2 48.158 48,145 48,259 RSD=0,17
48,056 48,220 48,165 RSD=0.17
48.271 48.089 48.162 RSD=0.16
Rd 51.618 50,958 51,468 RSD=0,58
51,528 51,012 50,995 RSD=0.59
51.745 51.126 51,450 RSQ=1.19
T ^ cvi DU'<?C HQC - 04/2013 (S6 444 NAM 53)
• Nghien cipu - Ky thugt
Rg1 16,500 S^ 16,110
^"^"^ 15 998 free ^^•^'>°
RSD=1.6
Re Ro
18,050 31,089 17,965 30,912 17,542 31.105 RSD=1.52 RSD=0.44 Bang 3: Thai gian luv twang doi cua cac Ginsenosid
Rgl Re Ro Rf
Thm gian itru (ti^
Rf 37.221 37.078 36,859 RSD=0,41 ginsenosid
Rbl 44,748 44,684 44,554 RSD=0,22
Re 46,442 46,356 46,512 RSD=0,17 cOa miu chuin r/iov gian iiru tuvng doi Ginsenosid
1.00*
1.12 1.95 2.30
Rbl Re Rb2 Rd
Rb2 Rd 48,185 50,317 48,023 50,985 48,110 51,156 RSD=0.13 RSD=0.87
Thin gian iiru tuong doi 2.78 2.90 3,00 3,2
* De xic dinh thir tv pic cua cic ginsenoid trong cao nhin sam, c/ipn thai gian luv cua Rg1 lam chat dinh dau, then gian luv tuvng doi cua cic ginsenosid lin lucd c6 tri so nhif trong bang 3.
Hinh 4: Sic /cj? d6 miu nwdv uong Korea Red Ginseng extract trong pha d^ng dim pH 6,8
Hinh 5: Sic ky dd miu Vien nang mim Koligin D-5240 trong pha dgng dim pH 6.8
Hinh 6: Sic ky do miu Keo hong sim Suger free trong pha dgng dim pH 6,8 TCr kdt qud cho thdy thdnh phdn ginsenosid
Ro CO gid trj khd cao trong cdc mau kd cd bdt cao nhdn sdm chudn, do d6 vide xac djnh cdc saponin ginsenosid trong eac ehe pham cd chda nhdn sdm khdng the bd qua thanh phdn ginsenosid Ro ndy.
Kk luin
NhU' vdy, chung tdi da xdy dyng dagc quy
trinh ^nh tinh cac ginsenosid trong mdt sd dang thyc phdm chiic nang c6 chda eao nhdn sdm bang phu'ang phap sde ky long hidu nang cao vdi dau dd UV. Quy trinh phdn tich dan gidn, ddp dng tdt cdc yeu eau cua phu'ang phdp phdn tieh djnh tinh gop phan giai quyet nhOng khd khdn trong kidm tra thye pham chdc nang c6 chda cao nhan sam tren thj trudng.
T ^ CHf DUgiC HQC - 04/2013 (S6 444 NAM 53)
• Nghien CLFU — Ky thuat Summary
Neutral ginsenosides Rgl, Re, Rf, Rbl, Re, Rb2, Rd and an acidic ginsenoside Ro in supplemented nutrient foods were determined by HPLC: Shimadzu chromatograph system equipped with an UV detector; Column:
Nueleodur pyramid RP18 (150 x 4.6 mm; 3 pm);
Mobile phase: Acetonitrile-phosphate buffer pH 6.8. The method proved desirably selective and appropriate for determination of ginsenosides in various formulations of supplemented foods.
Keywords: Ghsenosides F^l. Re, f^, Rbl, Re, F^Z f^i^adcScghsenoskieRc^ nutrient foods, HPLC.
Tai lieu tiiam ithao
1. Do Tdt Lpi (1999), NhQng cay thuSc vi vj thuoc vat Nam, NXB Y hoc Tp. Ho Chi lUlinh, 106 -107.2. Corbit R. M., Ferreira J. F. S., Ebbs S. D., Murphy L. L. (2005), "Simplified Extraction of Ginsenosides from American Ginseng (Panax quinquefolius L.) for High-Perfomiance Liquid Chromatography-Ultraviolet Analysis", J. of Agric.
Food Chem.. 53, 9867-9873.
3. Chong Z. W., Bin Z.. Wen X S., Anbao W., Ming N., Xiaoji L , Han H. A , Jing T. X., Robin T„
Tong C. H., Chun S. Y. (2006), 'Steamed American Ginseng Berry: Ginsenoside Analyses and Anticancer Activities", J. of Agric. Food Chem., 54, 9936-9942.
4. Fuzzati N., Gabetta B., Jayakar K., Pace R., Ramaschi G., Villa F. (2000), "Detennination of Ginsenosides in Panax ginseng Roots by Liquid Chromatography with Evaporafive Light-Scattering Detection". J. AOAC Inter, 83. 820-829.
5. United States Pharmacopeia! Convention USP 34"", 2011- National Formulary NF 2 9 ^
Ket qua phan lap va nhan dang taraxeryl acetat, taraxerol
va 7a-hydroxy sitosterol tu* la cay gao {Bombax malaharicum DC.)
Ho Thi Thanh Huyin, Thai Nguyen Hung Thu, Nguyin Thai An
Tnrdng Dai hoc Ehr^c Hd NoiDat van de
Cay gao eon du'p'c ggi la b6ng gao, m6c mien, gon... Id loai cay quen thuOc vdi T\Q<ja\
dan midn Bac Viet Nam. Theo kinh nghidm ddn gian, nhidu bO phan cua cdy nhu vd than, rd, hoa vd nhya duac sii dung de tri ede bdnh nha vidm loet da ddy. viem ioet ngodi da, thap khap, bo gay xaang, kidt ly...^^. Cdc nhd khoa hpc tren the gidi da tidn hdnh nghien ciiu hoa thye vdt cung nha thii tdc dung sinh hpc cua Id gao vd cho thay la ggo co tae dung bdo vd gan, ha huyet dp, ha dadng huydt...
Trong vdi nam gan ddy, mgt s6 nghien eiiu vd lodi cay nay da ghi nhan trong cay co cdc thdnh phan nha naphthoquinon, polysaccarid, shamimin, mangiferin, epicatechin va lupeoP^'.
Tuy nhien eac tai lieu tham khao cho thdy kdt qua nghien ciiu hod hoc vd lodi cdy ndy con chaa day du. Trong khudn kho bai bao nay, ehOng toi mo td qua trinh ehiet xudt, phdn lap vd nhdn dang mgt sd hap chdt tCr phan dogn eloroform chiet xudt Mr la cay gao.
Nguyen lieu, p l i u v n g phap ngiiien ci>u
Nguyen li^u
Mdu nghien ciiu la Id cda cay ggo thu hai t^i Haang San (My Biic, Hd Ngi) vdo thang 11 nam 2011, da dag'c TS. Tran Huy Thdi - Vidn Sinh thdi vd tdi nguydn sinh hge gidm djnh tdn khoa hge la: Bombax malabaricum DC, hg Ggo (Bombaeaceae).