CHIET TACH AXIT (-)-HYDROXY CITRIC TlT VO QUA BUfA BANG DUNG D|CH NH3
D A N G QUANG VINH, HUYNH SANG, DAO HIJNG CUdNG
•... '. Trudng D^i hpe SU ph$m - Dai hpe Di Ning
SUMMARY
By soaking with NH} solution, (-)-hydroxycitric acid has been extracted from rinds ofGarcinia ob- longifolia Champ, ex Benth. Experiments arul studies by soaking times, concentration ofNH} solution and the rate of solid/liquid phase fiave produced results as follow: The most effective time for extracting is 30 hours. The suitable concentration ofNHj solution is 15%. The rate of solidAiquidphase is 0.059 (approximately lOg of sample in 180ml solvent). The total amount of extracted acid and (-)-hydroxycitric acid in turn are 9.857g and 8.402gper lOOg dried rinds ofGarcinia oblongifolia Champ, ex Benth. This is the first result claimed the ability of (-)-HCA extracting from dried rinds ofGarcinia oblongifolia Champ, ex Benth by soaking with NH^ solution.
Keywords: Garcinia oblongifolia Champ, ex Benth, (-)-hydroxycitric acid, HPLC.
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M 6 DAU Axit (-)- hydroxy xitric (HCA) Id mpt thdnh phin chuylu trong Id, vd q i i bOa, G. Cowa, G. cambcaia, G. indica,...
[1], [3], [41 mOt logi cdy trdng khd pho biln 5 vung DOng Ngm A, An DO dupe dung tdm gia vj. (-)-HCA ed tdc dgng ngdn chgn qud trinh tfch md vd edi thign bitanee mP mdu. HCA se kilm ham q d trinh chuyen hda tupng dudng thQa trdng eo t h i thdnh md, giup ngdn chgn qud trinh b6o phi, dgc bigt td dP'i vPi nhQng ngudi du edn cd c h l dp dn q d nhilu bpt duPng. KhOng nhOng giup gidm cdn, (-)-HCA cdn cdi thign gidm cdc Icgi md xlu chd sQc khde nhu trygtycerid, LDL chctesterot, cho- lesterol todn phdn vd tdng HDL cholesterol Id logi md cd tdc dgng bdo vg thdnh mgch. (-)-HCA Idm gia tdng qud trinh tdng hpp glycogen vd tdng dp oxy hda, ddt chdy md thQa [5], [6], Cdc sdn phlm chiit x d t tQ cdy
' 3idr b d dung d l gii khd phd biln.
im edn dupc bdn trdn thj trudng hign nay
Cdc phuang phdp ehilt xuit (-)-HCA hign nay thudng dupc sQ dgng td phgdng phdp chiit sexhtet vd chong ninh treng ndi dp suit, thuPng tdn thdi gian vd sQ dgng nhigt dp cao d l tdm chuyin hod (-)-HCA thdnh dgng lacton cua nd Idm m i t khd ndng hogt tfnh sinh hpc. Trong khi dd, phuong phdp ngdm ehilt vdi tfnh thdng dgng, d l dp dgng, gid thdnh r l rd't ddng dupe quan tdm. Vigc nghign cQu mgt phuang phdp ehilt xuit mdi trgn ed sd phuong phdp ngdm ehilt bdng dung djch NH3 Id rit d n thig't, giup xgy dgng mpt quy trinh hodn chinh d l chiit xud't axit (-)-hydroxy citric tQ td, vd qud bQa.
Bdi bdo ndy trinh bdy quy trinh ehilt x d t axit (-)- hydroxy citric tQ vd qud bQa bdng phuong phdp ngdm ehilt bdng dung djch NH3 vd ede ylu tddnh hudng din higu s d t cua quy trinh.
II. NGUYEN Vi^T Ll|;U VA PHLfOfNG P H A P
1. Nguydn li^u va hda chSlt
Vd qud eua egy bUa dung tdm nguygn ligu dupe thu hdi tgi xa Hda Lign, huygn Hda Vang, TP. Dd Ndng. Tit cd ede dung mOi vd hda ehd't sQ dgng eho mdy sdc
ky Idng cao dp dupc cung d p bdi hang Merck. Chit chdn td (-)-Calcium threo-hydroxycitrate tribasic hydrate, BioChemika, Garcinia Cambogia Extract, ~ 19,5% as Ca, cung ed'p bdi hang Sigma-Aldrich, cdng thQc
Hda hpc & Lfng dung
Si 20 (jo4)/2oog
m
CzHioCaaOie.xHjO, khd'i IQpng phdn tQ 530,43, axit threo-hydroxycitric 67,3%. SQ dgng dung djch NH3 d l ehilt axit hQu cd vd nude elt 02 tin ed't dung che HPLC, methanol logi dung che sdc ky, H3PO4 logi PA.
2. May s i c ky long cao ap va di^u ki^n sSc ky
Hg thdng sdc ky idng eae dp dupe sQ dgng d l nghign cQg gdm mdy sdc kf idng cao dp hang Merck Hitachi D7000 trang bj vdi bom logi D7100 hang Merck Hi- tachi, vd ldp cpt sdc ki Lichrospher RP18 5pm x 4,6mm X 250mm. Qud trinh dd dupe thgc hign bdng deteetd D7240 Autosampter D7200, phin m l m Muiti - HSM manager. Pha dpng Id dung djch axit phetphorie 0,1%
vdi td'c dp ddng 1 .Omi/phut. Chd't chdn vd mdu dupc ipc qua mdng Ipc 0,45)im vd tigm vdo HPLC.
3. ChuS'n hi dung djch chu^n va \&y ddng dddng chud'n
Chuin bi HCA tU dd: Calcium three-hydrexyxitrat tribasic hydrate (15mg) cho vdo cd'c 50ml chQa 5,0mt nudc, va xQ ly vdi 500mg Ddvi/ex 50 [H*]. HOn hpp dupe khdy trong thdi gian 10 phut sQ dgng khiy tQ. Tdch lly phin dung djch, vd nhga dupc rQa vPi n_udc cd pH trung tinh. Nude rQa va dung djch dupc trpn idn vd tdm thdnh 50mt, khdy trdn, vd Ipc sQ dgng gily tpc.
Xiy dung dudng chuin: Phgong phdp xgy dgng dudng chuin dupc thgc hign bdng cdch dga vdo kit qui phdn tich mpt chudi cdc mdu (-)-HCA chuin. Ndm miu dung djch chuin chQa 37 - 87ppm (-)-HCA tg do dope tigm vdo HPLC, thae tde rQa gidi dope thgc hign nhu phdn thdc l d n d trgn, vd kit q d thu dupc cdc dign tfch pic. Dudng eong (-)-HCA dupc ve dga trgn bilu dign sg phg thupe giOa ndng dO eua (-)-HCA vd dign tfch pie (trung binh eua 03 tdn ehgy).
Khodng ndng dO miu chdn d n d l xdy dgng duPng chdn dupc xdc djnh dga vdo ndng dd thgc eua (-)-HCA ed trong mdu. Khodng ndng dd ndy tfnh tU gid trj gidi hgn dudi (LLOQ) din gid trj gidi hgn trgn (ULOQ).
4. Quy trinh ngSm chid't vdi dung djch NHj Vd qui bUa khd: Q d bUa sau khi rQa sgch, hong khd tiln hdnh bd qud d l logi bd rupt vd hgt. V6 qud dupc cdt nhd, sly khd trdng tij sly d nhigt dp 100°C thdi gian 24 gid din khi vd q d khO hodn todn, xay thdnh bOt mjn va sQ dgng d l ehilt axit hQu cd.
Cdn khodng lOg mdu vd khO, cho vdo binh tam gidc 500mt cd ndp vdi t h i tieh A dung djch NH3 ndng dO B trong thPi gian ngdm ehilt td C Sau khi hit thdi gian ngdm ehilt C, dung vdi tpc muslin d l Ipc dung djch trong binh tam gidc, d l tdch rigng phin djch ehilt vd bd vd q d . Dung nude d t d l rQa IQpng ba vd q d cho din khi pH cQa ngPc rQa bdng 7. Trdn cd nQPc rQa vdo djch ehilt. Djch ehilt cd mdu dd, dgc. Dun sOi djch ehilt d l dudi hit lupng khf NH3, eho din khi tupng hoi bay dupe kilm tra bdng gily quy tfm cho pH bdng 7.
Trpn tin dung djch chiit vdi 4 gram than hogt tfnh vd ngdm trong nude I m 30 phut. Sau dd, tiln hdnh tpc djch ehilt bdng phlu buchne vd gily Ipc, than hogt tinh dupc r d tgi 2 tdn, mdi ldn 10mt nuPc d t . Djch ehilt sau khi xQ ty bdng than hogt tfnh cd mdu vdng nhgt. Trgn tin djch ehilt vd dung djch nude rQg, cO dgc din 50mt, xQ ly vdi 100ml etgnet, d l trong vdng 30 phut d l kit tua hit pectin (kit tua mdu trdng sOa). Lpc chdn khIng d l tdch rigng phdn djch ndi vd kit tua. Phin kit tua afa 2 tin vdi 20mt ethanot vd tpc d l thu hdi axit. Trpn cdc djch ndi, cd dgc vd thgm nudc elt d l chdn din lOOmi.
Tiln hdnh chuin dd axit - bazp d l xdc djnh hdm tupng axit tdng ed trong mdu. Phin mdu sau khi chuin dg cdn Igi dupc luu giO d 4°C din khi sQ dgng cho HPLC d l xdc djnh (-)-HCA.
Thgc hign tgi nhilu lin thf nghigm trgn bdng cdch thay ddi cdc tham sd C, B, A d l khdo sdt higu suit ciia q d trinh ehilt x d t theo thdi gian ngdm ehilt, ndng dg dung djch NH3 vd ty tg rdn/tdng.
III. KET QUA VA T H A O L U ^ N
1. Kd't qua xSy ddng dddng chuS'n va xac djnh (-).HCA bing HPLC
Dudng chuin dupc xdc djnh bdng cdch thay ddi ndng dp eua 05 mdu chuin, vdi ndng dp tQ 37 din 87ppm, phuang trinh duPng c h d n : Y = 14971X - 9212.4 vdi Y Id dign tfch pic cua (-)-HCA, X td ndng dp (-)-HCA, hg sd tuong quan R^ = 0,9997.
Thanh phdn axit hOu eP xdc djnh bdng phuong phdp sdc kf tdng cao dp vd phuong phdp chudn dp dQpc the hign treng bing 1, 2, 3. Tdng axit xdc djnh bdng phQOng phdp chuin dp axit - bazp cd kit qud Idn hPn
tdng axit xdc djnh bdng HPLC. Gid trj thu dupc chu ylu ciJa phuong phdp HPLC dupe tfnh din chl td (-)-HCA, bdi vl gid trj thu dupc tQ dign tfch pic cua (-)-HCA tdn nhd't. Axit ehu ylu dupc tim thiy trong vd qud b d ngdm ehilt bdng dung djch NH3 bdng HPLC td (-)-HCA, dupc t h i hign trgn sdc kf dd hinh 2. Trdn sdc kf dd, (-)-HCA cho pic don trong t i t cd cdc miu ehilt. Xdc djnh pic (- )-HCA dga vdo pic c d axit (-)-HCA c h d n x d t hign d thdi gian tgu td 3,802 phut (hinh 1). ThPi gian tgtJ c d (-)-HCA dQpc tim thiy trong t i t cd d c mlo Id 3.82 ± 0,01 phut.
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Hda hpc & L^ng dungSi 20 (104)12oog
Chrom Typs: HPLC Channel : 1
0.00.5 1.0 1.5 2.02.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 Retention (min) STD-
Hinh I: Sic ki dd miu axit HCA chuS'n 2. Kd*^! qua chid't xuSit theo thdi gian Tdng tupng axft vd (-)-HCA ehilt tdch dupc tQ vd q d bQa bdng phuong phdp ngdm ehilt vdi dung djch NH3 tdno theo thdi gian dupe t h i hign tgi biiu dd 3. Theo bieu dd 3, tdng tupng axit vd (-)-HCA tdng theo thdi gian ngdm chidt den thdi gian Id 30 gid dgt gid tn ldn nhd't (tdng lupng gxit 8.384g/100g vd HCA 6.239g/100g) vd tigp tgc tdng thdi gian ngdm ehilt thi tdng IQpng axit gidm ddn. Dilu ndy ed t h i dupe gidi thfch nhQ sau: khi thdi gian ngdm ehilt tdng thi thdi gian tilp xuc giQa dung djch NH3 td mpt bazd vdi vd q d bQa cd ChQa axit cdng tdng ndn tupng axit treng vd q d bQa tham gia phdn Qng cdng nhilu thi q d trinh ngdm ehilt thu dupe nhilu axit. Tuy nhign, khi lupnq axit dupe ctiilt tdch ra vd tupng dung djch NH3 da ed du thdi j i a n de phdn Qng md klo ddi thdi gian nggm chigt thi dg tgo dilu kign cho vi khdn phdt triln, de modi cug (-)-HCA cd tfnh thgc phlm ngn tgpng axit thu dupc cd xu huPng gidm din. Do dd, thdi gian ngdm chigt tdi uu nhlt dupc tga chpn td 30 giP.
Retention Time (min)
Hinh 2: Sdc ki dd m§u vo qua bda khd ngSm chiet bSng dung djch NH3
3. Kg't qua chid't xufi't theo ndng dO dung djch NH3
Khi cd djnh thdi oian nggm chiit, thay ddi ndng dp dung djch NH3 thi tdng lupng axft vd (-)-HCA ehilt tdch dupc tQ vd qud bQa tdng theo ndng dp cua dung djch NH3 dupc t h i hign d biiu dd 4. Khi tdng ndng dO cua dung djch thi tdng tupng axit vd (-)-HCA tdng, khi tigp tgc tdng ndng dd eiJa dung djch tgn 20%, 25% thi lupng axit ehilt dupe cd xu hudng gidm din hay thay dot khflng ddng kg. Dilu ndy cd t h i dupe gidi thfch nhu sau: ndng dp dung djch NH3 thip thi tupng axit tham gia phdn Qng vPi dung djch NH3 thip ngn lupng axit dupe chiit tdch ra ft. Khi tdng ndng dp dung djch NH3, tQc Id tdng khd ndng phdn Qng vdi tupng axit treng vd q d bQa ngn tupng axit dupe ehilt tdch ra nhilu hdn.
Tuy nhign, khi tdng ndng dP c d dung djch NH3 q d cae thi khOng thugn tpi cho phdn Qng giQa IQpng axit treng vd qud bQa vd NH3 ndn gidm tupng axit dupe ngdm ehilt.
Bieu do 3: Dd thj bid'u dien s\f phy thuOc cua td'ng Id^ng axit va l\i(fng (-)-HCA
vao thdi gian ngSm chiet
DTungli/,^ngiixit BIICA
Bieu do 4: Dd th| bid'u diln sd phy thuQc cua td'ng \\i(fng axit va \\i(fng (-)-HCA
vao ndng dd dung djch
Hoa hpc & Lfng dung
Si 20 (io4)/20og
ceo
4. Kd't qua chidt xuS't theo ty 1$ rdn long Tdng lupng axft vd (-)-HCA ehilt tdch dupe tQ vd qgd bQa bdng phQdng phdp ngdm ehilt vdi dung djch NH3 thee ty lg rdn Idng dupc t h i hign d biiu dd 5. Khi tdng ty tg dung mdi hay gidm thee ty Ig rdn tdng thi tdng lupng axft vd (-)-HCA ehilt tdch tdng d i n , khi ty tg rdn/tdng dgt 0.059 thi lupng gxit dgt IPn nhlt vd nlu tilp tgc tdng tupng dung mOi (gidm ^ tg rdn/tdng) thi tupng axit vd (-)-HCA gidm vd thay dot khOng ddng k l . Dilu ndy ed t h i dupc gidi thfch nhu sau: khi tdng tupng dung mdi ehilt tdch trdn mpt khO'i tupng chit rdn dupc chigt tdch thi Chung ta tdng khd ndng hda tan cua axit dupe ehilt tdch tQ pha rdn sang pha tdng ngn tgpng axit thu dupe tdng lgn. Tuy nhign, khi tdng dung tfch cua dung mPi qud tdn thi gidm ndng dp cua cdc chd't phdn Qng, tdm gidm khd ndng phdn Qng ngn khdi tupng axit ehilt tdch dupc cd xu hudng gidm din. Ty Ig rdn Idng ehe kit qud eae nhlt td 0,059, tQc td khodng 180mt dung mOi che khodng lOg vd bQa khO.
0176 Omz 0.059 0.<»6 0.033
Bieu do 5: D 6 thj bid'u diin sd phy thu$c cua tdng Idgfng axit va Id^ng (-)-HCA
vao ty 1$ rdn long
IV. KET LUAN Tdm tgi, bdng phuang phdp ngdm ehilt vdi dung djch
NH3 da ehilt tdch dupc axit (-)-hydroxy citric tQ vd q d bQa khO. Kit qud khdo sdt qud trinh ehilt tdch axit hydroxy citric tQ vd qud bQa khO bdng phuong phdp ngdm chiit vdi dung djch NH3 thee thdi gian, ndng dO dung djch vd ty tg rdn tdng ehe thiy: thPi gian nglm chigt higu q d nhd't td 30 gid; ndng dO dung djch NH3 thfch hpp Id 15%; Ty tg rdn tdng thfch hpp Id: 0,059
(tgong duong 180ml dung mdi cho khodng 10g chit rdn); Tdng tupng axit vd axit (-)- HCA chiit dupe tin tgjt td 9.857g vd 8.402g/100g miu vd q d khd.
TQ kg't qgd nghign cQu ndy, cd t h i khdng djnh phuong phdp ngdm chiit vdi dung djch NH3 cd thi si!r dgng d l ehilt tdch (-)-HCA tQ nguygn ligu d l tdm cdc sdn phlm gidm cdn.
TAI LI|:U THAM K H A O 1. Bhabani S. Jena, Guddadarangawanahalty.
Jayaprakasha, Kunngmpurath K. Sakariah (2002), "Or- ganic Acids from Leaves, Fruits, and Rinds of Garcinia covja", Jpurnal pf Agricultural and Fcpd chemistry, 50(12), 3431-3434.
2. Dde Hung CuPng, Dgng Quang Vinh (2007), "Xie djnh axit hUu cd tU li, vd qui bUa bing sic ky Idng cad ip", Tgp ehf Khog hpc vd COng nghg - Dgi hpc Dd Ndng, 3(20), t r i 37-143.
3. Jayaprakasha, G. K.; Sakariah, K. K (1998), "De- termination of organic acids in Garcinia cambogia (Desr.) by HPLC", Journal of Chromatography A, 806(2), 337-339.
4. Jayaprakasha GK, Sakariah KK. (2002), "Determi- nation of organic acids in leaves and rinds of Garcinia indica (Desr.) by LC", Journal of Pharmaceutical and Bidmedieal Analysis, 28(2), 379-384.
5. Jena BS, Jayaprakasha GK, Singh RP, Sakariah KK. (2002), "Chemistry and biochemistry of f-j-.
hydroxycitrie acid from Garcinia', Journal of Agricultural' and Food chemistry, 50(1), 10-22.
6. Levi/is, Y. S. (1969), "Isolation and properties of hydroxycitrie acid". In Methods in Enzymology; Colowick, S. P., Kaplan, N. 0., Eds.; Academic Press: New Ydrk, 13, 613-619. V
Ngudi phan biin: TS PH^M VAN HAI
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Hda hpc & Lfng dyngSi 20 (io4)/20og
