Nghien CIPU • Ky t h u a t
Phytosome: Cac phircrng phap danh gia
Vu Thi Thu Giang, Dao Minh Huy, Pham Thi Minh Huf
Tnanig Dgi hgc Duac Hu \6i 'E-mail, [email protected]Summary
At present, researches on phytosome is still new to Vietnam due to insufficient understanding of manufacturing technology and evaluating methods. For getting effective approaches to this field, metiiods and means to evaluate phytosome charactenstics are essential, especially in evaluation ol the formations of active substance-phospholipid complex. Therefore, following our previous review on phytosome. this presents some methods, both in vitro and in vivo, for evaluation of phospholipid complex formations, and biological effects of phytosomes.
Keywords: Phytosome, evaluation.
Dat van de
Cdng nghe bao c h l phytosome duac bdt dau tLJ' nam 1989 tren ca s d quan sat Ihay rang cdc polyphenol co ai luc lien k i t manh vdi cac phospholipid trong cac md thyc vati""'- Tuy vay, cho den tan vai thap nien g i n day phytosome mdi duac tap trung nghien cuu manh me va ieng dung trong cdng nghe duyc pham. my phdm Phytosome da duyc chung minh nhieu uu diem noi bat nhu tang kha nang hdp thu, tang thdi gian ban thai va luu tru thulc trong c a the, cd the hudng dich va tang van chuyen vao ndi bao. keo dai tac dung va an toan, Bao che thanh cdng phytosome se k h i c phuc duac nhieu nhuoc diem ca ban cua cac hoat c h i t cd ngudn g l c t u duoc lieu nhu: khdng b i n , de bi phan hiiy bdi anh sang, nhiet dd. do am va dd tan han che. Phu'c hop duoc c h i t - phospholipid trong cau true phytosome cdn gop phan bao ve duoc c h i t chdng lai qua trinh chuyen hoa thudc trong dudng tieu hoa va tai gan, do do gop phdn cai thidn dang ke sJnh kha dung va hieu qua tac dung cua hoat chit, nhat (a nhung ch§t bi chuyen hda nhilu d gan, Phytosome co kha nang irng dung rdng rai trong ca duoc pham va my pham.
Hon nira, ky thuat bao c h l khdng qua phu'c tap nen phytosome d l tndn khai ifng dung trong san xudt nhieu dang bao che dung theo dudng uong, tiem va qua da '•'.
Cho d i n nay, bao che phytosome van la mot ITnh v y c nghien ciru mdi d nudc ta do chua co hieu biet ddy dii v l cdng nghd bao c h l va phuang
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phap danh gia, De tiep can iTnh v y c n a y mptcacii hieu qua, y l u t l cdn t h i l t ddi vdi cac nghien CLFU chinh la phuong phap va phuong tien danh gia cac dac tinh cua phytosome, trong dd quan Irqng nhat la danh gia kha nang tao phuc hop du'oc chdt - phospholipid. Chinh vi vay, t i l p theo bai tong quan v l phytosome '•", chiing tdi gioi thiesi ve mot sd phuong phap danh gia phuc iiop va tac dyng sinh hoc cua phytosome da du'pc nghien ciru trdn thd giai.
Phu-cyng phap hoa ly danh gia sif hinh thanh phii-c hp'p phytosome
Phytosome d u y c tao ra la do lidn ket iioa hoc true t i l p cua nhom chirc trong phan ii' hoat c h i t vdi phospholipid, Oe danh gia V3 xa:
nhan s y hinh thanh phirc hop, cd the s(y durq mot so phuong phap phan tich nhu, phan # nhiet (DSC, TGA), n h i l u xa tia X (XRD), phe hap thy hdng ngoai (IR/FTIR), va cdng hudng tu ('H-NMR, '^C-NMR),
Phan tich nhidt
Dua vao s u khac biet vd dac tinh nhiet cu;
phytosome va hdn h y p vat ly ciia duoc cii;
- phospholipid de danh gia v l s y hinh thar CLia phirc h y p K i t qua nghien cifu cua Zhan:
va cgng s u {hinh 1) khi nghien cii'u phirc ho;
curcumin-phosphatidylcholJn cho thdy, gian t nhiet cua phospholipid cd pic thu nhidt 145,1 ciia curcumin cd mdt pic thu nhiet tai 175,7'^C ciia phytosome cd mpt pic thu nhiet tai 122,5^C va cua hon hop vat ly x u i t hien ca 3 pjc tre'
T^P CHi DirgfC HQC - 9/2015 (SO 47^ \ T \ 1 S ^\H
Nghien CIPU - Ky thuat
O i l u ndy ed t h i ly giai r i n g khi dun ndng hdn hap, phospholipid chay Idng va mdt phan se tao phu-e vd'i hogt c h i t (phuang phap tgo phirc thdng qua dun chay) '^^i. Pic thu nhidt cua phytosome khac so vai ca phospholipid va hoat chat chCrng td da c6 s y hinh thdnh phire lam b i l n d l i tinh chat nhidt P ^ I J ) , Cac nghidn cuu vd'i naringenin 1'^, siiybin i^^, puerarin P°\ va cateehin P^I deu cho k i t lu|ln t u a n g t y .
Hinh 1; Giin dd nhipt cua (i) phospholipid, (ii) curcumin, (in) curcumin phytosome, vi (iv) hdn hgp v$t iy cua phospholipid vi liposome ''^'
Phdn t i c h p h o c o n g hu'O'ng tip NMR S y hinh thdnh phicc hyp d u y c xac nhgn
lien quan tai s y djeh ehuyen va gian rdng eac tin hieu dae tm-ng trong p h i NMR ^l Phytosome eua cateehin cd pho ^H-NMR g i l n g vdi hogt e h i t , ngogi trip eae pie lien quan tai nhdm - O H phenol. Cac proton phenolic tai vl tri 5; 7; 3'; va 4' ed pie tuang irng la 5 8,5; 8,6; 8,2; 8,04 bi djeh chuyen thanh 8,8; 8,9; 8,7; va 8,48 t u a n g ii-ng.
D i l u ndy ch! ra s y tuang tac cua phospholipid vdi nhdm - O H phenol cCia cateehin P^'. S y djch chuyen proton ndy cung tuang t y trong nghien ciru v l phytosome naringenin ^^i. Ngoai ra, trong pho H-NMR cua naringenin, pic tai 6 9,1 (H-4'), va 10,2 (H-7'-0H) b i l n m i t {hinh 2), chu-ng td s y t u a n g tae eiia phosphatidylcholin va nhdm - OH. D i l u nay cung phii hyp vai phan tich IR, khi polyphenol tgo phipc vdl d i u phan eye ciia phospholipid, dudi hydrocarbon mgch dai trd nen linh dgng han va se bao gdi p h i n phdn eye cua phospholipid, che I l p tin hidu cua d u y c chat t^', hien t u y n g nay cung tuang t y nhu phytosome curcumin '^^'. Cdc dudi hydrocarbon trd' ndn linh ddng han cung ly giai cho viec giam nhidt do chuyen pha eiia phytosome so vdi phospholipid khi danh gia thdng qua phan tich nhidt ' " ^ " i .
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Hinh 2: Phd 'H-NI\/IR ciJa (a) naringenin (trong DI^SO) va (b) phLFC naringenin-phospholipid (trong CDCIJ >^^i
• Nghien ciju - Ky thuat
Phan tich pho hap thu hong ngoai IR1
Pho se duyc theo ddi dya vao sy biln mit, chuyin djch, hogc gian rdng eiia cac pic dac trung cCia hogt chit, trong tnfdng hyp cua curcumin la 3413 em^ (-0H phenol), 1603cm^vdng benzen), 1510 em'(lien kit C=0) {hinh 3), cita phospholipid ed cac pic dgc tn^ng nhu 1737 cmHC=0), 1236 em-XP=0), 2854 cm ^-CHg). Cac pic nay khdng bi
Hinh 3: Phd FTIR cua (i) phospholipid, (ii) curcumin, (iii) phux; hgp curcumin-phosphoiipid, va (iv) hdn hop vit ly cua curcumin va phospholipid ''*' Phan tich nhieu xa tia X
Phuang phap nay khdng dac tru'ng bdng cac phuang phap tren khi xac nhan sy hinh thanh phirc hyp. Pho nhidu xg eua hoat chat thuang bao g i m cdc pic tinh t h i sac nhpn, cac pic nay vin t i n tai trong hdn hyp vdt ly tuy nhien biln m i t trong phuc hyp phospholipid {hinh 4) Dilu nay gai y rang cac phan tu duyc chat duyc phdn tan dudi dang phan tu- trong phospholipid hoac ton tai 6' dang vd djnh hinh I'^^'^K
Phuxyng phap danh gia sinh kha dung va hoat tinh cua phiPc hap phytosome
Danh gia sinh Itha dung
Mac dii duyc ehung minh mang lai nhilu tac dyng tot nhu ching oxy hda, chdng viem, bao vd vd khu ddc gan, thdm chf cd nhilu nghien ciru v l
bien mat va van giiF nguyen vj tri trong pho IR cua hon hop vat ly curcumin va phospholipid Pho cua phytosome cd s y djch chuyen so vdi curcumin nguyen lieu, vi du 3413-3263 cm-'(OH phenol), 1236-1240 (P=0) 1^5], CGng tuong t u nhu phuang phap NMR. lien k i t tao phuc gii>'a polyphenol va phospholipid duoc n h i l u tac gia xac nhan bao gdm chil y l u la lien k i t hydro i" is^^i.
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Hinh 4: Pho nhieu xa tia X do phan giai cao (HRXRDj cua (Ne) naringenin, (PC) phosphatidyl cholin, (NeCj
phLFC hop naringenin-phosphatidyl cholin'"' hieu qua chdng ung thu, nhung cac polyphenol trong d u y c lieu (silymarin, curcumin, cac cateehin trong tra xanh, hat nho,,,) d i u co nhuoc dilm lan la kha nang hap thu r i t kem, Chinh vi vay, d l Chiang minh duoc kha nang cai thien h§p tiiu thuoc qua dudng tieu hoa, danh gia sinh kha dung CLia phytosome bao c h l tren dong vat thi nghiem hoac ngudl tinh nguyen so vdi duyc chat d dang t u do ciing lieu luyng la c i n thiet trong cac nghien cuu danh gia phytosome, Nhidu nghien cuv cho k i t qua danh gia cac thdng s6 duyc dpng hpc CO ket luan chung la phifc hop phytosome chira duoc c h i t cho C^^__, va AUG Ion hon (242%
|SU25% I'", 434% 1''^') so vdi hoat c h i t gdc, T^^
ciia phytosome dao ddng, trong mot s6 trifdng hop nhd han hoat c h i t gdc (quercetin, kaemferol, isorhamnetin cua cao ginko) ^^\ mot s6 Idn han nhu oxymatrin, curcumin, silybin (hinh 5, 6 ) ' " " ' ^ '
Hinh 5: Ndng dd siiybin trong huyit tuxmg ngifoi tinh nguyen, n = 8'^'
z 0 15 30 60 120 Thoi gian (plljrl
Hinh 6: Ndng do curcumin trong huySt tuang chudt. n^3"3'
TAP CHI DUOC HOC - 9/2015 (80 473 NAM 55)
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Danh gia kha nang ch6ng oxy hoa in vitro Tdc dyng ehinh cua cac hyp c h i t polyphenol tir t h y c vdt la ehdng oxy hoa. Cd n h i l u phuang phap ed the lya ehpn d l danh gia kha nang e h i n g oxy hod eua hogt c h i t nhu do kha nang h i p thu g l c oxy (oxygen radical absorbance capacity:
ORAC), p h u a n g phap do eae chat e h i n g oxy hda bay hoan toan cdc goc (total radical trapping antioxidant parameter: TRAP), phuang phdp FRAP (Feme ion Reducing Antioxidant Power), p h u a n g phap loai goc t y do DPPH (1,1-diphenyl- 2-pierylhydrazyl) hode phuang phap do kha nang peroxld hda lipid. .
(>rrC
T Fe^iii) T
Phirong phap phan tich FRAP: D u y c thye hien dau tien bdi Benzie va Strain de do nang lyc khup trong h u y l t t u a n g '^' nhung cung d u y c sir dyng d l phdn tich cde e h i t c h i n g oxy hda trong thye vat. Phuang phap ndy d y a vdo s y khii' phirc Fe (III)-TPTZ (2.4,6-tripyridyl-s-triazine) thdnh phiFc Fe (II) - T P T Z b i n han bang mdt c h i t khu- ( c h i t e h i n g oxy hda) a pH t h i p . Fe (II) - TPTZ cd mdu xanh duang dam vd cd the do mat do quang d b u d c sdng Amax = 593nm. Tde dgng ehdng oxy hda d u y c danh gia bdi s y tang cudng do mdu cua phirc Fe (II) - T P T Z (Hinh 7).
|F«<l'KTPTZfel»*. Xm„ " S93 nm
Hinh 7: Phin Ong FFiAP"'!
Phwamg phip dinh gii kha nang lo^i gdc (If d o DPPH: Ld mgt phuang phdp d u y c diing pho b i l n vi dan gi§n, nhanh chdng vd I n djnh. DPPH la mpt goc t y do cd b u d c sdng c y c dgi hap thu tai 517 nm va cd mau tim. Cdc c h i t cd kha nang chong oxy hda se trung hda
goc DPPH bdng cdch cho hydro, Idm gidm do h i p thu tgi bude sdng c y c dgi va mdu cua dung djch phdn u-ng se nhat d i n , c h u y i n tip tim sang vang nhat. Phan irng trung hda g l c DPPH ciia cdc c h i t chong oxy hda duae minh hoa trong hinh 81*'.
-A- a.x3 "A-
DPPH Chfit cbdng oxy boa DPPH-K Hinh 8: Phan ting trung hda gdc DPPH
Phuvng phap do MDA: Peroxld hod lipid Id mOt ea c h l thudng g^p eua nhiing t l bao bj t i n t h u a n g trong thyc v§t vd dgng vgt, nd d u y c coi n h u Id c h i t chf thj cua qud trinh stress oxy hda trong t l bdo vd md. Viyc do MDA (Malondialdehyd) - Id sdn pham eua qua trinh peroxld hod lipid la mgt phuang phdp thudn tign vd nhgy eho vide ddnh gia n i n g dg lipid peroxld
trong mdu''!. Phuang phap do MDA d y a vdo phan ung vdi acid thiobarbituric. MDA phan irng vdi acid thiobarbituric (TBA) d QCC trong mdi trud'ng acid tgo phirc MDA-TBA cd mdu h i n g , 2 moi TBA k i t hyp vdi mgt moi MDA (hinh 9).
Phuc mau d u y c hod tan trong dung mdi hOu c a nhu butanol sau dd do pho hap thy d bude sdng 532 nm.
TAP CHI DlTQtC HQC - 9/2015 (SO 473 NAM 55)
• Nghien cifu - Ky thuat
O OHC—CHj—OHO O ° 'i
A
Phiit MDA-TBA mau biwig
Hinh 9: Phan img tao san phim MDA-TBA ^' Danh gid hoat tinh bao ve gan, chong oxy
hoa in vivo
Md hinh sir dung chit ddc gay ton thuang eho gan nhu aflatoxin, paracetamol, CCI^... cd t h i dp dung d l danh gia khd ndng bao ve t l bao gan cua hoat chat. Higu qua gay dgc va giai dgc trdn md hinh in vivo duyc danh gid bdng cdc thdng s l v l hinh thai hge, hda sinh nhu hoat tinh cua mgt s l cdc enzym ndi bao cua huylt tuang (phosphat kilm, glutamat oxaloacetat transaminase, vd glutamat pynjvat transaminase) va ehire nang gan. Phuang phap thir nghigm hoat tinh bao vd gan in vivo duyc chia thanh 2 hudng Id thir nghigm cdc hogt chit trdn chudt bj gay dgc gan elp tfnh (24-72 gid) va mgn tinh (tir 4-12 tuin).
C^c thi nghigm chung minh tac dung chong oxy hda va gidi ddc gan eua polyphenol thudng sir dyng ddng v$t thi nghidm la chudt, vd gay dgc gan bdng carbon tetraclond (CCI^). Vi dy vdi phytosome curcumin, chudt thi nghigm duyc ehia thdnh 6 nhdm va uing nudrc tinh khilt, hdn djch curcumin, hoac phuc hyp curcumin-phosphoiipid (2 mirc liiu) trong 7 ngay lien tyc. Tgi ngdy thu 7, ngoai trtr nhdm chu'ng (nhdm 1), eae nhdm edn lai duyc gay dde bing 1 iln tiem phuc mac cart»n tetradorid, sau do 1 ngdy, danh gid hogt tinh cac enzym dgc tni'ng trong gan. Kit qua eho may vdi nhdm enzym glutathion, nhdm ddng vgt sir dyng curcumin va phytosome deu cd hoat tinh, trong dd tac dyng cua phytosome tot han (P < 0,01). Muc enzym superoxid dismutase giam rd ret vdi nhdm chupt bj gdy ddc. Tgi liiu thdp (100 mgflcg), phytosome Idm tang mire enzym nay, trong khi curcumin ty do thi khdng (P < 0,01) '"i. Trdn md hinh ddng vdt tuang ty, kit qua vdi phytosome naringenin 1^=^', quercetin '"^cung tuang ty nhu curcumin.
Tedesco'^^ danh gid tae dyng bdo ve gan eiia phytosome silymann trdn gd cdng nghiep khi gdy dpc bdng aflatoxin Bl lilu 0,8 mg/kg. Gd duyc
chia thanh 3 nhdm: nhdm chung, nhdm gay dpc, va nhdm d u y c dieu tri bang phytosome silymarin (600 mg/kg). K i t qua cho thay trong ca qua trinh phat tnen ciia ddng vat, hoat tinh ciia alanin amino transferase d nhdm chirng va nhdm sir dyng phytosome khdng khac nhau.
Ket luan
Trong nghien ciru phytosome. danh gia die dac tinh hoa ly, sinh hgc cho san pham Id hit sire c i n thidt, giiip tao ra ca sd khoa hpc Vei thuc day viec phat trien irng dung thuc tien. Vi vay, trudc khi khdi ddng nghien cuu, viec hieu ro va lya chon phuong phap thich hop de danh gia phytosome tao thanh la r i t quan trong, C^c phuong phap hoa ly de danh gia kha nang tao phifc hop d u y c chat - phospholipid la phuong phap danh gia dac trung va d u y c thyc hien trong hau het cac nghien ciru Ngoai ra, de chirng mmh cho kha nang cai thien h i p thu, tang thdi gian ban thai va tuu tru thuoc trong co the thi c^n thirc hien cac phuang phap danh gia sinh kha dung cua t h u l c tren dgng vat thi nghiem hay ngudi tinh nguyen,
Doi vdi cac d u o c c h i t cd kha nang chdng oxy hda nhu cac polyphenol cd nguon goc tu thi;c vat, viec lua chgn md hinh danh gia tac dung sinh hgc cua d u y c c h i t trong phytosome so vdi dang t y do doi hdi nhung h i l u bilt chuyen sau v l ITnh vifc nay Dac biet trong md hinh danh gia in vivo, khdng nhung c i n t h i l t ke va kilm soat d i l u kien thi nghiem tot ma cdn phai thuc hi$n danh gia cac thdng so hda sinh tuang d l i phi>c tap. NhQ'ng phuang phap nay cdn kha mdi me cr Viet Nam, tuy nhien, de hgi nhap vdi xu hudng nghien ciru tren thd giai, thuc day nganh diip'C trong nudc ngay cang phat trien han, ndn bit tay vao xay dyng va phat tnen cac md hinh phii hop.
vdi s y phdi hop giua cac nha nghien ciru d nhieu ITnh v y c khac nhau.
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Nghien ciru - Ky thuat
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(Ngiy nh$n bii: 29/06/2015 - Ngiy duyet ding: 04/09/2015)
TAP CHi DU'OC HQC - 9/2015 (SO 473 NAM 55)
