Nghien
CLFU- Ky thuat
Thanh phan hoa hoc cua phan doan chiet bang n-butanol re loai dang sam Viet Nam (Codonopsis javanica (Blume) Hook.f.)
TrSn Thanh Ha'*, D6 Thi Ha', Ha Van Oanh^
' Vien Duac lieu -Truang Dgi hoc Duac Hd NSi 'E-mail: [email protected]
Summary
The phytochemistry of the n-butanol extracts from the roots of Codonopsis javanica (Blume) Hook was investigated. Four natural compounds were isolated and stmcturally identified by MS and NMR, as ampelopsin (^), hesperidin (2), adenosin (3), p-D-Fmctofuranose (2-^1) p-D-Fnjctofuranose (2-^1) p-D-Fmctofuranose (2—>1) a-D-Glucopyranose (4^1) a-L-(6-acetyi-rhamnopyranose) (A). The compounds 3, 4 were isolated from C. javanica for the first time.
Keywords: Codonopsis javanica (Blume) Hook.f, ampelopsin, oligosaccharide, adenosine.
Dat van de
Dang sam Viet Nam {Codonopsis javanica (Blume) Hook.f., ten ding danh Campanumoea javanica Blume; Hp Campanulaceae), la vj thuoc bo khi da dugc dting ttr lau dual dang thuic sing heac tam girng. Cay dugc sir dung treng diiu trj suy nhuge, thiiu mau, met moi, vang da, tieu chay, viem than, phu n i , tri, bang huyit va bach huyit.
Ngoai ra, no con dugc dting lam thuoc bo da day, chu'a ho, tieu 6am trong cae bai thuie phii hgp vai met s i vj thuie khac ^l Thee Do Huy Bich va cpng su, treng r i loai dang sam nam eo duang, chit bee, khOng eo saponin P'. Hoang Minh Chung va cpng su chi ra treng re dang sam nam ehi chira 17 acid amin, 3,12 ± 0,80 % saponin (thee phuang phap can), duang khir P "'. Tuy nhien cac nghien ciru tren mai chi mang tinh chit djnh tinh so bp nhtrng nhom chit co trong dang sam. Vi vay, nghien cu'u thanh phin hoa hpc, tim kiim cae hoat chit ttr dang sam la viec lam cin thiit cung cip them CO so khoa hpc chp viec sir dung mpt nguin dugc lieu c6 a nuoc ta lam thuoc. Trong nghien ciru mai day ciia chung t6i v i thanh phin hoa hpc cua loai nay buac d i u da phan lap dugc cac hgp chit yS-sitosterol, 2'-hydrexy-N-((E, 2R)-1,3,4- trihydroxyeetadec-S-en-2-yi) hexaeosanamid tu' can n-hexan, o-spinasterol 3-O-p-D-glucepyranesid ttr can ehiit ethyl acetat'^'.
Bai bao nay e6ng bo kit qua phan lap cac hgp chit ttr can chiit n-butanol re cay dang sam Viet Nam thu hai tai San La.
Nguyen lieu va phu'O'ng phap nghien ciru Nguyen lieu
Miu nghien ciru la re dang sam mpc hcang thu hai vao thang 5/2013 tai Son La, dugc TS. Pham Thanh Huyin - Khoa Tai nguyen duge lieu - Vien Dugc lieu tham djnh ten khea hpc la Codonopsis javanica (Blume) Hppk.f. Miu tieu ban dugc luu tai Khoa Tai nguyen duge lieu, Khea Hoa thuc vat - Vien Dugc lieu.
Phuang phap phan lap cac hop chit Phan lap cac hgp chit ttr re dang sam nam bang sic ky cpt Dung m6i, hoa chit dting de chiit xuit va phan lap gom methanol (MeOH), ethyl acetat dugc mua ciia cac cong ty hoa chit va dat tieu chuin cong nghiep. Theo del cac phan dean chiit bing sac ky lap mong (SKLM). Sic ky cpt dugc tiin hanh vol chit hip phu la silica gel pha thuang (0,040-0,063 mm, Merck); YMC (Fuji Silysia Chemical Ltd.). SKLM dugc thue hien tren ban meng Merck Kieselgel 60 F^^ (0,25 mm) va ban mong pha dao RP-1S resins (30-50 pm, Fuji Silysia Chemical Ltd.). Phat hien v i t chit bing den tir ngoai o hai buac song 254 nm va 365 nm heac dting thuie thir la dung djch H^SO^ 5% treng ein roi ho nong ban mong.
Phirong phap xac dinh cau true hoa hgc cac hop chat
Xae djnh c i u triic cae hgp chit phan lap duge dua tren cac thong so vat ly va cac phuang phap pho bae g i m : diem chay, pho cpng huang ttr hat nhan va p h i khii lugng. Diim chay dugc do tren may Beetius (Bite) tai Vien Dugc lieu.
TAP CHi DirOC HOC - 4/2016 (SO 480 NAM 56) 35
• Nghien - Ky thuat
Pho cpng huo'ng ttr hat nhan ghi bang may Bruker Avance-500 MHz (Dire) vo'i chit chuan npi la TMS tai Vien Hoa hpc, Vien Han lam khoa hpc va e6ng nghe Viet Nam. Pho khoi lugng de tren he thing LC/MS/MS - Waters xevo (My) tai Vien Hoa hpc, Vien Han lam khca hpc va cong nghe Viet Nam.
Chief xuat va phan lap cac hop chat finh khiet
3 kg dugc lieu nghiin nho duge ngam vol ein 90% 6' nhiet dp phong (3 lin, mii lin 4 ngay). Cae djeh chiit dugc gop lai va cit loai con nuac dual ap suit giam thu dugc can chiit ting (302,55 g).
Can chiit ting dugc them hen hgp ein-nuac (1:1) rii ehiit lin lugt trong eae dung moi eo dp phan cue tang din: n-hexan, ethyl acetat, n-butanci; lam khan cae djeh chiit bang Na^SO^, eit loai hit dung moi dual ap suit giam thu duge eae phan can chiit tuong irng: n-hexan (17,19 g), ethyl acetat (18,30 g), n-butanpl (256,46 g).
Phan tach can n-butancI (50 g) bang sac ky cpt silica gel vai dung m6i rira giai la ethyl acetat/
HO 7
methanol vo'i ty le ethyl acetat tang d i n tir 0 d i n 100% thu duge 25 phan doan ky hieu B-l^
B-XXV. Phan doan B-ll e6 cho bay hai hit dung m6i, xir ly loai mau r i i kit tinh lai trong he dung moi dichlomethan/ methanol (9:1,v/v) thu dugc hgp chit 1 (15 mg). Phan doan B-IV c6 cho bay hoi hit dung m6i, sau di loai bo chit mau, rol tiep tue phan tach tren cpt silica gel pha thuong, rira giai bang he dung mpi clereform - aceton - acid formic (35:2:1) thu dugc hgp chit 2 (13 mg). Phan dean B-XIV, e6 eho bay hai het dung moi tiip tuc phan tach tren ept silica gel pha dap vol chit hip phu YMC, he dung m6i rira giai MeOH/H20:1/1 thu dugc hgp chat 3 (8 mg) va 4 (43 mg).
Ket qua va ban luan
Tir phan doan n-butanol ciia dich chiet cin 90% r i dang sam da phan lap va xae djnh ciu true 4 hgp chit la ampelopsin (1), hesperidin (2), adenosine (3), /3-D-fruetofuranose (2^1)
;8-D-fructofuranese (2—>1) /3-D-fructefuranose (2—>1) o-D-glucopyranose (4-<^1) a-L-(6-aeetyl- rhamnopyranose) (4) (hinh 1).
QCH3
HO.,
OH - O H OH
O H / = 0 Rhap
Hinh1: Ciu triic Hap chat 1
Bpt mau trang nga; mp.: 253-255 °C; ESI- MS: m/z 319 [M-Hj* CTPT C,3H,P3 ^H-NMR (500 MHz, CD3OD) S (ppm): 11,7 (1H, s, OH-5), 6,57 (2H, s, H-2', H-6'), 5,94 (1H, d, J = 2,0 Hz, H-8), 5,90 (1H, d, J = 2,0 Hz, H-6), 4,86 (d, J = 11,5 Hz, H-2), 4,49 (1H, d, J= 11,5 Hz, H-3). "C-WMR(125 MHz, CD3OD) d (ppm): 85,1 (C-2), 73,5 (C-3) 198,1 (C-4), 165,1 (C-5), 97,3 (C-6), 168,5 (C-7)
OH "---I
OH HO loH L ^, Fruf2
Glcp 4
cac hap chat 1-4
96,2 (C-8), 164,3 (C-9), 101,7 (C-10), 129,3 (C-1'), 108,1 (C-2', C-6'), 146,7 (C-3', C-5'), 134,8 (C-4').
Hgp chit 1: Pho ESI-MS cho pic ion phan tir 6' m/z 319 [M-H]* tuong irng vol c6ng thire phan tir C,5H,Pg. Phan tieh pho 'H-NMR va "C-NMR cua hgp chit 1 vol hai tin hieu proton doublet a d„ 4,86 (d, J = 11,5 Hz, H-2), 4,49 (1H, d, J = 11,5 Hz, H-3) va tin hieu ciia nhom oxymethin 6" 8^
85,1 (C-2), 73,5 (C-3) cho du dean v i mpt khung
Nghien
CIFU- Ky thuat
cau triie 2, 3-dihydroflavonol cua chit 1. Su eo mat ciia hai vong tham eung duge the hien ro tren pho ^H-NMR vai cae tin hieu proton dac trung ciia vong tham t h i meta (vong A) vol 5^^ 5,94 (1H, d, J = 2,0 Hz, H-8), 5,90 (1H, d, J = 2,0 Hz, H-6) va t h i tetra 6' vong tham eon lai vol 5„ 6,57 (2H, s, H-2', H-6') eiing vai cac tin hieu tren pho "C-A//WR 5^ 165,1 (C-5), 97,3 (C-6), 168,5 (C-7), 96,2 (C- 8), 164,3 (C-9), 101,7 (C-10), 129,3 (C-1'), 108,1 (C-2', C-6'), 146,7 (C-3', C-5'), 134,8 (C-4'). Tir viec phan tieh eae du' kien pho nhu tren ta du doan hgp chit 1 la mpt flavonoid co cong thire ciu tao 5, 7, 3', 4', 5'-pentahydroxyl flavanonol). So sanh vol tai lieu tham khaoi^i cho ta kit luan chit 1 thu duge la dihydremyrieetin hay ampelopsin.
Hccp chat 2
Bpt mau trang; nhiet dp nong chay 253°C. ESI- MS: (m/z): 610 [M]*. 'H-A//MR (500 MHz, DMSO-d^) 5„ (ppm): 12,01 (1H, s, 5-OH), 9,08 (1H, s, 4'-0H), 6,94 (1H, d, J = 8,0 Hz, H-5'), 6,94 (1H, d, J = 2,0 Hz, H-2'), 6,90 (1H, dd, J = 2,0; 8,0 Hz, H-6'), 6,14 (1H, d, J = 2,0 Hz, H-6), 6,12 (1H, d, J = 2,0 Hz, H-8), 5,50 (1H, dd, J = 3,0; 12,5 Hz, H-2), 2,77 (1H, dd, J=3,0; 17,5 Hz, H-3a), 3,14 (1H, dd, J = 12,5;
17,5 Hz, H-Sb), 3,77 (3H, s, 5'-OCH3), Glc: 4,97 (1H, d, J = 7,5 Hz, H-1"), (5H, H-2", H-S", H-4", H-5", H-6"), Rha: 4,52 (1H, brs, H-1"'),(5H, H-2", H-S", H-4", H-5") 1,08 (SH, d, J = 6,0 Hz, H-6'"). "C-NIVIR (125 MHz, DMSO-dj) 5^ (ppm): 78,4 (C-2), 42,0 (C-3), 197,0 (C-4), 163,0 (C-5), 96,4 (C-6), 165,1 (C-7), 95,5 (C-8), 162,5 (C-9), 103,3 (C-10), 130,9 (C-1'), 114,1 (C-2'), 146,6 (C-S'), 148,0 (C-4'), 112,0 (C-5'), 117,9 (C-6'), 55,7 (5'-OCH3), glc: 100,6 (C-1"), 75,5 (C-2"), 72,1 (C-S"), 69,6 (C-4"), 76,3 (C-5"), 66,0 (C-6"), Rha: : 99,5 (C-1'"), 70,3 (C-2'"), 70,7 (C-3'"), 73,0 (C-4"'), 68,3 (C-5'"), 17,8 (C-6"').
Phi ESI-MS ciia hgp chit 2 che pic ien phan tir 6': m/z 610 [M]*, tuang irng vol cong thirc phan tir la C23H3^Q,5. Phi 'H-NMR eiia chit 4 ehe cac tin hieu cpng hup'ng dae tarng eiia met flavonoid glycesid. Trong do eo hai nhom-OH 6' 5„ 12,01 (1H, s, OH-5), 9,08 (1H, s, OH-4'); tin hieu eiia 5 proton treng hai vong tham gim hai proton ghep cap meta 6' 5„ 6,14 (1H, d, J = 2,0 Hz, H-6), 6,12 (1H, d, J = 2,0 Hz, H-S) thupc v i met vong tham (vong A), ba tin hieu proton con lai thupc v i vcng tham the 1,4, 5a'5„6,94(1H, d J = 2,0Hz, H-2'), 6,94 (1H, d J = 8,0 Hz, H-5'), 6,90 (1H, dd, J = 2,0, 8,0 Hz, H-6'). Met tin hieu eiia proton methyl chuyen djch ve truang thip a 3,77 (s, 5'-OCH3). Dang chii y la cae tin hieu eiia met nhom methin 0^ 5,50 (1H, dd, J = 3,0, 12,5 Hz, H-2) va met nhom methylen 5„2,77 (1H, dd, J= 3,0,17,5 Hz, H-3a) 3,14 (1 H,dd
J = 12,5, 17,5 Hz, H-Sp) cho ta du doan v i eau true mpt flavanon trong phin aglycon ciia chit 2.
Tren pho ^H-NMR con eho tin hieu ciia cac proton dac trung ciia 2 vong duang, trong do mpt duong la glucose voi proton anome 5,^ 4,97 (1H, d, J = 7,5 Hz, H-1"), duang cpn lai la rhamnese vol dac trung eua nhom methyl d n i o s (1H, d, J = 6,0 Hz, H-6'"), proton aneme 5„4,52 (1H, brs, H-1'"). Dieu nay dugc thiy ro net hon khi ta kit hgp khae sat cac pho "C-NMR va DEPT.
Khae sat pho "C-NMR va DEPT cho thay chit 2 bae gim 28 carbon trong dp cc mpt nhom carbonyl lien hgp eho tin hieu cpng huo'ng dac trung 6' 5^, 197,0 (C-4), mpt nhom methylen a 6(, 42,0 (C-S), mpt nhpm methin a 5^ 78,4 (C-2) cac tin hieu carbon eiia hai vong tham nam treng kheang 5^ 95,5 den 163,0. Nhom methyl chuyen v i truang thip a 5^ 55,7 (5'-OCH3). Tin hieu ciia 6' 5p 66,0 (C-6") dac trung che nhom methylen cua duang glucose, S^, 17,8 (C-6'") dac trung eho nhom methyl cua duang rhamnese.
Tir viec phan tich eae dti' kien pho, kit hgp so sanh vol tai lieu tham khao i^ eho phep khang djnh hgp chit 2 la mpt flavpnoid glycesid co ten gpi la hesperidin.
Hcyp chat 3
Bpt mau trang, nhiet dp nong chay 234 - 235 °C, tan trong MeOH, R,= 0,28 (CHCI,: MeOH = 3: 1);
vit CO mau xam 6' buoc song 254 nm. ^H-NMR (500 MHz, CD3OD), 5„ (ppm): 3,77 (1H, dd, J = 3 , 12,5 Hz, H-5'b), S,91(1H, dd, J = 2,5; 12,5 Hz, H-5'a), 4,19 (1H, q, H-4'), 4,35 (1H, m, H-S'), 4,76 (1H, t, J = 5,5, 11,5 Hz, H-2'), 6,00 (1H, d, J = 6,0 Hz, H-1'), 8,21 (1H, s, H-2), 8,33 (1H, s, H-8). "C-NMR (125 MHz, CD3OD), SJppm): 63,5 (C-5'), 72,6 (C-S'), 75,5 (C-2'), 88,2 (C-4'), 91,2 (C- 1'), 113,7 (C-5), 119,5 (C-8), 141,9 (C-4), 146,2 (C- 2), 153,5 (C-6).
Tren p h i ^H-NMR eiia 3 nhan thiy su xuit hien tin hieu eiia 2 tin hieu proton viing truang 8,21 (1H, s, H-2) va 8,33 (1H, s, H-S) dugc cho la do anh huang cua su co mat ciia hai nitre ben canh treng cong thirc phan tir. Mat khac su xuit hien eiia 5 proton 6' kheang 3,77 din 4,76 va mpt oxygenated proton 6- 6,00 the hien su xuit hien ciia vong 5 canh t h i hydroxyl a cac vj tri 2®5.
Tren pho "C-NMR va DEPT eiia 3 chi thiy xuit hien tin hieu eiia 10 nguyen tir carbon, treng do CO 1 nhom methylen, 6 nhom methin cting vai 3 carbon bae 4.
Ttr CO so nhu'ng dtr kien tren, kit hgp se sanh vai nhung dO' lieu phi trong tai lieu w nhan thiy hgp chat 3 ehinh la adenosin.
TAP CHI DirgfC HOC - 4/2016 (SO 480 NAM 56) 37
• Nghien - Ky
Hop chat 4 C h i t dee mau t r i n g
H1 (ppm) H2 H3 H4 H5 H6 CH3
Frufi
_
4,01 3,90
-
1.
Fruf2
_
401 3,90
_ -
thuat
Bang' Chat 4
Fruf3
_
4,01 3,90
- -
1: DUkien pho NMR eiia chit 4 Glcp
5.19
~ -
3,22 412
-
Rhap 5,19
~
4,58 2,20
Tinh toan bang phan mem chemdraw Fnifl
3,91:3,66 3,97 3,65 3,91 3,79; 3,54
Fruf2 3,76; 3,51
3,97 3,65 3,91 3.79; 3,54
Fnif3 3,76; 3,51
3,97 3,65 3,91 3,79; 3,54
Glcp 5,03 3,73 3,73 3.02 4,00 3.79; 3.54
Rhap 5,03 3,73 3,49 3,63 4,54 2,09 Cl (ppm)
C2 C3 C4 C5 C6 CH,
63,7 1057 75,8 69,1 82.7 64.3
60,5 102,9 76,3 69,1 82.7 64.3
62,4 99,1 77,2 69,1 82.7 64.3
97,7 74,5 73,3 77,5 82,9 65,5
93,6 70,8 72,7 71,5 83,7 211,5 30,8
62,0 110,8 77,0 72.1 80.7 62.3
59,8 109,2
77,3 72.1 80.7 62.3
60,1 103,0 77,6 721 80.7 62.3
1007 76,7 75,5 78,5 79,8 65,6
100,4 72,8 73,9 73,2 85,9 207,3 25,3 - chen lap tin hieu
Pho ' H va "C-NMR eiia hgp c h i t 4 cho tin hieu dae trung ciia mpt oligosaecharid
Tren pho ^H-NMR tin hieu proton trong viing 5„
4,57 -5,66 ppm dac trung cho tin hieu proton anome ciia vong duong. &^ 3,21 - 4,12 ppm dac trung cho cae proton methin treng nhom CH-OR. Tin hieu singlet ciia nhom methyl nam a 5^ 2,20 ppm.
Pho "C-NMR cho tin hieu ciia carbon co dp chuyen djeh nam trcng viing tin hieu ciia d u o n g , dang chii y la cac tin hieu S^, 93,6; 97,7; 99,1;
102,9; 105,7 dac trung che cac carbon anomeric, ngeai ra con co tin hieu o 5^211,5 dac trung che nhom carboxyl. Dae biet s u chuyen djch v i truong t h i p cua nhom methyl a 5^ 30,8 che ta gia t h i i t v i met lien k i t cua nhom nay vol nhom carbonyl dang acetyl CH3-C=0
Phan tich p h i DEPT cho t h i y treng 4 carbon anome eo ba tin hieu carbon bac 4 6' 5(, 99,1; 102,9;
105,7, hai tin hieu carbon methin 5^, 97,7, 93,6, ngoai ra con eo 14 tin hieu methin khac n i m treng vung 5^, 69,1-83,7. Sau carbon methylen nam treng viing 5j, 60,5-65,5; met nhom methyl 6" S^, 30,8.
Tir_ nhu'ng phan tieh 6- tren va tham khao tai lieu v i thanh p h i n hoa hpe mpt s i leal trong chi Codonopsis, c i u triie ciia eligesacharid '^i c i u true hgp c h i t 4 b u a c d i u d u g c xac djnh la met fructoeligesaceharid voi S fructofuranose, 1 glucepyranose va 1 rhamnopyranose bi acetyl hoa a C6. P h i H'-H' COSY che thay' proton anome eiia glucopyranese (5„ 5,20) t u a n g tae vai proton H-2-glu (5^^ 3,47). Proton aneme eiia rhamnopyranose (5„ 4,58 ) t u a n g tae vol H-2- rha (5„ 3,20 ppm ). Tin hieu carbon o 5^, 82,7 (a-Glcp-C-4 ) chuyen dieh ve truang y i u 9 ppm chimg to rang no lien k i t vol d u o n g rhamnese a v i t r i C-1.
Ttr cac du' kien p h i va tham khae cac tai lieu, so sanh tinh tpan vol p h i n m i m chemdraw (xem bang 1) chung toi d u a ra k i t luan ban dau v i c i u true ciia c h i t 4 la p-D-fructofuranose (2->1) (3-D-fnjetofuranese ( 2 ^ 1 ) p-D-fnjctofuranose (2—>1) a-D-glucepyranose (4<—1) a-L-(6-aeetyl- rhamnopyranese)
Ket luan
Bang eae phuang phap sac ky k i t hgp vol cac p h u a n g phap p h i , da phan lap va nhan dang c i u triic b i n hgp c h i t ttr cao c h i i t con eua r i dang sam. Treng do hai hgp c h i t adenosin (3), ;S-D- fructpfuranese (2—>1) p-D-fructcfuranose (2->1) p-D-fructefuranose (2—•I) a-D-glueopyranose ( 4 ^ 1 ) a-L-(6-acetyl-rhamnopyranose) (4) l i n d i u tien d u g c phan lap ttr cay nay.
Tai lieu tham khao
1. V o V a n Chi (2012), TCr diin Cay thuoc Viet Nam, NXB Y hoc, tr. 872-873.
2. B 5 Huy Bich va CS (2004), Cay thudc va ddng vat lam thuoc a Viet Nam, tap 1, tr. 739-743.
3. Hoang IVIinh C h u n g , Pham Xuan Sinh, Nguyen Manh Tuyen (2002), " B u o c dau nghien ciru thanh phan hoa hpc ciia vj t h u i c dang s a m Viet N a m " Tap chi Duoe //eu, 7 ( 1 ) , tr. 3-6.
4. Hoang Minh C h u n g , Pham Xuan Sinh (2002),
" B u o c dau nghien c i r u thanh phan saponin cua dang s a m Viet Nam", Tap chi Duac lieu, 7 (6), 163-165.
5. T r i n T h a n h Ha, B o Thi Ha, N g u y i n Minh Khai, N g u y i n Thj Ha, N g u y i n V a n Nghi (2014), "Thanh phan hoa hoc cua r i dang sam", Tap chi DLFCIC lieu, 19 (4), 211-216.
6. Venkata Sai Prakash Chaturvedula, Rue Huang (2013), "Isolation and N M R spectral studies of dihydromyricetin". Journal of Pharmacognosy and
Nghien
CIFU- Ky thuat
Phytochemistry, 2 (4), 113-115
7. Federica Maltese, Cornells Erkelens, Frank van der Kooy Young Hae Choi, Robert Verpoort (2009),
"Identification of natural epimeric flavanone glycosides by NMR spectroscopy", Foocf Chemistry, 116, 575-579.
8. Suk Whan Jung, Ae Jin Han, Hae Jin Hong, Myoung Gun Choung, Kwan Su Kim and Si Hyung Park (2006), "a-Glucosidase inhibitors from the roots
{Ngay nhan bai: 04/12/2015 •
of Codonopsis lanceolate Trautv.", Agric. Chem.
Biotechnol, 49(4), 162-164.
9. Cerantola S., Kervarec N., Pichon R., Magne C , Bessieres M. A, Deslandes E. (2004), "NMR characterisation of inulin-type fructooligosaccharides as the major water-soluble carbohydrates from Matricaria maritima (L.)", Carbohydr Res., 339(14), 2445-2449.
Ngay duyet dang: 04/04/2016)
Nghien cmi bao che... (TUP theo trang w)
v i phuang phap bao e h i , n h i i u nghien cu'u tRroc day t i i n hanh n h i i u phuang phap khae nhau de bao c h i phirc hgp, da s6 cac phuong phap nay la cae phuang phap bao c h i liposome'"'.
Hai phuong phap hay d u g e sir dung n h i t la hydrat hoa mang film va phuang phap thay doi dung m6i.
P h u a n g phap thay del dung moi kha don gian, d i thue hien, va khOng doi hoi nhieu t h i i t bj phirc tap.
Tuy nhien, v i n d i lua chpn dung moi d i hoa tan d u g c c h i t anh h u o n g r i t Ion den phirc hgp lipid tao thanh. AmB co dp tan cao n h i t trong dung moi DMSO ( 3 0 ^ 0 mg/ml), do vay chi e i n khoang 5 ml DMSO de hoa tan d u g c c h i t trong quy trinh bao e h i . Tuy nhien, dung m6i nay co nhuge diem khOng bay hoi trong qua trinh b i e hoi dung moi, de vay mpt p h i n d u g c c h i t v i n 6' trang thai hoa tan trong san pham h i n djeh e u i i cting. Hon nO'a, sir dung DMSO kh6ng bao e h i d u g c hen djch chira phirc hgp lipid dong n h i t do t u o n g tac cua no vol cac thanh p h i n khac trong c6ng thirc, hinh thanh kee von trong qua trinh p h i i hgp hai pha. Nhom dung moi alcol d u g e lua chpn vi li do re t i i n , d l k i i m , it dpc hai ban dung m6i co chira dor. Tuy nhien do dp tan cua d u g c c h i t trcng eae dung mci nay r i t t h i p , de do phai eai thien dp tan eiia d u g e c h i t trong cac dung moi nay. Amphotericin B la d u g c c h i t luong ti'nh, trong do eo nhom amin nen tan t i t hon trong moi truang acid. Do vay khi acid hoa alcol x u i n g pH 1,0, dp tan eua d u g c c h i t tang, giam lugng dung m5i can diing treng bae e h i , giam t h i i u thai gian b i c hoi dung moi va it anh huo'ng den m6i sinh.
Nhiet dp khi p h i i hgp hai pha d u g c thuc hien o" nhiet dp chuyen pha eiia phpsphelipid, kheang 60°C. Methanol eo nhiet dp SPI chi khpang 64°C, dp vay t i c dp bay hoi dung mai qua nhanh anh huo'ng d i n c i u tmc ciia phirc hgp lipid. Gia trj Span eiia m i u sit dung methanol truac va sau dong n h i t hpa cap hon dang k i so vol mau sir dung ethanol, chirng to khoang phan b i kich t h u a c ciia
{Ngay nhan bai: 24/02/2016
phirc hgp truac va sau dong nhat hoa rpng h o n so vol m i u sir dung ethanol.
ok chirng minh s u tao thanh phirc hgp lipid, trong nghien ciru da sir dung phuang phap chup hien vi dien tir b i mat d5ng lanh. Vol quy trinh da d u g c khao sat, phirc hgp lipid hinh thanh co hinh thai la dang sgi. Ngoai ra, d i chirng minh s u tao thanh phu'c hgp lipid, co the diing eae phuang phap danh gia nhiet dp chuyen pha eua phospholipid n h u phan tieh nhiet vi sai, n h i i u xa tia X hay phuang phap xac djnh lien k i t ( n i u co) giira d u g c c h i t va c h i t mang phospholipid bang each sir dung pho h i n g ngoai, hay p h i cpng h u a n g tir.
Nhom nghien ciru se thue hien cac phuang phap danh gia nay vae cac nghien ciru t i i p thee.
Kit luan
Da bao c h i d u g c phirc hgp lipid amphotericin B sir dung ta d u g c HSPC va DSPG vol ti le mel 7:3, b i n g phuang phap thay doi dung moi, sir dung ethanol da d u g e acid hoa d i n pH 1,0 lam dung m6i hoa tan d u g c c h i L boc hoi dung m6i bang phuang phap e i t quay 6' ap s u i t giam. D e lam nho kich t h u o c t i i u phan sir dung p h u a n g phap d i n g n h i t hoa ap s u i t cae o' 5000 psi.
Tai lieu tham khao
1. Adler-Moore J. P. and Proffitt R. T (2008),
"Amphotericin B lipid preparations: what are the differences?", Clin. Microbiol Infect, 14 SuppI 4, 25-36.
2. Fannakiotis D., Tverdek F P. va Kontoyiannis D.
P. (2013), "The safety of amphotericin B lipid complex in patients with prior severe intolerance to liposomal amphotericin B", Clin. Infect Dis., 56(5), 701-703.
3. Janoff A. S., Perkins W. R., Saletan L. et al (1993),
"Amphotericin B lipid complex (ABLC): A molecular rationale for the attenuation of amphotericin B related toxicities", J. liposome Res., 3(3), 451471A S Janoff, W R Perkins, 3(3), 4 5 1 ^ 7 1 .
4. Janoff A. S., Madden T. D., Cullis P. R., Keams J.
J D.A.G. (2002), "Method of preparing low toxicity drug- lipid complxes", US patent, 32 p.
Ngay duyet dang: 04/04/2016)
TAP CHI DirgfC HOC - 4/2016 (SO 480 NAM 56) 39