trang 53-57. 3. Vign Dm»c li§u (2006), Nghien diu phdt trien dupc liiu vd ddng dupc Viet Nam: Nghien aiu xdc
^ink ten khoa hgc, ddu vdn toy hda hpc,vd tdc dtfng sinh hgc cua mdt sd lodi ndm da nien thupc chi Ganoderma vi chi Phellinus. NXB Khoa hgc v i Ky Thuit, H i N0i, 331-343. 4. Abraham P, Sugumar E. (2008), Increased
^utathione levels and activity of PONi (phenyl acetate esterase) in the liver of rats after a single dose qf cyclophosphamide: a defense mechanisml Exp. Toxicol. Pathol., 59(5), 301-306. S. Chang ZQ, Oh BC, Lee SP, Rhee MH, Park SC (2008), Comparative immunomodulating activities of polysaccharides isolated from Phellinus spp. on cell-mediated immunity. Phytother Res., 22(10): 1396-1399. 6. Oltawa H., Oshisi N., Yagi K.
(1979), Assay for lipid peroxides in animal tissues by thiobarbituris acid reaction. Anal. Biochem., 95:351-358.
7., Park BJ, Lim YS, Lee HJ, Eum WS, Park J, Han KH, Choi SY, Lee KS (2009) Anti-oxidative effects of Phellinus linteus and red ginseng extracts on oxidative stress-induced DNA damage, BMB Rep., 42(8):500-505.
8. Tietze F. (1969), Enzymic method for quantitative determination of nanogram amounts of total and acidized glutathione: application to mammalian blood and other tissues. Anal. Biochem., 27, 502-522. 9. Zhu T, Kim SH, ChenCY (2008),^ medicinal mushroom: Phellinus linteus, Curr. Med. Chem., 15(13):1330-1335.
Tgp chiDirffc U$u, tap 18, so 1/2013 (Trang 31 - 38)
D O C T I N H D O I VCtt MOT S 6 TE BAO UNG THlT CUA CAC FLAVONOID PHAN LAP TlT N y VOI
Ddo Trong Tuin, Nguyin Thi Bich Thu, Nguyin Minh Khdi, Phuang Thien Thuang Khoa Hoa phdn tich - Tiiu chudn, Vien Duac lieu
(Nh^n bai n g i y 26 t h i n g 11 nam 2012) Summary
Cytotoxic Activities of Flavonoids Isolated from the Buds of Cleistocalyx operculatus Against some Cancer cell Lines
Fourteen flavonoids 1-14 were isolated from the buds of the plant Cleistocalyx operculatus by repeated column chromatography. Their structures were identified on the basis of physicochemical and spectroscopic analyses. Eleven compounds were tested for their cytotoxic activities against some human cancer cell lines. The most interesting finding is that compound 6 (5-hydroxy-7-methoxy-6,8-dimethylflavanone) exhibited significantly inhibitory effects against Hela cells with an ICjo value of 1.3 tig/ml.
Keywords: Cleistocalyx operculatus, Jlavonoid, cytotoxicity, cancer cell, 5-hyti-ox}>-7-meth(»^6,8-dimethy^avanone.
1. B^t v^n de ciiu ve tac dung sinh hoc cho bilt la, ny v6i C^y v6i e6 ten khoa hgc la Cleistocalyx va eac hoat chat eo tac dung khang khuan operculatus (Roxb.) Merr. et Perry, thupc hg [1], khang virus [4], ch6ng oxy hoa [6], [8], Sim (Myrtaceae) rSX quen thupc voi cac tinh ch6ng benh Alzheimer [7]. Hoat chit chinh ding bang Bic BO [1-3]. Tii lau nhan dan ta ciia ny voi la 2',4'-dihydroxy-6'-methoxy - da diing la vh ny vii hSm liy nuoc ulng, vira 3',5' - dimethylchalcone da dugc chiing minh c6 t6e dyng thanh nhi$t vira co tac dyng kien eo tac dyng chlng ung thu tren in vitro vk in t^, tieu thyrc, diing ngoJli chua myn nhot, gh6 vivo [9-11].
16 [1-3]. Den nay d3 co nhieu nghien ciiu ve Trong mpt nghien ciiu cua chiing toi cho cfiy vii eho biet thanh phin hoa hpc ehinh biet flavonoid la thanh phin hoa hpc chinh ctia la va ny vdl la tinh diu, triterpenoid [3], trong ny voi va chiing co tac dyng chlng vi chu ylu la cac flavonoid [4-8]. NghiSn virus [4]. Dl tim xem c6 them flavonoid nio
Tap chi Duac liiu, tap 18, s^ 1/2013 31
khac c6 tac dvmg chong ung thu ngoU ho?it chat chinh da i\it^ nghien ctiu, chiing toi thvc tac dyng ch6ng ung thu ciia cac flavonoid ti6n mpt s6 dong tS bao ung thu. Bai bao nay trinh bay kk qui thii tac dvmg dpc tinh cua 11 flavonoid phSn l?p tir nu v6i thu mua tai Vi$t Nam din su phat trien cua mpt so dong ts bao ung thu tten m6 hinh in vitro.
2. D^i tirong v^ phnong phap nghien cuu 2.1. Nguyen liiu, hoa chdt. dung mdi 2.1.1. Nguyen lipu:
Nu v6i dupc thu mua t^i cha D6ng Xuan, Ha N6i vao thdng 3 nam 2008 da duoc cii nhan Ng6 Van Trai xac dinh co ten khoa hoc la Cleistocalyx operculatus (Roxb.) Merr.
and Perry, thuoc hg Sim (Myrtaceae). Mau nghien ciiu hien dang duac luu giu" tai Khoa Hoa Phdn tich - Tieu chudn, ViSn Dupc iieu.
2.1.2. Hoa chit, dung m6i:
Dung m6i diing trong chiet xuat, phan lap boat chat la methanol (MeOH), n-hexan, aceton, ethyl acetat (EtOAc) deu dat tieu chuan cong nghipp vd dupc chung cdt lai truoc khi dung. Dung m6i phan tich g6m MeOH, «-hexan, aceton, EtOAc, H2O, acid formic... diing cho phdn tich sdc ky dSu dat tieu chudn phan tich. Bdn sdc ky 16p mong trdng sdn (silica gel GF254, RP-18 GF254) dupc mua cua hang Merck (Diic).
2.1.3. Te bdo vd nuoi cay tS bdo;
B6n dong tt bao ung thu dupc svt dung Id MCF-7 (dong tl bao ung thu vil), MCF- 7/ADR (dong tS bdo ung thu vii dd khdng Adriamycin), A549 (dong tS bdo ung thu phai), vd Hela (dong tS bao ung thu c6 til cung). Cac dong tS bao dupc nhdn tit Ngan hdng tJ bao cua Vien Cong nghe Sinh hpc Hdn Qu6c (Korea Research Institute of Bioseienee and Biotechnology).
Te bdo dupc nufli cdy bdng m6i truimg DMEM hoac RPMI (tuy theo loai tS bdo) trong tu nuoi cdy or nhiet dp 37°C vd 5%
CO2. Te bdo dupc nuoi cay 5n djnh tai khi du lupng te bdo cho thi nghiem thi dupc cdy chuySn.
2.2. Thm bi thi nghi$m
BSp chiSt hdi luu Memmert co sinh haa.
ruflt ga. Phdn tieh vd phdn Idp boat chit bdng, sic ky lop mong (silica gel, RP-18), sic 1^
cpt (silica gel, RP-18, sephadex-20), vd h^
th6ng sic ky long hipu ndng cao HPLC (Gilson). Ndng suit quay cue dupc do bdng mdy Rudolph Autopol IV. Pho tur ngo?i (UV) dupc do tren mdy Jasco V550 UVWIS.'Phl hflng ngo^i (IR) dupc do trSn may Impact 410-Nicolet. Phfl cflng huong tir hat nhan (NMR) dupc do tren mdy Avance 500- Bruker. Mdy do ELISA LB941 (Berthold Technologies) dupc sir dung de thir dflc tinh cua cac flavonoid doi vdi tS bdo.
2.3. Phdn lap vd xdc dinh cdu trdc cua cdcflavonoid
2.3.1. Phdn lap cac hoat chat:
Nu v6i khfl (2 kg), duac cho vdo binh cdu dung tich 10 lit. Chilt bdng cdeh dun h6i luu cdeh thiiy vdi MeOH trong hai gicr (3 linx7 L). Lpc, gpp cdc dich chiSt vd thu hoi MeOH bdng CO dudi ap suit giam dSn cao d}c (170 g). Cao ddc nay dupc hoa tan trong nu6c (2 L), loai t^p bang «-hexane (3x1,5 L), Idc phdn do?m voi EtOAc (3x1,5 L). Cdt thu h6i phan doan EtOAc dSn khfl dupc cao d|c EtOAc (75 g). Phan doan EtOAc co dflc tinh cao voi tS bdo tmg thu vii, dupc tdch phan doan bdng cflt silica gel (10x30 cm; kich thuoc hat 63-200 |im; Merck) vcri dung mfli chay cpt n-hexane/EtOAc (19:1, 18:2...dto 1:19, moi phdn doan 2,5 L). Sau khi ch?y cflt thu dupc 9 phdn dojn dua vdo kit qui phan tich bdng TLC (Fl: 10,6 g; F2: 3,5 g; F3:2,5 g; F4: 4,2 g; F5: 5,6 g; F6: 3,6 g; F7: 2,5 g;
F8: 5,6 _g; F9: 10,6 g). Phdn doan F3 dupc tinh chl bdng cpt Sephadex LH-20 (7x40 cm) vai dung mfli chay cflt la MeOH, kit qui thu dupc chit sfl 7 (3,8 g>. Phan dopi F4 vd phdn do£in F5 dupc tilp tyc tdch phdn do?n bdng cpt silica gel pha ddo RP18 (7x25 cm;
kich thudc hat 4 0 ^ 3 nm; Merck) vdi dung mfli chay cflt MeOH/H20, trudc khi tinh chS bing HPLC [Optima Pak Cig column (10x250
32 Tap chi Duac lieu, tdp IS, sg 1/2013
mm,cdh^nhfli 10nm,RS Tech, Korea); pha dflng MeOH/HzO vdi 0.1% formic acid; tflc dfl dflng 2 mL/min; detector UV phdt hipn o buflrc song kep 205 vd 254 nm]. Ket qua thu dupc them 13 chdt (1-6,8-14).
2.3.2. Xdc djnh cdu tnic ciia hoat chit phdn lap dupc:
KiSm tra dfl tinh khilt cua chit phdn lap bang sic ky ldp mflng vd HPLC. Xdc dinh ciu triic cdc chit phdn ldp duac thong qua phdn tich tinh chat ly hoa (cdm quan, nang suit quay cue), cdc phfl hflng ngoai (IR), phfl ttr ngo^ii (UV), phi cflng hudng tir h?t nhdn ('H- vd "C-NMR), phi khfli (MS) [4].
^ ^.7J^tlroxy-5^ne{hoxy-6, S-dimethjdisqflavone (l)iirttih thi mdu vdng; UV (MeOH) X^
nm Oog e) 255 (4.32), 298 (3.79); IR (KBr) Vm» 3386 (OH), 2926, 1637 (C=0), 1591, 1447, 1230, 1136 cm"';EIMS m/z (rel. int.):
296 ([M]*, 100), 295 (31), 281 (89), 278 (57), 266 (6), 265 (22), 250 (17), 195 (18), 77 (19); HREIMS m/z 296.1047 [M\* (Calcd for C,8His04, 296.1049); 'H-NMR (CDCI3, 500 MHz) ipp„ 7,88 (IH, s, H-2), 7,51 (2H, i,J= 8,4 Hz, H-2', 6'), 7,34 (3H, m, H-3', 4', 5'), 2,23 (3H, s, 6-CW3), 2,28 (3H, s, 8-C//3), 3,81 (3H, s, 5-OCHi); "C-NMR (CDCI3,125 MHz) ^pp„ 151,0 (C-2), 125,7 (C-3), 175,4 (C-4), 156,3 (C-5), 115,5 (C-6), 156,7 (C-7), 106,9 (C-8), 154,9(0-9), 113,1 (C-10), 132,2 (C-1'), 129,2 (C-2', 6'), 128,4 (C-3', 5'), 127,9 (C-4'), 8,2 (6-CH3), 8,1 (8-CH3), 61,7 (5-OCH3).
5,7-dihydroxy-6,8-
dirnethyldih'droflavonol (2): Tinh thi mdu ndu; [a]tf-24.0° (c 0.08, MeOH); UV (MeOH) \ ^ nm (log e) 297 (4.45), 340 (3.84); IR (KBr) v„„ 3423 (OH), 2927, 1639 (C=0), 1469, 1282, 1123 cm"'; EIMS m/z (rel. mt.): 300 ([Mf, 67), 285 (2), 271 (14), 270 (2), 181 (100), 152 (49), 77 (10);
HREIMS m/z 300.0999 [M]* (calcd for Ci,Hi60s, 300.0998). 'H-NMR (CD3OD, 500 MHz) «pp„ 5,04 (IH, d, J = 11,0 Hz, H-2), 4,51 (IH, d, y = 11,0 Hz, H-3), 7,56 (2H, d, J
= 8,0 Hz, H-2', 6'), 7,41 (3H, m, H-3', 4', 5%
1,97 (3H, s, 6-CH,), 2,02 (3H, s, 8-Cft);
'^C-NMR (CD3OD, 125 MHz) *pp„ 85,0 (C- 2), 74,1 (C-3), 198,9 (C-4), 158,9 (C-5), 104,5 (C-6), 164,7 (C-7), 105,5 (C-8), 160,3 (C-9), 101,8 (C-10), 139,1 (C-1'), 129,0 (C- 2', 6'), 129,6 (C-3', 5'), 129,9 (C-4'), 7,6 (6- CH3), 8,1 (8-CH3).
2,7-dihydro:(y-5-methoxy^,8-dimetfli^lavanone (3): Tinh thi mau ndu; UV (MeOH) >„„ nm (log 8) 294 (4.47), 338 (3.87); IR (KBr) v„„
3391 (OH), 2926, 1681 (C=0), 1621, 1410, 1338, 1114 cm"'; EIMS m/z (rel. int.): 314 ([M]*, 24), 299 (2), 223 (77), 195 (100), 152 (17), 77 (8); HREIMS tn/z 314,1152 (Mf (CisHisOs = 314,1154). 'H-NMR (CDCI3, 500 MHz) <Spp„ 3,16 (IH, i,J= 13,5 Hz, H- 3), 3,24 (IH, d, y = 13,5 Hz, H-3), 7,29 (2H, d, J = 8,0 Hz, H-2', 6'), 7,23-7,25 (3H, m, H- 3', 4', 5'), 2,03 (3H, s, 6-C//3), 2,09 (3H, s, 8- CHi), 3,97 (3H, s, OCi/3); '^C-NMR (CDCI3, 125 MHz) i5pp„ 103,8 (C-2), 42,0 (C-3), 193.7 (C-4), 155,2 (C-5), 109,3 (C-6), 162,3 (C-7), 101,6 (C-8), 167,9 (C-9), 104,8 (C- 10), 132,9 (C-1'), 130,6 (C-2', 6'), 128,3 (C- 3', 5'), 127,4 (C-4'), 7,2 (6-CH3), 7,9 (8- CH3),61,7(OCH3).
4,2',4'- trihydroxy - 6'- methoxy - 3',5'- dimethylchalcone (4): Tinh the mdu vdng;
UV (MeOH) X„ax nm (log e) 298 (3.91), 362 (4.47); IR (KBr) v„,x 3385 (OH), 2931, 1605 (C=0), 1545, 1437, 1229, 1164 cm"'; EIMS m/z (rel. int.): 314 ([M]*, 78), 313 (18), 299 (4), 284 (2), 221 (10), 194 (100), 166 (19), 136 (20); HREIMS m/z 314,1156 [M]*
(CisH.sOs = 314,1154); 'H-NMR (CD3COCD3, 500 MHz) Jppm 7,65 (2H, i,J= 8,5 Hz, H-2, 6), 6,94 (2H, d, J = 8,5 Hz, H-3, 5), 7,92 (IH, i,J= 15,5 Hz, H-a), 7,82 (IH, i,J= 15,5 Hz, H-yff), 2,09 (3H, s, V-CHi), 2,15 (3H, s, S'-Cft), 3,69 (3H, s, &-OCHi), 13,96 (IH, s, 2'-0H); "C-NMR (CD3COCD3, 125 MHz)
^pp,„ 128,1 (C-1), 131,4 (C-2, 6), 116,9 (C-3, 5), 160,7 (C-4), 124,4 (C-a), 144,3 (C-yS), 193.8 (C=0), 109,2 (C-1'), 162,7 (C-2'), 107,9
Tap chi Duac lieu, tdp IS, so 1/2013 33
(C-30, 161,4 (C-4'), 110,6 (C-5'), 159,8 (C- 6'), 8,3 (3'-CH3), 9,0 (5'-CH3), 62,6 (OCH3).
7-hydroxy-5-methoxy-6,S-dimethylflavone (S): Bot mdu vdng; 'H-NMR (CDCI3, 500 MHz) V 6,71 (IH, s, H-3), 7,90 (2H, m, H- 2', 6'), 7,52 (3H, m, H-3', 4', 5% 2,26 (3H, s, 6-CH3), 2,44 (3H, s, 8-CH3), 3,87 (3H, s, o c a ) ; "C-NMR (CDCI3, 125 MHz) ^ppm 160,9 (C-2), 108,2 (C-3), 177,8 (C-4), 155,8 (C-5), 115,3 (C-6), 156,9 (C-7), 107,2 (C-8), 154,9 (C-9), 112,3 (C-10), 131,9 (C-1'), 126,0 (C-2', 6'), 129,0 (C-3', 5'), 131,2 (C- 4'), 8,3 (6-CH3), 8,5 (8-CH3), 61,8 (OCH3).
5-hydroxy-7-methoxy-6,8-dimethylflavanone (6): Bpt mdu tring; ' H - N M R (CD3COCD3, 500 MHz) ^pp„ 5,50 (IH, dd, J = 13,0, 3,0 Hz, H-2), 2,74 (IH, dd, J = 16,0, 3,0 Hz, H- 3), 2,93 (IH, dd, J = 16,0, 13,0 Hz, H-3), 7,58 (2H, d, / = 8,0 Hz, H-2', 6'), 7,44 (2H, m, H-3', 5'), 7,38 (IH, m, H-4'), 2,10 (6H, s, 6-C//3 vd 8-C//3), 3,74 (3H, s, OC//3); '^C- NMR (CD3COCD3, 125 MHz) S^ 19A (C- 2), 46,3 (C-3), 189,1 (C-4), 158,5 (C-5), 109,6 (C-6), 160,8 (C-7), 112,7 (C-8), 160,4 (C-9), 108,2 (C-10), 140,9 (C-l'), 127,0 (C- 2', 6'), 129,5 (C-3', 5'), 129,1 (C-4'), 8,6 (6- CH3), 8,8 (8-CH3), 61,2 (OCH3).
2',4'-<iihydraxy-6'-met/Tcrxy-3',5Ulimeth)dchalcorte (7): Tinh tbl mdu vdng; UV Ka (MeOH):
205, 340 nm; EI-MS m/z: 298 [Mf; ' H - NMR (CD3COCD3,500 MHz) S„,„ 7,73 (2H, d,J= 8,0 Hz, H-2, 6), 7,45 (3H, m, H-3, H-4, H-5), 8,05 (IH, d, J= 16,0 Hz, H-^), 7,82 (IH, d, 7 = 16,0 Hz, H-a), 2,11 (3H, s, 3'- CH,), 2,16 (3H, s, 5'-C//3), 3,68 (3H, s, OCH3), 13,82 (IH, s, 2'-0H); '^C-NMR (CD3COCD3, 125 MHz) ^pp„ 136,4 (C-1), 127,8 (C-2, 6, C-a), 129,9 (C-3, 5), 131,2 (C- 4), 143,4 (C-yS), 193,9 (C=0), 110,7 (C-1'), 162.8 (C-2'), 107,9 (C-3'), 161,8 (C-4'), 159.9 (C-6'), 109,2 (C-5'), 8,3 (3'-CH3), 9,0 (5'-CH3), 62,4 (OCH3).
2'.4'-dit^droxy-3-methyl-6'-methoxychalcone (S): Dang ddu, mau vdng; UV Ka^ (MeOH):
310, 348 nm; EI-MS m/z: 284 [M]* (98), 207
(100), 181 (33), 122 (22), 77 (12); 'H-NMR (CD3OD, 500 MHz) S 8,01 (IH, i,J= 15,5 Hz, P), l.n (IH, d, y = 15,5 Hz, H-a), 7,67 (2H, d, y = 8,5 Hz, H-2, H-6), 7,42 (3H, m, H-3, H-4, H-5), 6,17 (IH, s, H.5'), 3,68 (3H, S, 6'-0CHs), 2,08 (3H, s, S'-Cft); "C-NMR (CD3OD, 125 MHz) S 194,4 (C=0), 165,4 (C-4'), 165,3 (C-2'), 162,8 (C-6'), 143,9 (C- yS), 136,9 (C-1), 131,5 (C-4), 130,2 (C-2, C- 6), 129,5 (C-3, C-5), 128,0 (C-a), 112,3 (C- 1'), 109,7 (C-3'), 99,0 (C-5'), .62,7 (6'- OCH3), 8,6 (3'-CH3).
6-formyl-8-methyl-7-0-methylpinocembrin (9): Bflt mdu cam; UV K„ (MeOH): 258, 345 nm; EI-MS m/z: 312 [M]*, 297, 284, 235, 208, 207, 180, 152, 104,' 77; 'H-NMR (CDCI3, 500 MHz) <5 10,23 (IH, s, 6-CHO), 7,45 (2H, m, H-2', H-6'), 7,42 (3H, m, H-3', H-4', H-5'), 5,54 (IH, ii,J= 12,5, 2,5 Hz, H-2), 3,91 (3H, s, 7-OCH3), 3,07 (IH, dd,/=
17,0, 12,5 Hz, H-3), 2,88 (IH, dd, 7 = 17,0, 2,5 Hz, H-3), 2,09 (3H, s, 8-CH3); '^C-NMR (CDCI3, 125 MHz) 5 192,7 (6-CHO), 187,4 (C=0), 167,9 (C-5), 166,3 (C-7), 165,1 (C- 9), 137,7 (C-1'), 129,1 (C-4'), 128,9 (C-3', C- 5'), 126,0 (C-2', C-6'), 114,0 (C-8), 107,5 (C- 6), 106,6 (C-10), 79,9 (C-2), 61,8 (7-OCH3), 44,9 (C-3), 7,1 (8-CH3).
(2S) - 8 -formyl - 5-hydroxy-7-methoxy-6- methylflavanone (10): Bflt mdu vdng; UV Ka (MeOH): 267, 335 nm; EI-MS m/z: 312 [Mf, 311, 235, 208, 180, 104; 'H-NMR (CDCI3, 500 MHz) S 12.67 (IH, s, 0J/-5), 10.21 (IH, s, CW0.8), 7.46 (2H, m, H-2', H- 6'), 7.40 (3H, m, H-3', H-4', H5'), 5.52 (IH, dd, J = 12.5, 2.5 Hz, H-2), 4.03 (3H, s, 7- OCH3), 3.01 (IH, dd, J = 17.0, 12.5 Hz, H- 3), 2.90 (IH, dd, J= 17.0, 2.5 Hz, H-3), 2.09 (IH, s, 6-Ci/j); "C-NMR (CDCI3,125 MHz) S 193,9 (8-CHO), 188,4 (C=0), 166,3 (C-9), 166,0 (C-7), 165,5 (C-5), 138,1 (C-1'), 128,9 (C-4'), 128,8 (C-3', C-5'), 125,8 (C-2', C-6'), 110,0 (C-8), 109,3 (C-6), 107,6 (C-10), 78,7 (C-2), 64,6 (7-OCH3), 45,2 (C-3), 7,3 (6-CHj).
7 - hydroxy -5-methoxy-8-methylflavanDm
34 Tgp chi Duoc liiu, tap 18, sg 1/2013
(11): Bflt mau tring; ' H - N M R (CD3COCD3, 500 MHz) <S 5,40 (IH, dd,J= 12,5, 2,5 Hz, H-2), 2,79 (IH, dd, J = 16,5, 2,5 Hz, H-3), 3,01 (IH, dd, J ^ 16,5, 12,5 Hz,), 6,29 (IH, s, H-6), 7,44 (4H, m, H-2', H-3', H-5', H-6'), 7,37 (IH, m, H-4'), 2,11 (3H, s, S-CH3), 3,84 (3H. s, 5-OCH3); " C - N M R (CD3COCD3, 125 MHz) S 79,0 (C-2), 45,7 (C-3), 189,2 (C- 4), 160,4 (C-5), 99,5 (C-6), 162,1 (-7), 112,5 (C-8), 160,9 (C-9), 109,3 (C-10), 138;8 (C- 1'), 126,1 (C-2', C-6'), 128.7 (C-3', C-4', C- 5'), 7,6 (8-CH3), 61.3 (OCH3).
S-methylpinocembrin (12): Bpt mdu trdng;
'H-NMR (CD3COCD3, 500 MHz).^ 5,40 (IH, dd, / = 12,5,2,5 Hz, H-2), 2,82 (IH, dd, J = 17,0, 2,5 Hz, H-3), 3,09 (IH, dd, J = 17,0, 12,5 Hz, H-3), 6,00 (IH, s, H-6), 7,46 (2H, m, H-2', H-6'), 7,43 (3H, m, H-3', H-4', H-5'), 2,06 (3H, s, 8-GH3); '^C-NMR (CD3COCD3, 125 MHz) S 79,2 (C-2), 43,5 (C-3), 195,9 (C-4), 161,7 (C-5), 94,8 (C-6), 162,3 (C-7), 104,4 (C-8), 160,6 (C-9), 103,0 (C-10), 138,4 (C-1'), 126,1 (C-2', C-6'), 128,8 (C-3', C-4', C-5'), 6,6 (8-CH3).
5,7~dihydroxy-6,8-dimethylfavanone (13):
D§ng diu mdu vang; UV Ka (MeOH): 297, 344 nm; EI-MS m/z: 284 [M]*, 266, 207, 180, 152; 'H-NMR (CD3COCD3, 500 MHz) 6 12,5 (IH, s, 5-0/f), 7,67 (2H, d, J = 8,0 Hz, H-2', H-6'), 7,54 (3H, m, H-3', H-4', H-5'), 5,62 (IH, dd, J = 12,5, 2,5 Hz, H-2), 3,19 (IH, dd, J = 17,0, 12,5 Hz, H-3), 2,93 (IH, dd, y = 17,0, 2,5 Hz, H-3), 2,15 (3H, s, 8- CH3), 2,13 (3H, s, 6-CH3); '^C-NMR (CD3COCD3, 125 MHz) d 197,4 (C=0), 163,1 (C-7), 160,1 (C-5), 158,6 (C-9), 140,1 (C-1'), 129,6 (C-3', C-5'), 129,3 (C-4'), 127,1 (C-2', C-6'), 104,5 (C-8), 103,5 (C-6), 103,2 (C-10), 79,6 (C-2), 43,7 (C-3), 8,3 (8-CH3), 7,6 (6-CH3).
2,2',4'- trihydroxy - 5 ' - methoxy - 3',5'- dimethylchalcone (14): Bflt mdu vdng cam;
UV K» (MeOH): 300, 366 nm; ' H - N M R {CD3COCD3, 500 MHZ) 5 8.20 (IH, i,J = 16.0 Hz, H-A 8.15 (IH, i.J= 16.0 Hz, H-
a), 7.67 (IH, d, y = 8.0 Hz, H-6). 7.27 (IH, m, H-4), 6.97 (IH, m, H-5), 6.92 (IH, m, H- 3), 3.09 (3H, s, 6'-OCH3), 2.14 (3H, s, H-5'), 2.08 (3H, s, H-3'); '^C-NMR (CD3COCD3, 125 MHz) S 194.2 (C=0), 163.2 (C-2'), 159.8 (C-4'), 158.7 (C-6'), 157.8 (C-2), 139.4 (C-P), 132.4 (C-4), 129.6 (C-6), 127.1 (C-o), 123.2 (C-1), 120.9 (C-5), 117.1 (C-3), 110.5 (C-1'), 109.1 (C-5'), 107.8 (C-3'), 62.6 (6'- OCH3), 8.9 (5'-CH3), 8.2 (3'-CH3).
2.4. Thic ddc tinh cua cdc chdt ddi vdi ti bdo ung thu
Cdc mau chdt dupc thu dflc tinh diu co dfl tinh khilt > 90%. Dpc tinh cua cdc flavonoid dli vdi cdc dflng tl bao ung thu dupc thir theo phuang phdp MTT [12]. Cu thi, tl bdo dupc nufli cay fln dinh trong tii nufli ciy d 37''C vd 5% CO2. Khi du lupng tl bdo cin cho thi nghiem tilp theo thi thu gom cdc tl bdo lai, hoa dSu trong mfli truflfng nufli ciy d mdt dfl 4x10^ tl bdo/ml rfli chuyin vdo ^ a 96 giSng, moi gieng cho 100 ^il moi truimg.
Sau 24 h fln djnh trong tii, thay mfli truimg nufli ciy bdng mfli trudng co chiia cdc miu thuflc thu dupc pha d cac nflng dfl khdc nhau.
Tilp tuc u te bdo trong 48 h, nhuflm mdu tl bao con song vdi mfli truimg nufli cay cfl chiia MTT [3-(4,5-dimethylthiazol-2-YL)- 2,5-diphenyl-tetrazolium bromide] d nflng dfl 2 mg/ml trong 2 h. Sau do lo^ bfl mfli truimg vd hoa tan cdc tinh thS formazan bang edch them vdo 200 jil dimethylsulfoxide PMSO).
Do dp hap thu cua dung dich ndy d 570 nm, dfl hdp thu cua dimg dich ty le thuan vcri sl tl bdo sflng sot [12]. Tdc dung ciia cdc chit dupc bieu hien bdng gia tn IC50, dupc hilu Id nflng dp md chdt do tic che dupc 50% s\r phdt triSn ciia tS bdo ung thu so vdi mau tring (mdu khflng thii thuflc) vd dupc tinh todn theo tdi lieu [13].
3. Kit qua vd ban lu^n
3.1. Xdc dinh cong thtic ctia cdc chdt phdn lap
Cite hoat chit 1-14 dupc phan ldp tir ny v6i dupc xdc djnh Id 7-hydroxy-5-methoxy-
Tgp cht Duac Uiu, tdp IS, sgI/2013 35
6,8-dunethylisoflavone (1); 5,7-dihydroxy-6,8 -dimethyldihydroflavonol (2); 2,7-dihydroxy- 5-methoxy-6,8-dunethylflavanone (3); 4,2',4' -trihydioxy-6'-methoxy-3',5'-dnnethylchalcone (4);7-hydroxy-5-methoxy-6,8-dBnethylflavqne (5); 5-hydroxy-7-melhoxy-6,8-dHnethylflavanone (6); 2',4' - dihydroxy - 6' - methoxy - 3',5' - dunethylchalcone (7); 2',4'-dihydroxy-3'-methyl -6'-methoxychalcone (8); 6-formyl-8-methyl- 7-0-methyIpinocembrin (9); (25)-8-formyl- 5-hydroxy-7-methoxy-6-methylflavanone (10);
7-hydroxy-5-methoxy-8-methylflavanone (11);
8 - methylpinocembrin (12); 5,7-dihydroxy- 6,8 dimethylfavanone (13) vd 2,2',4' trihydroxy-6'-methoxy-3',5'-dimethylchalcone (14). Cflng thirc hda hpc cua cdc chit 1-14
(Hinh 1) dirpc xac dinh dvra tien sir phdn tich c4c tinh chit \f hda vd cac phi UV, IR, %
NMR, " C - N M R , vd MS [4].
Trong sfl 14 flavonoid phan ldp dupc, chit s6 7 Id thdnh phdn chinh, cd 4 chit mdi Id cdc chit 1-4 va cdc chit S, 6, 8, 9, 11, 12 vd 14 dupc phdn l$p Idn diu tiSn tir cdy vii C operculattts. Tdt cd cdc flavonoid dupc phdn l^p diu CO nhdm methyl (CH3) hode formyl (CHO) liSn kit true tilp vdi vdng thom tjii vong A, tgi vj tri sfl 6 hode 8 (3' vd^6' dli vdi chalcon). Cdc flavonoid deu cd nhilu nhflm till (OH, CH3, OCH3) tai vflng A nhung rit it cfl nhflm thi t ^ vong B. Ddy Id cac ddc dilm ddc trung cua flavonoid phdn ldp dupc tir lodi Cleistocalyx operculatus.
IOv|A>S-Osl>J
S J.. L I H.C'VY'
2 OCHaO 3
OH O
'•U.O
Hinh 1. C6ng thflrc h6a hgc cda cdc flavonoid phSn l§p tir nu voi.
3.2. Tdc dtflig cda cdc chdt chiit duac Dflc tinh dfli voi t l bao ung thu ciia 11 trin cdc dong ti bdo ung thu flavonoid 1,2,4-7,9-13 dupc trmh biy trong
36 Tap chi Duac liiu, tap IS, sg 1/2013
Bing 1. Kit qua cho thiy cac flavonoid diu thi hien d^c tinh th6ng qua v i ^ lie ehl sir phit trien ciia cac te bao ung thu vdi cac gii tri ICso nSm trong khoing tir 1,3 - 27,7
^g/ml. Tuy tic dyng ciia cic flavonoid k6m hon so vdi eae ehat dang dupfc diing trong lim sang la camptothecin va adriamycin, chiing deu co tie dyng tit hon hai flavonoid thong thudng la quercetin vi eateehin (c6 cic gii tri ICso >30 p,g/ml, sl lieu kh6ng bao cio). Sd dl CO tic dyng manh hon cae flaVonoid nay vi da sl cac chit thii khong co nh6m thi a vong B (trir chdt sl 4) [14]. Kit qui ding chu y nhit la chat s6 6 co tic dyng lie che mgnh vi chpn lpe tren dong te bao ung thu CO tir cung (Hela), cho gia tri IC50 li 1,3 ng/ml (tuong duong 4,36 \xM < 5 |aM), trong khi cau thic chi khac di mpt nhom thi 6
Bang 1. D$c tinh cua cdc flavonoid phSn
v6ng A (cic chat s6 9-13) lim giam rit nhilu tic dyng lie chl dong tl bio nay. Vi§e co them nhom OH tai vj tri sl 2 (ehit sl 3) vi tM vj tri sl 3 (chit sl 2) kh6ng lam anh hu6n^ den d§c tinh cua cac chit. Nhu vgy co the kSt lugn cac nh6m thi theo thii tu 5- hydroxy-7-methoxy-6,8-dimethyl tai vong A dong vai tro quan trpng trong viec uc chl su phat triin ciia tl bao Hela. Cic nghiSn ciiu truoc diy cho thay flavonoid co nguin glc tu nhien eo tie dyng chlng ung thu tren ci mo hinh in vitro va in vivo [15]. Dilu nay gpi y rang CO thi nghien ciiu phat triin thulc ch6ng ung thu tii eae flavonoid ciia nu vii th6ng qua vipc nghiSn ciiu ea chl tic dyng cua chiing, dong thoi tong hpp ra eic hoat chit CO cau tnic tuong tu nhung cho tic dyng t6t hon.
lap dir^c tren mot so dong t^ bao ung thu Chit
1 2 4 5 6 7 9
lo
11 12 13 Camptothecin
Adriamycin
B$c tinh (ICso, jis/ttti) MCF7
10,9 11,5 11,1 9,4 12,6 8,7 12,9 9,3 12,3 14,9 10,1 1,09 0,S8
MCF7/Adr 12,0 11,7 7,7 7,4 4,3 6,7 9,4 6,8 13,5 10,5 4,9 0,62 4,78
A549 7,6 8,8 5,5 8,4 7,7 5,8 14,3 8,0 9,1 11,1 6,9 0,75 0,45
Hela 27,7 8,0 7,6 6,7 1.3 5,6 21,6 15,41 18,8 20,7 10,1 0,03 0,09
4. Kit lu^n
Trong nghien ciiu nay chiing t5i da phin 14p duoc 14 (1-14) flavonoid tir ny v6i va thir dpc tinh cua 11 flavonoid 1,2, 4-7, 9-13 trSn cic dong tl bio ung thu vii (MCF7 va MCF7/Adr), ung thu phii (A549), vi ung thu cl tu cung (Hela). Tit ci cac flavonoid diu c6 dpc tinh dli vdi cac dong tl bao ung thu
vdi gii tri IC50 nam trong khoang 1,3 den 27,7 fig/ml. Ket qui dang chii y nhit la chat sl 6 cho tie dung lic chS manh su phit triSn cua dong te bao ung thu co hi cung Hela vdi gii tri IC50 bing 1,3 fig/ml. Ket qua cua nghien ciiu niy gop phan dinh huong cho viec nghien ciiu phit trien cie hogt chat chlng ung thu co cau tnic li flavonoid.
Tap chi Duac lieu, tdp 18, se! 1/2013 37
L^i cam an: Cdc tdC gid chdn thdnh cdm an cho cdng trinh nghien cuu ndy thong qua Nhi$m Bd Khoa HQC vd Cong ngh4 da tdi tra k'fh phi vu Nghi djnh thu hgp tdc v&i Nhgt Bdn.
Tdi li^u tham khao
1. Do Tit Lpi (2001), Nhiing cdy thudc va vj thudc Viet Nam. NXB Y hgc, tr. 42?. 2. Va Vin Chi (1997), Tit diin cdy thudc Viet Nam. NXB Y hog, tr. 1330-1331. 3. Vi$n Duoc li^u (2004), Cdy thudc vd dpng vpt ldm thudc a Vi^t Nam. NXB Khoa hpc va KJ thu$t, T|p II, tr. 1065-1066. 4. Dao T.T., Bui T.T., Nguyen P,H„
Thuong P.T., Yoo S.S., Kim E.H., Kim S.K., Oh W.K. (2010), C-Methylated flavonoids from Cleistocalyx operculatus and their inhibitory effects on novel influenza A (HlNi) neuraminidase. J. Nat. Prod. 73, 1636- 1642. 5. Y C.L., Lu Y.H., Wei D.Z. (2004), Flavonoids from Cleistocalyx operculatus. Phytochemistry 65, 44Ji 447. 6. Min B.S., Thu C.V., Dat N.T., Dang N.H., Jang H.S., Hung T.M. (2008), Antioxidative flavonoids from Cleistocalyx operculatus buds. Chem. Pharm. Bull. 56, 1525-1528. 7. Min B.S., Cuong T.D., Lee J.S., Shin B.S., Woo M.H., Hung T.M. (2010), Cholinesterase inhibitors from Cleistocalyx operculatus buds. Arch. Bharm. Res.
33, 1665-1670. 8. TrSn Minh Ngpc, Do^n Cao Son, Pham Tudn Anh, 05 Quyen, Phircmg Thi#n Thuomg (2011), Nghien cihi tdc dung chdng oxy h6a cua nu v6i \Cleistocalyx operculatus (Roxb.) Merr. et Perry, Myrtaceacl.
Tpp dli Dupc hgc 51 (422), 35-38. 9. Ye C.L., Liu J.W., Wei D.Z., Lu Y.H., Qian F. (2004), In vitro anti-tunio?
activity of 2',4'-dihydroxy-6'-methoxy-3',5'-dimethyichalcone against six established human cancer cell lines>- Pharmacol. Res. 50, 505-510. 10. Ye C L . Liu J.W.. Wei D.Z., Lu Y.H., Qian F. (2005). in vivo antihimor activity by 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchaIcone in a solid human CEU'cinoma xenograft model.
Cancer Chemother. Pharmacol 55, 447-452. 11. Ye C.L., Liu J. W., Wei D.Z., Lu Y.H., Qian F. (2005), In vivo antitumor activity by 2',4'-dihydroxy-6'-methoxy-3',5'-dimethyIchalcone in a solid human carcinoma xenograft model. Cancer Chemother. Pharmacol. 56,70-74.12. Mosmann, T. (1983), Rapid colorimetric assay ftjr cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55-63. 13.
Wu L, Smythe A. M., Stinson S. F., Muliendore L. A., Monks A., Scudlero D. A.. Paull K. D., Koutsoukos A.
D., Rubinstein L. V., Boyd M. R., Shoemaker R. H. (1992), Multidrug-resistant phenotype of disease-oriented panels of human tumor cell lines for anticancer drug screening. Cancer Res. 52, 3029-3034.14. Loa J., Chow P., Zhang K. (2009), Studies of structure-activity relationship on plant polyphenol-induced suppression of human liver cancer cells. Cancer Chemother. Pharmacol. 63, 1007-1016. 15. Kandaswami C, Lee L.T., Lee P-P^
Hwang J.J., Ke F.C., Huang Y.T., Lee M.T. (2005), The antitumor activities of flavonoids. In vivo 19,895-909.
Tap chiDir^c iieu, tap 18, s61/2013 (Trang 38 - 43)
KHAO SAT TAC DUNG CUA POLYSACCHARID CHIET TlT NAM LINH CHI DO VA N A M LINH CHI VANG TREN VI THE TE BAO GAN,
LACH VA TUYEN iTC CUA CHUQT B? GAY SUY GIAM MIEN DICH Duang Nguyin Duy Tuyin', Nguyin Thi Thu Huang', Tran Minh ThSng'
Trung Tdm Sdmyd Duac lieu TP. Hd ChiMinh-Vien Duac li?u Khoa Gidi phdu benh - Benh vien Cha Rdy TP. Hd ChiMinh
(Nhan bai ngay 16 thing 01 nam 2013) Summary
Study on the Effects of Polysaccharide from Ganoderma lucidum and Ganoderma colossum on Microscopic Structures of Liver, Spleen and Thymus of Immunosuppressive Mice
Crude polysaccharides (PSP) were extracted from Ganoderma lucidum and Ganoderma colossum.
Cyclophosphamide (150 mg/kg, intraperitoneal injection) was used to induce immunosuppressive model in mice.
Hematoxylin-Eosin staining technique was applied for histological observation. The results revealed that structural damages were identified in liver, spleen and thymus of cyclophosphamide-immunosuppressed mice.
Either PSP from Ganoderma lucidum or PSP from Ganoderma colossum had no hepatoprotective effect on liver damage.fThe PSP from Ganoderma lucidum and Ganoderma colossum had proliferating effects on thymus"
central medulla and spleen pulp in cyclophosphamide-immunosuppressed mice. Our results support the 38 Tap chi Daac Uiu, tdp 18, s& 1/2013