BAI N G H I E N CLTU.
Danh gia kha nang ure che cua Pristimerin tach chiet tir r i cay Choc mau nam (Salada cochinchinensis Lour.)
tren cac dong te bao ung thu
EVALUATE THE PRISTIMERIN'S INHIBITORYABILITY FROM THE ROOT OF SALACIA COCHINCHINENSIS LOURON CANCERCELL LINES
Pham Thi Thu Hien\ Nguyen Duy Thuln^ Phuong Thi&n Thuong' ' B e n h vien Y H C T B o Cong an
^Hoc vien Y D u g c hoc co truyen Viet Nam
^Vien Duoc lieu - Bo Y te
TOMTAT
Cdc binh ung thu dang dUdc quan tdm nghien cHu do tf li mac binh tdng nhanh (33% tronggiai doqn tii nam 2005 din ndm 2015). Nhiiu hdp chdt thiin nhien co nguon gdc tii thtic vgt dUdc nghien ciiu desii dung thay thi cdc phUdngphdp hda trt, xg tried nhiiu tdc dung phu. Trong n^iin cHu ndy, chung tdi ddnh ^d khd ndngHc chitibdo ung thucua hdp chdt thien nhiin Pristimerin til ri cdy Chdc mdu nam (Salacia cochinchinensis Lour).Pristimerin CO tdc dgngiicchitdt trin 8 ddngtibdoungthUvdiICSOdcdcddngtibdoRD,HepG2,Hela, 0VCAR8, NCI-N87,AS49, PANC-1,MCF7 ldn ludtld: 0,73; 1,63; 1,67; 1,71; 2,15; 2,30; 2,77; 3,81 ^g/ml.
IC^g cua Pritimerin tUdng tti vdt chdt diiu tri ung thuDoxorubicin vdip = 0,89. Kit qua ndy cho thdy Pristimerin cd triin vgng trd thdnh chdt diiu tri ung thii.
TU khoa: Choc mdu nam, Pristimerin, chdng ung thu.
SUMMARY
The cancer is being researched due to a rapidly increasing incidence of disease (33% in the period from 2005 to 2015). Many natural plant-derived compounds have been studied to replace chemotherapy, radiation therapies have many side effects. In this study, we evaluated the ability ofthe natural compound Pristimerin extractedfrom
Ngdynhanbar 15/1/2019 Ngayphdn biin: 18/1/2019 Ngay chap nhan dang 18/1/2019
1221 TAP CHIY DUOC C 6 TRUYfiN VIET NAM ^ 3
the roots of Salacia cochinchinensis Lour to inhibit cancer cell lines. Pristimerin has a good inhibitory effect on 8 cancer cell lines with ICSO in RD, HepG2, Hela, 0VCAR8, NCI-N87, A549, PANC-1, MCF7 cell lines are 0.73; 1.63; 1.67; 1.71; 2.15; 2.30; 2.77; 3.81 ^g/ml, respectively Pritimerin IC^g is similar to Doxorubicin, a cancer treatment drug, with p = 0.89. This result shows that Pnstimerin has the potential to become a cancer treatment drug.
Keyword: Salacia cochinchinensis L., Pristimerin, anticancer
DAT VANDE
Ung thfl la mdt trong eic binh hiem ngheo vdi ty le tfl vong chiem hang thfl 2, chi sau benh tim maeh. Hiln nay, vile dieu tn benh ung thfl dang la mdt thach thfle ldn cho he thdng y tl vi xa hdi. Hiu het eie thude dieu tri ung thfl sfl dung trong lam sing thfldng ddc va gay ra tic dung khdng mong mudn eho cd the. Vi vay, xu hfldng hien nay li tim kilm, sing Ipc eac htfp chat thiln nhiin cd hoat tinh khang ung thfl de flng dung trong dieu tri. Qua eic cdng trinh nghiin cflu gan day cho thay, khi sfl dung eic loai thudc cd ngudn gde thfle vit it ed tic dung phu giy hai vi diy ehinh li Iy do quan trpng mi ngiy nay cac loai thudc cd nguon gde tfl thiln nhien ngay cing ehiem dfltfe Idng tin cua ngfldi tieu dung.
Theo cac nghiin cflu gin diy, mdt sd loai trong ehi Salacia L. dfltfe xem Ii ngudn cd gii tn vi giiu cac htfp chat cd hoat tinh sinh hpc. Nhilu loai trong sd niy da dfltfe chflng minh cd hogt tinh ehdng ung thfl nhfl Salacia oblonga [2], Salacia chinensis [3], [8], [9], [10]. Cac boat chat tach chiet dflpc tfl cac loii nay cd tie dung chdng ung thfl li Pristimerin, Mangiferin, Salacmol,... [d], [9]. Cdy Chdc mdu nam (Salaaa cochinchinensis L.) li loii dfltfe tim thay va d^e hiiu d Vi|t Nam, cd phin bd d khu vflc mien Trung vi Tiy Nguyin. Ciy Chdc mau nam mdi dfltfe phat hiln li eiy thudc quy cd kha nang hd trtf dieu tri ung thfl vi dii thio dfldng vi di dfltfe dung trong y hoe din gian de chfla cae binh nay. Tuy nhien, cho den nay cd rit it tii Heu nghiin
cflu ve thanh phin hda hpc vi tic dung chong ung difl eua eiy thudc niy Nham gdp phln ehflng mmh mdt cich khoa hpe cac tic dung ciia vi thudc tfl eiy Chdc miu nam, chung tdi tien hanh thflc nghiin cflu tach chiet vi dinh gii hoat tinh chdng ung thfl cua Pristmierm tfl eiy Chdc miu nam.
OOi TUDNG VA PHUDNG PHAP NGHIEN CUU
Ddi tfldng nghien cflu Nguyen vgt lieu
Re ciia eiy Chdc mau nam (Salaaa cochinchinensis L.) dfldc thu tai Vfldn Qudc gia Bach Mi, thdi gian tflthang6/2018.
Dong ti bdo ung thU
Cac ddng te bio ung thfl dfltfe eung eap bdi Ngin hing tl bio thupe Viln Smh hpe vi Cdng nghe Sinh hpe Hin Qudc (Korea Research Institute of Bioseience and Biotechnology), gdm cd: ddng te bio ung thfl gan HepG2, ddng t l bao ung thfl c6 tfl cung Hela, ddng tl bio ung thfl da diy NCI-N87, ddng tl bao ung thfl vii MCF7, ddng te bao ung thfl tuy PANC-1, ddng tl bio ung thfl budng trflng OVCAR-8, ddng tl bio ung thfl ctf tim RD va ddng te bao ung thfl phdi A549.
Phfltfng phap nghien cflu
Phdn ldp Pristimerin tU ri Chdc mdu nam Re khd aia cay Chdc miu nam (3,0 kg) dfldc cat nhd, ngam bang Edianol d nhilt do phdng 3 Ian (6 Ht X 3 Ian), mdi lin 4 ngay Cae dich chilt Edianol dfldc gdp 1^, cit thu hdi dung moi dfldi ap suit
TAP CHIYDUgC C 6 TRUYEN VIET NAM 1123
BAI NGHIEN COU •
giim d 60''C tren miy eat quay ehan khdng thu diidc can toan phan (45 l,56g) vdi dp amli 8,263%.
Phin tin can toan phan trong nflde. Sau dd, lac vi ehilt hdn dich tren vdi lan Ifldt eic dung mdi h€fu ctf (n-Hexan, Ethyl acetate vi n-Butanol) thinh cic phin doan cd chfla chit cd dp phan cflc khie nhau thu dfltfe cic phan doan n-Hexan (20,45g), phan doan Ethyl acetate (43,41g), phin doan n-Butanol (I21,73g), vi phan d o ^ H^O (l92,23g) vdi dp am lan Ifldt li: 1,611; 9,78; 9,603; 10,108%.
Tiln hanh sang loc kha nang flc ehe t l bio ung thfl cua eic phin doan dich chiet dl Ifla chpn phan doan cd kha nang flc che cao nhat.
Phin lip hdp ehat tfl phin doan da chpn bang sae ki cdt vdi cac chit hap phu sihea gel pha thfldng, pha dio (YMC, RP 18). Sac ky Ic^ mdng dfltfe dung de theo ddi vet eic ehit tfl dich chiet phin doan va kilm tra dp tinh klult cua cac chit phan lap.
Chat phin lip dflde d dang tinh khiet dflde xae dinh can cfl vao cac dfl lieu phd: phd hdng ngoai (IR), phd khdi (MS), phd edng hfldng tfl hat nhan ('H-NMR, '^C-NMR) va so sanh cac dfl lieu thu diidc tfl thflc nghiim vdi eie dfl lieu di cdng bd dl xae dinh.
Phtidngphdp thU tdc dung gdy doc tebdo ung thU Thfl dpc tinh te bio in vitro dfltfe thfle hien tai Khoa Dfltfe ly - Smh hda, Vien Dfldc hlu theo phfltfng phip MTT vi gia tri IC^^ dfldc tinh bang phin tich hdi quy khdng tuyIn tinh cua dfldng eong dip flng hlu tfltfng flng.
Cac ddng te bio dfldc Iflu gifl trong nittf Idng, danh thflc vi duy tri trong mdi trfldng DMEM (Dulbeceo's Modified Eagle Medium), hoic RPMI 1640 ed bd sung huylt thanh bl 10%, dung dich khing sinh va khang nim 1% (penicillin 50,000 units/L vi streptomycin 50 mg/L). Tl bio d\idc nudi cay eho phat triin tdi mfle khoang 70% diln tich dia thi thay mdi trfldng mdi va cay chuyin vao
dia 96 vdi mit dp 8.000 te bio/gieng, de t l bao bim dinh dn dinh qua dim rdi u vdi mau nghien cflu. Dilu kien nudi cay d nhi|t dp 37°C, khdng khi ehfla 5% CO^, 95% dp am trong 24 gid [7].
Tilp theo, miu nghiin cflu vi miu ddi chuiig dfldng (Doxorubicin) dfldc hda loang vdi mdi trfldng nudi eay thinh cac dai ndng dp (6,25 -100 [ig/ml) va bd sung vio eac gieng di ed ehfla tl bio de xae dinh ICjp
Tl bio vdi miu nghiin cflu trong 48 gid. Sau dd bd sung 20 [d MTT ndng dp 5 mg/ml vio mdi gieng vi u 3 gid trong dilu kiln nudi ciy. Tai cic t l bao sdng MTT cd mau vang bi bien ddi thanh tinh the formazan vi dflde hda tan trong dung mdi DMSO tao dung dich miu tim. Do mat dp quang d bfldc sdng 540 nm vdi bfldc sdng tham khao 630 nm xie dinh ty le t l bio sdng sdt.
Gia tri IC^p dfltfe tinh bing phan tich hdi quy khdng tuyen tinh cua dfldng cong dip flng lieu tfltfng flng [ l l ] , sfl dung phin mIm Graphpad Prism 5.0.
PhUdngphdp xU ly> s6 liiu
Cac sd lieu thflc nghiem dfltfe xfl Iy bang phin mem Graphpad Prism. Mdi mdt thi nghiim se dfltfe lap lai it nhat 3 lin. Cae kit qui thi nghiim dfldc bilu diln dfldi dang M ± SD (trong dd M: gii tri trung binh tflng Id, SD: dp lech chuan). Danh gia vi so sanh gifla cic 16 thfl vi Id ehflng bang phfldng phap thdng kl t - test. Sfl khie bilt cd y nghia thdng kekhip<0,05.
KET QUA
Sang Ipc phan doan c6 kha nang flc chi tl bio ung thfl tU dich chilt re cay Choc mau nam
Cac phin doan tfl djch chiet rl eiy Chdc miu nam (n-Hexan, Ethyl acetate, n-Butanol vi H^O) dfltfe thfl ddc tinh tren 8 ddng t l bio ung thfl d ndng dp lOO^ig/ml.
1241 TAP CHfY DUOC CO TRUYEN VIET NAM ^ C K !
^ i. I^ legdy chit tibdo ung thu cua cdc phdn dogn dich chUt til ri cdy Chdc mdu nam.
Dong te bao
HepG2
A549
MCF7
PANC4
NCI-N87
RD
Hela
OVCARS
Dich c h i a Ethanol
68,87 + 6,12
28,99 + 2,62
41,93 + 2,36
55,47+1,86
54,30 ±3,97
80,92 ±0,47
32,74 ± 9,73
64,80+ 3,13
Phan doan n-Hexan
94,36 + 0,47
90,61+0,32
93,28 + 0,19
88,73+2,82
93,08 ±0,14
95,20 + 0,15
92,43 + 0,62
91,35 ±1,23
P h ^ doan Ethyl acetate
34,68 + 2,62
7,21 +1,26
0
34,98+1,59
8,60 ±1,92
40,42 ±0,94
0
42,65 ± 1,11
Phan doan n-Butanol
7,49+1,06
9,35 + 0,83
0
0
6,05 ±0,63
22,54+1,37
5,98+1,06
1962 + 2,03
Phan doan HjO
8,59 ±1,02
973 ±2,55
0
0
4,12 ±1,56
18,75+1,55
3,27 ±1,46
4,69 + 0,40
Ket qui Bing 1 cho thiy phan doan n-Hexan cd kha nang gay chet te bio ung thfl cao nhat d ei 8 ddng te bio ung thfl (88,73 - 95,20%). Gia tri IC50 cua phan doan Hexan trin 8 ddng te bio ung thfl lin Ifldt tfl 4,86; 5,19; 7,53; 8,10; 9,00; 13,27;
17,65; 17,91 t*g/ml. Do do, phan doan n-Hexan dfltfe Ifla ehpn de phin lap htfp ehit cd kha nang chdng ung thfl.
Phan l|p va xac dinh clu tnic h ^ chit cd kha nang chdng ung thfl tfl phan doan Hexan
Htfp chit dfltfe phan lap tfl phin doan n-Hexan bang sac ki cdt vdi eac chat hap phu siliea gel pha thfldng, pha dao (YMC, RPiS). Cae phin doan thu dflde (18 phan doan) tiep tue dfltfe sing Ipc kha nang fle che eac ddng t l bao ung thfl. Phan doEin thfl 6 cd kha nang giy chet te bio ung thfl eao nhit vdi 1 htfp ehit dfldc phan lap (dfl lieu khdng dflde trinh biy).
Hdp chit phin lap dfldc do bd phd va xie dinh
cdng thflc cau tao. Ket qua xie dinh cho thay htfp ehit phin lip dfltfe la Pristimerin (Hinh l ) .
Hinh 1. Cdng thUc cau tgo ciia hdp chdt Pristimerin Kha nang flc che cac dong tl bao ung thfl cua Pristimerin
Khi ning fle ehi cae ddng te bao ung thfl cua Pristimerin dflde dinh gia vi so sinh vdi chat ddi ehflng dfltfng Doxorubicin, mdt chat dilu tri ung thfl da dfltfe difltfng mai hda, trin 8 dong te bio ung thfl (Bing 2).
TAP CHI Y DUOC C 6 TRUYEN Vl£T NAM SSSE 1125
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Bang 2. IC^i, cua Pristimerin vd Doxorubicin trin 8 ddng tibdo ungthu.
Mau thui
Pristimerin Doxorubicin
Tie dung lie che (lC,j, ^ig/ml) HepGl
1,63 2,42
A549 2,30 1,26
MCF7 3,81 1,74
PANC-l 2,77 4,13
NCI-N87 2,15 4,54
RD 0,73 0,41
Hela 1,67 0,43
OVCARS 1,71 2,57
Tac dung flc che cua Pristimerin trin 8 ddng te bio ung thfl tuin theo quy dinh ndng dp, tflc la khi ndng do tang thi tic dung flc che ting vi d ndng dp eao nhat thi tie dung fle ehe li tdt nhit (dfl Heu khdng dfltfe trinh bay). Gia tri IC;,, cua hdp chat Pristimerin trin 8 ddng te bio RD, HepG2, Hela, OVCARS, NCLN87, A549, PANC-lvi MCF7 tfltfng tif chit ddi ehiing dfldng Doxorubicin vdip = 0,89.
BANLUAN
Cay Chdc mau nam (Salacia cochinchinensis Lour) li loii die hiiu chi phin bd tai Viet Nam.
Hien nay, mdi ehi cd nghien cflu vl thanh phin hda hpc vi tic dung ha dfldng huyet eua cay Chdc miu nam [ 1]. Tuy nhien, cic nghiin cflu ve eae loii trong chi Salada da ehi ra rang eie hdp chat sinh hpe tach chil't tfl cae loii niy cd tac dung chdng ung thfl m,[8],[io].
Kit qui nghien cflu cho thiy, miu cao chilt phin doan bang dung mdi n-Hexan cd khi ning flc chi trin 80% vdi ndng dd sfl dung 100 ttg/ml tren ci 8 ddng t l bio ung thfl va gii tn ICj^trln 8 ddng tfl 4,86 -17,91 Hg/ml. Gii tri IC^j, tim dfldc cd thi thiy ring, phan doan niy cd kha ning giy ddc t l bio ung thfl vdi hoat tinh rit manh. Chinh vi vay, chiing tdi Ifla ehpn phin doan nay dl phin lip hdp chat cd khi ning flc chi t l bio ung thfl.
Kit qui phin l^p thu dflde hdp chit Pristimenn, tfldng tfl vdi eie nghiin cflu tren cac loii khae thudc ehi Salacia eflng thu dflde Pristimerin khi sfl dung
phfltfng phap chiet tfldng tfl [4], [5], [12].
Hdp ehit Pristimerin trflde diy da dfldc nghiin eflu tie dung gay ddc t l bio ung thfl trin mpt s6 ddng te bao va cd hi|u qua cao. Theo nghiin cflu cua Wu C. C. vi edng sfl nam 2005, Pristimerin thi hiln khi ning gay ddc t l bao ung thfl d eie ddng t l bio A549, MCF-7, MDA-MB-231, HepG2 vi Hep3B, vdi eie gii tri IC^,, nam trong khoing tfl 0,42 din 0,61 ^ig/ml [12]. Nghiin cflu cua Huanjie Yangvi edng sfl nim 2008 da chi ra rang: Pristimerin cd kha nang fle ehi boat ddng eua te bio ung thfl tien Hit tuyIn 20S cua thd va t l bao ung thfl tiln hit tuyIn d ngfldi 26S proteasome vdi IC^g lin Ifldt la 2,2 vi 3,0 ^iM/l [5]. Nim 2018, nghien cflu cua Hee Won Seo vi edng sfl da chi ra dfltfe tic dyng flc chi t l bio ung thfl rudt kit d ngfldi HCT116 vi SW480 va Iam sang td rang nd cd thi dfldc coi li mpt tie nhan tiem ning trong dilu tri ung thfl rudt kit [4].
Khi nghien cflu tren 8 ddng tl bio ung thfl thiie hiln trong thi nghiem nay, chiing tdi nhan thiy hpp chat Pristimerin the hiln ddc tinh manh (>80%) trin ca 8 ddng t l bio ung thfl vdi ndng dd sfl dyng 100 ng/ml va cd gii tri IC50 la 0,73 - 3,81 jig/ml.
Dieu niy cung hoin toan phu hdp vdi cae nghiin cflu trflde diy eua cic tie gii khac.
Nhfl viy, kit qui nghiin eflu trin mpt lin nfla Idling dinh khi nang flc che nhilu ddng t l bio ung thfl cua Pristimerin. Dieu nay cho thay Cay chdc miu nam cd tilm nang sfl dung trong dieu tri ung thfl vi cin dfldc nghiin cflu them.
1261 TAP CHI Y DUOC CO T R U Y E N V I £ T NAM 1333
4.
KET LUAN t h a p 0,73 - 3,81 ng/ml. Gia tri ICjo nay tfldng tfl K i t q u a nghien cflu c h o thay Pristimerin t i c h Doxorubicin, m p t ehat dflde sfl d u n g Iam t h u d c chiet tfl r l ciy Chdc mau n a m (Salacia cochinchinensis dieu trt n h i l u b e n h u n g thfl. D o dd, can cd t h e m Lour), m d t l o i i d^c hiiu eua \ ^ l t N a m ed t i e d u n g e i e nghien eflu de ed the sfl d u n g eiy C h d c mau fle c h i nhieu d d n g te bao u n g thfl vdi gia tri IC^g n a m trong d i l u tri u n g thfl.
TAI LIEU T H A M KHAO
1. D d I h i N g u y ? t Q u i ( 2 0 1 3 ) . "Nghiin cvlu tdc dung hg Glucose huyit cua ricdy Chdc mdu nam (Salacia cochinchinensis Lour., Celastraceae) trin thtic nghiim", Luan an t i l n si dfldc hpc, V i l n dfldc lilu, t r . 3 8 , 1 2 1 . 2. AnjaneyuluMusini, Jayaram Frakash Rao et al. ( 2 0 1 5 ) . "Phytochemicals of Salacia oblonga responsiblefor free radical scavenging and antiproliferative activity against breast cancer cell lines (MDA-MB-231)", Physiol
Moi Biol Plants, 21 ( 4 ) , pg.583-590.
3. U.A. D e o k a t e , S.S. Khadabadi et al. ( 2 0 1 2 ) . "Phytopharmacological aspects of Salacia chinensis".
Pharmacophore, Vol. 3 ( 3 ) , pg.156-163.
4. H e e W o n Seo et al. ( 2 0 1 8 ) . "Pristimerin, a Naturally Occurring Triterpenoid, Exerts Potent Anticancer Effect in Colon Cancer Cells", B i o m e d Sci Letters 2 4 ( l ) , p g . l 5 - 2 2 .
5. Huanjie Yang et al. ( 2 0 0 8 ) . "Pristimerin Induces Apoptosts by Targeting the Proteasome in Prostate Cancer Cells",} Cell B i o c h e m 1 0 3 ( l ) , pg.234 - 244.
6. Kaliappan, K a m m a l l a A K , R a m a s a m y M K e t al ( 2 0 1 4 ) . "LC-MS quantification of mangiferin inhydroalcoholic extract of Salacia oblonga, Salacia roxburghii and polyherbalformulation", I n t J Phytopharmacy Res4(l),pg.ll-15.
7. Larramendy M . L. e t al ( 2 0 1 8 ) . "Genotoxicity", I n t e c h O p e n , pg.1-17.
8. Jin S u n L e e e t al. ( 2 0 1 3 ) . "Anticancer Activity of Pristimerin in Epidermal Growth Factor Receptor 2'Positive SKBR3 Human Breast Cancer Cells", Biological a n d Pharmaeeutieal Bulletm, p g . 3 1 6 - 3 2 5 . 9. Ramakrishna. D., Shashank. A.Tidke. e t al. ( 2 0 1 6 ) . "A Source of Herbal Drug for Several Human Diseases and Disorders", International Journal of C u r r e n t Pharmaeeutieal Review and Research, 7 ( 3 ) , pg.122-133.
10. Tran Thi Minh, N g u y e n Thi H o a n g A n h e t a l . ( 2 0 1 0 ) . "Study on Chemical Constituents and Cytotoxic Activities of Salacia chinensis Growing in Vietnam Z", Naturforseh. 65b, pg.1284-1288.
11. W u L., S m y t h e A M et al. ( 1 9 9 2 ) . "Multidrug-resistant phenotype of disease-oriented paneh of human tumor cell lines used for anticancer drug screening". Cancer Res, 52, pg.3029-3033.
12. W u C. C. et al, ( 2 0 0 5 ) . "Pristimerin induces caspase-dependent apoptosis in .MDA-MB-23i cells via direct effects on mitochondria.", Moi cancer ther. 4 ( 8 ) , p g . l 2 7 7 - 1 2 8 5 .
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