I Nghien ciru - Ky thuat
i Danh gia mot so hoat tinh sinh hoc
o • • •
cua re cu cay ba bet lun (Mallotus nanus Airy-Shaw)
Phan Thi Hoa'', Nguyen Thj Minh Hang^
To Hal Tung^ Ngu>en Duy Thuan'
'Hoc vien Y Duac hoc c 6 n-uyeii Vi^l Nam -Vien Hoa sinh bien- Vien Hdn ldm Khoa hoc \a Cong nghe Vi^tSam
* E-mail. [email protected]
Summary
The extracts from the roots of Mallotus nanus, including the ethanol (MNT), n-hexane (MNH) and ethyl acetate extracts (MNE), were evaluated for antioxidant activity, cytotoxicity and antimicrobial activity, in term of the percentage of free radical DPPH scavenging potential, the antioxidant activity of MNE was the highest (83.59%), fowllowed by that of MNT at the concentration of 500 pg/mL (50.9 %), while the MNH showed no DPPH antioxidant activity. All of these extracts showed very strong cytotoxicity on KB cell lines with tC^ values of 0.79 pg/mL; 0.13 pg/mL and 0.30 pg/mL for for MNT, NMH, MNE.
respectively. The extracts all hardly exhibited activity against the tested microorganisms.
Keywords: Mallotus nanus, cytotoxicity, antioxidant activity, antimicrobial activity.
Dat van de
Chi Ba bet (Mallotus) g i m khoang 150 lodi, phdn b l tai cac khu vye tir An Op, Sri Lanka den Thdi Lan, Lao, Campuchia, Viet Nam va khap viing Malesian. Cdc lodi Mallotus la cae cay thuoc quy, chua n h i l u hoat tinh dang quan tam.
Mallotus dupe sir dung nhu nhung eay thude trong y hpc d truyin Viet Nam va ede n u d c Odng Nam A d l dieu In eae benh khdc nhau nhu nhiem tmng, roi logn lieu hda, benh gan, bdnh ngodi da, s i t , s i t ret va mdi loat cae bieu hien khde. Tuy nhien, tren t h l gidi eung nhu d Viet Nam, cdc loai thupe chi Mallotus edn chua dupe nghien euu nhieu ve thdnh phan hda hpc cung nhu hogt tinh sinh hpc.
Ba bet liin cd ten ktioa hoc la Mallotus nanus Airy-Shaw Id cay bui hay gd nhd, eao 30-70 em.
Ld hinh trai xoan rpng, hinh khien hay hinh tning d u ; dau cd mui nhpn hoac thudn nhpn; 7 gdn chinh dgng chdn vjt, khdng Idng. Cym boa cai dai 10-12 em, hoa cd 4-5 la dai; bdu ed gai ngc va Idng hinh sao. Qua nang, nhd, vd qua ed myn trdn vd rai rac ed ldng. Cdy ua sang, mpe trong rung thudng xanh, tren dat sa philn thach, d dp cao 100-500 mi'-^.
Trong ddn gian theo t i l n g dia phuang, loai eay nay d u p e gpi la cay dang cay. Ci vung Hda Binh, phan r i cu edy d u p c s d dung de Idm sang da cho cac m l , chj, em gai Irongnhirng ngay l l hpi. Cho den nay, mdi chT cd p h l n trdn mat dat d a loai cay nay d u p e nghien cdu v l thdnh phan hda hpc va hoat tinh ehong oxi hda. Phan r l aJ eay vdn edn chua d u p c nghien cuu ve thdnh phan hda hpc cung nhu hogt llnh sinh hpe
Vdi mye tidu nghien c u u cae hoat tinh sinh hpc nhdm lam sang td cae lac dung trong ddn gian cua cay ba bet lun (M. nanus), chiing tdi da tien hanh danh gid hoat tinh c h l n g oxi hda, hoat tinh gdy dde t l bdo va hoat tinh khang vi sinh vdl kiem djnh cua ede cao diiet tir phdn re cu d a I d i cdy nay
Nguyen lieu va phu^yng phap nghien ciju
Nguyen lieu thiPc vat
Cu eay ba bet lun dupe thu hdi tgi viing rtmg niii huyen Easup tinh Oakldk vao thdng 4 ndm 2014 vd d u p c PGS. TS. T r i n Van On - Bp mdn thye vdl - Trudng Oai hpc D u p c Ha Ndi tham djnh ten khoa hpc.
TAP C H I D U p c H p c - 7/2015 (SO 471 N.AM 55)
Nghien CIFU - Ky thuat
Dieu che cdc cao chiet tip r l cua cay ba bet liin (M. nanus Airy-Shaw)
Mdu cii eay ba bet lun {M. nanus Airy-Shaw) sau khi Ihu hdi d u p e rua sach, thai mdng, phai khd trong bdng ram rdi say d nhiet dp 55-60 "C d i n khd, sau dd xay thanh bdt. Mdu r l cdy dang bpt (4 kg) d u p c c h i l t bang ethanol 80% theo phuang phap c h i l l Scxhlet d i n h i t hoat c h i t . Djch e h i l t ethanol d u p e eat loai bdt dung moi xuong edn khoang 1/15 the tich ban dau. L i y 20 mL djeh ethanol, cd dae cat logi h i t dung mdi thu d u p c eao c h i l l ethanol (MNT). Phan djch ethanol cd dgc cdn lai d u p c pha loang vdi nude cat rdi ehiet phdn bo lan lupt vdi ede dung mdi n-hexan va ethyl acetat. Mdi dung mdi d u p c ehilt 3 lan, m i l l l n 500 mL. Gom djch eua 03 ldn c h i l l Igi vdi nhau, lam khd bdng Na^SO^ khan r l i e l l loai dung mdi d l thu d u p c eae egn c h i l l l u a n g ung Id n-hexan (MNH, 8,40 g), egn c h i l l ethyl acetat (MNE, 25,0 g) vd p h l n djch ethanol- n u d c cdn lai (1 L).
Phu'O'ng phdp ddnh gid hogt t i n h c h 6 n g oxi hda
Hogt tinh chong oxi hda cua cae cao chiet d u p c danh gid bang phuang phdp xac djnh hogt tinh quet g l c l y do DPPH (1,1- diphenyl 1-2 pierylhydrazyl).
M i u t h u d u p c pha trong DMSO. DPPH pha loang trong MeOH vdi nong dd thich hpp. 10 pL mau thCr dupe li vdi 190 pL dung djch DPPH, li d nhidt dd 37°C trong 20 phut vd do tren may ELISA d bude sdng 517 nm. Chat doi chdng acid ascorbic d u p c diing d l k i l m soal dp on djnh va danh gia hoat llnh Crc che tuang d u a n g . Cac phdp thu' d u p e lgp lai 3 lan.
Ket qua d u p c llnh theo cdng thdc sau:
% (FC ehe = [ (100 - OD^) / ( O D J x 100 - OD^: Mat dd quang trung binh cua mau thu- - OD^: Mat dd quang trung binh cua mau doi
chimg dm (khong cd mdu thd", chi cd DPPH, coi nhw gid tn Crc chi 0%) Nong dp u c c h l 50% (ICg^) d u p e xay dyng trdn 5 n i n g dd Ihir nghipm. Gid tn IC^^ d u p c xac djnh Iheo phuang phap h l i quy t u y i n tinh tren phan mem Graphpad Prism 5.0.
P h i r c n g phdp nghien CLPU hoat t i n h gay d o c t l bao trdn d d n g te bdo K B
Dong td bdo ung thw KB: do Trudng Ogi hpe Hawaii vd GS. Jeanette Maier, Trudng Ogi hpe Milan, Italia cung d p .
Phuvng phap nuoi cay te bao in vitro Cdc ddng t l bdo ung t h u d u p e nudi eay d u d i dgng dan ldp trong mdi trudng nudi c l y DMEM vdi thanh phan kem theo g i m 2 mM L-glutamin, 10 mM HEPES, va 1,0 mM natri pyruval, ngodi ra bo sung 10% h u y l t thanh thai nhi bd (fetal bovine serum - FBS, GIBCO).
T l bao d u p c cdy ehuyen sau 3-5 ngay vdi ti lp (1 ;3) va nudi trong tu am CO^ d d i l u kidn 37''C, 5% CO^.
Phep thO> sinh hgc xac djnh dgc tt'nh te bdo (cytotoxic assay) ^''^
Phuang phap thu dp dde te bao in vitro d u p c Vi#n Ung thu Quoc gia Hoa Ky (National Cancer Institute - NCI) xac nhgn Id phep thu dp ddc t l bao ehuan nham sang lpc, phat hidn cdc chat cd kha ndng kim ham s y phat trien hoae dipt t l bdo ung thu (TBUT) d d i l u kien in vitro. Phep t h u ndy d u p c thye hien theo phuang phap ciia Monks (1991) '^l Phep t h d tien hanh xac dinh ham lupng protein te bao long so dya vao mat dp quang hpc (OD - Optical Density) do d u p c khi thanh phan protein ciia te bao d u p c nhupm bdng Sulforhodamine B (SRB). Gia trj OD may do d u p e ti Id thuan vdi lupng SRB gan vdi phan t u protein, do dd lupng te bdo edng nhieu (lupng protein cang nhilu) thi gid tn OD cang ldn. Phep t h u d u p c thyc hien trong dieu kien cy the nhu sau:
Chdt thCr (10 pL) pha trong DMSO 10% d u p e dua vao cac g i l n g eua khay 96 g i l n g d l cd ndng dp sang Ipc la 20 |ig/mL. C h i t thu cd hogt tinh d u p e xde djnh IC^j, nhd ddi ndng dd 20 |ig/mL;
4 jig/mL; 0,8 ^g/mL; 0,16 ^glmL.
Trypsin hda l l bao thi nghiem d l lam rdi t l bao va d i m trong buong d i m de d i l u ehinh mat dp cho phil hpp vdl thi nghiem.
Them vdo cdc g i l n g thi nghiem lupng te bao phil hpp (trong 190 nL mdi Irudng) vd d l ehiing phat trien trong vdng I d 3-5 ngdy.
Mpt khay 96 gilng khdc khdng ed chat thu nhung ed TBUT (180 ^L) se dupc sir dung lam doi
JJ^? CHI DU"pC H p c - 7/2015 (SO 471 NAM 55)
Nghien ciPu - Ky thuat
ehCmg ngdy 0. Sau 1 gid, dfa ddi ehung ngay 0 se dupc d djnh te bao bdng aeid tridoracetic - TCA.
Sau giai dogn phat trien trong tu am CO^, t l bao d u p c CO dinh vao day g i l n g bang TCA Irong 30 phut, d u a c nhupm b l n g SRB trong 1 gid d 37 °C. Do bd SRB va eae g i l n g thi nghiem d u p c rua 3 lan bdng 5% aeid acetic rdi de khd trong khdng khi d nhiet dp phdng.
Cuoi cung, s u dyng 10 mM unbuffered Tris base d l hda tan lupng SRB da bam va nhupm ede phan l u protein, dua len may lac dta lde nhe trong 10 phut va s u dyng may ELfSA Plate Reader (Bio-Rad) d l dpc k i t qua v l ham lupng mau cua chat nhupm SRB qua pho hdp phu d bude sdng 515 nm. Kha nang song sdt eOa le bao khi cd mat chat thir se d u p c xdc ^ n h thdng qua edng thue sau:
OD(chit thu) - ODfngav 0)]i:10C
% song sot = ^r^-. ^ — — . , • '
0D\3di chung am) - OD(ngay 0)
% ue e h l = 100% - % song sdt.
Cde phep thu d u p e lap iai 3 lan d l dam bao tinh ehinh xde. Elliplicin (Sigma) ludn dupe sir dung nhu la chat ddi chung duang va d u p c thu nghipm d ede ndng dp 10 \xglrrL-, 2 jig/mL;
0,4 jig/mL; 0,08 pg/mL.
D M S 0 1 0 % ludn d u p c s u dyng nhu ddi ehirng dm. Gia bi \C^ (nong dp ue e h l 50% s y phat trien) se dupe xac dinh nhd vdo p h l n mem may tinh TableCurve. Chat Ihu nao cd I C ^ < 20 pg/mL (vdi ehat ehilt thd, hoac vdi phan dogn hda hpc) hogc ICjd £ 4 )ig/mL (vdi hogt c h l l tinh khill) se d u a c xem Id ed hogt tinh gay dpc t l bao va od kha nang Crc e h l s y phat trien hoae diet TBUT.
Hoat tinh khang vi sinh vat kiem djnh Hogt tinh khdng vi sinh vat kiem djnh d u p c t i l n hdnh de danh gid hogt tinh khang sinh cua cde mdu thu.
Cde ehung vi sinh vat kiem djnh bao gdm vi khuan Gr (-): Escherichia coli (ATCC 25922) va Pseudomonas aeruginosa (ATCC 25923), vi khuan Gr (+): Bacillus subtilis (ATCC 11774) va Staphylococcus aureus subsp. Aureus (ATCC 11632), n l m spi: Aspergillus niger (439) va Fusarium oxysporum (M42) vd n l m men: Candida albicans (ATCC 7754) va Saccharomyces cerevisiae (SH 20).
Hoat tinh khang vi sinh vat kiem dinh dupc l i l n hanh d l danh gia hoat tlnh khang sinh cua cae m i u chilt, dupc thyc hien tren philn vi lupng 96 g l ^ theo phuang phap hien dgi eiia Vander Berghw va Vlietiinck (1991). va McKane L. &
Kandel (1996). Chdng duang la Streptomycin cho vi khuan Gr (+), tetracyclin cho vi khuan Gr(-) va nystatin hoac amphotericin B cho ndm spi va nlm men. Khang sinh pha trong DMSO 100% vdi ndng dp thteh hpp. Chung am la vi sinh vat kiem djnh khong trpn khang sinh va c h i t thir. Mdi tn/dng duy tri va bao t i n gidng Id Saboraud Dextrose Broth (SDB-Sigma) cho nam men va nam mdc, va Trypease Soya Brolh (TSB-Sigma) cho vi khuan Mdi tnidng thi nghiem la Eugon Broth (Difco. My) eho vi khuan va Mycophil (Difco, My) cho nlm.
Tiin hdnh thi nghiem: Cac chung kilm dinh d u p c hoat hoa va pha loang tdi nong do 0,5 dan vi Mc Fland rdi tien hanh thi nghiem. Cac philn thi nghidm trong tii am 37^C/24 gid cho vi khuan vd 30'^C/48 gid d l i vdi n l m sai va nam men.
Tinh kit qua: Cac mdu du'Oc pha loang theo eac thang nong dp thap dan, de tinh ndng dp ire ehe t i l friilu (MIC) la ndng dp ma d do vi sinh vat bi ire e h l g i n nhu hoan toan. Mdu thd co IVIIC < 200 pg/ml, m i u tinh cd MIC £ 50 pg/mL la cd hoat tinh.
Ket qua va thao luan Hoat tinh c h l n g oxi hda
Cac cao chiSt t6ng ethanol (MNT), n-hexan (MNH) va ethyl acetat (MNE) da duac danh gia hogt tinti chdng oxi hoa theo phuang phap quet goc t y do DPPH. K i t qua (bang 1) cho t h i y chi cd cao MNT va MNE the hien hoat tinh chong oxi hda d muc dp y l u thdng qua tac dyng quet gdc t y do vdi gia tr; phan tram uc c h l l l n lupt la 50,59% va 83.59% a ndng do 500 pg/mL,
Bdng 1 : Hoat tinh quet gdc tu- do DPPH STT
2
3
4 Ten mau
MNT
MNH
MNE
Acid ascorbic
Wong do thip 100 500 100 500 100 500 50
% t i t che 11,69 50,53
29,83 83,59
*(,35
TAPCHi DL OC HOC - 7/2015 (80 471 NAM 55)
• Nghien CLFU - Ky thuat
Tir k i t qua tren ed the nhgn I h l y rang eac chat ed hoat tinh ehdng oxi hda d r l cu cay ba bdt lun (M. nanus Airy-Shaw) Id eae ehat cd dp phdn c y c trung binh va lgp Irung chu yeu trong cao c h i l t ethyl acetat (MNE).
Hoat tinh gay ddc te bdo
Cde eao c h i l t ethanol (MNT), n-hexan (MNH) vd ethyl acetat (MNE) da d u p e ddnh gia hoat tinh gdy dde l l bdo doi vdi ddng l l bao ung t h u bieu md mieng KB. Cd 3 eao c h i l l d i u the hien hoat tinh u e c h l s y phat trien eua te bdo ung t h u KB r i t manh vdi gid trj iC^^ l l n lupt id 0,79 pg/mL d l i vdi MNT, 0,13 pg/mL ddi vdi MNH va 0,30 pg/mL doi vdi MNE; trong khi gia tri \C^ cua ellipticin la 0,57 pg/ml. N h u vgy, cao c h i l t n-hexan (MNH) vd ethyl acetat (MNE) cd tde dyng dc c h l s y phat trien cua te bdo ung t h u KB manh hon ed c h i t doi Chung duang ellipticin. K i t qua nay cho thay r l eii cay ba bet liin {M. nanus Airy-Shaw) rat cd tiem nang d l phat t r i l n thdnh che pham ho t r p d i l u tri ung t h u bieu md, tuy nhien e i n tiep tuc ddnh gid khd ndng gay ddc t l bao cua cac eao c h i l l nay doi vdi cac ddng t l bdo lanh linh va nghien cuu sdu han d l i vdi ddng le bao ung t h u KB cung nhu cdc ddng t l bdo ung thu khdc.
Bang 2: Ket qua danh gid hoat tinh gdy ddc te bdo doi vai dong te bao ung thw KB Nong do
(fig/mt) 100
20 4 0,S 0 , « 0,032
/C„
MNT 100,11 100,02 91,55 51,06 2,63 -0,52 0,79
•/.(Tcctii MNH 100,15 100,05 99,70 92,28 55,04 14,99 0,13
MNE 101,23 100,54 98,22 82,66 23,37 -1,31 0,30
Ellipticin 99,77 76,43 40,58 12,78
0,57
Bdng 3: Kit qua danh gid hoat
Hoat tinh khang vi sinh v a t kiem dinh Cae cao ehilt tong ethanol (MNT), n-hexan (MNH) va can chiet ethyl acetat (NME) d i u khdng ed tdc dung uc c h l cdc chimg vi khuan Gr(+), vi khuan Gr(-) va nam men thu nghiem. Tuy nhien, cde cao c h i l l da t h l hidn hoat tinh tren 2 ehiing nam mdc thu nghiem. Ca ba cao ehilt deu uc che s y phat t r i l n ciia ehiing n l m moc F. oxysporum a mue dp yeu vdi gia tri MIC la 200 pg/mL eho ea 3 logi eao c h i l t Odi vdi chung ndm moc A. niger thi chi cd MNH va MNE the hipn hoat linh y l u vdi gid trj MIC deu la 200 pg/mL
tinh khang vi sinh vat kiim djnh
su '^"f"
1. MN 2. MNH 3. MNE
N6ng d^ mau (pg/mt)
200 200 200
VtliltuanGr(-) E. coll P. aeniginosa
(-) (-)
H (-)
(-) (-)
Ndng dgucclTetdi thiiu (MIC pg/mt)
Vt tthuin Gr(*) Nim moc Nam men B sutAitis S. aureus A niger F. oxysporum S. cerevisiae C. albicans
(-) (-) (-) 200 (-) (-) (-) (-) 200 200 (-) (-) 0 H 200 200 (-) (-)
Ket luan
Cde eao c h i l l tong ethanol (MNT), n-hexan (MNH) va ethyl acetat (MNE) cua r§ cii cay ba bet liin {M. nanus Airy-Shaw/) da dupc ddnh gia ve cdc hogt tinh chong oxi hda, gay ddc l l bao vd khang vi sinh vat k i l m djnh. Oang chu y han cd la kha ndng gdy dpc t l bdo eua ede cao chilt. Ca 3 cao ehilt deu the hipn hoat linh uc c h l s y phat triln eua t l bao ung t h u KB rat mgnh vdi gid tii iCg^ la 0,79 pg/fTiL d l i vdi MNT 0,13 pg/mL doi vdi MNH va 0,30 pg/mLdoi vdi MNE, trong khi gid tri IC^jjCua ellipticin Id 0,57 pg/mL. Cao chilt MNT va MNE cd tac dyng chong oxi hda qudt goc t y do y l u vdi ty le
% uc e h l d ndng dp 500 pg/mL lan lupt la 50,59%
va 83,59%. Cdc cao chilt khdng hoac the hien hoat tinh rdt y l u trong cac t h u nghiem ddnh gid hoat tinh khdng vi sinh vgt kiem djnh. Cac k i t qua tren cho thdy cdc hoat chat trong re cii cdy ba bet liin (M. nanus Airy-Shaw) chii y l u la eac chat cd dp phan cyc tmng binh tap tnjng trong cao chilt ethyl acetat (MNE).
L d i cdm a n : Cac tac gia chan Ihdnh cam an Ban chli nhiem d l tai d u p c phe duyet theo q u y l t djnh so 251 BYT/2014.
Tai lieu tham khao
1 N g u y e n N g h i a T h i n ( 2 0 0 7 ) , " T a x o n o m y o f E u p h o b i a c e a e in V i e t n a m " , Vietnam National University Publishers, p. 1 7 9 - 2 0 2 .
