Nghien CLPU - K y t h u a t
Tac dung chong oxy hoa va bao ve gan cua hat vai (Semen Litchi) tren thirc nghiem
Pham Thj AnhS H6 Thanh NgaS Nguyen Manh 'Riyen^'
'TrucmgBai hoc YDuoc Hai Phdng,^Truang Bai hoc Duac Hd Npi
* E-mail:[email protected]
Summary
Anti-oxidant bioactivities of the ethanol extracts of the Litchi seeds were studied by the DPPN free radical scavenging mechanism on the experimental CCI4-induced hepatitis in mice. The extracts inhibited the increase in serum AST and ALT activities, but exhibited no antioxidant effects through the quantitative analysis ofMDA in the mouse liver homogenates.
Keywords: Antioxidant effects, hepatoprotective, semen Litchi, Lichi seed.
Dat van d§
Cay vai {Litchi chinensis Sonn.) la loai eay nhiet dd-i va can nhiet doi d u g c t r i n g rat pho b i i n 6" nhieu n u o c tren t h i gid'i. O Viet Nam, vai la loai eay an qua duge t r i n g d' h i u h i t cae tinh m i i n B i c , cd gia tri kinh t i va dmh duo'ng eao. Mot trong nhung d u pham trong qua trinh e h i b i i n , s u dung vai la hat vai. Tren thye t i , p h i n Ion hat vai duge xem la rac thai mae du hat vai cung la mot vj thude vd-i ten goi le ehi hach trong y hpc co truyen ed edng nang hanh khi tan k i t , k h u han, chi thong, nhung muc do s u dung eon han ehe.
Tren t h i gioi, hat vai dang duge eae nha nghien cuu quan tam v i tac dung ha dudng huyit, ha lipid mau, ehdng ung t h u , cung n h u tac dyng ehdng oxy hda'^'"*'. Tuy nhien, eac nghien cuu v i hat vai Viet Nam cdn t h i y ft diegc cdng bo, dac biet ehua t h i y cong bo nao v i nghi§n euu tac dung ehdng oxy hda va bao ve gan cua hat vai Viet Nam. De ed the khai thac, s u dung hat vai, mdt ngudn nguyen lieu kha ddi dao d Viet Nam, n h i m phat huy hon n&a gia trj eua cay vai Viet Nam trong viee tao ngudn nguyen lieu lam thuic, Chung tdi t i i n hanh nghien cuu tac dung ehdng oxy hoa va bao ve gan cua hat vai.
Nguyen vat lieu va phu-cng phap
nghien CLPUNguy§n lieu nghien cu'u
Hat vai 6\jgc thu hai o huyen Thanh Ha - tinh Hai D u o n g vao thang 5 - 6 nam 2012, M i u cay
CO hoa, d u g c TS. Nguyen Quoc Huy (86 mdn Thuc v§t, Truong Dgi hpc D u g c Ha Noi) giam djnh ten khoa hoc la: Litchi chinensis Sonn., Hp Bd hdn (Sapindaceae). Tieu ban d u g c luu giu' tgi phong tieu ban, Bd mdn Thuc vat, Trudng Dai hoc D u g e Ha Ndi (Ma tieu ban: HNIP/17853/13).
D o n g vat t h i n g h i e m
Chupt n h i t t r i n g chung Swiss, ca 2 giing, khoe manh, trong lugng 25,0 ± 2,0 g do Vien Ve sinh Djeh t i Trung u o n g cung c i p . Ddng vat thi nghigm d u g e nudi trong d i i u kien day du thuc an va n u o c udng tai phdng thi nghiem 5 ngay trudc khi nghien euu va trong s u i t thoi gian nghien cuu tai Khoa D u g c ly - Vlen D u g e lieu.
Hoa chat t h u o c thCp
- Hoa chit, thudc thCr. KCl, acid tricloracetic, acid thiobarbituric, HCI 0,1 N ehuan, acid acetic, n- butanol, CCI^, d i u olive ... dat tieu ehuan thi nghiem.
- Vien nen Silymarin (Safegan) 70mg, nha san xuat Amphareo USA (Sd 16: A3001).
- Kit dinh luung cic enzym va chit chuyen hoi trong mau: Kit djnh lugng ALT, AST va bilirubin (Human).
P h u c n g phap nghien c u u
Hat vai sau khi d u g c lam sach, s i y khd cf nhiet dd khdng qua 70°C, dem xay thd. B6t tho hgt vai (4 kg) d u g c chiet x u i t bang ethanol 50% 5 l i n , moi l i n 15 lit bang phuong phap ngam Ignh d nhiet do phong (25 - 32°C). L i n 1 ngam 30 gio'.
TAP CHI DI/OC HOC - 5/2014 (SO 457 NAM 54)
• Nghien CCFU - Ky thuat
cac lan t i i p theo ngam 24 gid'/lin, rut djch ehiit.
Ggp eac djeh c h i i t , c i t thu hoi dung mdi d u d i ap s u i t giam. Thu d u g c 412 g cao dac hgt vai toan p h i n CO dg am 17,4% (eao HVT1).
Khao sat tic dung chong oxy hoa in vitro bing thu" nghiem don goc DPPH (1,1-diphenyl- 2-picryl-hydrazyl)
Cho 10 pi dung dieh cao dae hat vai chiet bang ethanol 50% (HVT1) da duge pha trong methanol thanh eac dung djch cd nong do t u 12,5-200 pg/ml vao 90 pi DPPH 150 pM (pha trong methanol). H6n hgp phan ung d u g c de trong bong t i l 30 phut o- 30°C. Do mat do quang (OD) bang ELISA cua hang Labsystems tai bude sdng 517nm. Mau chung sCr dung 10 pi methanol Lap Igi 3 l i n , lay gia tri trung binh. Xae dinh IC^, b i n g each xay d y n g d i thj lien quan giua ndng dp mau t h u va % uc ehe DPPH, so sanh vo'i e h i t d i i chieu la quercetin theo phuong phap thud-ng quy'^'.
Khio sat tac dung bio ve gan tren mo hinh gay ton thwong gan cap bing carbon tetra clorid (CCIJ
Chugt nhat t r i n g d u g c chia n g i u nhi^n thanh 5 Id, mdi 16 12 eon:
L5 1 (chung sinh hgc): Uong nud-c c i t Lo 2 (ehung bgnh ly): Uong n u o c c i t Ld 3 (chung duong): U i n g silymarin 70 mg/
kg the trgng (kg TT)
L6 4 (thCr thuoc HV5): U i n g cao hat vai d- l i i u t u o n g d u o n g 5 g d u g c li&u (gDL)/kg TT.
Lo 5 (thup thuoc HV10): Uong eao hat vai d"
lieu t u o n g d u o n g 10 g DL/kg TT.
Chugt 6 cae 16 thu d u g c gay ton thuong gan bang each tiem phue mgc dung dieh CCI^ 7%
pha trong dau olive vo'i lieu 0,1 ml/10 g cliugt.
Bang 1 : Tic dung bao ve gan cua hat vai
c u mdi 2 ngay tiem mdt l i n . Lo chung sinh ly tiem dau olive d i n g the tieh. Cho uong thudc t h u , t h u i c doi chieu hoac nude c i t vd'i the tich tuong d u o n g lien tue trong 8 ngay ^. Ngay thu 8, sau khi uong thudc thu 1 gid, l i y mau tmh mach eo chudt, ly tam l i y huyet thanh dem dinh lugng AST, ALT, bang may dinh lugng smh hda t y ddng.
G i i t chudt, l i y gan de djnh lugng MDA (malonyl dialdehyd) theo phuong phap Wasowich va Balahoroglu ^'-^^
X u ly so lieu
Theo phuong phap t h i n g ke dung trong y sinh hgc bang p h i n m i m Microsoft Office Excel 2007.
K i t qua nghien CLPU Tac dung don goc tu> do DPPH
Hinh 1 : Do thi ket qua hoat tinh don gdc tu- do cua hat vii
Cao chiet ethanol 50% hat vai ed tac dung ehdng oxy hda theo c o c h i dpn g i c t y do DPPH vo'i gia tri IC^^ xac dinh d u g c la 36,99 pg/ml, tuy nhien hoat tfnh nay y i u hon so vdi quercetin (IC^Q = 5,10 pg/ml), tuong t y nhu eae k i t q u i nghien euu da edng b i tren t h i gioi '^'.
Tac dung bao ve gan
TT 1 2 3 4 5
t o thi nghiem L6 1 (chijng sinh hpc) Lo 2 {chOrg benh ly) L6 3 (silymarin 70 mg/kg TT) Lo 4 (hat vSi 5 g DL/kg TT) L6 5 ( h a t v a i 1 0 g D U k 3 T T )
n 10 12 11 12 12
Hoat do AST (Ul/I) 185,31179,6 4064,32 ± 374,32"
2730,66+1483,5";AA 3196,8 ±1297,76",A 3029,30 ±1504,80";A
Hoat do ALT (Ul/I) 44,015 ±18,87 4177,06 ±921,17"
2364,79 ± 1613,55";AA 3217,80 ±1523,73"
2697,075 ±1434,03",A Ghi chu: So vai 16 chCeng sinh ly, **: p < 0,001; So v&i Id chirng benh ly, AA; p < 0,01; A p < 0,05
T^P CHI DlTgfC H p c - 5/2014 (SO 457 NAM 54)
• Nghien CCFU - Ky thuat
AST va ALT deu giam so vo'i 16 chung b^nh ly (15 2) CO y nghTa thong ke (p < 0,05), trCF hogt dd ALT d-16 t h u cao hat vai l i i u 5 g DL/kg TT (16 4). Tac dung giam boat d6 AST va ALT cua hgt vai c6 xu huong phu thuge vao l i i u .
Ham l y v n g MDA trong gan Bang 2: Anh hu^ng cua cao chiit h^t vai lin ham Iwgng MDA gan chudt Ket qua thye nghiem cho thay, boat dp AST
va ALT d 16 2 (mo hinh) va cac Id t h u (16 3, 4 , 5) gay doc b i n g CCI^ tang cao rd ret so vd-i enzym AST va ALT (p<0,01). Thude d i i ehung duong cd tac dung giam hogt dp AST va ALT rd ret so vol Id chLPng b?nh ly (p < 0,01). 0 eae Id t h u , hoat do
TT 1 2 3 4 5
Ld thi nghiem L6 1 (chl>ng sinh hpc) Lo 2 (chLKng benh ly) Lo 3 (silymarin 70 mg/kg TT) Lo 4 (hat vai 5 g DL/kg TT) LP 5 (hat vai 10 g DL/kg TT)
n 10 12 11 12 12
Nong do MBA (nmol/IOOmg gan) 27,04 ± 5,41 52,56 ±12,42 37,98 ± 8,73"
49,45 ± 9,89 47,09 ±10,07 Ham lugng MDA d ea 4 16 so vd-i Id chung
sinh ly tang manh boi CCI^. Ngoai t r u Id chung duong s y giam ham hugng MDA ed y nghTa thong ke so vdi 16 chung benh ly (p < 0,01), cac 16 thCp cao hat vai l i i u 5 g DL/kg TT va 10 g DL/kg TT ehi cd xu hud'ng giam ham lugng MDA so vol 16 chung (p > 0,05).
Ban luan
Ket qua thf nghigm eho t h i y , hat vai cd tac dung chong oxy hda theo c o c h i dpn goc t y do DPPH voi gia tn \C^ xac dinh d u g c la 36,99 pg/
ml. Dieu nay ehung td, hat vai Viet Nam cd hoat tinh c h i n g oxy hda ro ret. K i t qua nghien c u u nay t u o n g t y vdi nghien cuu eua Lu Zhihe va Li Shen (2009) khi nghiSn cuu ve tac dung dgn goc t y do cija hat vai Trung Qudc. Theo do, 6 nong do 1 mg/ml, hgt vai cd kha nang dgn goe t y do DPPH manh hon dung djch rutin cung nong do.
Kha nang nay tang dan theo s y tang len eua nong dp t h u va duy tri on djnh d" ndng do 5 mg/ml. 6 nong dg nay, kha nang dgn gdc t y do DPPH cua hat vai dat 76,42% w.
D i i voi t h u nghiem chong oxy hoa va bao ve gan in vivo tren chugt n h i t t r i n g gay ton thuong gan bang CCl^, hat vai cd tac dung lam giam ro ret hoat dp AST va ALT trong mau v a c d x u hudng phu thupc vao lieu. Vo'i lieu t h u tuong d u o n g 5 g DL/kg TT, h^t vai the hien cd kha nang lam giam hoat dp cua enzym AST (p<0,05) va cd xu hud'ng giam hoat dp ALT, Hoat dp giam di rd ret 6 m u c lieu t h u 10 g DL/kgTT doi vo'i ca 2 enzym AST va ALT ed y nghTa thong ke (p < 0,05). Tuy nhien anh hud'ng cua hat vai len ham lugng MDA trong
to ehuc gan chi the hien xu h u o n g giam so vdi 16 Chung benh ly (p > 0,05).
K i t qua nghien c u u nay c6 s y t u o n g dong vd'i nghien c u u eua Luo Weisheng va epng sy (2013) khi nghien euu tac dyng c h i n g oxy hda va bao ve gan cua flavon toan p h i n c h i i t tach t u hat vai tr§n md hinh chupt c i n g trang gay ton t h u o n g gan b i n g CCI^'^'. Theo do, hoat do AST va ALT trong h u y i t thanh chudt 6" cac 16 thu giam ro ret voi s y khac biet cd y nghTa thong k§
so voi 16 chung benh ly (p < 0,05). Tuy nhi§n, khi danh gia ve kha nang giam ham lugng MDA trong gan cua hat vai, k i t qua nghien cuu cOa Chung tdi lai khong the hien s y khac bigt ro ret so vd'i Id Chung benh ly n h u nghien euu eua Luo Weisheng va epng s y , Phai chang nguyen nh§n eua s y khae biet nay la do lieu luong CCl^ g§y ton t h u o n g gan va l i i u dCing eua m i u thCp chua t u o n g thich? Cd t h i do s u dung l i i u CCI^ qua eao d i tao mo hinh benh ly, nhung lieu m l u t h i edn 6 mue do t h i p nen chua the hien duge tac dung mot each ro ret. Mpt diem khac biet nua giu-a 2 nghien CLPU la dgng vat thi n g h i f m . Trong nghien c u u nay, chung tdi s u dyng chugt nhat t r i n g cdn trong nghien c u u cCia Luo Weisheng, ddng vat thi nghiem d u g c s u dung la chudl cong trang. Phai chang tdn tgi m u c dp dap i>ng khac nhau vd'i m i u t h u eua dgng vat t h i nghiem? Tuy nhien can cd cac nghien c u u tiep theo de kiem ehung eae gia t h i i t nay,
Ket luan
Ket qua nghien euu tac dung chong oxy hoa va bao ve gan cua eao chiet ethanol hat vai cho thay:
T^P CHi D i r a c HOC - 5/2014 (SO 457 NAM 54)
• Nghien CIFU - Ky thuat
- Cao c h i i t ethanol 50% hat vai cd tac dung chong oxy hoa theo c o ehe dgn gdc t y do DPPH, - Tren m6 hinh gay viem gan cap d chudt n h i t trang b i n g CCI^, hat val cd tac dung ue c h i s y tang hoat do AST va ALT trong h u y i t thanh o miFC l i i u t u o n g d u o n g 10 g DL/kg TT
- Hat v i i khdng t h i hi^n tac dyng chong oxy hda qua viec djnh lugng MDA trong djeh dong the gan chupt nhat trang,
Tai lieu tham khao 1. Balahoroglu R, Dulger H, Ozbek H. (2008),
"Protective effects of antioxidants on the experimental liver and kidney toxicity in mice", Eur J. Gen Med., 5 (3), pp, 157-164.
2. K. Nagendra Prasad, Bao Yang, Shaoyu Yang et al (2009), "Identification of phenolic compounds and a praisal of antioxidant and antityrosinase activities of from Litchi (Litchi sinensis Sonn) seeds", Food Chemistry, 116, pp 1-7.
3. Lijun Wang, Guodong Lou, Zhongjum Ma et ai (2011), "Chemical consitituents with antioxidant activities from litchi (Litchi chinensis Sonn) seeds".
Food Chemistry, 126, pp. 1081-1087.
4. Lu Zhihe, Li Shen (2009), "Antioxidant capacity
and preliminary active components of Lichi seed extract", Shipin Kexue. 30 (23), pp. 110-113 (Tleng Trung),
5. Luo Weisheng, Ou Shiyu. Jin Yaling etai (2013),
"A research of the effect of total flavones from Litchi chinensis Sonn on hepatic fibrosis in rats", Chongqing Yixye, 2 (42), pp, 373-378 (Tleng Trung)
6, Philip Molyneux (2004), "The use of the stable free radical diphenylpicryl-hydrazyl (DPPH) for estimating antioxidantactivity", Songklanakann J. Set.
Techno.. 26 (2). pp. 2^^-2^Q.
7. Turner R. A. (1965), "Screening methods in pharmacology". Test for hematocity, 1, pp, 299-300,
8, Wasowich W., Neve J., Peretz A. (1993),
"Optimized steps in fluorometnc detemiination of thiobarbituric acid-reactive substances in serum importance of extraction pH and Influence of sample presen/ation and storage", Clin. Chem., 39, pp. 2522- 2526
9, Xiong A. H., Shen W. J., Xiao L. Y, Lv. J, H., (2008), "Effect of Semen Litchi containing serum on proliferation and apoptosts of HepG2 cells", Zhong Yao Ca;, 31(10), pp. 1533-6
10. Xu X., Xie H., Wang Y, Wei X. (2010), "A-type proanthocyanidins from lychee seeds and their antioxidant and antiviral activities", J. AgnC Food.
Cftem., 58(22), pp. 1667-72, {Ngiy nhin bii: 19/02/2014 - Ngiy duyet dang: 12/05/2014)
Nghien cvtu da hinh di truyen tap doan cac giong dinh lang (Polysciasfruticosa L. Harms)
CO 6" Viet Nam bang ky thuat RAPD
Nguyen Thi Ngoc Tram'*, Dang Tr^ng LiroTig^
' Cong ty TNHH Thien Duac
^Vien Di truyen Nong nghiep 'E-mail: [email protected]
Summary
Polyscias fruticosa L. Harm (Araliaceae), commonly used as a good hematopoietic and restorative, was researched by detecting DNA in specific area on the genome as FiAPD molecular marker (random amplified polymorphic DNA). A close variation among the samples 1; 16; 17 and 18 of Polyscias was demonstrated with genetic similarity coefficient of 0.90 - 0.98. Especially, the highest value was up to 0.98 for the samples 17 and 18(i,e. there was variation of 0.02 in genetic molecular structure). Thanks to the similarity coefficient, classification trees were set up to indicate the relationship among the studied Polyscias samples. The use of RAPD markers also revealed a close variation among them. Evaluated by using 33 RAPD primers, the highest difference value at the molecular level was only 6%. This may
TAP c m DirOC HQC - 5/2014 (SO 457 NAM 54)
