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CHNG 2 PHNG PHP NGHIN CU2.1 Nguyn liu v i tng nghin cu2.1.1 Nguyn liu nghin cuNm Linh chi Vit Nam (LCVN), Linh chi sm (LCS) (Ganoderma lucidum), Vn chi nu (VCN), Vn chi vng (VCV) (Trametes versicolor) c cung cp bi ThS. C c Trng, Trung tm Nghin cu Linh chi v Nm dc liu. Dc liu c xay nh, bo qun ni kh mt. Cc mu th c chit t bt nm. Cao cn c chun b bng cch chit hi lu Soxhlet bt nguyn liu vi cn 96 % theo t l 1 : 15 (dc liu : dung mi), dch chit c c gim p thu c cao cn vi hiu sut chit theo bng 2.1. Cao nc c chun b bng cch un nguyn liu 3 ln vi nc nng, mi ln trong 1 gi 100C theo t l 1 : 15 (dc liu : dung mi), dch chit c c cch thy thu c cao nc vi hiu sut chit theo bng 2.1. Bng 2.1. Hiu sut chit cc cao ca cc nm dc liu Nm dc liu Linh chi Vit Nam Cao Cao cn Cao nc Cao cn Cao nc Cao cn Cao nc Cao cn Cao nc Hiu sut (%) 8,2 4,8 5,4 8,8 6,6 11,8 8,4 11,2

Linh chi sm

Vn chi vng

Vn chi nu

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2.1.2 ng vt nghin cuChut nht trng c, Mus musculus var Albino, 5 - 6 tun tui, trng lng trung bnh 20 2 g, c cung cp bi Vin Vc xin v Sinh phm Y t TP. Nha Trang v c n nh t nht mt tun trc khi th nghim. Chut c nui y bng thc phm vin, gi, rau x lch v nc ung y . Th tch cho ung hay tim l 10 ml/kg th trng chut.

2.1.3 Ha chtEndoxan 500 mg: cha thnh phn hot cht: 534,5 mg cyclophosphamid momohydrat tng ng vi 500 mg cyclophosphamid khan (k hiu CY), Baxter Oncology GmbH, Germany. L sn xut 8K593E, sn xut 11/2008, hn dng 11/2011. Methanol (MeOH). 1,1-diphenyl-2-picryhydrazyl (DPPH). Trolox (Calbiochem Ltd. Co.). Acid ascorbic (Merck Co. Germany). Malonyl dialdehyd (MDA) (Merck Co. Germany) m Phosphat (PBS) Dung dch Dimethylsulfoside (DMSO) (Merck ) Kali clorid 1,15% (KCl). Natri clorid 0,9% (NaCl). m Tris-HCl (Merck Co. Germany). Acid tricloacetic (TCA). Acid thiobarbituric (TBA) (Nacalai Tesque, Kyoto, Japan). Cc dung mi v thuc th thng thng khc

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2.1.4 My mc-Thit bT sy Memmert (Germany). Cn k thut Mettler Toledo AB204. My c gim p Buchi waterbath B-480. My siu m Bransonic ultrasonic cleaner. n soi UV Desaga. My o pH Meter Schott. Thit b nghin ng th (KA Works, Asia Sdn. Bh, Malaysia). My n nhit hiu Desconectar. My ly tm lnh Sartorius (Germany). My o quang ph t ngoi kh kin Unicam, HE 10 SY vision 32 Software, thermo Spectrorius. My phn tch huyt hc t ng Abbott Cell-Dyn 3700

2.2. Kho st thc vt2.2.1. Kho st hnh thi bn ngoiQuan st dc liu bng mt thng, m t c im hnh thi qu th: hnh dng, mu sc, kch thc, mt trn, mt di.

2.2.2. Kho st vi hc 2.2.2.1. Kho st bt dc liuQuan st bt bng cm quan. Soi bt: dc liu c ct nh, sy nhit khong 60 0C, tn nh, nghin nt, soi trong dung dch KOH bng knh hin vi, chp hnh.

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2.2.2.2. Kho st vi phuTin hnh ct vi phu, soi di knh hin vi v chp hnh

2.3. Th tinh khit [3]2.3.1. Xc nh mt khi lng do lm kha nhm ng mu c sy kh n khi lng khng i 105 C. Cn chnh xc 2 g bt dc liu trn a, em sy 105 C khong 8 gi n khi lng khng i. Ly ra ngui trong bnh ht m khong 15 pht. Cn v ghi li kt qu. Tip tc sy v cn li nh trn vi ln n khi no kt qu hai ln cn lin tip chnh lch nhau khng qu 0,5 mg. Thc hin trn 3 mu v ly gi tr trung bnh. a - b Cng thc xc nh m : X = a X: t l m dc liu (%)

100

a: khi lng bt dc liu hay khi lng cao trc lc sy b: khi lng bt dc liu hay khi lng cao sau khi sy kh n khi lng khng i

2.3.2. tro 2.3.2.1. Tro ton phnTro ton phn l lng tro cn li sau khi nung mt dc liu. Cc ion trong tro ton phn di dng carbonat hay oxid. Cch tin hnh: Cn chnh xc 1 g nguyn liu cho vo chn nung nung n khi lng khng i. Tri u dc liu y chn v t trn bp in cho n khi dc liu chy hon ton v chn khng cn bc khi.

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t chn vo l nung 300 - 600 C cho n khi v c ha hon ton (tro khng cn mu en). Dng kp st ly chn nung ra, ngui khong 30 pht trong bnh ht m. Cn v ghi kt qu. t chn tro vo l nung tip tc nung nhit trn trong 1 gi v em ra cn. Tip tc lm nh vt cho n khi no kt qu hai ln cn lin tip chnh lch nhau khng qu 0,5 mg. Tnh kt qu : Tro ton phn (%) =G2 - G (G1 - G)(1-X)

100

G: Trng lng chn nung (g) G1: Trng lng chn nung v mu th (g) G2: Trng lng chn v tro trng sau khi nung v cn n trng lng khng i (g) X : T l m dc liu (%)

2.3.2.2. Tro khng tan trong acidCch tin hnh Ha tan tro ton phn trn vo 25 ml HCl 4N, un cch thy trong 10 - 15 pht. Thnh phn tro khng tan c lc trn giy lc khng tro. Ra c lc v tro bng nc ct nng n khi dch lc cho phn ng trung tnh (th bng giy qu). Sau em giy lc cha cht khng tan trong HCl cho vo chn nung nung trn bp in n khi thy khng cn khi trng bay ln. Tip tc a vo l nung v thc hin nh cch tin hnh ca tro ton phn. Tnh kt qu: Tro khng tan trong acid (%) = m1 : Khi lng chn nung (g) m2 : Khi lng chn nung v tro khng tan trong HCl (g) m : Khi lng mu th (g)(m2 m1 ) 100 m

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2.4. Phn tch s b thnh phn ho thc vt [3]2.4.1. Nguyn tcQuy trnh dng xc nh nhanh mt s nhm hp cht thng gp trong nguyn liu thc vt bng cc phn ng ha hc (thng c gi l phn tch s b thnh phn ha thc vt) da trn nguyn tc: phn tch hn hp cc cht trong nguyn liu thnh 3 phn on n gin da theo phn cc tng dn: km phn cc, phn cc trung bnh v phn cc mnh bng cch chit nguyn liu ln lt qua cc dung mi: ether ethylic, ethanol, v nc, dng cc phn ng ha hc c trng (thng l cc phn ng kt ta, phn ng mu) pht hin cc nhm hp cht c trong dch chit. Tin hnh phn tch theo quy trnh phn tch thnh phn ha thc vt ca B mn Dc liu Khoa Dc Trng i hc Y Dc TP.HCM da trn c s quy trnh phn tch ca Ciuley (Trng i hc Dc khoa Rumani) c sa i.

2.4.2. Chit xutCn khong 10 g dc liu v chit ln lt qua cc dung mi ether ethylic, ethanol 96 %, nc ct, lng dch chit ca mi loi c cn khong 50 ml nh tnh.

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Dc liu

Ether ethylic/ soxhlet

Dch chit ether

B dc liu

Ethanol hi lu

Dch chit cn

B dc liu

Nc/ cch thy

Dch chit nc

B dc liu

S 2.1. Tm tt quy trnh chit xut trong phn tch thnh phn ha thc vt

2.4.3. nh tnh bng phn ng ho hc

Chit bng ether Dc liu Dch ether

Acid bo Tinh du Carotenoid Tritepenoid t do Coumarin Alkaloid Anthraquinon Flavonoid Alkaloid Coumarin Glycosid tim Flavonoid pyron Anthocyanidin Proanthocyanidin Tannin Saponin Acid hu c Hp cht kh Alkaloid Glycosid tim Flavonoid pyron Anthocyanidin Proanthocyanidin Tannin Saponin Acid hu c Hp cht kh Polyuronic

M giy lc Cn c mi thm Phn ng Carr-Price Phn ng Liebermann-Burchard Pht hunh quang/ OHThuc th chung Phn ng Borntrger Phn ng Cyanidin Thuc th chung Pht hunh quang/ OHThuc th Raymond-Marthoud, xanthydrol Phn ng Cyanidin HCl HCl/t0 ta FeCl3 5%, gelatin mui Phn ng Liebermann-Burchard v tnh to bt trong nc Na2CO3 si bt Thuc th Fehling Thuc th chung Thuc th Raymond-Marthoud, xanthydrol Phn ng Cyanidin HCl ta HCl/t0 FeCl3 5%, gelatin mui Phn ng Liebermann-Burchard v tnh to bt trong nc Na2CO3 si bt Thuc th Fehling Pha long trong cn ta

B dc liu

Chit bng cn

Dch cn

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Chit bng B dc liu

Dch nc

Bng 2.2. Phn tch s b thnh phn ha thc vt

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2.5. Kho st hot tnh chng oxy ha in vitro [13]2.5.1. Kho st hot tnh chng oxy ho in vitro bng th nghim DPPH (test DPPH) 2.5.1.1. Nguyn tcCc cht nghin cu c tc dng chng oxy ha s lm gim mu ca 1,1diphenyl-2-picrylhydrazyl (DPPH). Xc nh kh nng ny bng cch o quang bc sng c hp thu cc i ti = 515 nm.

NO2 NO2 N NO2 N

Hnh 2.1. Cng thc DPPH

2.5.1.2. Tin hnhPha dung dch DPPH 0,6 mM trong methanol (MeOH). Cc mu th cao cn v cao nc c tin hnh nghin cu 7 nng : 2000 g/ml, 1500 g/ml, 1000 g/ml, 500 g/ml, 100 g/ml, 50 g/ml, 10 g/ml (cc mu pha trong dd DMSO). 0,5 ml mu th cc nng kho st c cho phn ng vi 0,5 ml dung dch DPPH. Thm MeOH va 4 ml. Hn hp sau khi pha c nhit phng 30 pht. o quang bc sng = 515 nm. Acid ascorbic (Merck, Germany) c s dng lm cht i chiu.

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2.5.2. Kho st hot tnh chng oxy ho in vitro bng th nghim malonyl dialdehyd (MDA test) 2.5.2.1. Nguyn tcXc nh kh nng c ch peroxy ho lipid thng qua vic xc nh hm lng MDA theo phng php ca E.A.Makarova, 1989; Robak v cng s, 1988. MDA c sinh ra trong qu trnh peroxy ha lipid, khi cho phn ng vi acid thiobarbituric, mt phn t MDA phn ng vi hai phn t acid thiobarbituric to phc mu hng hp thu cc i bc sng 532 nm. Phn ng thc hin mi trng pH 2 - 3, nhit 90 - 100 0C trong vng 10 - 15 pht. o cng mu ca phc suy ra lng MDA c trong mu. Phng trnh phn ng nh sau:

Hnh 2.2. Phng trnh to phc mu gia MDA v acid thiobarbituric

2.5.2.2. Cch tin hnhPha thuc th TBA 0,8 %. Mi mu th cao cn v cao nc c tin hnh nghin cu 7 nng : 2000 g/ml, 1500 g/ml, 1000 g/ml, 500 g/ml, 100 g/ml, 50 g/ml, 10 g/ml (cc mu pha trong dd DMSO). Tch no chut v nghin ng th trong dung dch m phosphat 5 mM theo t l 1 : 10 (no : dung dch m) nhit 0 - 5 0C. Ly 0,5 ml dch ng th,

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thm vo 0,1 ml cc nng mu th v 1,4 ml m phosphat, 37 0C trong 15 pht. Kt thc phn ng bng 1 ml acid tricloacetic 10%, ly tm 10000 vng/pht, ly 2 ml dch trong cho phn ng vi 1 ml acid thiobarbituric 0,8% 1000C trong 15 pht v o mu = 532 nm. Trolox (Calbiochem Ltd. Co.), ng phn ca vitamin E c s dng lm cht i chiu.

2.5.3. Tnh kt quHot tnh chng oxy ha HTCO (%) c tnh theo cng thc: HTCO (%) = [(ODChng - ODTh) / ODChng] 100 ODchng: mt quang ca dung mi DMSO ODth: mt quang ca mu th

2.6. Nghin cu in vivo2.6.1. Gy suy gim min dch bng cyclophophamid (CY) [11], [24] 2.6.1.1. Nguyn tcCyclophosphamid l mt cht gy c ch min dch c s dng ph bin trong ha tr liu ung th trn lm sng v l mt cht gy c c ch cc ch tiu lin quan n chc nng min dch trong cc nghin cu thc nghim trn ng vt. Ngoi ra, cyclophosphamid cn th hin tc dng ph trn cc c quan khc nh gan, thn, tiu ha, tit niu,... Gan c xem l hng ro chn ca c th, ngn cc sn phm c hi thm nhp vo qua ng tiu ha, ng thi lm gim c tnh v thi tr mt s cht cn b do chuyn ha trong c th to nn. Trong qu trnh chuyn ha gan, cyclophosphamid c bin i thnh nhng cht c kh nng gy c, lm gia tng qu trnh peroxy ha lipid ti gan.

2.6.1.2. Cch tin hnhChut th nghim c chia thnh hai nhm: nhm chut bnh thng CY(-) v nhm b gy suy gim min dch [CY(+), tim phc m liu duy nht Endoxan

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ha tan trong nc mui sinh l vi liu tng ng cyclophosphamid 150 mg/kg th trng]. Cc l th nghim c b tr theo bng 2.3. Bng 2.3. B tr th nghim Nhm L (N = 10-12) CY(-) Chng Th Chng Th Tim phc mc CY (i.p.) 150mg/kg 150mg/kg Mu th Ung Nc ct Cao Cao Nc ct

CY(+)

CY: cyclophosphamid Bng 2.4. Liu tng ng vi 5 g dc liu/kg th trng cho cc cao th nghim Nm Cao Liu ung (g/kg) Linh chi Vit Nam C.nc 0,44 C.Cn 0,41 Linh chi sm C.nc 0,24 Vn chi vng Vn chi nu

C.cn C.nc C.cn C.nc C.cn 0,27 0,56 0,42 0,59 0,33

2.6.2. Kho st trng lng c th trn chut nht trng b gy suy gim min dch bng CYTin hnh cn trng lng c th chut trc khi tim CY v sau 5 ngy, 10 ngy c 2 nhm bnh thng v nhm gy suy gim min dch.

2.6.3. Kho st huyt hc trn chut nht trng b gy suy gim min dch bng CYTin hnh ly mu ui chut sau 5 ngy v 10 ngy cc nhm chut. Mu chut c kim tra huyt hc ti Bnh vin Bnh Dn bng h thng phn tch huyt hc t ng Abbott Cell-Dyn 3700.

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2.6.4. Kho st trng lng tng i ca cc c quan trn chut nht trng b gy suy gim min dch bng CYSau 10 ngy, chut hai nhm bnh thng v nhm gy suy gim min dch c tch ly gan, lch, tuyn c, tuyn thng thn xc nh trng lng tng i ca cc c quan bng cng thc sau: g % = Pcq /Pct * 100 Pcq: trng lng ca c quan Pct: trng lng c th chut ti thi im kho st

2.6.5. Kho st hm lng malonyl dialdehyd (MDA) trong gan sau khi gy suy gim min dch bng cyclophosphamidNgy 1Cn trng lng Tim CY

Ngy 3

Ngy 5

Ngy 8

nh lng MDA nh lng MDA nh lng MDA

S 2.2. Kho st hm lng MDA trong gan sau khi tim cyclophosphamid nh lng hm lng MDA trong gan Tch gan chut v nghin ng th trong dung dch m KCl 1,15 % theo t l 1 : 10 (gan : dung dch m) nhit 0 - 5 0C. Ly 2 ml dch ng th, thm vo 1 ml dung dch m Tris - HCl, 37 0C trong 1 gi. Kt thc phn ng bng 1 ml acid tricloacetic 10 %, ly tm 10000 vng/pht, ly 2 ml dch trong cho phn ng vi 1 ml acid thiobarbituric 0,8 % 100 0C trong 15 pht v o mu = 532 nm. [13]

2.6.6. Kho st tc dng bo v gan theo hng chng gc t do qua vic nh lng malonyl dialdehyd (MDA) trong ganSau khi gy suy gim min dch v iu tr theo s 2.3 , tin hnh nh lng hm lng MDA nh phn 2.6.5.

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Ung mu th nghim (10 ml/kg) Ngy 1 2 3 4 5 6 7 8

Cn trng lng v tim CY

Cn trng lng v nh lng MDA

S 2.3. Kho st tc dng bo v gan theo hng chng gc t do.

2.6.7. Tnh kt quHm lng MDA (nM/ml) c tnh theo phng trnh hi qui cht chun MDA: y = 0,0637x - 0,0029 (R2 = 0,9981). CnMMDA/ml dch ng th = (ODth + 0,0029)/0,0637. Sau khi tnh c hm lng MDA (nM/ml dch ng th) hm lng MDA (nM/g protid).

2.7. nh gi kt quCc s liu c biu th bng tr s trung bnh M SEM (Standard Error of Mean sai s chun ca gi tr trung bnh) v x l thng k bng phn mm Jandel Scientific SigmaStat-98 da vo php kim One-Way ANOVA hay Student t-test vi P < 0,05.