Hoi nghi Khoa hqc ky- niem 35 ndm Vien Khoa hqc vd Cong nghe Viet Nam - Hd NqilO 2010
NGHIEN CLTU NUOI TRONG MOT SO LOAI NAM AN VA NAM DlTOC LIEU VIET NAM, THU NHAN, CHUYEN HOA VA KHAO SAT HOAT TINH KHANG U THU^C NGHIEM CAC POLYSACCARIT CUA CHUNG
Le Mai Huung'. C6 Thi Thuy Van^ Ha Phuong Thu', Nguyen Bich Thuy', Tran Thj Hong Ha', TrSn Thi Nhu H^ng\ Do Huu Nghj', Pham Quoc Long',
Nguyen Xuan Phue^, Rene Ulrich^, Martin Hofrichter^
1- Vien Hda hoc cac hgp chat thien nhien, 18 - Hoang Qudc Viet, C§u Gidy, Ha Ndi 2- Vien Khoa hoc vat lieu, 18 - Hoang Qudc Viet, Cau Gidy, Ha Ndi
3- Vien Di truyen ndng nghiep, Dudng Pham Van Ddng, Cdu gidy, Ha ndi 4- Trudng Dai hoc qudc tk Zittau, Markt 23, Zittau 02763, CHLB Dire
Email : lehuongOO(fl)gmail.com Tom tat:
Trong qud trinh dm kiim cdc chdt cd hogt tinh sinh hgc tir ndm l&n, todn bd cdc cdng vlic nghien ciru di tu phdn lap ndm tir thiin nhiin, nudi trdng chung bdng hai con du&ng khdc nhau: len men dich the vd nudi trdng trin ca chdt rdn di thu nhdn qud thi, sau dd cdc sdn phdm dugc tdch chiit hda hgc di thu nhgn cdc sdn phdm Ihu cdp cd ddnh gid hogt tinh khdng u. Trong bdi ndy, chung tdi lap trung vdo md td cdc qud trinh lin men, phdn lap cdc polysaccarcit ddc biel tu ndm Id l,3-[i-D glucan, chirng minh rdng sdn phdm ndy cd khd ndng bao bgc curcumin, su bao bgc ndy ldm Idng khd ndng hda tan cua curcumin vd hon the nira, sdn phdm kit hgp ndy cd khd ndng uc chi sir phdt triin khdi u khi nudi cdy li bdo ung thu thuc nghiem bdng kt thudt nudi cdy ti bdo ung Ihu di tgo Ihctnh khdi u 3 chieu trong mdi tru&ng thgch mim ddc biit.
Abstract:
In the course of seeking for bioaclive compounds from mushroom, all research works have been Implemented including how to isolate mushroom from natural resource, plant them by h\>o ways: liquid and solid fermentations, then chemical extraction obtain their secondary metabolites and biological assessment including antitumor activity. In this report, MC focused on mushroom fermentation, isolation the polysaccharides l,3-[3-D glucan from the fruiing bodies and liquid fermented products, demonstrated that l,3-/3-Glucan is able lo encapsulate curcumin. Upon encapsulation, the solubility of Curcumin increased. Moreover, encapsulated Curcumin revealed enhanced anti-tumor promotion activity in vitro compared to Curcumin or l,3-f3-Glucan alone by the 3Dtumor promotion assay in vitro.
I. DAT VAN DE
Cac polysacarit, dac biet la P-D glucan la thanh phdn khang u chii yeu ciia ndm ldn.
Cac nguyen ly can ban ciia tac dung khang u va dilu bidn miin dich ciia cac polysaccharit tir nam ldn la: (1) ngan chan qua trinli gay phat sinh benh ung thu bdng sir dung cac san phdm nay theo dirdng an udng; (2) Khang u true tilp hang loat cac u ddng gen va di gen: (3) tang cudng mien dich khang u khi ket hgp vdi hda dilu tri; (4) ngan chan di can cac khoi u.
(Wasser, S.P..2000) [1].
Tiiu ban: cdc chdt co hogi tinh sinh hqc ISBN: 978-604-913-012-0
Ben canh viec sir dung true tiep cac san pham ciia nam Idn cd chua cac polysacarit da neu tren, dk nang cao hieu qua sir dung nam, viec nghien cuu chuyen hda cac polysacarit cua ndm thanh cac thanh phan cd trong Iugng phan tir thap hon, dl hdp thu trong co the va lam tang hieu qua phdng va chdng khdi u gay ung thu la mot hudng di hit sire cd y nghia cho cac nghien cim theo hudng di mdi nay d Viet Nam.
Trong thdi gian 5 nam trd lai day, phdng Sinh hoc thuc nghiem da tap trung nghien cim vl ndm ldn Basidiomycetes, bdt ddu tir phan lap, nudi cay theo hai con dudng khae nhau dl thu nhan san phdm, tach ehilt, sang loc hoat tinh sinh hoc cac chat trong ndm Idn va so sanh ham Iugng va hoat tinh cac chdt khi nudi trong theo cac phuong phap khae nhau. Trong phdn bao cao nay, chimg tdi so luge lai mot sd kit qua co ban vl nghien ciiru nudi trdng nam, tap trung vao ndi dung nghien ciiu tach ehilt cac polysaccharit dac biet tir nam la 1,3, (3- glucan; cho tuong tac vdi nano curcumin, nghien cim chi tao vat lieu nano Cur-GIu va ket qua thir nghiem hoat tinh ciia nd bdng phuong phap irc chi hinh thanh khdi u 3 chieu tren thach mim (3D anti-tumor promoting assay) in vitro.
II. HOA CHAT, THIET BI VA PHU-ONG PHAP NGHIEN CLTU
Phuong phap Ien men chim dk thu sinh khdi ndm theo phuong phap cua Mizuno 1996, 1999a [2]. Nudi trdng ldy qua thi theo Nguyin Lan Dung va CS, 2003 [3].
Phuong phap tach ehilt dl thu nhan polysaccharit theo phuong phap cua Mizuno 1996, 1999a [2], tach chiit thu nhan 1,3 - p- glucan theo Zhaojing Wang et al., 2004 [4] va Xiaoyu Chen e t a l , 2010 [5].
Ddng tl bao cho ca hai thir nghiem: Ddng ung thu gan ngudi HepG2 (Hepatocellular carcinoma) hien dang luu giir tai phdng Sinh hoc thuc nghiem - Vien Hda hoc cac hgp chat thien nhien.
Phuong phap thir nghiem sinh hoc:
Thir nghiem gay doe te bao (cytotoxycity assay): Dugc tien hanh theo phuong phap ciia Likhiwitayawuid va cs., 1993 dang dugc tien hanh tai Vien nghien cim ung thu Qudc gia cua My (NCI), dugc thuc hien tai phdng Sinh hoc thuc nghiem - Vien Hda hgc cac hgp chat thien nhien tir nam 1996 den nay [6,7].
Phuong phap lie che hinh thanh khdi u 3 chieu tren thach mem (anti-tumor promoting assay) in vitro Theo tac gia Jong Bin Kim( 2005) [8] va Huiyuan Gao & CS, 2007 [9], hien dang dugc trien khai thuc hien tai phdng Sinh hgc thuc nghiem - Vien Hda hoc cac hgp chat thien nhien.
Nguyen ly: Thir nghiem irc che hinh thanh khdi u (anti-tumor promoting assay) in vitro tren mdi trudng thach mem la phep ban dinh lugng va do sir bien ddi hinh thai cua khudn lac te bao do anh hudng ciia cac hda chat. Sir bien ddi nay gan lien vdi viec bam dinh ciia cac te bao rieng le (cac te bao tao thanh khdi u 3 chieu tren thach mem). Qua trinh diln ra sir bien ddi hinh thai dugc cho la cd lien quan mat thie'; vdi qua trinh ung thu hda in vivo. Nhin chung, giira qua trinh bien ddi in vitro va qua trinh ung thu hda in vivo cd mot sir lien quan nhat dinh, mac dii mdi lien quan nay thay ddi tiiy thudc vao he nghien cuu. Kit qua duong tinh ciia thir nghiem nay la nhiing tin hieu dang tin cay thdng bao trudc hoat ddng in vivo, dugc coi la chi thi ciia qua trinh ung thu hda in vivo.
Quy trinh boc Curcumin bdng I,3-6-Glucan theo Bisht S et al., 2007 [10], Phuong Thu Ha&CS.,2010[lI].
Hoi nghi Khoa hqc ky niem 35 ndm Vien Khoa hqc vd Cong nghe Viet Nam - Hd Not 10/2010
III. KET QUA NGHIEN ClTU a. Nuoi trong nam
Phirong phdp lin men chim di thu sinh khdi ndm dirge tdm tdt nhir sau: Ndm dugc nudi cay chim tren mdi truong dinh dudng cd cac thanh phdn sau: Bdt do tuong - 20g; Dudng kinh- 20g; (NH4)2S04 - 2,5g; CaCOa 2g; MgS04 l-2g; nhiet do thich hgp nhdt la 22°C, pH thich hgp nhat la pH 6, thdi gian thich hgp nhat de thu sinh khdi la sau 7 ngay nudi cdy. Quy trinh nudi trdng tren co chdt rdn thu nhan qua thi dugc tiln hanh theo quy trinh d hinh 1.
Mun cua tao am bang nude voi trong. Thdi gian u 4-5
Bong hat tao am bang nude vdi trong. Thai gian u tir 24-
Phoi tron nguyen lieu
Ddng nguyen lieu
Khir triing
z
Cay giong
LTom bich (Nuoi
Cham sdc
Thu nhan qua the
Hinh 1: Quy trinh nudi trdng ndm tren ca chdt rdn di thu nhdn qud thi
Hinh 2: Phdng uom sgi Hinh 3: Phdng chdm sdc qud thi
Tieu ban: cdc chdl co hog! tinh sinh hoc ISBN: 978-604-913-012-0
b. Quy trinh ehiet tach polysaccaride
Thdng qua ket qua nghien cu'u ehiet tach polysaccharide va tdi uu hoa dilu kien ehiet, chiing tdi kien nghi qui trinh cdng nghe ehiet tach polysaccharide theo 3 giai doan. nliu sau:
Cho 200 g nam khd da nghien nhd cd do am 4% va 1600 ml cdn 80% vao mot binh cau 2 lit cd lap sinh han nguge, d nhiet do dudi 60 C Sau 20 h loc lay dich cdn, cdt quay chan khdng thu hdi cdn va dugc san pham WI. Phan khdng tan, cho them 1500 ml nude ndng, lac deu rdi ehiet d 100°C trong 2h. Loc lay dich ehiet, sau dd diing cdn ti le 3:1 (v/v) tiia tach dugc W2.
Phan khdng tan tiep tuc cho them NaOH 4%, ti le 10:1 (v/w), khuay deu d nhiet do 50°C, trong 90 phut, loc lay dich (lap lai 2 lan). Dich loc thu dugc gom lai chinh ve pH= 7^8 bang axit axetic, de yen tinh trong 30 phiit, loc tach lay phan ket tiia W3. Hieu suat san pham thu dugc khoang 23-^25% (theo so dd dudi d hinh 4).
Nam 200 g, do am
Nghien
Chiet I -Cdn 80%, tile 10:1 (v/w), < 60 °C, 20 h Dichl
Loai
Phan khdng
WI Chiet II . Nude, tile 10:1 (v/w), 100°C, 2 h
Tiia cdn, ti le 3:1
Dich 2
1 '
W2
Phan khdng Chiet 111
Chinh pH =7, Dich 3 tiia cdn, ti le 3:1 (v/v)
Ba
NaOH 4 %, ti le 10:1 (v/w), 50 °C, 90 phut
Chinh pH, bd W3 Phan bdn vi
Hinh 4: Sa dd cdng nghi chiit tdch polysaccaride c. Phan tich cau triic cua polysaccharide
^ San pham polysaccharide W3 sau khi sdy phun dugc chup phd Cong hudng tir hat nhan ( H-NMR, '^C-NMR) va phd Hdng ngoai. Cac dir lieu phd cho thdy san phdm cd cdu true hda hoc cd mach chinh la ciia chudi mach chinh 1,3-p-D-glucan (hinh 5):
Hoi nghi Khoa hqc ky niem 35 ndm Vien Khoa hqc vd Cong nghe Viet Nam - Hd NqilO 2010
OH
Hinh 5: Cdu true 1,3-P-D-glucan
d. Boc Curcumin bang 1,3-P-Glucan
Curcumin tinh khiet dugc hoa tan trong ethanol tuyet ddi dem trdn Ian vdi dung dich I,3-P-Glucan (da dugc hoa tan trong nude cat 2 lan). Hdn hgp dugc khuay deu bang may khuay tir va dn dinh d nhiet do phdng trong 12 gid. Sau dd, dung mdi dugc bde bay bang he thdng say chan khdng. Phan Curcumin khdng dugc boc se hi loai bang each quay Iy tam hon hgp huyen phii. Mixel Curcumin da gan ket 1,3-P-GIucan (Cur-Glu) dugc nghien cim bdng phuong phap FESEM, UV-Vis, FL va PL.
Ket qua khi hda tan Cur-Glu trong nude, Cur-Glu tan tdt tao thanh dung dich cd mau vang trong sudt, khae hoan toan vdi curcumin khi chua hoc 1,3-p-GIucan
Cau triic, hinh thai hgc ciia Cur-Glu
Khi tien hanh do phd hap thu dien tir ciia Cur-GIu bang may VARIAN spectrophotometer Carry 5000 UV-Vis-NIR. Hinh 2 la phd hap phu ciia Cur trong ethanol va Cur-Glu trong H2O cho thay: Cur trong ethanol cd dinh hap thu cue dai tai 428 nm, trong khi dd Cur da dugc boc bang Giu cd cudng do hap thu tang vot va cd dinh hap thu cue dai tai 415 nm. Sir dich chuyen dinh hap thu cue dai ve bude sdng ngan va tang cudng do hap thu ciia phd chimg td cd sir tuong tac giGa Curcumin vdi 1,3-P-GIucan. Tren cac phan tir Cur va Giu deu cd nhieu nhdm OH- nen cac phan tir nay cd the tuong tac vdi nhau theo lien ket hydro.
<
415 428
Cur Cur-Glu
300 400 500
Wavelength (nm)
— I —
600 700
Hinh 6: Phd hdp thu ciia Cur (dd) vd Cur-Glu (xanh)
Dung dich Cur cd dinh hdp thu cue dai tai 538 nm, trong khi dd dung dich Cur-GIu cd dinh hap thu cue dai tai 530 nm vdi cudng do huynh quang tang cao hon so vdi dung dich Curcumin. Nhu vay, phd huynh quang cung mot ldn niia chiia dung nhirng ddu hieu khdng dinh Curcumin da dugc boc 1,3-P-Glucan.
Tiiu ban: cdc chdt co hogi linh sinh hqc ISBN: 978-604-913-012-0
c 0) 10000
9 0 0 0 - 8000 7000 6 0 0 0 - 5000 4000 3000 2 0 0 0 - 1000
0 -1000
530
/"S 538 Cur Cur-Glu
350 400 450 500 550 600 650 700 750
Wavelength (nm)
Hinh 7: Phd huynh quang Cur (dd) vd Cur-Glu (xanh)
e. Thu nghiem sinh hoc
Kit qua: Kit qua thir nghiem gay ddc te bao ciia cac san pham curcumin, glucan va hon hgp phdi trdn giira Cur-Glu diu khdng bilu hien hoat tinh gay ddc te bao Hep-G2 khi nudi cdy in vitro. Tuy nhien, trong thir nghiem hoat tinh khang hinh thanh u thi cd su khae biet rd ret giiia cac san phdm vl kha nang lic che hinh thanh u ciing nhu kich thudc cac khuan lac. Cac kit qua dugc tdng ket trong bang 1. va cac anh 6a, 6b, 6c va 6d .
Bdng 1. Kit qud thir nghiim hogt tinh gdy ddc ti bdo vd hogt tinh uc chi tgo u tren thgch mem ciia cdc sdn phdm
Mau thir
Cur Giu Cur-GIu
Ndng do ban dau 8mg/ml 8 mg/ml 8mg/ml
HT gay ddc te bao ( %TB sdng sdt)
99,3 ± 0,5 93,3 ± 0,9 90,7 ± 0,7
Ty le tao u tren thach mem so vdi
DC(%) 80 ± 1,3 75,5 ± 1,1 30,2 ± 1,5
Kich thudc khuan lac tmng binh giam so vdi
DC (%) 20-30 23-30 40-50
Hoi nghi Khoa hqc ky niem 35 ndm Vien Khoa hqc vd Cong nghe Viet Nam - Hd NqilO 2010
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Hinh 8: Hinh dnh thu nghiim khd ndng uc chi hinh thdnh khdi u ciia ddng ti bdo Hep-G2 tren thgch mem sau 2 tudn nudi cdy. Mdu ddi chung (hinh a), mdu Cur-Glu (hinh b), mdu Giu
(hinh c) vd mdu Cur (hinh d)
Hinh anh sir hinh thanh khdi u 3 chilu tren thach mim cho thdy hieu qua lic chi rd ret ciia hdn hgp cur-glu vdi ty le 4:1 len sir hinh thanh khdi u ciia ddng tl bao nay tren thach mim va dang chii y hon la nd da gan dugc vao tl bao lam cho tl bao phat quang.
IV. KET LUAN
Cd the tien hanh nudi trdng nam dugc lieu bang ca hai phuong phap: Nudi trdng truyen thdng tren co chat ran va len men dich thi dl thu nhan cac chdt cd hoat tinh sinh hoc phuc vu nghien ciiu va tao thuc pham chiic nang.
Da nghien ciiu qui trinh tach chiit 1,3-P-Glucan tir ndm dugc lieu va bude ddu chuyen hda san pham bang thir nghiem hoc curcumin bdng san phdm 1,3-P-GIucan. San phdm hda tan rat tdt trong nude va cd tinh phat quang manh. Kit qua nghien cihi hira hen kha nang ling dung trong chiia tri benh ung thu va danh ddu quang hoc va bilu hien hoat tinh lie chi rd ret Ien sir hinh thanh khdi u cua ddng tl bao ung thu gan HepG2 tren thach mim md ra hy vong mdi trong hudng nghien ciiu iing dung cac san phdm thien nhien trong phdng va dilu tri ung thu dudng tieu hda.
Loi cam on:
Cdng trinh dugc ho trg kinh phi trich tir NDT Viet nam-CHLB Dirc(2006-2008) va dl tai "Khao sat hoat tinh khang u thuc nghiem cua cac polysacarit dac biet tir ndm an va ndm dugc lieu va nghien cim san phdm chuyin hda sinh hoc cac polysacarit nay thanh cac san phdm cd gia tri sir dung cao hon" quy NCCB Nafosted 2009-2011.
Tiiu ban: cdc chdt co hogt linh sinh hqc ISBN: 978-604-913-012-0
TAI LIEU THAM KHAO
1. S.P. Wasser, " Medicinal mushrooms as a source of antitumor and immunomodulaling polysaccharides'\ Received: 2 May 2002 / Revised: 17 June 2002 / Accepted: 20 June
2002 / Published online: 10 September 2002 © Springer-Verlag 2002.
2. Mizuno M, et al, 'Anti-tumor polysaccharide from the mycelium of liquid cultured Agaricus blazei milV\ Biochem Moi Biol Int, vol. 47 (4) (1999) pp. 707-714.
3. Nguyin Lan Diing , " Cdng nghe nudi trdng ndm", lap 1, tap 2, NXB Ndng Nghiep HaNdi, 2003.
4. Zhaojing Wang, Dianhui Luo, Zhongyan Liang. J, " Carbohydrate polymers", vol. 57 (2004) pp. 241-247.
5. Xiaoyu Chen, Lina Zhang, Peter Chi Keung Cheung. J, " International immunopharmacologf" vol. 10 (2010) pp. 398-405.
6. Skehan. P, Storeng. R, Scudiero. D, Monks. A, Mc Mahon. J, Vistica. D, Warren. J.T, Boskesch. H, Kenney. S, and Boyd. M.R - J. Nat. Cancer Inst, vol. 82 (13) (1990) pp.
1170.
7. Likhitwitayawwuid. K, Angerhofer. CK, Cordell. G.A, Pezzuto. J.M, and Ruanggrungsi. N - J. Nat. Prod, vol. 56 (I) (1993) pp. 30- 38.
8. Jong Bin Kim. "Seminar in Cancer Biology'" vol. 15 (2005) pp. 365-377.
9. Huiyuan Gao, Bailing Hou, Masonori Kuroyanagi, Lijun wu , Asian J. of Trad. Medi, vol. 2 (3) (2007) 104-109.
10. Bisht S, Feldmann G, Soni S, Ravi R, Karikar C, Amornath M and Aniirban M J.
Nanobiotechnol. vol. 5 (3) (2007).
11. Phuong Thu Ha, Thi Minh Nguyet Tran, Hong Duong Pham, Quang Huan Nguyen, and Xuan Phuc Nguyen - Adv. Nat. Sci. Nanosci. Nanotechnol. vol. 1 (2010) trang.
7.