1

Phn 1: M U1.1 T VN Theo chng ta bit th Linh chi l mt trong dc tho thin nhin c xp vo loi thng dc. Cch y hng ngn nm, nm Linh Chi c dng lm thuc, cc sch dc tho ca nhiu triu i Trung Quc u ghi nhn Linh Chi c s dng lm thuc t lu i. Gi tr dc liu ca linh chi dc ghi chp trong cc th tch c ca Trung Quc, cch nay hn 4000 nm (Zgao, J.D., 1994). T nhng kinh nghim lu truyn trong nhn gian, loi ngi bit s dng Linh chi theo nhiu cch khc nhau. n nay khoa hc k thut pht trin, nm Linh chi cn c cc nh khoa hc trn th gii chng minh c tc dng hu ch trong vic iu tr bnh: ung th, cao huyt p, tiu ng, tim mch, HIV, vim gan siu vi, suy nhc thn kinh [3, 9] Hin nay Linh chi khng cn khan him nh lc trc do con ngi c th p dng k thut nui trng Linh chi trong mi trng nhn to v ngy cng pht trin mnh trn th gii v t n quy m cng nghip. Trong hai thp nin gn y, cc nh khoa hc nc Nht Bn, Trung Quc, Hn Quc, Singapore, Hongkong tng cng nghin cu sn xut nm Linh Chi v m rng hiu qu s dng dc liu ny. Ti th trng Vit Nam, nhu cu s dng nm Linh chi lm thuc cha bnh trong nc cng nh xut khu ngy cng tng. Nhiu c s tin hnh nghin cu nui trng, ch bin v thm d cc hot cht sinh hc c trong nm Linh chi. Cc thnh phn ha hc c trong nm Linh chi rt phong ph bao gm cc nhm: acid bo, steroid, alcaloid, protein, polysaccharide [19]. Trong thnh phn c tc dng dc l qu bu, c trng cho nm Linh chi phn ln thuc nhm triterpenoid [2]. Vit Nam vi kh hu nhit i gi ma v ngun nguyn liu cellulose di do, to iu kin cho ngh trng nm khng ngng pht trin. Hin nay c nc c 32/61 tnh thnh c c s nui trng nm dc liu (thng 12/2001) [6]. Da vo tnh hnh ngy cng pht trin ca ngnh nm Vit Nam v s cho php ca b mn cng ngh sinh hc ca Trng Cao ng Kinh t Cng ngh TP. H ChNguyn V Duy Khanh

2

Minh chng ti quyt nh thc hin ti Xy dng quy trnh sn xut sinh khi si Ganoderma lucidum. 1.2 MC CH Xy dng quy trnh sn xut sinh khi nm Linh chi 1.3 YU CU Xc nh cc yu t dinh dng n tc pht trin ca si nm Linh chi Xc nh cc yu t pH, xc nh ng cong tng trng ca si nm Linh chi Ti u ha iu kin nui cy si nm theo quy trnh thc nghim Phn tch thnh phn hot cht c trong si nm 1.4 HN CH TI Cha kho st ht cc yu t dinh dng nh hng n mi trng nui cy

Nguyn V Duy Khanh

3

Phn 2: TNG QUAN TI LIU2.1 NM LINH CHI [2, 5, 6, 12] 2.1.1 Khi qut chung Nm Linh chi c tn khoa hc l Ganoderma lucidum, ngi min Bc xa cn gi l nm lim. Trong th tch c nm Linh chi cn c gi vi tn khc nh Tin tho, Nm trng th, Vn nin nhung Nm Linh chi thng phn b vng nhit i v cn nhit i, chng thng pht trin trn gi th l g mc hoc cc nguyn liu c cht s Hnh thi qu th nm Linh chi c m t nh sau: Tai nm ha g, hnh qut hoc thn. Mt trn m c vn ng tm v bng long, mu vng cam cho n mu m hoc nu en. Mt di phng, c nhiu l nh li ti, l c quan sinh bo t. Cung nm c v cng, sm mu v bng long. Gi tr dc liu ca Linh chi dc ghi chp trong cc th tch c ca Trung Quc, cch nay hn 4000 nm (Zgao, J.D., 1994). Trong sch Thn nng bn tho cch y khong 2000 nm thi nh Chu v sau c nh dc hc ni ting Trung Quc L Thi Trn phn ra thnh Lc Bo Linh Chi thi nh Minh vi cc khi qut cng dng dc l khc nhau, ng theo tng mu (L Thi Trn, 1590). Theo L Thi Trn th nm Linh chi c 6 mu khc nhau: Xch chi (Linh chi cn gi Hng chi) Hc chi (Linh chi en cn gi Huyn chi) Thanh chi (Linh chi xanh cn gi Long chi) Bch chi (Linh chi trng cn gi Ngc chi) Hong chi (Linh chi vng cn gi Kim chi) T chi (Linh chi tm)

Cho n nay Linh chi khng cn gii hn trong phm vi t nc Trung Quc, m mang tnh ton cu. Hin ti c khong 250 bi bo ca cc nh khoa hc lin quan n dc tnh v lm sng ca Linh chi c cng b.Nguyn V Duy Khanh

4

Cu trc c o ca Linh chi chnh l thnh phn khong vi lng loi, trong mt s khong t nh germanium, vanadium, crm... Chng c s dng l nhn t quan trng cho nhiu loi phn ng chng ung th, d ng, lo ha, x va, ng mu ni mch, gip iu chnh dn truyn thn kinh, bo v cu trc ca nhn t bo vi hm lng rt thp cc nc Chu , c bit l Trung Quc, Nht Bn, Hn Quc, i Loan, vic nghin cu pht trin v s dng Linh chi ang c cng nghip ha vi quy m ln v phn loi, nui trng ch ng, ch bin v bo ch dc phm. ng thi nghin cu ha dc cc hot cht c tc dng dc l v phng php iu tr lm sng. Vit Nam, trong cc ti liu lu li ca Hi Thng Ln ng, L Hu Trc (1720-1791) cng thy cp nllinh chi. Sau , L Qu n cn khng nh, y l ngun sn vt qu him ca t rng i Nam. Trong quyn Cy thuc v v thuc Vit Nam (1991), gio s Tt Li cn m t chi tit v trnh by v c tnh tr liu ca loi nm ny, ng thi cho rng y l loi Siu thng dc. 2.1.2 V tr phn loi [5, 7] Nm Linh chi c v tr phn loi c tha nhn rng ri hin nay: Ngnh: Ngnh ph: Lp: Lp ph: B: H: H ph: Ging: Eumycota Basidiomycotina Hymenomycetes Hymenomycetidae Aphyllophorales Ganodermataceae Ganodermoidae Ganoderma

2.1.3 c im hnh thi v chu trnh sng ca nm Linh chi 2.1.3.1 c im hnh thi nm Linh chi (Ganoderma lucidum) Linh chi thuc nhm nm ln v rt a dng v chng loi. T khi xc lp thnh mt chi ring l Ganoderma Karst (1881), n nay tnh ra c hn 200 loi c ghi nhn, ring Ganoderma lucidum c 45 loi. [3]Nguyn V Duy Khanh

5

Nm Linh chi l mt trong nhng loi nm ph g, c bit trn cc cy thuc b u (Fabales). Nm xut hin nhiu vo ma ma, trn thn cy hoc gc cy. Vit Nam Nm Linh chi c gi l nm Lim v c pht hin min Bc bi Patouillard N.T (1890 n 1928).

Hnh 2.1: Nm Linh chiNm Linh chi (qu th) cy nm gm 2 phn cung nm v m nm (phn phin i din vi m nm). Cung nm di hoc ngn, nh bn c hnh tr ng knh 0,53cm. Cung nm t phn nhnh, i khi c un khc cong queo. Lp v cung mu , nu , nu en, bng, khng c lng, ph sut ln mt tn nm. M nm khi non c hnh trng, ln dn c hnh qut. Trn mt m c vn gch ng tm mu sc t vng chanh - vng ngh - vng nu - vng cam - nu - nu tm nhn bng nh lng vecni. M nm c ng knh 2 - 15cm, dy 0,8 - 1,2cm, phn nh cung thng g ln hoc hi lm. Khi nm n tui trng thnh th pht tn bo t t phin c mu nu sm.

Nguyn V Duy Khanh

6

2.1.3.2 Chu trnh sng ca nm Linh chi

Qu th Si nm song nhn m

Si nm n nhn m v bo t m

Phi nhn trong m

Hnh 2.2: Chu trnh pht trin ca nm Linh chi [5] 2.2. THNH PHN HA HC V C TNH DC L CA NM LINH CHI (GANODERMA LUCIDUM) Cc phn tch ca G-Bing Lin chng minh cc thnh phn ha dc tng qut ca nm Linh chi nh sau : [18] Nc: Cellulose: Lignine: Lipid: Monosaccharide: Polysaccharide: Sterol: Protein: Thnh phn khc: 12 13% 54 56% 13 14% 1.9 2.0% 4.5 5.0% 1.0 1.2% (chng hot ng khi u, tng sc khng v h min dch cho c th) 0.14 0.16% 0.08 0.12% K, Zn, Ca, Mn, Na, khong thit yu, nhiu vitamin, amino acid, enzyme v hp cht alcaloid.

Nguyn V Duy Khanh

7

T nhng nm 1980 n nay, bng cc phng php hin i: ph k UV (t ngoi), IR (hng ngoi), ph k khi lng - sc k kh (GC MS), ph cng hng t ht nhn v c bit l k thut sc k lng cao p (HPLC) cng ph k plasma (ICP), xc nh chnh xc gn 100 hot cht v dn xut trong nm Linh chi. [2] Bng 2.1: Cc hot cht sinh hc v dn xut trong nm Linh chi (Ganoderma lucidum) [19] Thnh phn hot cht ARN **(Khng xc nh) ** Adenosine Nhm cht Nucleic acid Alkaloid Glycoprotein Nucleotide Hot tnh dc l Kch thch h min dch Chng virt B tim c ch khi u Tng s lu thng mu Th gin c, gim au Chng khi u Kch thch h min dch Gim lng ng huyt B tim Chng d ng Ganoderic Acids Triterpenoid Bo v gan c ch tng hp cholesterol Ganodermadiol Adenosine Triterpenoid Nucleotide Gim huyt p c ch ACE Tng s lu thng mu Th gin c, Gim au Chng khi u Tng sn sut khng thNguyn V Duy Khanh

Loi m nm Bo t Qu th Qu th Qu th

Beta D - glucans

Polysaccharide

Qu th

Qu th

Qu th H si nm

Beta D -glucans

Polysaccharide Kch thch h min dch

H si nm

8

Uridine, Uracil Cyclooctasulpher Ling Zhi 8 Ganodosterone Ganoderic Acids Ganodermic Acid T O Oleic Acid

Nucleoside

Phc hi s do dai Chng d ng

H si nm H si nm H si nm H si nm H si nm H si nm H si nm

Protein Steroid Triterpenoid Triterpenoid Acid bo

Chng d ng quang ph iu ho huyt p Bo v gan Bo v gan c ch tng hp cholesterol Chng d ng

Nm 2001, Masao Hattori ly trch c 10 triterpene mi, bao gm lucidumol A v B, cc ganoderic acid: A, B, E, F, H, K, Y v R. Trong kiu Lanostane triterpene c thnh phn chnh l lipophilic. C khong 130 hp cht c ly trch t qu th, h si v bo t nm Linh chi. Thnh phn v hm lng triterpene ph thuc vo ngun ging, yu t mi trng. Vai tr ca triterpene c ngha quan trng trong phng chng cn bnh HIV. [22] Hng lot cc nghin cu ca Shufeng Zhou chng minh rng polysaccharide v triterpene ca nm Linh chi c kh nng cha tr bnh vim gan mn tnh. Ganopoly c ch qu trnh dch m ca ADN polymerase ca virt gy bnh HBV, ngn chn s hot ng ca virt. Ngoi ra polysaccharide v triterpene tc ng hu hiu trong vic iu tr bnh i ng loi 2 (type II diabetes mellitus) cho cc bnh nhn. [14] Nm 1994, Lin Zhibin v Lei Lin Sheng xc nh c trng lng phn t ca Polysaccharide t G.lucidum l khong 7.100 9.300. Nhng tng kt v vai tr sinh dc hc ca nhm polysaccharide cc loi nm Linh chi c gii thiu ti Hi tho Bc Kinh vi cc bo co ca cc tc gi i Loan, Trung Quc, Hoa k.

Nguyn V Duy Khanh

9

He, Y. et al (1992) kho cu cc BN3B - gm 4 polycaccharide ng nht c hot tnh tng min dch. Trong BN3B1 c xc nh l glucan (ch cha glucose) v BN3B3 l mt arabinogalactan mang cc lin kt glycoside. Hikino, H.et al t 1985 n 1989 chng minh hot lc h ng huyt ca nhiu polysaccharide. l cc heteroglycan c c hot tnh chng ung th. Cc ganoderan B c tc dng lm tng mc insuline trong huyt tng, gim sinh tng hp glycogen v gim hm lng glycogen trong gan. y chnh l c s tr liu trn cc bnh nhn i tho ng. Cc phc hp polysaccharide protein c hot tnh chng khi u v tng tnh min dch. Nm 1994, Byong Kak Kim tin hnh lai h si nm bng phng php dung hp Protoplast gia chng G.lucidum vi G.applanatum, thm ch vi c nm hng (Lentinus edodes), qua tng cng hot tnh chng khi u sarcom 180 ca cc phc polysaccharide protein ln ng k. Lei L.S v Lin L.B (1993) chng minh tc dng tng sinh tng hp IL 2 (Interleukine-2) v hot tnh ADN polymerase chut gi tui bi polysaccharide, cng soi sng thm kh nng tr ha, tng tui th ca cc nm Linh chi. Nhng nghin cu v polysaccharide khng tan trong nc cng chng t hiu lc chng khi u rt r, thm ch lm tan khi u vi t l cc loi G.lucidum v G.applanatum (Takashi, 1985; Liu G.T, 1993). [2] C l a dng nht v c tc dng dc l mnh nht l nhm Saponine, triterpenoide v cc acid ganoderic. Vai tr ca cc cht ny ch yu l c ch gii phng histamine, c ch Angiotensine Conversino emzyme (ACE), c ch sinh tng hp Cholesterol v h huyt p. [2, 3, 19] 2.3 KH NNG CHA BNH CA NM LINH CHI Linh chi c dng nh mt thng dc khong t 4000 nm nay Trung Quc v ngi ta cha thy tc dng xu hay c tnh ca Linh Chi. Chng c khng nh l nhn t quan trng cho nhiu loi phn ng chng ung th, d ng, lo ha, x va, ng mu ni mch, gip iu chnh dn truyn thn kinh, bo v cu trc ca nhn t bo.Nguyn V Duy Khanh

10

Ti liu c nht ni tng i c th v kh nng tr liu ca nm Linh chi cng ca L Thi Trn (1595). Theo tc gi trong 6 loi Linh chi th mi loi c c tnh ring. Bng 2.2: Lc bo Linh chi v tc dng tr liu (L Thi Trn, 1590) Tn gi Thanh chi Hng chi Hong chi Bch chi Hc chi Mu sc Xanh Vng Trng en c tnh dc l V chua, tnh bnh, khng c ch tr sng mt, b gan kh an thn, tng tr nh. V ng, tnh bnh, khng c, tng tr nh, dng tim, b trung, cha tr tc ngc. V ngt, tnh bnh, khng c, an thn ch t kh. V cay, tnh bnh, khng c, ch phi, thng mi, an thn, cha ho nghch. V mn, tnh bnh, khng c, tr chng b tiu

Theo cch din t truyn thng ca ngi phng ng, cc tc dng ln ca nm Linh chi nh sau: [1, 7, 8] Kin no (lm sng sut, minh mn) Bo can (bo v gan) Cng tm (thm sc cho tim) Kin v (cng c d dy v h tiu ho) Cng ph (thm sc cho phi, h h hp) Gii c (gii ta trng thi d cm) Trng sinh (tng tui th)

Qua phn tch cc hot cht v mt dc l v s dng nm Linh chi, ngi ta thy Linh chi c tc dng vi mt s bnh: i vi cc bnh tim mch: Nm Linh chi c tc dng iu ho, n nh huyt p. Khi dng cho ngi huyt p cao, nm Linh chi khng lm tng m lm gim bt, dng nhiu th huyt p n nh. i vi nhng ngi suy nhc c th, huyt p thp th nm Linh chi c tc dng nng huyt p ln gn mc d chu nh ci thin, chuyn ho dinh dng. i vi bnh nhim m, x mch, dng nm Linh Chi c tc dng gim cholesterol ton phn, lm tng nhm lipoprotein t trng cao trong mu, lm gim h s sinh bnh. Nm Linh chi lm gim xu th kt b ca tiu cu, gim nng m trongNguyn V Duy Khanh

11

mu, gim co tc mch, gii ta cn au tht tim. Nhiu nghin cu pht hin ra vai tr ca cc nguyn t khong vt him. Vanadium (V) c tc dng chng tch ng cholesterol trn thnh mch. Germanium gip lu thng kh huyt, tng cng vn chuyn oxy vo m. Hin nay, ch s Ge trong cc dc phm Linh chi c xem nh l mt ch tiu quan trng, c gi tr trong iu tr tim mch v gim au trong tr liu ung th. [2, 3] i vi cc bnh v h hp: nm Linh Chi em li kt qu tt, nht l vi nhng ca iu tr vim ph qun d ng, hen ph qun ti 80% c tc dng gim v lm nh bnh theo hng khi hn.[7, 8] Hiu qu chng ung th: Bng vic kt hp cc phng php x tr, ho tr, gii phu vi tr liu nm trn cc bnh nhn ung th phi, ung th v v ung th d dy c th ko di thi gian sng trn 5 nm cao hn nhm khng dng nm. Nhiu thng tin i Loan cho bit nu dng nm Linh chi trng trn g long no iu tr cho cc bnh nhn ung th c t cung t kt qu tt - khi u tiu bin hon ton. Cc nh khoa hc i hc Haifa, Israel khng nh nm linh chi - mt loi nm di thng dng trong ng Y Trung Quc (v Vit Nam) - c th cha ung th tuyn tin lit. Ung th tin lit tuyn l mt trong nhng dng ung th ph bin nht n ng, vi hn 543.000 ngi c chn on mi nm trn ton th gii. Kh nng khng HIV: kho st kh nng khng HIV ca cc hp cht trong nm Ganoderma lucidum, ngi ta s dng dch chit t qu th trong th nghim khng virt HIV 1 trn cc t bo lympho T ngi. S nhn ln ca virt c xc nh qua hot ng phin m ngc trn b mt cc t bo lympho T c gy nhim HIV 1. Kt qu cho thy c s c ch mnh m hot ng sinh sn ca loi virt ny (Gau J.P, 1990; Kim, 1996). Do , nhiu quc gia a Linh chi vo phc iu tr tm thi, nhm tng cng kh nng min dch v nng th trng cho cc bnh nhn trong khi AZT, DDI, DDC, cn him v rt t. Cc nghin cu ti Nht Bn chng minh cc hot cht t nm Linh chi c tc dng nh sau: (Masao Hattori, 2001) Ganoderiol F v ganodermanontirol c hot tnh chng HIV 1

Nguyn V Duy Khanh

12 Ganoderderic acid B v lucidumol B c tc ng c ch hu hiu protease

HIV 1 Ganodermanondiol v lucidumol A c ch pht trin t bo Meth A

(mouse sarcoma) v LLC (mouse lung carcinoma). Ngoi ra cc ganoderma alcohol l lanostane triterpene vi nhm hydroxol (-OH) v tr C25 c kh nng chng HIV 1, Meth A v LLC chut. [2, 22] Kh nng antioxydant: Nhiu thc nghim ch ra vai tr ca cc saponine v triterpenoid, m trong Ganoderic acid c coi l hiu qu nht (Wang C.H, 1985). Nhng nghin cu gn y ang y mnh theo hng lm giu Selenium - mt yu t khong c hot tnh antioxydant rt mnh vo nm Linh chi. Chnh v vy con ngi c th ch i vo mt dc phm tng tui th, tr ho t nm Linh chi ni chung v Linh chi Vit Nam ni ring. [2] Cc hot cht sinh hc trong nm Linh chi c kh nng kh mt s gc t do trong c ch chng no ha, chng ung th. Bo v v chng nh hng ca cc tia chiu x. Linh chi cng c tc dng gip c th thi loi nhanh cc cht c, k c cc kim loi nng nh: Ch, Germanium. 2.4 MT S NG DNG LM SN Tr suy nhc thn kinh: Bnh vin Hoa sn thuc Vin Y hc s 1 Thng hi bo co: Dng c 2 loi Linh chi nhn to v Linh chi hoang di ch thnh vin ( mi vin tng ng 1g thuc sng), mi ln ung 3 vin, ngy 3 ln, mt liu trnh t 10 ngy n 2 thng. Tr 225 ca, t l kt qu 83,5 - 86,3%, nhn xt thuc c tc dng an thn, iu tit thn kinh thc vt v tng cng th lc ( theo bo Tn y hc, s ph chuyn v bnh h thng thn kinh 1976,3:140). Tr chng cholesterol mu cao: Bo co ca S nghin cu khng khun t cng nghip T xuyn, dng lin tc t 1 n 3 thng cho 120 ca thuc c tc dng h cholesterol huyt thanh r rt, t l kt qu 86% ( theo bo co ng trn bo thng tin Trung tho dc 1973,1:31). Tr vim ph qun mn tnh: T nghin cu Linh chi tnh Qung ng bo co dng siro Linh chi v ng Linh chi, tr 1.110 ca c kt qu v c nhn xt l thuc cNguyn V Duy Khanh

13

tc dng i vi th hen v th h hn ( theo t bo co t liu Y dc Qung ng 1979,1:1). Tr vim gan mn tnh: Tc gi dng polysaccarit Linh chi chit xut t Linh chi hoang di ch thnh thuc bt ha nc ung, tr cc loi bnh vim gan mn hot ng, vim gan mn ko di v x gan gm 367 ca, c nhn xt phn ln triu chng ch quan c ci thin, men SGOT, SGPT gim t l 67,7% ( Tp ch Bnh gan mt 1985,4:242). Tr chng gim bch cu: dng polysaccarit ch thnh vin (mi vin c 250mg thuc sng) cho ung, theo di 165 ca, ghi nhn t l c kt qu 72,57% ( Bo co ca Lu ch Phng ng trn tp ch Trung hoa huyt dch bnh 1985,7:428). Tr bnh x cng b, vim da c, bnh liput ban , ban trc: dng Linh chi ch thnh dch, tim bp v vin ung. Tr x cng b 173 ca, t l kt qu 79,1%, vim da c 43 ca, c kt qu 95%, Liput ban 84 ca c kt qu 90%, ban trc 232 ca, c kt qu 78,88% ( Thng tin nghin cu Y hc 1984,12:22). Theo sch Trung dc ng dng lm sng: thuc c tc dng i vi bnh lot bao t, ri lan tiu ha ko di, thng dng phi hp vi Ng bi t, ng sm, Bch trut, Trn b, K ni kim, Sa nhn, Sinh khng. Tr x cng mch, cao huyt p, tai bin mch no: thng phi hp vi K huyt ng, Thch xng b, n b, Cu tch, trng, Th ty t, Hong tinh. Thuc cn dng cha bnh ng mch vnh, au tht ngc. Dng gii c cc loi khun: phi hp vi Cam tho, Gng, To. Ngoi ra sch Nhng cy thuc v v thuc Vit nam ca tt Li c ghi: Thuc cha bnh ph n thi k mn kinh, gip thng minh v tr nh, dng lu ngy gip cho nh ngi, tng tui th. Nhiu ngi mua nm Linh chi v nu canh, nu sp lm mn n cao cp. 2.5 GII THIU S LC V HOT CHT C TRONG NM LINH CHI 2.5.1 Ganoderma polysaccharide (GLPs) [15, 16] C trn 200 loi polysaccharide c ly trch v thu nhn t nm Linh chi nhng polysaccharid gm 2 loi chnh: GL-A: Gal: Glu: Rham: Xyl (3,2: 2,7: 1,8; 1,0)Nguyn V Duy Khanh

M= 23.000 Da

14

GL-B: Glu: Rham: Xyl (6,8: 2,0: 1,0) chnh l Glu, nn gi l Glucan.

M= 25.000 Da

GL-A c thnh phn chnh l Gal, nn gi l Galactan, cn GL-B c thnh phn b (1-3) -D-glucan, khi phc hp vi mt protein, c tc dng chng ung th r rt (Kishida & al., 1988). . Hu ht cc GLPs hnh thnh t 3 chui monosaccharide, c cu trc xon c 3 chiu, ging cu trc ca ADN v ARN. Cu trc xon ny ta trn khung sn cacbon, lng khung sn t 100,000 1000,000, a s chng tn ti pha trong vch t bo (CWM). Mt phn polysaccharide phn t nh khng tan trong cn cao , nhng tan trong nc nng. Ngoi polysaccharide t qu th, polysaccharide cng c thu nhn t qu trnh nui cy trong mi trng dch lng v rn, chng vn c hot tnh sinh hc trong vic cha tr. Polysaccharid c ngun gc t Linh chi dng iu tr ung th c cng nhn sng ch (patent) Nht. Nm 1976, Cty Kureha Chemical Industry sn xut ch phm trch t Linh Chi c tc dng khng carcinogen. Nm 1982, Cty Teikoko Chemical Industry sn xut sn phm t Linh Chi c gc glucoprotein lm cht c ch neoplasm. Bng sng ch M 4051314, do Ohtsuka & al. (1977), sn xut t Linh Chi cht mucopolysaccharid dng chng ung th. 2.5.2 Ganoderic Acid Ganoderic acid c nh hng l mt cyclopropene hoc cyclopentene. Hm lng G.acid thay i theo ging Linh chi, mi trng nui trng, giai on bo t ganodermal. Chnh s thay i ny lm cho mc ng b nh hng. Hm lng G.acid cao th c nhiu v ng. [18, 19] Triterpenoid l nhng hp cht c tng hp t 6 n v isopren. Cc triterpen c b khung chnh t 27 30 nguyn t carbon (C38H48) rt thng gp trong thc vt. Cc triterpenoid tn ti di dng t do (khng c phn ng), c cu trc vng, mang mt s nhm chc nh: -OH; -Oac; eter -O-; Carbanil C=O; ni i C=C. c tnh chung l c tnh thn du (tan tt trong eter du ha, hexan, eter ethyl, cloroform), t tan trong nc ngoi tr khi chng kt hp vi ng to thnh glycosid. [10, 11]Nguyn V Duy Khanh

15

Bng 2.3: Cc hot cht triterpenoid c tc dng cha bnh trong nm Linh chi (Ganoderma lucidum) (L Xun Thm, 1996) Hot cht Ganoderic acid R,S Ganoderic acid B, D, F, H, K, S, Y Ganodermaldiol Ganodermic acid Mf Ganodermic acid T.O Ganodermic acid Hot tnh c ch gii phng histamin H huyt p H huyt p c ch tng hp cholesterol c ch tng hp cholesterol c ch tng hp cholesterol

Triterpenoid c bit l acid ganoderic c tc dng chng d ng, c ch s gii phng histamin, tng cng s dng oxy v ci thin chc nng gan. Hin nay, tm thy trn 80 dn xut t acid ganoderic. Trong ganodosteron c xem l cht kch thch hot ng ca gan v bo v gan. 2.5.3 Ganoderma Adenosine [18, 19] Adenosine thuc nhm purine v l thnh phn chnh trong cu trc nucleic acid. Nm Linh chi c nhiu dn xut adenosine, tt c chng u c hot tnh dc liu mnh. Chc nng ca adenosine: Gim nht mu c ch kt dnh tiu cu Ngn chn hnh thnh cc nghn Tng lng lipoprotein 2 3 phosphricglycerin Gia tng kh nng vn chuyn oxygen, tng lng mu cung cp cho no

Lc mu v tng tun hon mu trong c th 2.5.4 Alcaloid [10, 11] Alcaloid l nhng hp cht hu c c cha nit, a s c nhn d vng, c phn ng kim, chng c cu trc ha hc v hot tnh sinh hc rt a dng.Nguyn V Duy Khanh

16

Cc alcaloid dng t do hu nh khng tan trong nc, thng tan trong dung mi hu c: cloroform, eter diethyl, alcol bc thp. Cc mui ca alcaloid th tan trong nc, alcol v hu nh khng tan trong dung mi hu c nh: cloroform, eter, benzen. Chnh v th, tnh ha tan ca cc alcaloid ng vai tr quan trng trong vic ly trch alcaloid ra khi nguyn liu v trong k ngh dc phm iu ch dng thuc ung. Alcaloid l nhng cht c hot tnh sinh hc, nhiu ng dng trong ngnh y dc v nhiu cht rt c. Cc alcaloid c tc dng rt khc nhau ph thuc vo cu trc ca alcaloid. Tc dng ln h thn kinh Tc dng ln huyt p Tc dng tr ung th 2.5.5 Hp cht Saponin [21, 23] Saponin l mt loi glycosid, c cu trc gm hai phn: phn ng gi l glycon v phn khng ng gi l aglycon. - Saponin triterpenoid: Phn aglycon ca saponin triterpenoid c 30 cacbon, cu to bi 6 n v hemiterpen v chia lm 2 nhm: Saponin triterpenoid pentacyclic: phn aglycon ca nhm ny c cu trc gm 5 vng v phn ra thnh cc nhm nh: olean, ursan, lupan, hopan. Phn ln cc saponin triterpenoid trong t nhin u thuc nhm olean. Saponin triterpenoid tetracyclic: phn aglycon c cu trc 4 vng v phn thnh 3 nhm chnh: dammanran, lanostan, cucurbitan. - Saponin steroid: Gm cc nhm chnh: spirostan, furostan, aminofurostan, spiroalan, solanidan. Hp cht Saponin c nhiu cng dng trong y hc nh: Tr long m, cha ho L cht ph gia trong mt s vc xin Tc dng thng tiu Tc dng khng vim, chng khi u 2.5.6 Germanium hu c [18, 19]Nguyn V Duy Khanh

17

Gemanium l nguyn t him, do nh khoa hc ngi c khm ph vo nm 1885. Germanium c th cung cp mt lng ln oxygen v thay th chc nng ca oxygen. N kch thch kh nng vn chuyn oxygen tun hon mu trong c th ln n 1,5 ln. V th, lm tng mc trao i cht v ngn chn qu trnh lo ha. C th con ngi l thnh phn ca cc electron. Khi mc nng lng tng hoc gim thp, dn n s xo trn cn bng v biu l tnh trng bnh l. Gemanium hu c s duy tr mc nng lng mt cch bnh thng trong c th v bo v sc kho. Khi t bo ung th xut hin, chng lm xo trn qu trnh trao i cht. Gemanium s iu ho v kim sot qu trnh ny, t ngn chn t bo ung th pht trin. Chc nng ca Germanium: Tng cng kh nng mang oxygen v gim nguy c xut hin ung th Gim nguy him t kt qu tr liu phng x Ngn chn bnh thiu mu cc b

Phn 3: VT LIU V PHNG PHP TH NGHIM3.1 THI GIAN V A IM LM TH NGHIMNguyn V Duy Khanh

18

ti c thc hin t 27/09/2008 n 1/1/2009 ti Trng Cao ng Kinh t Cng ngh TP. H Ch Minh 3.2 I TNG NGHIN CU Nm Linh chi (Ganoderma lucidum) 3.3 VT LIU TH NGHIM 3.3.1 Thit b Cn phn tch Knh hin vi My chp nh k thut s T cy v trng Ni hp thanh trng T sy 3.3.2 Ha cht Cc ha cht bao gm: MgSO4, KH2PO4, Glucose, Agar. 3.3.3 Mi trng s dng 3.3.3.1 Mi trng dinh dng Mi trng PGA (Potato glucose agar) Khoai ty: 200 (g) Glucose: 20 (g) Agar: 20 (g) Nc ct va : 1000 (ml) Cc mi trng u c kh trng 1210C trong 15 pht. 3.3.3.2 Mi trng nhn ging Chn loi la go tt, nu cho la va nt nanh, vt ra ro, sau phi trn vi cm bp. 3.4 PHNG PHP TH NGHIM 3.4.1 Quan st hnh thi qu th nm Ganoderma lucidum 3.4.1.1 Hnh thi qu th 3.4.1.2 Phng php quan st h si nm [9] Ly mt t si nm dn u vo 1 git nc c trn lam knh

Nguyn V Duy Khanh

19 C nh si nm trn lam knh bng cch h nh mt di lam knh qua li

trn ngn la n cn Nhum bng dung dch fuchsin trong 5 10 pht Ra thuc nhum bng cn 95% Ra ngay vi nc ct chm dt cng on ty mu Quan st mu vt di vt knh x1003.4.1.3 Phng php quan st bo t nm Linh chi [13, 17]

Ly qu th nm Linh chi trong giai on phng thch bo t, t ln

1 t giy trng thu bo t Dng khuyn cy ly t bo t ri dn u vo 1 git nc trn lam knh

C nh bo t trn lam knh bng cch h nh mt di ca lam knh qua ngn la n cn n kh Quan st bo t nm di vt knh x100

3.4.2 Kho st sinh trng h si nm Linh chi trn cc mi trng lng Cch tin hnh th nghim: Cc mi trng u c pha vi nng nh sau, sau mi trng u hp kh trng 121oC/15 pht. Sau 9 ngy ta thu sinh khi v sy nhit 500C. Sau tin hnh cn trng lng kh Tng s chai cy: 52 chai 3.4.2.1 nh hng ca hm lng MgSO 4 n tc tng sinh khi trong mi trng lng Th nghim c b tr 4 nghim thc, mi nghim thc 4 chai, mi chai cha 20ml mi trng Nghim thc Mi trng nui cy 1 PG + 1 g/l MgSO4 2 PG + 2 g/l MgSO4 3 PG + 3 g/l MgSO4 4 PG + 4 g/l MgSO4 3.4.2.2 nh hng ca hm lng KH2PO4 n tc tng sinh khi trong mi trng lng Th nghim c b tr 4 nghim thc, mi nghim thc 4 chai, mi chai cha 20ml mi trngNguyn V Duy Khanh

20

Nghim thc 1 2 3 4

Mi trng nui cy PG + 0,5 g/l KH2PO4 PG + 1g /l KH2PO4 PG + 1,5 g/l KH2PO4 PG + 2 g/l KH2PO4

3.4.2.3 nh hng ca hm lng Glucose n tc tng sinh khi trong mi trng lng Th nghim c b tr 5 nghim thc, mi nghim thc 4 chai, mi chai cha 20ml mi trng Nghim thc 1 2 3 4 5 Mi trng nui cy P + 10 g/l Glucose P + 15 g/l Glucose P + 20 g/l Glucose P + 25 g/l Glucose P + 30 g/l Glucose

3.4.3 Kho st s nh hng ca pH ln mi trng nui cy PH nh hng rt ln n tc tng trng ca si nm trong mi trng lng. cc gi tr pH khc nhau th tc sinh trng ca si nm khc nhau. iu ny dn n lng sinh khi thu c s ph thuc vo gi tr pH. Do , trong th nghim ny chng ti tin hnh kho st nh hng ca pH n tc sinh trng v pht trin ca si nm Linh chi tm c gi tr pH thch hp cho moi trng lng Cch tin hnh: pha mi trng lng PG (potato glucose) vi nng pH khc nhau v vo chai thu tinh 100ml mt lng mi trng 20 ml. Mi trng c hp kh trng 121oC/15 pht, ngui v cy mt lng ging nht nh vo (mi ln cy gp 1 ht la), nhit phng. Tin hnh thu nhn sinh khi sau 9 ngy. Sinh khi c sy n kh nhit 50oC v tin hnh cn trng lng kh. Th nghim c thc hin vi 7 nghim thc, mi nghim thc 2 chai. Vy tng s chai cy l 14 chai 3.4.4 Nghin cu ng cong sinh trng ca Ganoderma lucidum Chng ti kho st s tch ly sinh khi si nm Linh chi trn mi trng lng PG

Nguyn V Duy Khanh

21

Cch tin hnh: pha mi trng v vo chai thu tinh 0,5 lt mt lng mi trng 20 ml. Hp kh trng 121oC/15 pht, ngui v cy mt lng ging nht nh vo (mi ln cy gp 1 ht la), nhit phng. Tin hnh thu nhn sinh khi cc ngy 3, 6, 9, 12, 15, 21. Sinh khi c sy n kh nhit 50 oC v tin hnh cn trng lng kh. Trong qu trnh th nghim, gm 5 nghim thc, mi nghim thc gm 2 chai. Tng s chai cy l 10 chai. 3.5. PHNG PHP QUY HOCH THC NGHIM 3.5.1. Mc ch Tm iu kin ti u thc hin cc qu trnh hoc chn la thnh phn ti u ca nhiu phn t trong qu trnh nui cy vi sinh vt. Phng php quy hoch thc nghim nhiu yu t cho php thay i ng thi nhiu yu t v xc nh c tng tc gia cc yu t, nh gim bt s th nghim. 3.5.2. Phng php x l s liu Phng trnh hi quy c xy dng t cc kt qu thc nghim. Cc h s trong phng trnh hi quy m t nh hng ca cc yu t nghin cu n mt t bo. nh hng ca cc yu t c nh gi da trn xc xut tin cy hay mc ngha p ca chng. nh hng c coi l rt ln khi p 0,0001, nh hng l ng k khi 0,0001 < p 0,05 v khng ng k khi p > 0,05. Ch c nhng h s biu din nh hng rt ln v ng k mi c gi li trong phng trnh hi qui cui cng. Khi nh hng ca cc yu t hay nh hng tng h gia cc yu t l ng k th h s biu din nh hng ca cc yu t lun c gi li trong phng trnh. Phng trnh hi quy Yi = b0 +b1 x1 + b2 x2 + b3 x3 + b12 x1 x2 + b13 x1 x3 + b23 x2 x3 + b123 x1 x2 x3 Trong Yi: Sinh khi nm thu c (g/l)

Nguyn V Duy Khanh

22 xi (i= 1 k): l cc yu t nh hng, mi yu t c hai mc l hai gii hn

bin ca n. bo, bi, bj, bij: l cc h s trong phng trnh hi quy

n gin trong tnh ton, chuyn t h trc t nhin sang h trc khng th nguyn (h m ho). Xt yu t c k hiu l Zi Zio = ( Zi max + Zimin)/2 Vi: + Zimax: mc cao + Zimin: mc thp + Zio: mc c s im c ta (Z1o, Z2o, Z3o,Zko) gi l tm ca phng n

Vic m ho c thc hin d dng nh chn tm ca min c nghin cu lm gc ta . Phng php x l s liu t cc kt qu thc nghim. Cc h s trong phng

trnh m t nh hng ca cc yu t nghin cu n kh nng tng sinh khi. Phng trnh hi quy Y = b0 +b1 x1 + b2 x2 + b3 x3 + b1b2 x1 x2 + b1b3 x1 x3 + b2b3 x2 x3 + b1b2b3 x1 x2 x3 Vi + Y: sinh khi (g/l) + b0, b1, b2, b3, b12, b13, b23, b123 : cc h s trong phng trnh hi quy + x1 : nng MgSO4 (g/l) + x2 : nng KH2PO4 (g/l) + x3 : nng Glucose (g/l) Bng 3.1: Cc yu t th nghim Cc mc Mc c s Khong bin thin Mc trn Mc diNguyn V Duy Khanh

Cc yu t (g/l) x1

x2

x3

23

Bng 3.2: B tr th nghim S TN 1 2 3 4 5 6 7 8x1 x2 x3

y1

y2

y3

Ytb

+ + + + -

+ + + + -

+ + + + -

3.6 XC NH DC CHT C TRONG NM LINH CHI 3.6.1 Phng php nh tnh alcaloid [11] 3.6.1.1 Chun b dch th pht hin s hin din ca alcaloid trong dc liu ngi ta thng p dng nguyn tc th ca Webb vi cch th gm 2 phn nh sau: - Phn 1: Bt nm Linh Chi xay nhuyn (10 20 gam) v dung dch nc 1% H2SO4 c cho vo erlen, un nh trong 1 gi. Lc v ly dch lc th nghim vi c 2 loi thuc th: Mayer, Dragendorff. Quan st kt ta, nu c kt ta theo qui nh l dng tnh. Tuy nhin, nu khng c kt ta, cha th kt lun l khng c alcaloid m phi tip tc th nghim phn 2. - Phn 2: Bt xay nhuyn (10 20 gam) ngm trong dung dch prollius l hn hp gm: chloroform:ethanol 95o:NH4OH m c, theo t l l 8:8:1 (mi trng phi c tnh baz). Ngm ngui trong 24 gi, nhit phng, thnh thong lc trn. Lc v ui dung mi n cn, thu c cn. Ha tan cn trong dung dch HCl 1% un m cho d tan. Lc v ly dch lc th nghim vi 2 loi thuc th: Mayer, Dragendorff.3.6.1.2 Thuc th nh tnh alcaloid

- Thuc th Mayer: Ha tan 1,36 gam HgCl2 trong 60 ml nc ct v 5 gam KI trong 10 ml nc ct. Thu hn hp 2 dung dch ny li v thm nc ct cho 100 ml.

Nguyn V Duy Khanh

24

Nh vi git thuc th Mayer vo dung dch acid long c cha alcaloid, nu c alcaloid s xut hin ta mu trng hoc vng nht. Cn lu v ta to thnh c th ha tan tr li trong lng tha thuc th hoc ha tan bi ethanol c sn trong dung dch th. - Thuc th Dragendorff: Ha tan 8 gam Nitrat bismuth Bi(NO3)3 trong 25 ml HNO3 30% (D=1,18). Ha tan 28 gam KI v 1 ml HCl 6N trong 5 ml nc ct. Hn hp 2 dung dch ny li yn trong t lnh 5oC s thy ta mu sm xut hin v tan tr li, lc v thm nc cho 100 ml. Dung dch mu cam c cha trong chai mu nu che sng, ct trong t lnh, c th gi lu vi tun. Nh vi git thuc th Dragendorff vo dung dch acid long c cha alcaloid, nu c alcaloid s xut hin ta mu cam nu. 3.6.2 Phng php xc nh hp cht saponin [11] Chit 10 gam dc liu vi cn 70% bng cch ngm trong 24 gi ri lc. C dch lc bc hi n cn kh. Dng cn lm cc phn ng nh tnh. 3.6.2.1 Th nghim tnh to bt Mt c tnh quan trng ca saponin l tnh to bt, nn y l mt trong nhng phng php chnh xc nh tnh s hin din ca saponin. Cch tin hnh: Ha tan mt lng cn tng ng vi 1 gam dc liu vo 5 ml nc nng. Lc vo mt ng nghim 1,6 16 cm v ngui, thm nc cho 10 ml, dng ngn tay ci bt ming ng nghim v lc mnh theo chiu dc ng nghim trong 1 pht (khong 30 ln lc). yn ng nghim, quan st lp bt v nh gi kt qu: Bt bn trong 15 pht: Bt bn trong 30 pht: Bt bn trong 60 pht: 3.6.1.2 Th nghim Fontan Kaudel Ly mt lng cn tng ng vi 1 gam bt dc liu, un nng nh trn cch thy ha tan vi 10 ml nc. Chia u vo 2 ng nghim. ng 1: thm 2 ml HCl 0.1N (pH =1) ng 2: thm 2 ml NaOH 0.1N (pH =13) Bt ming ng nghim v lc mnh theo chiu dc c 2 ng trong 1 pht v yn, quan st cc ct bong bng trong c 2 ng nghim.Nguyn V Duy Khanh

+ ++ +++

25

Nu ct bt trong c 2 ng cao ngang nhau v bn nh nhau, th s b xc nh l c saponin triterpenoid. Nu ng pH = 13 c ct bt cao hn nhiu so vi ng pH = 1, s b xc

nh l c saponin steroid. 3.6.3 nh tnh triterpenoid (bng phn ng Liebermann Burchard) Chit 10 20 gam bt dc liu bng diethylether lc trong bnh nn, trong 10 20 pht, chit cho ti khi dch ether sau khi bc hi khng cn li lp cn m trn mt knh ng h, gp cc dch chit, lc v c li n khi cn khong 50 ml dch chit ether. Ly 5 ml dch chit ether cho vo chn s, bc hi ti cn. Ha tan cn vi 0,5 ml anhydrid acetic, ri thm vo dung dch 0,5 ml chloroform. Chuyn dung dch vo 1 ng nghim nh kh, dng pipet pasteur thm cn thn 1 2 ml H2SO4 m c ln thnh ng nghim nghin cho acid chy xung y ng nghim. Ni tip xc gia 2 lp dung dch c mu nu hay n tm, lp pha dung dch trn dn dn chuyn thnh mu xanh lc hay tm. Kt lun c triterpenoid 3.6.4 nh tnh acid hu c Ly 2 ml dch chit nc cho vo mt ng nghim. Thm vo dung dch mt t tinh th natri Na2CO3. Nu c cc bt kh nh si ln t cc tinh th Na2CO3 th kt lun l c acid hu c. 3.7 NH LNG POLYSACCHARIDE (GLPs) [15, 16] Polysaccharide c nhiu dng v nhiu qui trnh chit khc nhau. Chit GLPs 100oC trong 16 gi, cho nng sut ly trch cao, nhng lm bin i cu trc sinh hc cc polysaccharides c trong nm Linh chi. Mt qui trnh th hai c ng dng rng ri chit cc GLPs nhit thp, nhm n nh cu trc sinh hc ca cc GLPs.

Nguyn V Duy Khanh

26 Sinh khi nm Linh Chi

Nghin -Ngm nc 70oC trong 3 gi Hn hp chit rt ln 1

Dch chit ln 1

B chit ln 1 -Ngm trong nc 70oC trong 3 gi Hn hp chit rt ln 2

Dch chit ln 2

B chit ln 2 -Ngm trong cn 80% 70oC trong 2 gi Dch chit ln 3

Hnh 3.1: Qui trnh chit sut polysaccharides t nm Linh chi (Yihuai Gao v ctv, 2001) Thu nhn c 3 dch chit v em i lc. Ly phn cn (dch l lng sau khi lc) em i sy kh v cn trng lng. T nh gi hm lng polysaccharide th c trong qu th nm Linh chi en. 3.8 PHNG PHP X L S LIU S liu c x l thng k v v biu bng phn mm Excell. Tc sinh trng trung bnh ca h si nm trn cc mi trng c tnh nh sau: Xi = (x1 + x2 + x3 + + xi)/ni Vi: - xi: Tc sinh trng trn cc mi trng (cm/ngy)Nguyn V Duy Khanh

27

- ni: S quan st S dng phn mm Statgraphics Ver. 7.0 so snh s khc bit v tc tng trng h si nm trn cc nghim thc th nghim mc = 0,05 hay LSD (95%) (Least Significant Difference).

Nguyn V Duy Khanh

28

Phn 4: KT QU V BN LUN4.1 QUAN ST HNH THI GII PHU CA NM LINH CHI (GANODRMA LUCIDUM) 4.1.1 Hnh thi qu th nm Linh chi

Hnh 4.1 : Hnh thi nm Linh chi Nhn xt: Th qu ca nm Linh chi thu hi c c cung ngn, nh bn. cung nm mp kho (ng knh 2- 3 cm), lp v cung lng , nu. M nm hnh qut, i khi c hnh d dng, mt m nm lng, nu, c vn gn ng tm v c tia phng x, mu sc khi non th c th vng chanh vng cam cho n ti, cng v gi th mu cng sm li. Kch thc t 3 12 cm, dy t 1 3,5 cm. Phn nh cung g ln hoc lm xung. Mt di th qu thng mu trng kem hi vng, c nhiu l nh. y l lp bo tng sinh sn ca nm, chnh nhng l ny l ni phng thch bo t khi qu th trng thnh. Mt l khong 3 5 ng/mm2, ming l gn trn. Khi b i qu th ra th thy y l nhng ng thng, chiu dy t 0,3 1,5 cm. Theo cc nh nghin cu th bn trong thnh ng c th quan st thy m bo t cha ra ngoi di knh hin vi. Khi bo t m chn th n c phng thch dc theo ng ny v thot ra ngoi qua ming l ng. Cn phn tht nm trn th c mu vng nu n nu . Phn cch gia phn trn v phn di (tng sinh sn) l kh r rng v phn trn th cc lp tia si hng ln, cn phn di th l ng thng hng xung.

Nguyn V Duy Khanh

29

4.1.2 H si nm Ganoderma lucidum

Hnh 4.2 : Hnh thi si nm Linh chi (vt knh du x100)4.1.3 Cu trc bo t nm Ganoderma lucidum

Bo t m c dng trng, ta c thy r trn c 2 hnh u c u chp trn - nhn, phng cng. chnh l chp ph l ny mm ca bo t. Hnh trn cn cho thy r lp v ca chng c cu trc v kp, c kin to l thng (porus, lacunae) trn b mt v ngoi ng nh cc m t v loi nm Linh chi chun. Theo cc nh khoa hc th y l c im c trng ca cu trc bo t nm Linh chi. Mu sc ca bo t 2 hnh l tng nhau l mu vng nu, chnh gia khi ni cht t li dng git du c mu nht hn. Kch thc bo t khong 8 11 x 6 8 m

Hnh 4.2 : Cu trc bo t nm Ganoderma lucidum4.2 S SINH TRNG V PHT TRIN CA NM LINH CHI (GANODERMA

LUCIDUM) 4.2.1 Tc sinh trng h si nm Ganodrma lucidum trn cc mi trng lng 4.2.1.1 nh hng ca hm lng MgSO4 n tc tng sinh khi nm Linh chi trong mi trng lng Mg2+ l khong cht cn thit cho s hot ng mt s loi enzym, gip qu trinh sinh tng hp xy ra nhanh hn. Trong th nghim ny chng ti s dng MgSO4 ccNguyn V Duy Khanh

30

hm lng khc nhau tm c hm lng MgSO4 cn thit qu trnh to sinh khi xy ra nhanh nht. Th nghim c b tr 4 nghim thc, mi nghim thc 4 chai, mi chai cha 20ml mi trng Bng 4.1 : nh hng ca MgSO4 ln mi trng Nghim thc NT1 NT2 NT3 NT40.5 khi lng sinh khi (g/20ml) 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 NT1 NT2 NT3 NT4 Nghim thc

Mi trng nui cy PG + 1 g/l MgSO4 PG + 2 g/l MgSO4 PG + 3 g/l MgSO4 PG + 4 g/l MgSO4

Khi lng (g/20ml) 0,178 0,346 0,466 0,379

Biu 4.1: nh hng ca MgSO4 ln tng sinh khi Nhn xt: Da vo biu 4.1 chng ti nhn thy khi s dng MgSO4 mc 1g/l th sinh khi pht trin chm nht v khi tng dn hm lng MgSO4 ln 3g/l th lng sinh khi thu c cao nht 0,466 g/20ml v khi tng hm lng MgSO4 ln 4g/l th lng sinh khi gim ng k ch cn 0,379 g/20ml iu ny c th khi tng Mg2+ vt qu mc chu ng ca t bo, s gim cc phn ng tng hp trong t bo nm, c ch s hot mt s enzyme cn thit trong qu trnh tng hp t bo

Nguyn V Duy Khanh

31

4.2.1.2 nh hng ca hm lng KH2PO4 n tc tng sinh khi trong mi trng lng KH2PO4 l ngun cung cp kali v photphat cho nm. Ngun kali ng vai tr lm ng yu t (cofactor), cung cp cho cc loi enzym hot ng. ng thi ng vai tr cn bng khuynh (gradient) bn trong v ngoi t bo. Ngun phosphate tham gia tng hp ATP, acid nucleic, phospholipid mng. V vy chng ti tin hnh kho st hm lng KH2PO4 thch hp cho mi trng nui cy sinh khi nm Linh chi Th nghim c b tr 4 nghim thc, mi nghim thc 4 chai, mi chai cha 20ml mi trng Bng 4.2: nh hng ca KH2PO4 n tc tng trng Nghim thc NT1 NT2 NT3 NT40.5 khi lng sinh khi (g/20ml) 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 NT1 NT2 NT3 NT4 Nghim thc

Mi trng nui cy PG + 0,5g/l KH2PO4 PG + 1g/l KH2PO4 PG + 1,5g/l KH2PO4 PG + 2g/l KH2PO4

Khi lng (g/20ml) 0,322 0,475 0,449 0,364

Biu 4.2: nh hng ca KH2PO4 n tc tng sinh khi Nhn xt : Da v biu 4.2 chng ti nhn thy khi tng hm lng KH2PO4 ln dn th lng sinh khi tng. Mt khc, khi hm lng KH2PO4 s dng vt qu mc chu ng ca t bo nm th lng sinh khi thu c gim. Lng sinh khi thu c cao nht lNguyn V Duy Khanh

32

0,475 g/20ml khi lng KH2PO4 l 2 g/l. Khi lng KH2PO4 l 1 g/l th lng sinh khi thu dc thp nht 0,322 g/20ml 4.2.1.3 nh hng ca hm lng Glucose n tc tng sinh khi trong mi trng lng Glucose c cha ngun cacbon dng tng hp nn cc cht: hydratcacbon, amino acid, acid nucleic, lipid cn thit cho s pht trin ca nm. Trong sinh khi nm, cacbon chim na trng lng kh, ng thi ngun cacbon cung cp nng lng cho qu trnh trao i cht. i vi cc loi nm khc nhau th nhu cu cacbon cng khc nhau. Th nghim ny chng ti kho st hm lng Glucose nh hng ti tc d to sinh khi si nm Linh chi trn mi trng lng Th nghim c b tr 5 nghim thc, mi nghim thc 4 chai, mi chai cha 20ml mi trng Bng 4.3: nh hng ca Glucose n tc tng trng Nghim thc NT1 NT2 NT3 NT4 NT5 Mi trng nui cy P + 10 g/l Glucose P + 15 g/l Glucose P + 20 g/l Glucose P + 25 g/l Glucose P + 30 g/l Glucose Khi lng (g/20ml) 0,389 0,414 0,435 0,247 0,215

Nguyn V Duy Khanh

33

Khi lng sinh khi (g/20ml)

0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 NT1 NT2 NT3 Nghim thc NT4 NT5

Biu 4.3: nh hng ca Glucose n tc tng sinh khi Nhn xt: Da vo biu ta thy khi tng hm lng Glucose ln th sinh khi tng theo n gi tr Glucose = 20g th sinh khi thu c 0,4345g, Hm lng Glucose cng tng th lng sinh khi thu c cng gim. Vy hm lng Glucose = 20g l gi tr tt nht cho mi trng lng nui cy sinh khi Kt lun : Da vo kt qu ghi nhn c th i vi hm lng MgSO4= 3g, KH2PO4 = 1g, glucose = 20g th hm lng sinh khi pht trin nhiu nht. Nh vy i vi mi trng lng c b sung hm lng khong cht trn ta s thu c lng sinh khi t gi tr cc i 4.3 NH HNG CA PH LN MI TRNG CY Hu ht mi trng nui cy u c pH ti thch cho qu trnh pht trin ca tng ging. S tng gim pH (chnh l s tng hoc gim H+) trong mi trng nui cy s nh hng n s pht trin ca h si t nm Linh chi. Chng ti tin hnh cy ging trong chai thu tinh 100ml, cha mt lng mi trng l 20ml. Trc khi hp thanh trng, ta chnh pH mi trng trong cc mu v cc

Nguyn V Duy Khanh

34

gi tr : pH =3,5 ; pH =4; pH =4,5; pH =5; pH =5,5; pH =6,8; pH =7. Sau tin hnh cy ging bng cch gp mt ht la cho vo chai. Bng 4.4 : nh hng pH ln tc to sinh khi ca Linh Chi pH TN 1 2 TB 3,5 0,193 0,111 0,152 4 0,225 0,241 0,233 4,5 0,348 0,290 0,319 5 0,333 0,366 0,349 5,5 0,344 0,386 0,365 6,8 0,346 0,336 0,341 7 0,278 0,270 0,274

Khi lng sinh khi (g/20ml)

0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 3,5 4 4,5 5 pH 5,5 6,8 7

Biu 4.4: nh hng ca pH ln s tng sinh khi ca Ganoderma lucidum Nhn xt Da vo biu trn chng ti nhn thy sinh khi t nm c kh nng tng trng tt trong khong pH = 5,5. Khi nui cy trong m trng c pH = 3,5 th khi lng sinh khi thp nht 0,152 g/20ml. Sinh khi tng dn theo gi tr ca pH, nhng khi tng gi tr pH 6,8 th sinh khi gim 0,341 g/20ml v gim dn cho n pH = 7 trong cng mt iu kin nui cy. Kt lunNguyn V Duy Khanh

35

Nh vy chng ti chn c gi tr pH = 5,5 l gi tr pH ti thch cho s pht trin ca t nm vi lng sinh khi cao nht t 0,365 g/20ml 4.4 NG CONG SINH TRNG CA NM LINH CHI Qu trnh sinh trng ca t nm phi tri qua 4 giai on: giai on thch nghi, giai on pht trin, giai on cn bng v giai on gi ci. Trong th nghim ny, chng ti tin hnh nui cy sinh khi trong trong nhng chai nh 100ml, cha 20ml mi trng PG + MgSO4 + KH2PO4. iu chnh v pH 5,5 ri sau em i hp thanh trng. Mi ln cy gp 1 ht la cho vo chai. Khong thi gian ly mu l cch nhau 3 ngy. Mu c sy kh 500C - 600C sau i cn v ghi nhn s liu. Bng 4.5: Khi lng sinh khi nm Linh chi thay i theo thi gian khi nui cy trong mi trng lng Thi gian (ngy) TN LN 1 LN 2 TB 3 NGY 0,114 0,144 0,129 6 NGY 0,167 0,148 0,158 9 NGY 0,254 0,319 0,286 12 NGY 0,533 0,451 0,492 15 NGY 0,751 0,809 0,780 18 NGY 0,814 0,863 0,839 21 NGY 0,754 1,009 0,881

Nguyn V Duy Khanh

36

Khi lng sinh khi (g/20ml)

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 3 6 9 12 Ngy 15 18 21

th 4.1: ng cong sinh trng ca Ganoderma lucidum Nhn xt ng cong sinh trng l mt yu t quan trng trong qu trnh nui cy sinh khi si nm Linh chi. Da vo th ta bit chnh xc c khong thi gian no sinh khi pht trin mnh nht, lc no sinh khi n nh v lc sinh khi bt u gi i. T ng cong sinh trng chng ti nhn thy : Trong 6 ngy u, t nm thch nghi vi mi trng, sinh khi tng ln rt t

n ngy th 12, sinh khi pht trin nhng khng nhanh, t nm dn dn chuyn sang mu vng

T ngy 12 n ngy 15, sinh khi pht trin vt bc T ngy 15 tr i sinh khi tng rt chm, t nm t vng nht chuyn sang mu vng m 4.5 TI A HA THNH PHN MI TRNG LNG NUI CY H SI GANODERMA LUCIDUM BNG PHNG PHP THC NGHIM Chng ti xc nh in kin ti a thu c sinh khi cc i, chng ti tin hnh ti a ho thnh phn mi trng nui cy vi sinh vt theo phng php qui hoch thc nghim v tin hnh nh sau :

Nguyn V Duy Khanh

37

Xt k=3 nhn t nh hng dn s pht trin ca nm (Glucose, KH2PO4, MgSO4) v mi th nghim c lp li 3 ln Hm mc tiu Y l sinh khi Ganoderma lucidum Bng 4.6 : Mc bin thin ca cc nhn t sinh trng Cc mc Mc c s Khong bin thin Mc trn Mc di Trong Cc yu t (g/l) x1 3,0 0,5 3,5 2,5

x21,0 0,2 1,2 0,8

x320 2 22 18

x1 : Hm lng MgSO4 b sung vo mi trng khoai ty ( g/l) x2 : Hm lng KH2PO4 b sung vo mi trng khoai ty ( g/l) x3 : Hm lng glucose b sung vo mi trng khoai ty ( g/l) y1 : Khi lng sinh khi Ganoderma lucidum ln lp th 1 (g/20ml) y2 : khi lng sinh khi Ganoderma lucidum ln lp th 2 (g/20ml) y3 : khi lng sinh khi Ganoderma lucidum ln lp th 3 (g/20ml) Ytb : l khi lng trung bnh ca sinh khi Ganoderma lucidum trong 3 ln lp li (g) Bng 4.7 : kt qu th nghim theo phng php thc nghim S TN 1 2 3 4 5 6 7 8 S2 = GTT = f = K-1=2 => Gtb = => GTT< Gtbx1 x2 x3

3,5 2,5 3,5 2,5 3,5 2,5 3,5 2,5

1,2 1,2 0,8 0,8 1,2 1,2 0,8 0,8

22 22 22 22 18 18 18 18

y1 0,165 0,170 0,170 0,166 0,175 0,191 0,176 0,158

Y2 0,167 0,171 0,174 0,181 0,176 0,170 0,192 0,180

Y3 0,172 0,177 0,176 0,180 0,18 0,179 0,190 0,181

Ytb 0,168 0,173 0,173 0,176 0,177 0,180 0,187 0,173

Nguyn V Duy Khanh

38

4.6 XC NH HP CHT C TRONG NM LINH CHI (GANODERMA LUCIDUM) 4.6.1 nh tnh alkaloid Th nghim 1 Cho dch chit bt t nm Linh chi tc dng vi thuc th Mayer v thuc th Dragendoff ng 1 : dch chit t nm Linh chi + thuc th Dragendoff ng 2: dch chit t nm Linh chi + thuc th Mayer 1 2

Hnh 4.5: nh tnh alcaloid vi thuc th Mayer v Dragendoff Kt qu Da v kt qu th nghim trn ta thy ng 1: Khi dch chit tc dng vi thuc th Dragendoff th dung dch chuyn sang mu nu nhng khng c kt ta ng 2: Dch chit c mu vng nht, xut hin kt ta trng Th nghim 2

1

2

Nguyn V Duy Khanh

39

Hnh 4.6: nh tnh alcaloid vi thuc th Mayer v Dragendoff Kt qu ng 1: xut hin mu vng nhng khng c kt ta ng 2: mu hi c xut hin kt ta nhng vi hm lng rt t Nhn xt: Dch chit nm Linh chi en phn 1 em th nghim vi 2 loi thuc th Mayer v Dragendorff th nhn thy kt qu l dng tnh. i vi dch chit phn 2 em th nghim th kt qu l m tnh: Bt dc liu trch vi nc acid: c th trch ht tc c cc alcaloid dng baz t do (N s bin thnh NH+ tan trong nc), alcaloid dng th cp N+, dng N oxid (N+ -> O), dng glycosid, alcaloid loi c tnh phn cc mnh, nhng s trch lun nhng hp cht c cha nit (protein, glycoprotein, nucleotide) l nhng hp cht tuy khng phi l alcaloid nhng c th cho kt qu dng tnh vi thuc th. Bt dc liu trch vi dung mi hu c kim s khng trch c nhng alcaloid dng N oxid, dng N t cp, dng tan tt trong nc. Phng php ny trch tt cc alcaloid dng baz t do c tnh phn cc km v tnh baz yu, cng nh cc alcaloid c cu trc c th C=C N . Kt lun: hot cht alcaloid c trong t nm Linh chi vi hm lng rt thp nn nhng phng php nh tnh khng thy c s hin din ca chng 4.6.2 nh tnh saponin 4.6.2.1 Th nghim tnh to bt

Nguyn V Duy Khanh

40

Hnh 4.7: th nghim tnh to bt t sinh khi nm Lnh chi Kt qu: bn ca bt Kt qu Sau 15 pht + Sau 30 pht ++ Sau 60 pht +++ bn ca bt u bn vng sau 60 pht. Nh vy dc liu t sinh khi c cha hot cht saponin 4.6.2.2 Th nghim Fontan Kaudel ng 1: dch chit sau khi cho acid vo ng 2: dch chit sau khi cho bazo vo 1 2

Hnh 4.8: Th nghim saponin ton phn theo Fontan Kaudel Kt qu Bt trong 2 ng nghim u bn hn 15 pht, bt trong ng nghim 2 cao hn gp 3 ln ct bt trong ng nghim 1 Nh vy, trong mi trng kim bt bn hn trong mi trng acid. S b kt lun l c saponin steroid.Nguyn V Duy Khanh

41

4.6.3 nh tnh triterpenoid 1 2

Hnh 4.9: nh tnh triterpenoid bng phn ng Liebermann Burchard Kt qu th nghim nhn thy sinh khi v qu th nm Linh chi c cha hot cht triterpenoid (mi trng chuyn sang mu xanh lc sau khi cho acid H2SO4 m c vo t t). ng 1(i chng): nc ct + acid ng 2: Dch chit + acid m c 4.6.4 nh tnh acid hu c

Hnh 4.10 : nh tnh acid hu c c trong qu th Linh chi. Sau khi cho dch chit nc bt t v qu th nm Linh chi tc dng vi tinh th Na2CO3 v h nng th nhn thy c bt kh xut hin rt t v bay ra. Nh vy, dch chit nc t t nm Linh chi c cha hm lng rt t thnh phn acid hu c.

4.6.5 nh lng polysaccharide t sinh khi nm Linh chi

Nguyn V Duy Khanh

42

Tin hnh ly trch polysaccharides t 10 gam nm Linh chi sy kh theo qui trnh th nghim. Sau qu trnh lc v sy kh thu nhn c 0.3144 gam polysaccharide th. Nh vy hm lng polysaccharide th c trong qu th nm Linh chi ch t khong 0.31%.

Hnh 4.11 : hnh nh polysaccharide kt ta sau khi lc

Phn 5: KT LUNNguyn V Duy Khanh

43

5.1. KT LUN

Mi trng lng vi hm lng glucose (18g), KH2PO4 (0,8g), MgSO4 Vi gi tr pH= 5,5 sinh khi pht trin tt nht Thi gian thua hoch l 14 ngySinh khi h si (GAnoderma lucidum) c cha cc thnh phn ha hc:

(3.,5g) thch hp cho sinh khi Ganoderma lucidum t dc hiu sut cao

Saponine, saponin triterpenoid, acid bo v hm lng polysaccharide th ly trch c trong sinh khi nm Linh chi l khong 0,31% 5.2 NGH

V thi gian thc hin ti c hn nn ti cha th nghin cu y v nm Linh chi (Ganoderma lucidum), ngh nghin cu thm v: Kho st su hn tc dng ca mt s khong cht khc v thnh phn ha hc, tc dng dc l, lm sng. Nhm hon thin quy trnh nui cy trong mi trng lng v c th ng dng trong ngnh dc v ngnh sn xut tr tho dc.

Phn 6: TI LIU THAM KHONguyn V Duy Khanh

44

Ti liu ting vit1. on Sng, 2003 Linh chi nguyn cht v bnh thi nay. Nh xut bn Y hc, H

ni, Vit nam.2. L Xun Thm, 1996. Nghin cu c im sinh hc v c in hp thu khong

nm Linh chi Ganoderma lucidum (Leyss.ex Fr).Karst. Lun n ph tin s khoa hc sinh hc, i hc Khoa Hc T Nhin, i hc Quc Gia H ni, Vit nam.3. L Xun Thm, 1996. Nm Linh chi - dc liu qu vit nam. Nh xut bn mi

c mau.4. L Duy Thng, 2001. K thut nui trng nm n, tp 1. Nh xut bn Nng

nghip.5. Nguyn Ln Dng, 2001. Cng ngh nui trng nm, tp 1 v 2. Nh xut bn

Nng nghip, H ni.6. Nguyn Hu ng, 2003. Nui trng ch bin nm n v nm lm thuc cha

bnh. Nh xut bn Nng nghip, H ni.7. Nguyn Hu ng v inh Xun Linh, 2000. Nm n nm dc liu - cng dng

v cng ngh nui trng. Nh xut bn H ni.8. Nguyn Hu ng, Nguyn Th Sn v Zani Federico, 2002. C s khoa hc v

cng ngh nui trng. Nh xut bn Nng nghip, H ni.9. Nguyn c Lng, Phan Th Huyn v Nuyn nh Tuyt, 2003. Th nghim

cng ngh sinh hc, tp 2, th nghim vi sinh vt hc. Nh xut bn i hc Quc Gia TPHCM.10. Nguyn Phc Nhun, 2001. Gio trnh sinh ho hc, phn 1. Nh xut bn i

hc Quc Gia TPHCM.11. Trn Hng, 2004. Phng php nghin cu dc liu. i hc Y Dc TP.HCM. 12. Trn Vn Mo, 2004. Nui trng ch bin nm n v nm lm thuc cha bnh. Nh xut bn Nng nghip, H ni.

Ti liu ting anh

Nguyn V Duy Khanh

45 13. Steyaert R.L, 1972. Species of Ganoderma and related genera mainly of the boyor

and leiden herbaria. National de Beigique, Burxelle.14. Shufeng Zhou, A clinical Study of a Ganoderma lucidum extract in patients with

type II diabetes mellitus. Division of Pharmacology and Clinical Pharmacology, Faculty of Medicine and Health Science, Auckland University, Auckland, New Zealand.15. Yihuai Gao, Guoliang Chen, Jin Lan, He Gao and Shufeng Zhou, 2001. Extractoin

of Ganoderma polysaccharides at relatively low temperature. Froc Int Symposium Ganoderma Sci, Auckland.16. Yihuai Gao, Jin Lan and Zhifang Liu, Extraction and determination of Ganoderma

polysaccharides. Int Med Complement Med Vol 1, Supplement 1,00-00.17. Zhaoji Ding, 1980. The Ganodermataceae in chine. Berlin Shiffigart.

Ti liu internet 18. http://www.About Lingzhi.htm19. http://www .activeCompoundsReishi.html 20. http://www. Biomed~1.htm 21. http://www.bfa-ernaehrung.de/Deutsch/Information/pflanzenstoffe/artikel3.pdf 22. http://www. Mushroom Information Gano HIV.mht 23. http://203.162.44.79/khcn/magazine/Tap7/So6/06.htmnh5

Nguyn V Duy Khanh