Dibenzilbutirolakton lignánok azonosítása, bioszintézisük nyomon követése Arctium, Centaurea, Cirsium fajok terméseiben és mennyiségi meghatározása Centaurea termésekben és in vitro sejttenyészeteiben Szokol-Borsodi Lilla Témavezetők: Dr. Böddi Béla tanszékvezető egyetemi tanár az MTA doktora és Dr. Gyurján István† professor emeritus az MTA doktora ELTE Biológia Doktori Iskola Vezetője: Dr. Erdei Anna Kísérletes Növénybiológia Doktori Program Programvezető: Dr. Szigeti Zoltán ELTE Növényszervezettani Tanszék Budapest 2013 Id. Dr. Béres József iránymutató gondolata: RÖVIDÍTÉSEK JEGYZÉKE ················································································································· 4 II. IRODALMI BEVEZETŐ ··················································································································· 6 II.1. A lignánok általános jellemzői ···································································································· 6 II. 2. A lignánok előfordulása ·············································································································· 9 II.3. A lignánok bioszintézise ············································································································ 12 II.4. A lignánok gyógyászati hatásai és hatásmechanizmusai ··························································· 14 II.5. Glikozidok és aglikonok in vivo: a növényi β-glikozidáz enzim ·············································· 18 II.6. Kémiai analízis ·························································································································· 19 II.6.1. Extrakció ····························································································································· 19 II.6.2 Minőségi meghatározás ······································································································· 19 II.8. A lignánok mennyiségi és minőségi változása a termés érése során ········································· 23 II.9. Centurea fajok in vitro szövettenyészete ··················································································· 23 II.10. A hatóanyagtermelés és az organogenezis összefüggései ······················································· 25 III. CÉLKITŰZÉSEK ··························································································································· 27 IV. ANYAGOK ÉS MÓDSZEREK ····································································································· 28 IV.1. Növényi minták ···························································································································· 28 IV.1.1. Növényi minták ················································································································· 28 IV.1.2. A termésérés folyamata ····································································································· 29 IV.1.3. A csírázás folyamata ········································································································· 29 IV.1.4. A kaszattermés részeinek szétválasztása ··········································································· 30 IV.2. Vegyszerek, reagensek ············································································································· 30 IV.3. Növényi szövettenyésztés ········································································································ 30 IV.3.1. A különböző Centaurea fajok kallusztenyészeteinek létrehozása és fenntartása ············· 30 IV.3.2. A munkában használt táptalajok összetétele ····································································· 31 IV.3.4. A sejkultúra biomassza növekedésének vizsgálata ··························································· 31 IV.4. Mikroszkópos vizsgálatok ········································································································ 32 IV.4.1. Transzmissziós elektronmikroszkópos vizsgálatok ······························································ 32 IV.4.2. Pásztázó elektronmikroszkópos vizsgálatok ····································································· 32 IV.4.3. Fénymikroszkópos és sztereomikroszkópos vizsgálatok ·················································· 32 IV.4.4. A mikroszkópos minták értékelése ··················································································· 32 1 IV.5. Kémiai analízis ························································································································· 33 IV.5.1. Lignán extrakció ··············································································································· 33 IV.5.2. Nagyhatékonyságú folyadékkromatográfiás (HPLC) elválasztás ultraibolya spektrofotometriás (UV) és tömeg spektrometriás (MS) detektálással ········································· 33 IV.6. Glikozidáz enzimes hidrolízis ·································································································· 33 IV.7. Savas hidrolízis ························································································································ 34 IV.8. Az eredmények statisztikai értékelése ····················································································· 34 V. EREDMÉNYEK ······························································································································ 35 V.1.A lignánok azonosítása és mennyiségi meghatározása ······················································ 35 V.1.1. A lignánösszetétel folyadékkromatográfiás meghatározása ··············································· 35 V.2. A lignánösszetétel változása a termés érése és csírázása során ················································· 41 V.2.1. A lignánösszetétel változása a termésérés során ································································ 41 V.2.2. A lignánösszetétel változása a csírázás során és a lignánok megoszlása a kaszattermés termésfala és az embrió részei között: natív, csíráztatott, és glikozidáz enzimmel hidrolizált minták ············································································································································ 43 V.3. A Centaurea fajok terméseinek lignánösszetétel vizsgálata ······················································ 48 V.3.1. A natív minták lignánösszetétele ························································································ 48 V.3.2. A hidrolizált minták lignánösszetétele ··············································································· 51 V.4. A Centaurea fajok szövettenyészeteinek létrehozása, lignán összetételének és mennyiségének vizsgálata, valamint morfológiája ····································································································· 51 V.4.1. A Centaurea fajok szövettenyészeteinek létrehozása ························································ 52 V.4.2. A Centaurea adjarica biomassza termelése ······································································· 52 V.4.3. Az in vitro kallusz kultúrák differenciációja ······································································ 53 V.4.3. Az in vitro kallusz kultúrák lignán összetételének és mennyiségének vizsgálata, valamint az egyes kultúrák differenciálódott részeinek lignán tartalma ······················································ 61 VI. AZ EREDMÉNYEK MEGVITATÁSA ························································································· 65 VI.1. A lignánok azonosítása és mennyiségi meghatározása ···························································· 66 VI.1.1. A lignánösszetétel folyadékkromatográfiás meghatározása ············································· 66 VI.2. A lignánösszetétel változása a termés érése és csírázása során ··············································· 67 VI.2.1. A lignánösszetétel változása a termésérés során ······························································· 67 2 VI.2.2. A lignánösszetétel változása a csírázás során és a lignánok megoszlása a kaszattermés termésfala és az embrió részei között: natív, csíráztatott, és glikozidáz enzimmel hidrolizált minták ············································································································································ 68 VI.3. A Centaurea fajok terméseinek lignánösszetétel vizsgálata ····················································· 69 VI.3.1. A natív minták lignánösszetétele ······················································································ 69 VI.3.2. A hidrolizált minták lignánösszetétele ·············································································· 71 VI.4. A Centaurea fajok szövettenyészeteinek létrehozása, lignán összetételének és mennyiségének vizsgálata, valamint morfológiája ····································································································· 71 VI.4.1. A Centaurea fajok szövettenyészeteinek létrehozása ······················································· 72 VI.4.2. Az in vitro kallusz kultúrák differenciációja ····································································· 72 VI.4.3. Az in vitro kallusz kultúrák lignán összetételének és mennyiségének vizsgálata, valamint az egyes kultúrák differenciálódott részeinek lignán tartalma ······················································