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Afrikkalaisten Combretum- Ja Terminalia-Lajien Uutteet, Fraktiot Ja Yhdisteet Ja Niiden Antimikrobiset Ominaisuudet

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Afrikkalaisten Combretum- Ja Terminalia-Lajien Uutteet, Fraktiot Ja Yhdisteet Ja Niiden Antimikrobiset Ominaisuudet AFRIKKALAISTEN COMBRETUM- JA TERMINALIA-LAJIEN UUTTEET, FRAKTIOT JA YHDISTEET JA NIIDEN ANTIMIKROBISET OMINAISUUDET Satu Helenius Pro gradu Helsingin yliopisto Farmasian tiedekunta Farmaseuttisen biologian osasto Joulukuu 2013 SISÄLLYS Lyhenteet ......................................................................................................... 3 I KIRJALLISUUSKATSAUS .................................................................... 1 1. JOHDANTO ................................................................................................ 1 2. AFRIKKALAINEN TERVEYDENHUOLTO ................................................ 2 2.1. Johdatus afrikkalaiseen terveydenhuoltoon ................................................. 2 2.2. Perinteinen lääketiede Afrikassa ................................................................. 5 2.3. Kasvilääkinnän merkitys afrikkalaisessa terveydenhuollossa ........................ 8 2.3.1. Perinteisen kasvilääkinnän heikkouksia ...................................................... 11 2.3.2. Perinteisten lääkekasvien ekologinen käyttö ja luonnon monimuotoisuuden säilyttäminen .................................................................................................. 12 2.4. Perinteisen lääketieteen asema Afrikan perusterveydenhuollossa ................ 15 3. COMBRETACEAE-HEIMO ...................................................................... 18 3.1. Combretaceae-heimon systematiikka, morfologia, levinneisyys ja kansanlääkinnällinen käyttö ........................................................................... 18 3.1.1. Combretum-suku .................................................................................... 18 3.1.2. Terminalia-suku ..................................................................................... 20 3.2. Combretum- ja Terminalia-sukuihin kuuluvien lajien biologisesta aktiivisuudesta ja niiden sisältämistä yhdisteistä .............................................. 21 3.2.1. Antimikrobisesti aktiiviset uutteet ja yhdisteet ............................................. 21 3.2.2. Kombretastatiinit .................................................................................... 22 3.2.3. Ellagitanniinit ......................................................................................... 24 3.2.4. Tanniinien erotusdiagnostiikasta ............................................................... 27 3.3. Taustaa työssä käytetyistä mikrobeista ...................................................... 29 4. TYÖN TARKOITUS JA TAUSTA .............................................................. 31 II KOKEELLINEN OSUUS .................................................................... 32 5. MATERIAALIT JA MENETELMÄT ......................................................... 32 5.1. Kasvimateriaali ....................................................................................... 32 5.2. Uuttomenetelmät ..................................................................................... 32 5.2.1. Soxhlet-uutto .......................................................................................... 32 5.2.2. Neste-neste-uutto .................................................................................... 33 5.3. Kromatografiset menetelmät .................................................................... 34 5.3.1. Analyyttinen ohutkerroskromatografia ....................................................... 34 5.3.2. Pylväskromatografia ................................................................................ 34 5.3.3. Preparatiivinen ohutkerroskromatografia .................................................... 36 5.4. Antimikrobiset kokeet .............................................................................. 36 5.4.1. Bakteerikannat........................................................................................ 37 5.4.2. Agardiffuusiomenetelmä .......................................................................... 37 5.4.3. Mikrodiluutiomenetelmä .......................................................................... 38 6. TULOKSET JA TULOSTEN TARKASTELU ............................................. 41 6.1. Raakauutteiden ja neste-neste-uutto fraktioiden saannot ............................ 41 6.1.1. Soxhlet-uuttomenetelmän saannot ............................................................. 41 6.1.2. Neste-neste-uuton saannot ........................................................................ 42 6.2. Kromatografisten menetelmien tulokset .................................................... 45 6.2.1. Tulokset tutkittujen Combretum- ja Terminalia-lajien uutteiden kvalitatiivisesta koostumuksesta käyttäen analyyttistä ohutkerroskromatografiaa ............................. 45 6.2.2. Terminalia kaiserana -lajin juuren kuoren raakauutteesta preparatiivisella Lobar RP-8 -pylväskromatografialla eristetyt fraktiot ..................................................... 46 6.2.3. Terminalia kaiserana -lajin juuren kuoren raakauutteesta preparatiivisella ohutkerroskromatografialla saadut fraktiot ........................................................... 48 6.3. Antimikrobiset vaikutukset ...................................................................... 50 6.3.1. Agardiffuusiomenetelmällä saadut tulokset neste-neste-uuttofraktioiden antibakteerista vaikutuksista .............................................................................. 50 6.3.2. Mikrodiluutiomenetelmällä saadut tulokset neste-neste-uuttofraktioiden antibakteerista vaikutuksista .............................................................................. 54 6.3.3. Mikrodiluutiomenetelmällä saadut tulokset Terminalia kaiserana -lajin juuren kuoren raakauutteesta preparatiivisella Lobar RP-8-pylväskromatografialla eroteltujen fraktioiden antimikrobisuudesta ......................................................................... 60 7. JOHTOPÄÄTÖKSET ................................................................................ 63 Kirjallisuusluettelo ........................................................................................... 66 LYHENTEET AAMPS Association of African Medicinal Plant Standars AI Activity index, aktiivisuusindeksi ATCC American Type Culture Collection BuOH Butanoli CA-1 Kombretastatiini A-1 CA-4 Kombretastatiini A-4 CA-4P Kombretastatiini A-4 fosfaatti CB-5 Kombretastatiini B-5 Cfu Colony forming unit, pesäkkeen muodostava yksikkö CHCl3 Kloroformi Cr Crude extract, raakauute GC Growth Control, kasvukontrolli H2O Deionisoitu vesi H3PO4 ortofosforihappo HHDP Heksahydroksidifenyyliyksikkö HIV-1 Human Immunodeficiency Virus, ihmisen immuunikatovirus, tyyppi 1 HPLC High Performance Liquid Chromatography, korkean erotuskyvyn nestekromatografia IC50 50 % Inhibitory Concentration, pitoisuus, joka estää tutkitun mikrobin kasvua 50 % verrattuna kasvukontrolliin MDR Multidrug Resistant, useille antibiooteille resistentti MeOH Metanoli MIC Minimum Inhibitory Concentration, pienin mikrobin näkyvää kasvua estävä pitoisuus vuorokauden kasvatuksen jälkeen MRSA Methicillin-resistant Staphylococcus aureus, metisilliinille resistentti S. aureus MTB Mycobacterium tuberculosis NaCl Natriumkloridi NC Negative Control, negatiivinen kontrolli NMR Nuclear Magnetic Resonance, ydinmagneettinen resonanssi OAU Organisation of African Unity, Afrikan yhtenäisyysjärjestö Pmy Pesäkkeen muodostava yksikkö, colony forming unit Rf-arvo Retardation factor, retardaatioarvo; ohutkerroskromatografiassa yhdisteen kulkeman matkan suhde liuotinrintaman kulkemaan matkaan Rpm Rounds per minute, kierrosta minuutissa SC Solvent Control, liuotinkontrolli SEM Standard Error of Mean, keskiarvon keskivirhe T Tutkittava yhdiste TLC Thin Layer Chromatography, ohutkerroskromatografia tR Retention time, retentioaika UNDP United Nations Development Programme, Yhdistyneiden kansakuntien kehitysohjelma UV max Ultraviolettivalon absorbtiomaksimin aallonpituus V/V Volume/Volume, pitoisuus ilmoitettuna tilavuus/tilavuus WHO World Health Organization, Maailman terveysjärjestö Wi Water insoluble, veteen liukenematon Ws Water soluble, veteen liukeneva 1 I KIRJALLISUUSKATSAUS 1. JOHDANTO Luonnonaineet ovat yhdisteitä, joita syntetisoidaan luonnossa – kasveissa, eläimissä tai mikrobeissa. Kaikki nämä tuottavat primaarimetaboliitteja, kuten nukleiinihappoja, hiilihydraatteja tai rasvoja, energia-aineenvaihduntaan. Kasvit tuottavat myös sekundaarimetaboliitteja, jotka eivät ole elämän kannalta välttämättömiä, mutta kuitenkin hyödyllisiä kasvin kannalta. Sekundaarimetaboliitit saattavat esimerkiksi suojata kasvia bakteereita ja kasvinsyöjiä vastaan tai houkutella pölyttäjiä huolehtimaan kasvin lisääntymisestä. Monet kasvien tuottamat yhdisteet ovat myös osoittautuneet toimiviksi hoitovaihtoehdoiksi ihmisten tai eläinten sairauksien hoidossa, ja nykyään noin 50 % kaikista kaupallisista lääkkeistä on alun perin kasveista peräisin tai muokattu kasvien sisältämistä yhdisteistä (Drewes 2012). Kasvit ja niiden sisältämät yhdisteet ovat edelleen tärkeä osa terveydenhuoltoa maailmanlaajuisesti, ja erityisen tärkeää tämä on kehitysmaissa, joissa ne voivat olla ainoa mahdollisuus hoitaa vakaviakin sairauksia (Singh ja Kumar 2012). Jopa 80 % Aasian, Afrikan ja latinalaisen Amerikan väestöstä käyttää kasveja sairauksiensa hoitoon, ja yhtä suuri osa afrikkalaisista luottaa perinteisen lääketieteen ja kansanparantajien apuun terveytensä hoidossa (WHO 2002). Kasvilääkinnän pitkästä historiasta, rohdosten yleisyydestä ja luonnon monimuotoisuudesta huolimatta afrikkalaisista lääkekasveista
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