The standard extract of Ginkgo biloba leaves (EGb761) is being clinically used in Europe for the treatment of impaired cerebral function in primary degenerative disorders such as Alzheimer disease (AD) and vascular dementia. The abnormal production and aggregation of amyloid β peptide (Aβ) and the deposition of fibrils in the brain is considered as key steps in the onset of AD. For this reason, the inhibition of Aβ aggregation and the destabilization of preformed fibrils have been identified as effective approaches for the prevention and treatment of AD. EGb761 mainly contains three categories of components: flavonol glycosides (FGs), terpene trilactones (TTLs), and catechins and procyanidins. Among them, only TTLs have been evaluated for the inhibition towards Aβ aggregation, and the effects were much weaker than that of the extract. It was suggested that EGb761 should contain several other compounds that are responsible for this activity. In the current study, we have conducted a comprehensive investigation of the generally chemical and bioactive properties of the compounds belonging to all three of the major component categories present in EGb761. We aimed to identify the compounds responsible for the inhibitory activity exhibited by EGb761 towards Aβ42 aggregation and the destabilization of preformed fibrils. The results can be summarized as follows. Seven major FGs were isolated from EGb761 and determined to be quercetin 3-O-α-(6′′′-p-coumaroyl glucopyranosyl-β-1,2-rhamnopyranoside (1), kaempferol 3-O-α-(6′′′-p-coumaroyl glucopyranosyl-β-1,2-rhamnopyranoside) (2), quercetin 3-O-β-D-rutinoside (3), quercetin 3-O-α-L-(β-D-glucopyranosyl)-(1,2)-rhamnopyranoside (4), isorhamnetin 3-O-β-D-rutinoside (5), kaempferol 3-O-β-D-rutinoside (6), and kaempferol 3-O-α-L-(β-D-glucopyranosyl)-(1,2)-rhamnopyranoside (7) by structural analysis. Four catechins and two procyanidins were also found in EGb761 and identified as catechin, epicatechin, gallocatechin, epigallocatechin, procyanidins B1 and B3. The inhibitory activities of these compounds and four major TTLs (i.e., ginkgolides A, B, and C and bilobalide) towards Aβ42 aggregation were evaluated using a thioflavin T fluorescence assay. The results revealed that catechins and procyanidins compounds exhibited significant inhibitory activities with IC50 values in the range of 3-16 μM, and those of the procyanidins were stronger. Three of the FGs (FG1, FG3 and FG4) showed moderate inhibitory activities, with IC50 values in the range of 30-70 μM, whereas the other four FGs (i.e., FG2, FG5, FG6 and FG7) and the four TTLs showed much weaker activity. The catechins and procyanidins also showed potent activities towards the destabilization of preformed Aβ fibrils. Based on these results, we established several structure-activity relationships (SARs) that specific structural groups, as well as the number and position of hydroxyl groups, the linking sequences of the sugar moieties, and the three dimensional conformations of the compounds were important to their inhibitory activity. We also established quantitative analysis of all these tested compounds and simultaneously determined their contents in the Ginkgo extracts. In addition, total FGs, total TTLs, and total catechins and procyanidins were prepared by column chromatography. Similarly, they were also quantitatively analyzed and their inhibitory activities towards Aβ42 aggregation were evaluated. The results showed that total catechins and procyanidins exerted much stronger activity than total FGs and total TTLs. Comprehensive assessment of their activities and contents in the extract indicated that the contribution of the FGs was almost twice that of the catechins and procyanidins, which indicated that they played an important role in the effect of the extract. TTLs alone could barely contribute to the EGb’ effect and probably exerted their influence through the synergistic effect with FGs, which was speculated from the study. In conclusion, the current study has shown that the catechins and procyanidins present in EGb761 possessed potent ability to inhibit Aβ aggregation and destabilize preformed fibrils. FGs made a significant contribution to EGb761’s inhibitory activity towards Aβ aggregation and its neuroprotective effects. Furthermore, the SAR study provide evidences for further research and development of Ginkgo products and drugs designed to target Aβ aggregation or the destabilization of preformed fibrils. Despite their low contents in EGb761 and related products, catechins and procyanidins, and even proanthocyanidins deserve further study because of their potent effects towards Aβ aggregation and the destabilization of preformed fibrils, which could have a significant impact on the quality control of Ginkgo leaves and Ginkgo products.

Orientador: Mary Anne Heidi Dolder, Sergio Luis Pinto da Matta / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T09:49:19Z (GMT). No. of bitstreams: 1 Predes_FabriciadeSouza_M.pdf: 1559316 bytes, checksum: e206924da33f15ad0daf61b7e2a85726 (MD5) Previous issue date: 2007 / Resumo: o cádmio (Cd) é um dos poluentes ambientais importantes que causam danos a vários órgãos e tecidos, incluindo os testículos. O objetivo deste trabalho é investigar o efeito protetor e curativo do extrato de G biloba (GbE) nos testículos de ratos adultos Wistar tratados com dose única de Cd. Trinta animais foram divididos aleatoriamente em 5 grupos que receberam: Grupo 1: água por 56 dias ( controle); Grupo 2: GbE por 56 dias; Grupo 3: CdClz (dose única) e água por 56 dias; Grupo 4: CdCh (dose única) e GbE por 56 dias (Cd + GbE); Grupo 5: pré-tratamento com GbE por 30 dias e CdCh no 31° dia (GbE-Cd). O GbE foi administrado diariamente na dose de 100 mg/Kg de peso corporal por gavagem. O cloreto de cádmio (CdCh) foi injetado intraperitonealmente em dose única de 3 umol/Kg de corporal. Após a coleta de sangue, os ratos foram fixados por perfusão com Kamovsky modificado. Fragmentos de testículo incluídos em metacrilato foram secionados na espessura de 3um e corados com azul de toluidina/borato de sódio 1% para estudo morfométrico em miCroscopia óptica. Para observação em microscópio eletrônico de transmissão fragmentos de testículo foram refixados com tetróxido de ósmio 1 % no mesmo tampão a 4°C, desidratados em acetona e incluídos em Epoxy. Cortes ultrafinos foram feitos com navalha de diamante e contrastados com acetato do uranila 2% e citrato de chumbo 2%. Os níveis de testosterona plasmática obtida por RIA não foram alterados neste estudo. Não houve alteração do peso corporal e testicular, do epidídimo e das glândulas acessórias, vesícula seminal e próstata além do índice gonadossomático. A proporção volumétrica e o volume absoluto dos componentes testiculares não alteraram após os tratamentos. O grupo 3 que recebeu somente Cd teve redução significativa no volume individual das células de Leydig. Entretanto, animais que receberam GbE como pré tratamento ou GbE após a dose de Cd não apresentaram esta redução. Isto sugere que GbE é eficaz em manter o volume das células de Leydig em ratos tratados com Cd. O número de células de Leydig não variou nos grupos estudados. Em microscopia eletrônica foram visíveis os efeitos do Cd nos testículos. Nas células de Sertoli foram observados vacuolização citoplasmática, alterações nas junções celulares e acúmulo de gotículas de lipídio e corpos residuais. Espaços intercelulares expandidos foram observados no epitélio germinativo. Cromatina irregularmente condensada e acrossoma anormal foram encontradas em espermátides alongadas. Algumas células endoteliais sofreram alterações morfológicas no núcleo e perderam algumas junções. As células de Leydig mostraram o citoplasma denso com organelas, como retículo endoplasmático liso e mitocôndrias, mal definidas. A administração de GbE após a dose de Cd mostrou-se eficaz em manter a ultraestrutura normal dos testículos do rato. Diferentemente, o pré-tratamento com GbE não foi eficaz em proteger o epitélio seminífero da toxicidade do Cd. Entretanto, as células de Leydig apresentaram morfologia normal com o retículo endoplasmático liso bem definido e numerosas mitocôndrias. Conclui-se que a administração de GbE por 56 dias após dose única de 3 umol de CdCh é capaz de proteger todo o parênquima testicular dos efeitos tóxicos do Cd. Entretanto, o pré-tratamento com GbE não protegeu o epitélio seminífero, mas foi eficaz em proteger as células de Leydig dos efeitos tóxicos do cádmio / Abstract: Cadmium is one of the important environmental pollutants that affect various organs and tissues, including the testis. The aim of this study was to investigate the protective and curative effect of G. biloba extract (GbE) on cadmium-induced testis damage of adult Wistar rats. Thirty rats were randomly divided in five groups that consisted in :Group 1: water for 56 days (control); Group 2: GbE for 56 days; Group 3: CdClz (single dose) and water for 56 days; Group 4: CdCh (single dose) and GbE for 56 days (Cd + GbE); Group 5: pre-treatment with GbE for 30 days and CdCh on the thirty-first day (GbE-+Cd).The GbE was administered daily in a dose of 100 mg/Kg BW by gavage. The cadmium chloride (CdCh) was injected i.p. in a single dosage of 3 umol/Kg BW. After blood collection, rats were fixed by whole-body perfusion in Karnovsky fixative. Methacrylate-embedded testis fi-agments were sectioned at 3um thickness and stained with toluidine blue/sodium borate 1 % for morphometric study in the light microscope. For electron microscopy transmission examination, testis fragments were post-fixed with 1 % osmium tetroxide in the same buffer at 4°C, dehydrated in acetone and embedded in epoxy resin. Ultrathin sections were cut with a diamond knife and stained with 2% uranyl acetate and 2% lead citrate prior to observation. Plasma testosterone values obtained by the RIA method were not modified in this study. No change occurred in the average of corporal and testicular weight, gonadosomatic index, epididymis and accessory glands weight. The volumetric proportion and absolute volume oftesticular componenfs did not change after the treatments. Group 3 had a significant reduction in Leydig cell individual volume. However, animals that received GbE as pre-treatment or GbE after a cadmium dose did not present this reduction of Leydig cell volume. This suggests that GbE is effective in maintaining Leydig cell volume in cadmium-damaged testis. The number ofLeydig cells did not vary in any group studied. In electron microscopy observations, this Cd dose caused visible alterations in the testis. Cytoplasmic vacuolation, disruption oftight junctions, accumulation oflipid droplets and residual bodies were observed in Sertoli cells. Expanded intracellular spaces were observed between the tubule cells. Irregularly condensed chromatin and abnormal acrosomes were found in late spermatids. Endothelium was affected, showing some disruption of tight junetions and morphologieally altered nuclei. Leydig eells showed a dense eytoplasm with poorly defined organelles sueh as SER and mitoehondria. The administration of GbE after the Cd dose is effeetive in maintaining almost normal rat testis ultrastructure. However, GbE pretreatment was not able to protect testis from Cd toxicity, since toxicity signals were observed in the seminiferous epithelium. However, Leydig cells showed the normal morphology with well-defined and abundant smooth endoplasmic reticulum and mitochondria. It can be concluded that, GbE administration for 56 days after a single dose of 3 umol of CdCh could protect the testicular parenchyma from Cd toxic effects. However, GbE pretreatment did not protect the seminiferous epithelium, although it was effective in protecting Leydig cells from Cd toxic effeets / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

<p>U okviru ove disertacije izvr&scaron;eno je preparatvno izolovanje etarskog ulja li&scaron;ća ginka (<em>Ginkgo biloba</em> L.) destilacijom pomoću vodene pare, u cilju određivanja pojedinih fizičko-hemijskih parametara. Oficinalnim postupkom određen je sadržaj etarskog ulja u li&scaron;ću ginka i iznosi 0,0083%. Li&scaron;će ginka je ekstrahovano klasičnim rastvaračima tj. sme&scaron;om alkohol-voda, pri čemu je koncentracija alkohola iznosila 40% (m/m). Primenom navedenog rastvarača, izvr&scaron;ena je vi&scaron;estupna protivstrujna ekstrakcija (u pet stupnjeva) pri čemu je dobijen tečni ekstrakt (<em>Extracta fluida</em>) sa relativno visokim sadržajem ekstraktivnih materija (17,06%). Tečni ekstrakt je direktno kori&scaron;ćen za dobijanje suvog ekstrakta li&scaron;ća ginka (<em>Extracta sicca</em>)primenomvsu&scaron;nie sa raspr&scaron;ivanjem (spray dryer). Kvalitativna i kvantitativna karakterzacija izvr&scaron;ena je primenom postupka tečne hromatografije na tankom sloju (HPTLC) i određen sadržaj ukupnih flavonoida sračunatih na rutin, a i na katehin, a određen je i sadržaj ukupnih fenola sračunatih na hlorogensku kiselinu. Glavni deo doktorske disertacije predstavlja ekstrakcija sistema li&scaron;će ginka-ugljenik (IV)-oksid pod pritiskom, pri čemu je ispitivan uticaj stepena usitnjenosti droge na prinos ekstrakcije, i kori&scaron;ćen koeficijent brze i spore ekstrakcije kao mera kinetičkog pona&scaron;anja ekstrakcije. Dobijeni rezultati ispitivanja su pokazali značajan uticaj stepena usitnjenosti droge na brzinu ekstrakcije, pogotovo, kod ekstrakcije natkritičnim ugljenik (IV) oksidom. Radi izbora optimalnog protoka ekstragensa ispitivani su sledeći protoci 0,095, 0,194 i 0,277 kg/h . Na osnovu prinosa ekstrakcije usvojeno je da je protok ekstragensa od 0,194 kg/h optimalan. Prinos ekstrakcije je ispitivan kori&scaron;ćenjem dva postupka ekstrakcije, i to: ekstrakcija tečnim ugljenik (IV) -oksidom (temperatura ispod kritične temperature Tc= 31,1^oC, a pritisak ne&scaron;to ispod ili iznad kritičnog pritiska p_c=73,8 bar), i ekstrakcija natkritičnim ugljenik (IV)- oksidom (pritisak i temperatura iznad kritičnih vrednosti pritiska i temperature). U natkritičnoj oblasti promenom pritiska se značajno menjaju svojstva ekstragensa, povećava se sposobnost rastvaranja, dielektrična konstanta i dr. Ispitivan je uticaj temperature na prinos ekstrakcije (izotermni proces), pri čemu se dobijaju rezultati koji se ne mogu objasniti jednostavno, analizirajući samo uticaj gustine rastvarača na moć rastvaranja, već se za obja&scaron;njenje dobijenih rezultata uključuje i uticaj napona pare ekstrahovane komponente, tako da rastvorljivost komponente može da se poveća, smanji ili ostane ista sa povećanjem temperature na konstantnom pritisku, u zavisnosti koji uticaj je dominantniji. Kod izotermnih postupaka prinos ekstrakcije raste sa povećanjem pritiska ekstrakcije, &scaron;to je u saglasnosti sa teoretskim principima. S druge strane, ekstrakti dobijeni pri vi&scaron;im pritiscima imaju manji sadržaj etarskog ulja &scaron;to se obja&scaron;njava činjenicom da veći pritisci imaju veću moć rastvaranja glavnih komponenata kao i komponenata kao &scaron;to su smole, voskovi i masna ulja. Kvalitativnom i kvantitativnom analizom selektovanih ekstrakata dobijenih tečnim (130 bar, 20^oC, 3 h) i natkritičnim (100 bar, 40^o C, 4 h) ugljenik (IV)-oksidom, kao i etarska ulja izolovana iz ovih ekstrakata nađeno je da ispitivani uzorci sadrže nalkane, račvaste alkane i jedinjenja sa kiseonikom, među kojima posebno mesto zauzimaju fenoli sa zasićenim i nezasićenim alkil ostacima.<br />Izvr&scaron;ena je uporedna anliza etarskog ulja dobijenog destilacijom li&scaron;ća ginka pomoću vodene pare i etarskih ulja dobijenih iz selektovanih ekstrakata. Interesantan je podatak koji se odnosi na sadržaj etarskog ulja u drogi određen direktnom destilacijom droge pomoću vodene pare i izdvajanjem etarskih ulja iz ekstrakata takođe destilacijom pomoću vodene pare. U ovom drugom slučaju dobija se, preračunavanjem, sadržaj etarskog ulja u drogi vi&scaron;estruko veći (4-10 puta) od sadržaja, određenog oficinalnim postupkom, u drogi. Ova pojava se obja&scaron;njava uvođenjem pojma &bdquo;vezano&ldquo; etarsko ulje i &bdquo;slobodno&ldquo; etarsko ulje. Ekstrakcijom ugljenik (IV)-oksidom pod pritiskom tzv. vezano etarsko ulje se oslobađa voskova i masnog ulja &scaron;to se odražava na njegovu povećanu količinu u CO₂-ekstraktima. Na kraju u ovoj disertaciji izvr&scaron;eno je modelovanje ekstrakcionog sistema li&scaron;će <em>Ginkgo biloba</em> &ndash; ugljenik (IV)-oksid pod pritiskom kori&scaron;ćenjem model jednačine Naika i saradnika, modifikovane model jednačine Reverchon i Sesti-Osseo kao i model, če&scaron;će kori&scaron;ćen, koji je predložila Sovov&aacute;. Kori&scaron;ćeni modeli mogu relativno uspe&scaron;no da se koriste za opisivanje ekstrakcionog sistema li&scaron;će <em>Ginkgo biloba</em> &ndash; ugljenik (IV)-oksid pod pritiskom. Radi iznalaženja najpovoljnijih uslova ekstrakcije primenjen je metod odzivne povr&scaron;ine variranjem parametra ekstrakcije (pritisak, temperatura i vreme ekstrakcije). Na osnovu eksperimentalnih rezultata dobijen je polinom drugog reda za izračunavanje optimalnog prinosa ekstrakcije i određeni su najpovoljniji uslovi ekstrakcije kao i međusobni uticaji pojedinih parametra.</p> / <p>Within this thesis preparative isolation of essential oil from leaves of ginkgo (<em>Ginkgo biloba</em> L.) by steam distillation was carried out, in order to determine some physico-chemical parameters. The essential oil content in the leaves of ginkgo wass determined by an officinal procedure and its value is 0.0083%. Ginkgo leaves were extracted with conventional solvents, i.e. alcohol-water mixture, where the alcohol concentration was 40% (w/w). The forementioned solvent was used to carry out a multistage counter-current extraction (five stages) by which the liquid extract (<em>Extracta fluida</em>) with a relatively high content of extracts (17.06%) was obtained. The liquid extract was directly used to obtain a dry extract of ginkgo leaves (<em>Extracta sicca</em>) by spay drying. The qualitative and quantitative characterisation was based on the proceedings of liquid thin layer chromatography (HPTLC), the content of total flavonoids expressed as rutin, and as catechin, and the total phenol content expressed as chlorogenic acid was determined. The main part of the doctoral thesis is the extraction system of ginkgo leaves-carbon (IV) oxide under pressure, in which the effect of the drug particle size on the extraction yield was studied, and as a measure of the kinetic behavior of extraction the coefficient of fast and slow extraction was used. The obtained results showed a significant effect of the drug particle size on the speed of extraction, particularly, the extraction with supercritical carbon (IV) oxide. For the purpose of selecting the optimal flow of the solvent several flowrates were investigated 0.095, 0.194 and 0.277 kg/h. Based on the yield of extraction the solvent flowrate of 0.194 kg/h was assumed as optimal. The extraction yield was investigated using two extraction procedures: extraction with liquid carbon (IV) oxide (temperature below the critical temperature Tc = 31.1 ⁰C and pressure slightly below or above the critical pressure p_c = 73.8 bar), and extraction with supercritical carbon (IV) oxide (pressure and temperature above the critical values of pressure and temperature). In the supercritical area the change in pressure significantly the influences the properties of the solvent, increases the ability of dissolving, dielectric constant, etc. The effect of temperature on extraction yield was examined (isothermal process), where the obtained results can not be explained simply by analyzing only the effect of solvent density on the power of dissolution, but to explain these results the effect of vapor pressure of the extracted components must be included, so that the solubility of the component may increase, decrease or remain the same with increasing temperature at constant pressure, depending on which influence is dominant. In isothermal processes the extraction yield increases with increasing pressure of extraction, which is consistent with theoretical principles. On the other hand, extracts obtained at high pressures have a lower content of essential oils which can be explained by the fact that higher pressures have a greater power of dissolution of the main components as well as components such as resins, waxes and fatty oils. The qualitative and quantitative analysis of the selected extracts obtained with liquid (130 bar, 20 ⁰C, 3 h) and supercritical (100 bar, 40 ⁰C, 4 h) carbon (IV) oxide, and essential oils isolated from these extracts showed that the studied samples contain n-alkanes, branched alkanes and compounds with oxygen, including phenols with saturated and unsaturated alkyl residues. A comparative analysis of the essential oil obtained by distillation of ginkgo leaves by steam and essential oil obtained from selected extracts was carried out. An interesting fact concerning the essential oil content in the drug determined by direct distillation of drugs by steam and separating the essential oils from extracts also produced by steam distillation. In the other case, by calculation, the obtained essential oil content in the drug is several times higher (4-10 times) of content, determined by an fficinal procedure, in the drug. This phenomenon is explained by introducing the notion of &quot;linked&quot; essential oil and &quot;free&quot; essential oil. The extraction with carbon (IV) oxide under pressure the so called linked essential oil is released from waxes and fatty oils which is reflected in its increased amount in CO₂-extracts. At the end of this dissertation the modeling of the extraction system leaves of <em>Ginkgo biloba</em> - carbon (IV) oxide under pressure was carried out using the model equation of Naik and associates, the modified model equation of Reverchon and Sesti Osseo and also like a frequently used model proposed Sovov&aacute;. The mentioned models can be relatively used to describe the extraction system leaves of <em>Ginkgo biloba</em> - carbon (IV) oxide under pressure. In order to find the most favorable conditions of extraction, the response surface methodology varying extraction parameters was used (pressure, temperature and extraction time). Based on the experimental results a second order polynom for the calculation of the optimum yield of extraction was obtained and the extraction conditions and the mutual influence of some parameters were determined.</p>