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Title: Chemical analysis of fruit bodies of and in vitro cultures of Sarcodon imbricatus (L.) P. Karst. and biological activity of polysaccharides fractions

Abstract:

The object of the study was Sarcodon imbricatus, a fungus from the Basidiomycota division occurring in spruce forests, covered with strict protection in Poland. In vitro cultures were driven from the hymeneal layer of the sporocarp. A pure line of mycelium cells was obtained in the culture. PCR-RFLP DNA analysis of the obtained mycelium was performed to confirm genetic purity and compliance with the cells used to initiate the culture. To optimize the culture medium and conditions, various sources of carbon and nitrogen were tested, along with combinations of incubation temperature and initial pH of the medium. Highest increases in biomass were obtained using the optimized Lubiński medium (Turło et al., 2004) containing: fructose (50g/l) and casein hydrolysate (10g/l). The determined optimum culture conditions were 20°C and initial pH of 6.0. The second stage of the study covered chemical analyses of mycelium collected in natural sites and of the mycelium from in vitro cultures. Chemical analyses included the following groups of compounds: fatty acids, sterols, amino acids, indole compounds, phenolic acids, polysaccharides and mineral compounds. Chromatographic methods (partition, column and thin-layer chromatography) were used in isolation and/or purification of the analyzed compounds. High-performance liquid chromatography (HPLC) was used for quantitative analysis. Spectral methods (1H NMR, EI-MS) were used for identification of the compounds. The performed chemical analyses identified: · Eight fatty acids, both saturated and unsaturated in the original sporocarps (including three unsaturated acids: oleic acid, linoleic acid and α-linoleic acid) and 11 fatty acids in the in vitro culture cells (including eight unsaturated acids, such as, among others, the α-linoleic acid), · Ergosterol, both in natural mycelia and in vitro cells. This compound has been quantitated, isolated and identified using spectral methods (1H NMR, EI-MS, UV-VIS). Additionally, ergosterol peroxide was identified in the sporocarps. · Presence of proteinogenic amino acids (endo- and exogenous). 14 proteinogenic amino acids were identified and quantitated in the sporocarps (including eight that are exogenous to humans). Seven compounds were identified in the in vitro cultures (including 4 amino acids exogenous to humans). · Indole derivatives – tryptophan, tryptamine and serotonin were identified in both sporocarp and in vitro mycelium. Additionally, trace amounts of melatonin were found in the sporocarps. Those compounds were identified using spectral methods (1H NMR, EI-MS,UV-VIS), · Three phenolic acids were found in both the sporocarps and in vitro mycelium (p-hydroxybenzoic acid, protocatechuic acid (3,4- dihydroxybenzoic acid) and syryngic acid (4-hydroxy-3,5 dimethoxybenzoic acid) and identified using spectral methods (1H NMR, EI-MS), · Three polysaccharide fractions (FOI, FOII and FOIII) were isolated from the sporocarps and two fractions (FKI and FKII) from the in vitro mycelium. Qualitative analysis of the isolated polysaccharide fractions identified galactose and fucose (FOI, FKI) and glucose and fucose (FOII, FKII). The FOIII fraction from the sporocarps was glucan, · Total percentage content of sugars, percentage content of uronic acids and molecular masses in the isolated fractions, · Qualitative and quantitative composition of the mineral compounds in both in vivo and in vitro material. In the third part of the study, biological activity of selected polysaccharide fractions obtained from the sporocarps and in vitro mycelium was assessed using in vitro tests, covering: antibacterial activity against five G(+) and G(-) bacterial strains, anti-viral activity against the HPV-1 virus and cytotoxicity against animal cancer cells (melanoma B16 and sarcoma XC) and human cancer cells (breast cancer). Fractions isolated from both the sporocarps and in vitro cultures demonstrated a low level of anti-bacterial activity. ; On the other hand, they slowed down replication of the HPV-1 virus and showed cytotoxic to the exposed cancer cell lines. This was the first analysis of this type, covering both sporocarp and in vitro culture isolates. The analyses performed under this study allowed more specific identification of the metabolite composition in Sarcodon imbricatus sporocarps and determination of the biosynthetic abilities of the in vitro mycelium cultures. It has been demonstrated that in vitro cultures do maintain ability to synthesize a range of metabolites occurring in the sporocarps, like for example ergosterol, indole compounds and phenolic acids. On the other hand, in vitro cultures have the ability to produce compounds, which are not present in the sporocarps. This fact is demonstrated by the difference in qualitative composition of fatty acids between the sporocarps and the in vitro mycelium. Results of the performed biological activity analyses lead to the conclusion that Sarcodon imbricatus may be qualified as a species with potential therapeutic properties and that the polysaccharide fractions isolated from both the sporocarps and in vitro cultures may be deemed as compounds responsible for therapeutic effects. The results of this study are not only cognitive but they also have practical application potential. It has demonstrated that in vitro cultures may be an alternative, high-yield source of a variety of biologically active metabolites.

Level of degree:

2 - studia doktoranckie

Degree discipline:

farmacja

Degree grantor:

Wydział Farmacji

Promoter:

Halina Ekiert

Date issued:

2010

Format:

application/pdf

Identifier:

oai:dl.cm-uj.krakow.pl:810

Call number:

ZB-113478

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Access rights:

tylko w bibliotece

Location of original object:

Biblioteka Medyczna Uniwersytetu Jagiellońskiego - Collegium Medicum

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Last modified:

Dec 10, 2019

In our library since:

Nov 21, 2012

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http://dl.cm-uj.krakow.pl:8080/publication/810

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