Antimicrobial activity of crude extracts prepared from fungal mycelia

Synytsya, A.,Monkai, J.,Bleha, R.,Macurkova, A.,Ruml, T.,Ahn, J.,Chukeatirote, E. China Humanity Technology Publishing House 2017 Asian Pacific journal of tropical biomedicine Vol.7 No.3

Objective: To evaluate the in vitro antimicrobial property of three different partitioned extracts (petroleum ether, ethanol and water) prepared from some fungal mycelia. Methods: Seven fungal mycelia were prepared, initially extracted with acidified ethanol (0.2 mol/L HCl in 80% ethanol), yielding the raw crude extracts. The obtained extracts were then further partitioned with petroleum ether (F1), ethanol (F2) and water (F3). All the fractions were tested for antimicrobial activity using the disc diffusion assay. Results: Our data showed that all the fractions could inhibit the testing bacteria. However, the inhibitory activity was found to be dependent on (i) the fungal strains used; (ii) the solvent extracted; and (iii) the testing bacteria assayed. In general, the ethanolic extracts (F2) derived from all fungi displayed highest inhibitory activity against the testing bacteria except for Chaetomium sp. Conclusions: The findings of the present study concluded that the extracts prepared from the fungal mycelia had the bioactive compounds with antibacterial property. This study is a pioneering work and further study should be carried out for development of the new drug leads.

Isolation and characterization of bioactive polysaccharides from algae and medicinal mushrooms

Andriy Synytsya,GordonK.Gomba,Radek Pohl,Peter Capek,Jana Čopíková,Yong-Il Park 한국당과학회 2011 한국당과학회 학술대회 Vol.2011 No.1

Algae and mushrooms are rich sources of bioactive natural components. Among these compounds, polysaccharides are interesting due to their immunomodulating, antitumor, antiviral, coagulating and other properties. Polysaccharides play an outstanding role in the construction of algal and mushroom cell walls. First step of the strategy of polysaccharide isolation from such raw material is fractionation of individual cell wall components by subsequent extractions. The second step is rigorous purification of obtained fractions by using of preparative chromatography, chemical reagents and specific enzymes. Finally, individual polysaccharides within a fraction can be separated from each other by procedures based on the difference in their solubility in various media, sensitivity to chemical or enzymatic destruction, affinity to specific molecules and complexation with metal cations. Structural analysis of isolated algal or mushroom polysaccharides is based on combination of separation, chemical and spectroscopic methods. Total hydrolysis followed by HPLC or GC analysis gives the information about the monosaccharide composition. Gel chromatography equipped with various detectors is used for the molecular mass estimation. FTIR spectroscopy is effective for detection of functional groups, estimation of fraction purity as well as type of polysaccharide. Correlation NMR spectroscopy together with sugar linkage analysis is a powerful tool for detail analysis of polysaccharide structure (types of linkage and substitution, branching, anomeric configuration etc.). Polysaccharide fractions isolated from fruiting bodies of medicinal mushrooms Pleurotus ostreatus, Pleurotus eryngii, Agaricus blazei and Piptoporus betulinus were defined mainly as specific glucans. Branched 1,3-1,6-β-glucan and amylose-like 1,4-α-glucan were found in water soluble fractions, while linear 1,3-α-glucan in alkali soluble fractions. Effective deproteinisation and separation of α- and β-glucans was achieved by the treatment with phenolic reagent. Insoluble fractions contained some amount of chitin as a component of cell wall chitin–glucan complex. Fruiting bodies of wood decay fungi of genera Phellinus and Inonotus contains polyphenols integrated with cell wall polysaccharides. Most of polyphenols, which are interesting due to their strong antioxidative activity, were successfully removed from raw material by extraction with acidic ethanol. Soluble and insoluble polysaccharide fractions isolated from these mushrooms still contained colored products of polyphenol degradation. Polysaccharides were purified by the treatment with acidic or alkali solutions of hydrogen peroxide and defined mainly as branched β-glucans (soluble fractions) and chitin–glucan complex (insoluble part). Korean seaweeds Undaria pinattifida and Capsosiphon fulvescens are sources of bioactive polysaccharides, which were isolated and defined respectively as sulfated/acetylated galactofucan (fucoidan) and sulfated glucuronorhamnoxylan. These polysaccharides have shown various biological activities which make them interesting compounds for medicinal applications. This work was supported by Czech Science Foundation (Project No. 525050273), Ministry of Education of the Czech Republic (Projects No. CEZ: MSM6046137305) and Gyeonggi-do province, Korea (GRRC program of the Catholic University of Korea)

Structure and anticoagulant activity of a sulphated polysaccharide isolated from Maesaengi ( Capsosiphon fulvescens)

Andriy Synytsya,Ye Seul Na,Ju Hee Ko,Woo Jung Kim,Jae Kweon Park,Hang Soo Jo,Doo Jin Choi,Sung Min Kim,Yong ll Park 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1

Characterization and Immunostimulating Activity of a Water-soluble Polysaccharide Isolated from Haematococcus lacustris

박제권,김지훈,이철균,Andriy Synytsya,조항수,김성욱,박주웅,박용일 한국생물공학회 2011 Biotechnology and Bioprocess Engineering Vol.16 No.6

A water-soluble polysaccharide was isolated and purified from the culture filtrate of the photosynthetic green microalgae Haematococcus lacustris by 75% ethanol precipitation and Sepharose CL-6B column chromatography. The molecular mass of the purified polysaccharide (named HCP) was estimated to be approximately 135 kDa by sizeexclusion HPLC and its monosaccharide composition was galactose, glucose and mannose at a relative molar ratio of 2.0, 1.0, and 4.1, respectively, suggesting that HCP is a galactomannan. Fourier-transform infrared and elemental analysis revealed that the purified HCP contains sulfate esters by 1.08% (in mass) and no detectable level of protein. The HCP significantly stimulated murine macrophage RAW264.7 cells to secrete the pro-inflammatory cytokine,TNF-α, in a dose-dependent manner and also enhanced the expression of COX-2 and iNOS genes at a concentration of lower than 10 μg/mL HCP. These results indicated that the sulfated HCP of H. lacustris has potent early innate immune stimulating activities.

Low Molecular Weight Mannogalactofucans Derived from Undaria pinnatifida Induce Apoptotic Death of Human Prostate Cancer Cells In Vitro and In Vivo

Lee, Jisun,Lee, Seul,Synytsya, Andriy,Capek, Peter,Lee, Chang Won,Choi, Ji Won,Cho, Sarang,Kim, Woo Jung,Park, Yong Il Springer-Verlag 2018 Marine biotechnology Vol.20 No.6

Structure and Immune Modulating Activity of a Polysaccharide Purified from Mulberry Fruits Pericarp

Ji Sun Lee,Andriy Synytsya,Hyun Bok Kim,Woo Jung Kim,Seong Jae Jang,Mi Ja Chung,Ju Hee Ko,Doo Jin Choi,Ha Na Choi,Yong Il Park 한국당과학회 2011 한국당과학회 학술대회 Vol.2011 No.1

In the present study, we investigated structure and immune modulating activity of a polysaccharide (tentatively named JS-MP-1), previously purified from Mulberry fruits (Morus bombycis Koidz) pericarp. Based on HPAEC-PAD analysis, major monosaccharides of JS-MP-1 were shown to be galactose (37.6% in mole percentage), arabinose (36.3%) and rhamnose (18.4%), respectively. EA analysis did not show the presence of sulfur (0.07%). Biochemical analysis showed neutral sugars (35.25%), uronic acid (11.25%), sulfate (0.16%) and proteins (0.3%). FT-IR spectra of JS-MP1 confirmed the presence of carboxyl groups (uronic acid – α-GalAp in RG1) and COC, CO and CC stretching vibrations at 948 – 1150 cm-1 corresponding to polysaccharides. Peak at 894 cm-1 indicates the presence of β-Galp units. 1H-NMR spectrum (in D2O) confirmed the presence of α-1,2-Rhap, α-Araf, β -Galp and α-1,4-GalAp units. The most intense HMQS signals were observed for terminal α-Araf. The presence of methylester groups in the residues of α -1,4-GalAp was confirmed by the HMQS peak at C 54.3/H 4.01 ppm (OCH3). Taken collectively, the JS-MP-1 is probably a rhamnogalacturonan type 1 (RG1), which contains: (a) partially methyl esterified α-1,4-D-GalAp and α -1,2-L-Rhap units in backbone, and (b) α-L-Araf and β-Galp are bound to Rha as terminal units or more complex side chains (arabinan, galactan and/or arabinogalactan). The ELISA assay showed that JS-MP-1 remarkably stimulates the release of the proinflammatory cytokines, TNF-α and IL-6, in a dose-dependent manner, from the RAW 264.7 cells. RT-PCR analysis also showed a significant induction of the iNOS and COX-2 gene expression. These results suggest that JS-MP-1 isolated from the Mulberry fruits has immune modulating activities.

Structure of fucoidan isolated from sporophyll of Korean Brown Seaweed Undaria pinnatifida

Woo Jung Kim,Andriy Synytsya,Doo Jin Choi,Hang Soo Jo,Ji Sun Lee,Ji Won Choi,Yong-Il Park 한국당과학회 2011 한국당과학회 학술대회 Vol.2011 No.1

Polysaccharides from brown seaweeds are interesting due to their various biological activities. A water-soluble polysaccharide was isolated and purified from the sporophyll of Korean Undaria pinnatifida by dilute acid extraction, CaCl2 precipitation to remove alginates, and purified DEAE-cellulose chromatography. Structure of this polysaccharide was characterized by various methods such as elemental analysis, HPAEC-PAD, HPLC analysis, FT-IR, FT-Raman, and 1H and 13C NMR. The average molecular mass of this polysaccharide was estimated about 2.1×103 kDa by HPLC. The polymer showed lower electrophoretic mobility (RSH 0.127) than carrageenans (0.096-0.100) and chondroitin sulphate A (0.103), but higher than alginate (0.170). It is consisted of neutral sugar (52.34 % in mass) and sulfate esters (7.4 % in mass), no uronic acid was observed. From the HPAEC-PAD analysis, the monosaccaride composition was shown to be fucose, galactose, xylose, and mannose, with a molar ratio of 1, 0.8, 0.08, and 0.005, respectively. The major monosaccharide was fucose (50.9 mol%), suggesting that it is a fucoidan, a general name of seaweeds polysaccharides containing fucose residues. Other sugars, galactose (44.6 mol%), xylose (4.2 mol%), and mannose (0.3 mol%), were present as minor component. Very intense and broad FT-IR band at 1258-1259 cm-1 were attributed to S=O stretching of sulfate esters. The IR band centered at 840-842 cm-1 was attributed to COS bending of sulfate substituents. Elemental analysis (EA) of this polysaccharide resulted in C (23.03%), H (3.07%) and S (9.18 %). Taken together, these results clearly demonstrated the presence of sulfate groups in this polysaccharide. The degree of sulfation was determined to me 0.93 by DS (moles of SO3- per mole of sugar units) value, which shows about one sulfate group per sugar unit. Consequently the purified polysaccharide was defined as O-acetylated sulphated galactofucan.

Partial Purification of Fucoidanolytic Enzyme from Sphingomonas Paucimobilis PF-1 (KCTC 11130BP) and Preparation of Anticoagulant of Low-Molecular Weight Fuco-oligosaccharides from Korean Undaria pinnatifida Sporopphyll Fucoidan

Woo Jung Kim,Yean Kyoung Koo,Andriy Synytsya,Hye Sook Yun-Choi,Sung Min Kim,Yong-Il Park 한국당과학회 2009 한국당과학회 학술대회 Vol.2009 No.1

Fucoidan was purified from the sporophyll of Korean Undaria pinnatifida and low-molecular weight fuco-oligosaccharides (LMFOs), ranging from 305 to 3,749 Da, were obtained by enzymic digestion of the purified fucoidan by Sphingomonas paucimobilis PF-1 (KCTC 11130BP). The enzyme was partially purified through 3 steps, including ammonium sulfate fractionation and affinity column chromatography. The enzyme was purified at 2.9% (activity/activity) yield from the concentrated supernatant of the sonicated PF-1. SDS-PAGE analysis of the partially enzyme showed three main band (34, 80 and 100 kDa) but still not purified to homogeneity. However, Native electrophoresis gel shown in gave the two main band at 30 and 60 kDa. The results presented here suggest that the 60 kDa subunit forms 2 species, namely 80-, 100 kDa subunits. The enzyme activity was assayed at different pH ranging from 3.0 to 8.0. The optimal activity was absorbed at pH 5.6. The enzyme was active in the range of pH 5-6, but the activity decreased at above pH 7 and below pH 4. When assayed at pH 5.6, crude enzyme showed its optimal activity at 30 ℃, and then sharply decreased by 30 ℃. When the incubation temperature was increased to 37, 42 ℃, there was decreased about 40-50 % of original activity the retained under the same conditions. The intact fucoidan and its LMFOs were compared for their anticoagulanting activities. Intact fucoidan prolonged activated partial thromboplastin time (APTT) at concentrations of 5 and 10 ug/ml, which was about 2.3- and 4.2- fold of the control (clotting time 90 and 168.4s each). LMFOs showed at concentrations of 100 and 200 ug/ml, which was about 2.3- and 3.9- fold of the control. Intact fucoidan prolonged thrombin time (TT) at concentrations of 5 and 10 ug/ml, which was about 42 and 68.7 s, respectively. It was about 2.8- and 4.6- fold. LMFOs approximately 1.7- and 2.6-fold at 50 and 100 ug/ml compared to that of control (14.8 s), respectively. LMFOs did not show any affect on prothrombin time (PT) at different concentrations compared with control but intact fucoidan effect increased with increasing concentration (10, 20, 50, 100 μg).

Isolation and Characterization of Renewable Extracellular Polysaccharides from Microalga

Jae Kweon Park,Yong Il Park,Andriy Synytsya,Choul Gyun Lee 한국생물공학회 2013 한국생물공학회 학술대회 Vol.2013 No.10

Macromolecules Isolated from Phellinus pini Fruiting Body: Chemical Characterization and Antiviral Activity

이새미,JINCHENGMIN,이윤희,김우정,박제권,박용일,장원종,신현동,Andriy Synytsya 한국고분자학회 2010 Macromolecular Research Vol.18 No.6

Two major polymers, EP-AV1 and EP-AV2, were purified from the hot-water extract of the Porodaedalea pini (Brot.) Murrill (syn. Phellinus pini) fruiting body by 75% ethanol precipitation and size-exclusion chromatography. These polymers were shown to be polysaccharides consisting of glucose as the main sugar residue as well as other minor sugars, such as galactose, xylose and mannose, and are variants of β-1,3-glucans, with high molecular masses,1,006 and 100 kDa, respectively. Fourier-transform infrared and elemental analysis suggested that these polymers contained certain level (~10% in dry mass) of phenolic compounds. Both polymers inhibited plaque formation in Vero cells caused by herpes simplex virus 1 (HSV-1) by 91% and 93% at 5 g/mL, and by the coxsackie virus B3 (CVB3) in HeLa cells by 32% and 84% at 1 mg/mL, respectively. On the other hand, their cytotoxicity to both cells was negligible. Their selectivity indices (ratio CC50/EC50) against the HSV-1 virus were quite high, >25,000 (EP-AV1) and >16,670 (EPAV2),respectively. These polysaccharides also inhibited the neuraminidase activity in a dose-dependent manner but only at relatively higher concentrations. These results suggest that these water-soluble polysaccharides are promising agents for the treatment of HSV-1 infections.