Kumar Sapkota

Purification and Characterization of a Fibrinolytic Protease from a Culture Supernatant of Flammulina velutipes Mycelia

In this study we purified a fibrinolytic enzyme from the culture supernatant of Flammulina velutipes mycelia by ion exchange and gel filtration chromatographies, it was designated as F. velutipes protease (FVP-I). This purification protocol resulted in 18.52-fold purification of the enzyme at a final yield of 0.69%. The molecular mass of the purified enzyme was estimated to be 37 kDa by SDS–PAGE, fibrin-zymography and size exclusion by FPLC. This protease effectively hydrolyzed fibrin, preferentially digesting α-chain over β-and γ–γ chain. Optimal protease activity was found to occur at a pH of 6.0 and a temperature of 20 to 30 °C. The protease activity was inhibited by Cu2+, Fe2+ and Fe3+ ions, but was found to be enhanced by Mn2+ and Mg2+ ions. Furthermore, FVP-I activity was potently inhibited by EDTA and EGTA, and it was found to exhibit a higher specificity for chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 20 amino acid residues of the N-terminal sequence of FVP-I were LTYRVIPITKQAVTEGTELL. They had a high degree of homology with hypothetical protein CC1G_11771, GeneBank Accession no. EAU86463.

Purification and characterization of fibrinolytic metalloprotease from Perenniporia fraxinea mycelia

In this study we purified and characterized a fibrinolytic protease from the mycelia of Perenniporia fraxinea. The apparent molecular mass of the purified enzyme was estimated to be 42kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), fibrin zymography and size exclusion using fast protein liquid chromatography (FPLC). The first 20 amino acid residues of the N-terminal sequence were ASYRVLPITKELLPPEFFVA, which shows a high degree of similarity with a fungalysin metallopeptidase from Coprinopsis cinerea. The optimal reaction pH value and temperature were pH 6.0 and 35-40 degrees C, respectively. Results for the fibrinolysis pattern showed that the protease rapidly hydrolyzed the fibrin alpha-chain followed by the beta-chain. The gamma-gamma chains were also hydrolyzed, but more slowly. The purified protease effectively hydrolyzed fibrinogen, preferentially digesting the Aalpha-chains of fibrinogen, followed by Bbeta- and gamma-chains. We found that protease activity was inhibited by Cu(2+), Fe(3+), and Zn(2+), but enhanced by the additions of Mn(2+), Mg(2+) and Ca(2+) metal ions. Furthermore, the protease activity was inhibited by EDTA, and it was found to exhibit a higher specificity for the chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The mycelia of P. fraxinea may thus represent a source of new therapeutic agents to treat thrombosis.

Neuroprotective effect of Rosmarinus officinalis extract on human dopaminergic cell line, SH-SY5Y.

Hydrogen peroxide (H2O2) is a major Reactive Oxygen Species (ROS), which has been implicated in many neurodegenerative conditions including Parkinson’s disease (PD). Rosmarinus officinalis (R. officinalis) has been reported to have various pharmacological properties including anti-oxidant activity. In this study, we investigated the neuroprotective effects of R. officinalis extract on H2O2-induced apoptosis in human dopaminergic cells, SH-SY5Y. Our results showed that H2O2-induced cytotoxicity in SH-SY5Y cells was suppressed by treatment with R. officinalis. Moreover, R. officinalis was very effective in attenuating the disruption of mitochondrial membrane potential and apoptotic cell death induced by H2O2. R. officinalis extract effectively suppressed the up-regulation of Bax, Bak, Caspase-3 and -9, and down-regulation of Bcl-2. Pretreatment with R. officinalis significantly attenuated the down-regulation of tyrosine hydroxylase (TH), and aromatic amino acid decarboxylase (AADC) gene in SH-SY5Y cells. These findings indicate that R. officinalis is able to protect the neuronal cells against H2O2-induced injury and suggest that R. officinalis might potentially serve as an agent for prevention of several human neurodegenerative diseases caused by oxidative stress and apoptosis.

Antioxidant and Antimelanogenic Properties of Chestnut Flower Extract

In this study, we analyzed the antioxidant and antimelanogenic properties of a variety of solvent extracts of pre-bloom and full-bloom chestnut flowers. Among the solvent extracts, a pre-bloom methanol extract (preM) and an ethanol extract (preE) showed the highest amounts of phenolics (467.92±0.45 and 456.24±5.88 mg of gallic acid equivalent/g of extract) and flavonoids (60.96±1.86 and 41.59±8.57 mg of quercetin equivalent/g of extract). These extracts exhibited the highest DPPH radical and reducing activities, as well as the greatest mushroom tyrosinase inhibition activity. In addition, preE effectively protected the skin against ultraviolet (UV) rays. Further, extracts were tested for cytotoxicity on human melanoma cells (SK-MEL-2), and we observed that all the extracts were non-cytotoxic for the cells. Their effects on tyrosinase and melanin inhibitory action were further assessed, and we found that all the extracts reduced the tyrosinase activity and melanin formation of SK-MEL-2 cells as effectively as arbutin. Moreover, the protein level expression of tyrosinase decreased dramatically. However, the protein levels of the other melanogenic enzymes, tyrosinase-related protein 1 (TRP1) and dopachrome tautomerase (DCT), were not altered significantly. Therefore, the antimelanogenic effects of chestnut flower extracts were attributable to their inhibitory effects on tyrosinase via their anti-oxidative action, making them a strong candidate for use in food, cosmetics, and pharmaceutical applications.

Expression of human growth hormone gene in Pleurotus eryngii

Recombinant human growth hormone (rhGH) has been widely used in many medical applications. Large scale production of rhGH is important for a wider application of this molecule in the field of proteomics. This investigation reports a modified Agrobacterium-mediated method for transfer and expression of human growth hormone gene in the popular edible mushroom Pleurotus eryngii. A binary vector pCAMBIA1304 containing the hGH2 gene was constructed and introduced into Pleurotus eryngii via Agrobacterium tumefaciens-mediated transformation. Integration of hGH2 into mushroom genome was detected by PCR and expression was confirmed by Western blot assay. The successful expression of the human growth hormone gene in mushroom suggests that the proposed modified transformation system is probably useful for the production of transgenic mushrooms.

Enhancement of tyrosine hydroxylase expression by Cordyceps militaris

Cordyceps militaris is a popular medicinal mushroom, and has received extensive attention for medical application because of its various physiological activities. However, there is limited information about the function of Cordyceps militaris on dopaminergic system. This study has attempted to evaluate the effect of cultured fruiting bodies of Cordyceps militaris extract (CME) on the expression of the tyrosine hydroxylase (TH) gene in PC12 cells and rat brain and stomach. Related mRNA levels were determined by the RT-PCR. Protein levels were measured by Western blot and immunohistochemistry. Our results demonstrated CME induced TH gene expression both in vitro and in vivo. Treatment of 10 µg/ml and 20 mg/kg CME to PC12 cells and rat cells yielded significant increases of TH protein levels. Significantly, TH immunoreactive neurons were detected not only in the brain but also in the stomach. TH-immunohistochemical staining was markedly enhanced in animals treated with CME compared to those in the untreated control. These results suggest that CME can upregulate the dopaminergic (DArgic) system, and may contribute to neuroprotection in neurodegenerative diseases.

Biosynthesis of Organic Germanium Using Cordyceps militaris

본 연구에서는 동충하초 균주를 이용하여 SDAY 배지 액체배양과 번데기배지 배양을 이용하여

PURIFICATION AND CHARACTERIZATION OF FIBRINOLYTIC ENZYME FROM CULTURED MYCELIA OF ARMILLARIA MELLEA

100{\sim}5,000\;ppm

Antioxidant Activity and Protective Effects of Tripterygium regelii Extract on Hydrogen Peroxide-Induced Injury in Human Dopaminergic Cells, SH-SY5Y

농도에서 유기게르마늄 함유 동충하초 생산방법을 연구하여 게르마늄 동충하초 생산 가능성을 확인하였다. 액체배양과 번데기 배지 배양에서 게르마늄 첨가 농도를 100 ppm으로 조정했을 때, 생산되는 유기게르마늄의 양이 각각 442.4 ppm/g과 284 ppm으로 가장 높은 생산수율을 보여짐을 확인하였다. 또한 게르마늄의 양을 최대 5,000 ppm(액체배지)과 1,000 ppm(번데기배지)으로 첨가 했을때 각각 4,509.7 ppm/g과 1,058 ppm/g의 유기게르마늄이 생산 되어지는 것도 확인할 수 있었다. 이러한 결과로 비추어 볼 때, 무기게르마늄이 동충하초가 생장함에 있어 영양물질로 공급되어 양질의 유기게르마늄으로 변환 생산 되어진다는 것을 보여주는 것으로 해석할 수 있었다. 또한 본 연구 개발의 결과로 얻어진 게르마늄 함유 동충하초는 기존의 동충하초 효능에 유기게르마늄이라는 약리활성 물질이 첨가됨으로써 기능성식품, 화장품 원료 및 의약품 원료로도 사용될 수 있을 것으로 사료되어진다. 【In the present study, an attempt has been made to produce a high quality medicinal mushroom Cordycops militaris supplemented with organic Ge. Cordycops militaris was cultivated in SDAY liquid medium containing yeast extract 10 g, peptone 10 g, glucose 40 g per liter and chrysalis media supplemented with inorganic Ge at 100, 500, 1000, and 5000 ppm concentrations. The greatest organic Ge production of 442.4 ppm/g and 284 ppm/g were observed in SDAY liquid and Chrysalis media cultures supplemented with 100 ppm inorganic Ge respectively. Similarly, 4,509.7 ppm/g and 1,058 ppm/g of organic Ge were obtained from liquid and chrysalis media cultures supplemented with 5,000 ppm and 1,000 ppm inorganic Ge, respectively. In addition, higher concentration of organic Ge was obtained in mycelia than fruiting bodies. These results indicate that the concentration of organic Ge increase with decreasing inorganic Ge concentration in the medium. This is the first report on production of high valuable Cordyceps militaris contained with organic Ge.】