• Korean Journal of Food Science and Technology
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• v.38 no.4
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• pp.475-482
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• 2006

The quality characteristics and functional properties of fermented soybean by lactic acid bacteria (FSB) were investigated and compared with those of soybean (control) and commercial cheonggukjang Powder (CGP). The crude protein, lipid, and fiber contents of FSB were similar to those in CGP. The vitamin $B_2$ content in FSB (1.4 mg%) was similar to the control (1.3 mg%) whereas it was remarkably low in the CGP (0.2 mg%). The bright yellow color of FSB determined by Hunter's colormeter was quite different from the dark reddish brown color of the CGP. The pH of FSB was the lowest and thee amino-nitrogen was 517.2 mg%, which was higher than that in CGP (468.1 mg%). Glutamic acid, aspartic acid and leucine were the most abundant amino acids. In particular, the increase in the glutamic acid level was noticeable in FSB. The fatty acid compositions of FSB and CGP were similar to the control. However, the sucrose and fructose levels were lower after fermentation but the glucose level was higher. The results of isoflavone analysis by HPLC showed that the levels of daidzein and genistein in FSB were as much as 48.33 and 52.82 mg%, respectively, which is higher than that found in CGP. The DPPH free radical scavenging effects of FSB and CGP were 1.8 times higher an those of the control. The fibrinolytic activity determined by the diameter of the lysed area on the fibrin plate was the most effective in FSB. In conclusion, it is believed that FSB has a similar or higher quality than CGP. Therefore, FSB is expected to be good functional food material.

• Journal of Microbiology
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• v.45 no.2
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• pp.158-167
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• 2007

In this study, we have cloned a novel cDNA encoding for a papain-family cysteine protease from the Uni-ZAP XR cDNA library of the polychaete, Periserrula leucophryna. This gene was expressed in Escherichia coli using the T7 promoter system, and the protease was characterized after partial purification. First, the partial DNA fragment (498 bp) was amplified from the total RNA via RT-PCR using degenerated primers derived from the conserved region of cysteine protease. The full-length cDNA of cysteine protease (PLCP) was prepared via the screening of the Uni-ZAP XR cDNA library using the $^{32}P-labeled$ partial DNA fragment. As a result, the PLCP gene was determined to consist of a 2591 bp nucleotide sequence (CDS: 173-1024 bp) which encodes for a 283-amino acid polypeptide, which is itself composed of an 59-residue signal sequence, a 6-residue propeptide, a 218-residue mature protein, and a long 3'-noncoding region encompassing 1564 bp. The predicted molecular weights of the preproprotein and the mature protein were calculated as 31.8 kDa and 25 kDa, respectively. The results of sequence analysis and alignment revealed a significant degree of sequence similarity with other eukaryotic cysteine proteases, including the conserved catalytic triad of the $Cys^{90},\;His^{226},\;and\;Asn^{250}$ residues which characterize the C1 family of papain-like cysteine protease. The nucleotide and amino acid sequences of the novel gene were deposited into the GenBank database under the accession numbers, AY390282 and AAR27011, respectively. The results of Northern blot analysis revealed the 2.5 kb size of the transcript and ubiquitous expression throughout the entirety of the body, head, gut, and skin, which suggested that the PLCP may be grouped within the cathepsin F-like proteases. The region encoding for the mature form of the protease was then subcloned into the pT7-7 expression vector following PCR amplification using the designed primers, including the initiation and termination codons. The recombinant cysteine proteases were generated in a range of 6.3 % to 12.5 % of the total cell proteins in the E. coli BL21(DE3) strain for 8 transformants. The results of SDS-PAGE and Western blot analysis indicated that a cysteine protease of approximately 25 kDa (mature form) was generated. The optimal pH and temperature of the enzyme were determined to be approximately 9.5 and $35^{\circ}C$, respectively, thereby indicating that the cysteine protease is a member of the alkaline protease group. The evaluation of substrate specificity indicated that the purified protease was more active towards Arg-X or Lys-X and did not efficiently cleave the substrates with non-polar amino acids at the P1 site. The PLCP evidenced fibrinolytic activity on the plasminogen-free fibrin plate test.

• Journal of Life Science
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• v.21 no.10
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• pp.1422-1427
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• 2011

This study was performed to develop effective antithrombotic agents from traditional herb extracts. Prunella vulgaris L. has been used traditionally as a medical resource in cancer therapy, as well as treatment of hypertension and inflammation, and as a diuretic. However, the effects of Prunella vulgaris on thrombosis and platelet activation have not been clearly understood. Antithrombotic and antiplatelet activities of oriental medicinal herbs were investigated by evaluating the effect of the aqueous extract from Prunella vulgaris on the blood coagulation, platelet aggregation and fibrinolysis. Prunella vulgaris extracts showed effective anticoagulant activity in coagulation times such as activated partial thromboplastin time (APTT) and prothrombin time (PT). Prunella vulgaris also inhibited adenosine diphosphate (ADP)- and collagen-induced platelet aggregation. In addition, evaluation of fibrinolytic activity showed that the Prunella vulgaris extracts have high solubility. From these results, it is suggested that Prunella vulgaris can be a potential candidate for anticoagulants and antiplatelets, as well as fibrinolytic agents.

• Journal of Mushroom
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• v.13 no.3
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• pp.192-198
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• 2015

This study was performed in order to analyze the fibrinolytic, thrombin inhibitory, anti-oxidative, acetylcholinesterase inhibitory, and immuno-enhancing activities of the water extract and solvent fractions isolated from Grifola frondosa. Fibrinolytic activity was analyzed using the fibrin plate method, and thrombin inhibitory activity was assayed using the substrate H-D-Phe-piparg- pna. Anti-oxidative activity was estimated using the DPPH assay, and AChE inhibitory activity was measured using the spectrophotometric method. Immuno-enhancing activity was examined using the nitric oxide (NO) production in RAW 264.7 macrophage cells. Cell viability was determined using the MTS assay. Fibrinolytic activities were the highest in water extract (1.55 plasmin units/mL) followed by water fraction (0.85 plasmin units/mL). The thrombin inhibitory activities of the water and ethyl acetate fractions were determined to be 76.43% and 72.59%, respectively. The acetylcholinesterase inhibitory activities of chloroform and hexane fractions exhibited values of 95.14% and 94.74%, respectively. The butanol fraction showed the highest anti-oxidative activity at 94.47%. Anti-proliferating activity against Raw 264.7 cells showed no cytotoxicity. The production of NO in Raw 264.7 cells increased up to 2-fold by adding the water fraction compared to the untreated control. These results suggest that Grifola frondosa may serve as a useful functional food for the enhancement of immune function and the prevention and therapy of cardiovascular diseases.

• Korean Journal of Food Science and Technology
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• v.38 no.5
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• pp.691-698
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• 2006

The purpose of this study was to investigate how herb extracts may improve blood circulation. Twenty-six extracts from nine different herbs (marjoram, lavender, dill, rosemary, hyssop, rose, lemon balm, pineapple sage, and echinacea) were evaluated for their anti-hypertensive effects via angiotensin I converting enzyme (ACE) inhibition. Their cholesterol-lowering effects via hydroxy-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibition and their fibrinolytic activity via fibrin-plate method were also evaluated. Both water extraction of rose flowers and 70% EtOH extraction of pineapple sage leaves effectively reduced the ACE activity with inhibition rates of 133.8% and 91.2%, respectively. Similarly, both water and 70% EtOH extracts of rose flowers strongly inhibited the enzymatic activity of HMG-CoA reductase by 48.9% and 80.5%, respectively. Water and 70% EtOH extracts of rose flowers also showed relatively high fibrinolytic activity. Based on these observations, rose flower extracts can be developed as a functional tool for use in the improvement of blood circulation.

• Journal of Life Science
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• v.23 no.12
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• pp.1525-1531
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• 2013

Baegilju is a famous traditional Korean wine made over the course of 100 days. The physiological functionalities of Baegilju were evaluated using different tests. The spectrophotometric method was used to determine the total concentration of polyphenolics and flavonoids and DPPH and ABTS radicals. A nitrite scavenging assay was used to evaluate antioxidant activity. The fibrin plate method was used for fibrinolysis and to evaluate angiotensin I converting enzyme (ACE) inhibitory activity; finally, the colorimetric determination method was used to evaluate acetylcholinesterase (AChE) inhibitory activity. The total polyphenolic content of non-sterilized Baegilju and sterilized Baegilju were 391.59 ${\mu}g$ and 401.33 ${\mu}g$ tannic acid equivalents/ml, respectively; and the flavonoids contents were 284.75 ${\mu}g$ and 308.35 ${\mu}g$ quercetin equivalents/ml, respectively. Baegilju exhibited more excellent antioxidant activities (DPPH and ABTS radicals, nitrite scavenging activity) than did Cheongju. In addition, the fibrinolytic activity and AChE inhibitory activity were found to be higher in Baegilju than they were in Cheongju. The ACE inhibitory activity of non-sterilized Baegilju, sterilized Baegilju, and Cheongju were 23.62%, 19.99%, and 38.91%, respectively. Therefore, these results suggest that Baegilju has potential as an antioxidant agent and anti-thrombosis agent.

• Journal of Plant Biotechnology
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• v.36 no.1
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• pp.30-37
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• 2009

Tissue-type plasminogen activator (t-PA) is a thrombolytic agent important in fibirn clot lysis. T-PA causes fibirn-specific plasminogen activation. Six binary vectors harboring t-PA and its derivative genes were cloned and expressed in transgenic alfalfa plants. The insertion of the t-PA and its derivative genes in genomic DNA of alfalfa plants was confirmed by PCR. The presence of the t-PA and its derivative transcripts in total RNAs of the transgenic alfalfa leaves was verified by RT-PCR. ELISA experiments demonstrated that the highest level of recombinant t-PA expression was $75.1{\mu}g$/ total soluble protein (mg) in alfalfa plants. The amount of recombinant t-PA and its derivative proteins in transgenic plants was estimated to range from 9.7 to $39.5{\mu}g$/ total soluble proteins (mg). Western blot analysis of the transformed alfalfa leaves revealed bands of approximately 68-kDa recombinant t-PA and its derivative proteins. The fibrinolysis of recombinant t-PA and its derivative proteins was confirmed by a fibrin plate assay (range from 3.2 to 8.1 cm). The results presented provide information for the development of an additional production of recombinant human proteins having pharmaceutical applications using transgenic plants.

• Journal of the Korean Society of Food Science and Nutrition
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• v.39 no.8
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• pp.1087-1096
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• 2010

This study was conducted to investigate the physiological activity of extracts of fresh mushrooms. The components were extracted by hot water; subsequently, the hot-water extract was subjected to 60% ethanol precipitation to yield high-molecular-weight (HMW) and low-molecular-weight (LMW) fractions. Total polyphenol contents, $\beta$-glucan contents, electron-donating ability (EDA), superoxide dismutase (SOD)-like activity, nitrite-scavenging activity, fibrinolytic activity, nitric oxide (NO) production, and inhibition of NO production of the mushroom extracts were measured using lipopolysaccharide (LPS)-stimulated murine macrophages, RAW 264.7 cells. The extracts of Lentinus edodes (Berk.) Singer and Pleurotus ostreatus (Fr.) Kummer contained the highest levels of $\beta$-glucan (33.5% and 25.57%, respectively). Further, the LMW fractions of the Phellinus linteus contained the highest levels of polyphenols (233.23 mg/g). The EDA of LMW fractions (10 mg/mL) of the Phellinus linteus and Agaricus bisporus were 80.74% and 51.35%, respectively. Further, SOD-like activities of the LMW fractions were high as compared to those of the HMW fractions. Nitrite-scavenging activities of the LMW fractions (pH 1.2; concentration, 10 mg/mL) of the Phellinus linteus and Pleurotus ostreatus (Fr.) Kummer were 75.95% and 41.05%, respectively. The fibrinolytic activity of the LMW fractions of all mushrooms showed no enzyme activity by fibrin plate assay. The fibrinolytic activity of the extracts of Tricholoma matsutake was the greatest inhibitory activity at 60.4%. Further study revealed that the mushroom extracts exhibited anti-inflammatory effects on RAW 264.7 cells. The LMW fraction ($500\;{\mu}g/mL$) of the Phellinus linteus considerably inhibited NO production (100%).

• Journal of Life Science
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• v.23 no.1
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• pp.15-23
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• 2013

AprE51 from Bacillus amyloliquefaciens CH51 is a 27 kDa subtilisin-like protease with fibrinolytic activity. AprE51-6 showing increased catalytic activity was produced previously. To enhance the thermostability of AprE51-6, 2 residues, Gly-166 and Asn-218 based on B. subtilis subtilisin E were mutated by site-directed mutagenesis. The results of the mutational analysis showed that substitution of arginine for Gly-166 (AprE51-7) increased the fibrinolytic activity 1.8-fold. An N218S mutant (AprE51-8) also increased the fibrinolytic activity up to 4.5-fold in a fibrin plate assay. Purified AprE51-7 and AprE51-8 mutants had a 1.9- and a 2.5-fold higher $k_{cat}$, respectively, and a 2.1-1.9-fold lower $K_m$, respectively. This resulted in a 3.8- and a 4.7-fold increase in catalytic efficiency ($k_{cat}/K_m$), respectively, relative to that of wild-type AprE51. AprE51-8 had a broader pH range than AprE51-6 and nattokinase, especially at an alkaline pH value. In addition, AprE51-8 showed higher thermostability than AprE51-6 at $60^{\circ}C$. The half-lives of AprE51-7 and AprE51-8 at $50^{\circ}C$ were 21.5 and 27.3 min, respectively, which are 2.0 and 2.6 times longer, respectively, than that of the wild-type AprE51.