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BACKGROUND Osteoarthritis (OA), a chronic age-related disease characterized by the slowly progressive destruction of articular cartilage, is one of the leading causes of disability. As a new strategy for treatment of OA, mesenchymal stem cells (MSCs) have the potential for articular cartilage regeneration. Meanwhile, thrombospondin 2 (TSP2) promotes the chondrogenic differentiation of MSCs. AIM To investigate whether TSP2 induces chondrogenic differentiation of human adipose-derived MSCs (hADMSCs) and potentiates the therapeutic effects of hADMSCs in OA rabbits. METHODS We investigated the chondrogenic potential of TSP2 in hADMSCs by analyzing the expression of chondrogenic markers as well as NOTCH signaling genes in normal and TSP2 small interfering RNA (siRNA)-treated stem cells. Anterior cruciate ligament transection surgery was performed in male New Zealand white rabbits, and 8 wk later, hADMSCs (1.7 × 106 or 1.7 × 107 cells) were injected into the injured knees alone or in combination with intra-articular injection of TSP2 (100 ng/knee) at 2-d intervals. OA progression was monitored by gross, radiological, and histological examinations. RESULTS In hADMSC culture, treatment with TSP2 increased the expression of chondrogenic markers (SOX9 and collagen II) as well as NOTCH signaling genes (JAGGED1 and NOTCH3), which were inhibited by TSP2 siRNA treatment. In vivo, OA rabbits treated with hADMSCs or TSP2 alone exhibited lower degree of cartilage degeneration, osteophyte formation, and extracellular matrix loss 8 wk after cell transplantation. Notably, such cartilage damage was further alleviated by the combination of hADMSCs and TSP2. In addition, synovial inflammatory cytokines, especially tumor-necrosis factor-α, markedly decreased following the combination treatment. CONCLUSION The results indicate that TSP2 enhances chondrogenic differentiation of hADMSCs via JAGGED1/NOTCH3 signaling, and that combination therapy with hADMSCs and TSP2 exerts synergistic effects in the cartilage regeneration of OA joints.

Anti-atherosclerosis effects of perilla oil were investigated, in comparison with lovastatin, in rabbits fed a high-cholesterol diet (HCD). Hypercholesterolemia was induced in rabbits by feeding the HCD containing 0.5% cholesterol and 1% corn oil, and perilla oil (0.1 or 0.3%) was added to the diet containing 0.5% cholesterol for 10 weeks. HCD greatly increased blood total cholesterol and low-density lipoproteins, and caused thick atheromatous plaques, covering 74% of the aortic wall. Hyper-cholesterolemia also induced lipid accumulation in the liver and kidneys, leading to lipid peroxidation. Perilla oil not only attenuated hypercholesterolemia and atheroma formation, but also reduced fat accumulation and lipid peroxidation in hepatic and renal tissues. The results indicate that perilla oil prevents atherosclerosis and fatty liver by controlling lipid metabolism, and that it could be the first choice oil to improve diet-induced metabolic syndrome.

Since multiple sclerosis (MS) is featured with widespread demyelination caused by autoimmune response, we investigated the recovery effects of F3.olig2 progenitors, established by transducing human neural stem cells (F3 NSCs) with Olig2 transcription factor, in myelin oligodendrocyte glycoprotein- (MOG-) induced experimental autoimmune encephalomyelitis (EAE) model mice. Six days after EAE induction, F3 or F3.olig2 cells (1 × 106/mouse) were intravenously transplanted. MOG-injected mice displayed severe neurobehavioral deficits which were remarkably attenuated and restored by cell transplantation, in which F3.olig2 cells were superior to its parental F3 cells. Transplanted cells migrated to the injured spinal cord, matured to oligodendrocytes, and produced myelin basic proteins (MBP). The F3.olig2 cells expressed growth and neurotrophic factors including brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), and leukemia inhibitory factor (LIF). In addition, the transplanted cells markedly attenuated inflammatory cell infiltration, reduced cytokine levels in the spinal cord and lymph nodes, and protected host myelins. The results indicate that F3.olig2 cells restore neurobehavioral symptoms of EAE mice by regulating autoimmune inflammatory responses as well as by stimulating remyelination and that F3.olig2 progenitors could be a candidate for the cell therapy of demyelinating diseases including MS.

Stroke is one of the most-devastating brain diseases causing acute death or permanent disability. Although tissue-type plasminogen activator was approved by Food and Drug Administration for early reperfusion of the occluded vessels, oxidative injury may cause extensive brain infarction. Accordingly, there is a need for effective neuroprotection during reperfusion, and stem cell-based therapeutic approaches should fulfill this requirement. We established human neural stem cells (NSCs) encoding gene of choline acetyltransferase (F3.ChAT), an acetylcholine-synthesizing enzyme, and investigated whether infusion of the F3.ChAT cells attenuate the ischemia-reperfusion brain damage in a rat model of middle cerebral artery occlusion (MCAO). F3.ChAT cells were found to produce much higher amounts of ChAT as well as neuroprotective and anti-inflammatory neurotrophins than their parental F3 NSCs. After 2-h occlusion, the artery was reperfused, along with intravenous infusion of the stem cells (1 × 106 cells/rat). Administration of the F3.ChAT cells markedly reduced the infarction volume and improved both the cognitive dysfunction and behavioural deficits of MCAO animals, in which F3.ChAT cells were superior to F3 cells. F3.ChAT cells not only restored microtubule-associated protein-2, a neuronal cytoskeletal protein, and preserved microvessels, but also suppressed lipid peroxidation, pro-inflammatory cytokines, glial fibrillary acidic protein, and intercellular adhesion molecule-1 in the brain tissues. The results demonstrate that early intravenous infusion of NSCs expressing ChAT and neurotrophins attenuate brain and capillary injuries and restore neurobehavioural functions via neuroprotective and anti-inflammatory activities, and that F3.ChAT cells could be a candidate for the neuroprotection and functional recovery of acute stroke patients.

In order to assess inhibitory potentials of white rose petal extracts (WRPE) on the activities of enzymes related to dermal aging according to the extraction conditions, three extraction methods were adopted. WRPE was prepared by extracting dried white rose (Rosa hybrida) petals with 50% ethanol (WRPE-EtOH), Pectinex® SMASH XXL enzyme (WRPE-enzyme) or high temperature-high pressure (WRPE-HTHP). In the inhibition of matrix metalloproteinase-1, although the enzyme activity was fully inhibited by all 3 extracts at 100 µg/mL in 60 min, partial inhibition (50-70%) was achieved only by WRPE-EtOH and WRPE-enzyme at 50 µg/mL. High concentrations (≥250 µg/mL) of all 3 extracts markedly inhibited the elastase activity. However, at low concentrations (15.6-125 µg/mL), only WRPE-EtOH inhibited the enzyme activity. Notably, WRPE-EtOH was superior to WRPE-enzyme and WRPE-HTHP in the inhibition of tyrosinase. WRPE-EtOH significantly inhibited the enzyme activity from 31.2 µM, reaching 80% inhibition at 125 µM. In addition to its strong antioxidative activity, the ethanol extract of white rose petals was confirmed to be effective in inhibiting skin aging-related enzymes. Therefore, it is suggested that WRPE-EtOH could be a good candidate for the improvement of skin aging such as wrinkle formation and pigmentation.

Aging brings about the progressive decline in cognitive function and physical activity, along with losses of stem cell population and function. Although transplantation of muscle-derived stem/progenitor cells extended the health span and life span of progeria mice, such effects in normal animals were not confirmed. Human amniotic membrane-derived mesenchymal stem cells (AMMSCs) or adipose tissue-derived mesenchymal stem cells (ADMSCs) (1 × 106 cells per rat) were intravenously transplanted to 10-month-old male F344 rats once a month throughout their lives. Transplantation of AMMSCs and ADMSCs improved cognitive and physical functions of naturally aging rats, extending life span by 23.4% and 31.3%, respectively. The stem cell therapy increased the concentration of acetylcholine and recovered neurotrophic factors in the brain and muscles, leading to restoration of microtubule-associated protein 2, cholinergic and dopaminergic nervous systems, microvessels, muscle mass, and antioxidative capacity. The results indicate that repeated transplantation of AMMSCs and ADMSCs elongate both health span and life span, which could be a starting point for antiaging or rejuvenation effects of allogeneic or autologous stem cells with minimum immune rejection. Significance This study demonstrates that repeated treatment with stem cells in normal animals has antiaging potential, extending health span and life span. Because antiaging and prolonged life span are issues currently of interest, these results are significant for readers and investigators.

Helicobacter pylori-eliminating effects of FEMY-R7, composed of Laminaria japonica and Oenothera biennis extracts, were investigated in mice and humans. Male C57BL/6 mice were infected with the bacteria by intragastric inoculation (1×10(9) CFU/mouse) 3 times at 2-day intervals, and simultaneously, orally treated twice a day with total 20, 64 or 200 mg/kg/day FEMY-R7 for 2 weeks. In Campylobcter-like organism (CLO)-detection tests on gastric mucosa and feces, FEMY-R7 reduced the urease-positive reactivity in a dose-dependent manner; i.e., the positivity ratios were decreased to 70, 20, and 10% for gastric mocosa and to 80, 50, and 20% for feces. In a clinical sudy, human subjects, confirmed to be infected with Helicobacter pylori, were orally administered twice a day with capsules containing total 100, 320 or 1,000 mg/man/day FEMY-R7 (matching doses for 20, 64 or 200 mg/kg/day, respectively, in mice from a body surface area-based dose translation) for 8 weeks. FEMY-R7 decreased the positivity ratios in feces to 70, 40, and 30%, respectively. In bacterial culture, H. pylori was identified from the CLO-positive stools of mice and humans. The bacterial identification ratios exhibited a good correlation between the matching doses in mice and humans. It is suggested that FEMY-R7 could be a promising functional food without tolerance as an adjunct to reduce the dosage of antibiotics for the treatment of recurrent H. pylori infection.

Anti-adipogenic and anti-obesity effects of a green coffee been extract (GCE) from unroasted Coffea canephora Robusta beans were investigated in vitro and in vivo. Insulin-induced adipogenesis in 3T3-L1 preadipocytes was inhibited by 25.6 and 87.9% following treatment with 100 and 500 μg/mL GCE, respectively. Expression of adipogenesis-specific genes was down-regulated. Body weight gains of C57BL/6 mice fed a high-fat diet (HFD) were suppressed in a concentration-dependent manner using GCE (0.3 or 1% in the diet), exhibiting a marked lower gain in 1% GCE-fed mice than in normal diet-fed animals. Decreases in body fat weights and the size of adipocytes were confirmed. GCE supplementation reduced concentrations of blood lipids and leptin, and attenuated hepatic steatosis. GCE inhibits adipogenesis by modulating cell signaling and improves dietary obesity by reducing lipid accumulation and the size of adipocytes.

Helicobacter pylori-eliminating effects of FEMY-R7, composed of fucoidan and evening primrose extract, were investigated in mice and humans. Male C57BL/6 mice were infected with the bacteria by intragastric inoculation (1×10 9 CFU/mouse) 3 times at 2-day intervals, and simultaneously, orally treated twice a day with 10 or 100 mg/kg FEMY-R7 for 2 weeks. In Campylobcter-like organism-detection test, FEMY-R7 markedly reduced the urease-positive reactivity. In a clinical sudy, human subjects, confirmed to be infected with Helicobacter pylori, were orally administered twice a day with a capsule containing 150 mg FEMY-R7 for 8 weeks. FEMY-R7 significantly decreased both the Delta over baseline-value in urea breath test and the serum pepsinogens I and II levels. The results indicate that FEMY-R7 not only eliminates H. pylori from gastric mucosa of animals and humans, but also improves gastric function.

Since oils and fats can induce metabolic syndrome, leading to cardiovascular and cerebrovascular diseases, the present study was performed to find out whether the plant oils affect the cerebral hemorrhage in stroke-prone spontaneously hypertensive (SHR-SP) rats.From 47 days of age, male SHR-SP rats were given drinking water containing 1% NaCl to induce hypertension, and simultaneously fed semi-purified diets containing 10% perilla oil, canola oil, or shortening. The onset time of convulsion following cerebral hemorrhage was recorded, and the areas of hemorrhage and infarction were analyzed in the stroke brains.In comparison with 58-day survival of SHR-SP rats during feeding NaCl alone, perilla oil extended the survival time to 68.5 days, whereas canola oil shortened it to 45.7 days. Feeding perilla oil greatly reduced the total volume of cerebral hemorrhage from 17.27% in the control group to 4.53%, while shortening increased the lesions to 21.23%. In a microscopic analysis, perilla oil also markedly decreased the hemorrhagic and infarction lesions to 1/10 of those in control rats, in contrast to an exacerbating effect of shortening. In blood analyses, perilla oil reduced blood total cholesterol and low-density lipoproteins which were increased in SHR-SP, but canola oil further increased them and markedly lowered platelet counts.Perilla oil delayed and attenuated cerebral hemorrhage by improving hyperlipidemia in hypertensive stroke animals, in contrast to the aggravating potential of canola oil and shortening. It is suggested that perilla oil should be the first choice oil for improving metabolic syndrome in hypertensive persons at risk of hemorrhagic stroke.

In Alzheimer disease (AD), amyloid-beta (Aβ) peptides induce the degeneration of presynaptic cholinergic system, in which decreased activity of enzyme choline acetyltransferase (ChAT) responsible for acetylcholine synthesis is observed. Cereboost™, an extract of American ginseng extract, contains a high concentration of Rb1 ginsenoside which is a well-known ingredient improving human cognitive function. We investigated the effects of Cereboost™ on learning and memory function of mice challenged with an Aβ1-42 peptide and the underlying mechanisms in vitro. Cereboost™ protected against Aβ1-42-induced cytotoxicity in F3.ChAT stem cells, and enhanced the ChAT gene expression. Aβ1-42 injection into the mouse brain impaired the cognitive function, which was recovered by oral administration of Cereboost™. In addition, Cereboost™ restored brain microtubule-associated protein 2 and synaptophysin as well as acetylcholine concentration. The results demonstrate that Cereboost™ administration recovered the cognitive function of AD model animals by enhancing acetylcholine level via ChAT gene expression and neuroprotection.

White rose (Rosa hybrida) petals were extracted with ethanol (EtOH) or butanol (BuOH), and tested for their antimicrobial activities against two species of Gram-positive bacteria, six species of Gram-negative bacteria, and two species of fungi. On in vitro antimicrobial assays, Helicobacter pylori and Propionibacterium acnes were highly susceptible to white rose petal extract (WRPE)-EtOH and WRPE-BuOH, leading to minimal inhibitory concentrations of 100 and 10 μg/mL for H. pylori and 400 and 40 μg/mL for P. acnes, respectively. In in vivo experiments, C57BL/6 mice were infected with H. pylori by intragastric inoculation (1 × 10(8) CFU/mouse) 3 times, and orally treated twice a day for 14 days with WRPE-EtOH and WRPE-BuOH. On a CLO kit assay, 200 mg/kg of WRPE-EtOH fully eliminated the bacteria from the gastric mucosa, and the effect of 100 mg/kg of ethanol fraction was similar to pantoprazole (30 mg/kg), displaying 75% elimination. WRPE-BuOH was more effective, exhibiting 75% elimination at 20 mg/kg. The CLO test results were confirmed by bacterial identification. WRPE-EtOH and WRPE-BuOH inhibited the growth of various bacteria and fungi, and in particular, they effectively killed H. pylori and eliminated the bacteria from the mouse stomach. The results indicate that WRPE-EtOH and WRPE-BuOH could be good candidates for the elimination of H. pylori.

Objective. Since oligodendrocyte progenitor cells (OPCs) are the target cells of neonatal hypoxic-ischemic encephalopathy (HIE), the present study was aimed at investigating the protective effects ofN-acetyl-L-cysteine (NAC), a well-known antioxidant and precursor of glutathione, in OPCs as well as in neonatal rats.Methods. Inin vitrostudy, protective effects of NAC on KCN cytotoxicity in F3.Olig2 OPCs were investigated via MTT assay and apoptotic signal analysis. Inin vivostudy, NAC was administered to rats with HIE induced by hypoxia-ischemia surgery at postnatal day 7, and their motor functions and white matter demyelination were analyzed.Results. NAC decreased KCN cytotoxicity in F3.Olig2 cells and especially suppressed apoptosis by regulating Bcl2 and p-ERK. Administration of NAC recovered motor functions such as the using ratio of forelimb contralateral to the injured brain, locomotor activity, and rotarod performance of neonatal HIE animals. It was also confirmed that NAC attenuated demyelination in the corpus callosum, a white matter region vulnerable to HIE.Conclusion. The results indicate that NAC exerts neuroprotective effectsin vitroandin vivoby preserving OPCs, via regulation of antiapoptotic signaling, and that F3.Olig2 human OPCs could be a good tool for screening of candidates for demyelinating diseases.

Effects of FEMY-R7, composed of fucoidan and evening primrose extract, on the bacterial growth and intragastric infection of Helicobacter pylori as well as gastric secretion were investigated in comparison with a proton-pump inhibitor pantoprazole. For in vitro anti-bacterial activity test, H. pylori (1×10(8) CFU/mL) was incubated with a serially-diluted FEMY-R7 for 3 days. As a result, FEMY-R7 fully inhibited the bacterial growth at 100 µg/mL, which was determined to be a minimal inhibitory concentration. In addition, 6-hour incubation with H. pylori, FEMY-R7 inhibited urease activity in a concentration-dependent manner, showing a median inhibitory concentration of 1,500 µg/mL. In vivo elimination study, male C57BL/6 mice were infected with the bacteria by intragastric inoculation (5×10(9) CFU/mouse) 3 times at 2-day intervals, and simultaneously, orally treated twice a day with 10, 30 or 100 mg/kg FEMY-R7 for 7 days. In Campylobcter-like organism-detection test and bacterial identification, FEMY-R7 exerted a high bacteria-eliminating capacity at 30-100 mg/kg, comparably to 30 mg/kg pantoprazole. In contrast to a strong antacid activity of pantoprazole in a pylorus-ligation study, FEMY-R7 did not significantly affect gastric pH, free HCl, and total acidity, although it significantly decreased fluid volume at a low dose (10 mg/kg). The results indicate that FEMY-R7 eliminate H. pylori from gastric mucosa by directly killing the bacteria and preventing their adhesion and invasion, rather than by inhibiting gastric secretion or mucosal damage.

Since scalp hair loss has increased recently even in young people, seriously affecting individual's quality of life, the hair growth-stimulating effects of Laminaria japonica extract (LJE) and Cistanche tubulosa extract (CTE) were investigated. After confirming anagen phase of follicles under shaving, male C57BL/6 mice were dermally applied with 3% Minoxidil or orally administered with the combinations of LJE and CTE for 21 days. Minoxidil promoted the hair regrowth and increased γ-glutamyl transpeptidase (γ-GTP) and alkaline phosphatase (ALP) activities. In addition, Minoxidil up-regulated epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) levels. Co-administration of LJE and CTE at 54 mg/kg LJE plus 162 mg/kg CTE exerted synergistic promoting effects on the hair regrowth, comparable to 3% Minoxidil. LJE preferentially enhanced ALP activity, while CTE increased both γ-GTP and ALP activities as well as EGF and VEGF expressions. In vivo air pouch inflammation model, carrageenan-induced vascular exudation and increased nitric oxide and prostaglandin E2 concentrations in the exudates were synergistically suppressed by co-administration of LJE and CTE. In addition, inflammatory cell infiltration was substantially inhibited by the combinational treatment. The results suggest that combinational oral treatment with LJE and CTE in appropriate doses and ratios prevent hair loss and improve alopecia, which might be in part mediated by their anti-inflammatory activities.

Effects of FEMY-R7, composed of fucoidan and evening primrose extract, on the bacterial growth and intragastric infection of Helicobacter pylori as well as gastric secretion were investigated in comparison with a proton-pump inhibitor pantoprazole. For in vitro anti-bacterial activity test, H. pylori (1×108 CFU/mL) was incubated with a serially-diluted FEMY-R7 for 3 days. As a result, FEMY-R7 fully inhibited the bacterial growth at 100 µg/mL, which was determined to be a minimal inhibitory concentration. In addition, 6-hour incubation with H. pylori, FEMY-R7 inhibited urease activity in a concentration-dependent manner, showing a median inhibitory concentration of 1,500 µg/mL. In vivo elimination study, male C57BL/6 mice were infected with the bacteria by intragastric inoculation (5×109 CFU/mouse) 3 times at 2-day intervals, and simultaneously, orally treated twice a day with 10, 30 or 100 mg/kg FEMY-R7 for 7 days. In Campylobcter-like organism-detection test and bacterial identification, FEMY-R7 exerted a high bacteria-eliminating capacity at 30-100 mg/kg, comparably to 30 mg/kg pantoprazole. In contrast to a strong antacid activity of pantoprazole in a pylorus-ligation study, FEMY-R7 did not significantly affect gastric pH, free HCl, and total acidity, although it significantly decreased fluid volume at a low dose (10 mg/kg). The results indicate that FEMY-R7 eliminate H. pylori from gastric mucosa by directly killing the bacteria and preventing their adhesion and invasion, rather than by inhibiting gastric secretion or mucosal damage.

The effects of an ethanolic extract of Angelica gigas (EAG) on the vascular smooth muscle cell (VSMC) proliferation and high-cholesterol diet-induced hypercholesterolemia and atherosclerosis were investigated. Rat aortic VSMCs were stimulated with platelet-derived growth factor-BB (25 ng/mL) for the induction of DNA synthesis and cell proliferation. EAG (1-10 µg/mL) significantly inhibited both the thymidine incorporation and cell proliferation in a concentration-dependent manner. Hypercholesterolemia was induced by feeding male New Zealand white rabbits with 0.5% cholesterol in diet for 10 weeks, during which EAG (1% in diet) was given for the final 8 weeks after 2-week induction of hypercholesterolemia. Hypercholesterolemic rabbits exhibited great increases in serum total cholesterol and low-density lipoproteins (LDL) levels, and finally severe atheromatous plaque formation covering 28.4% of the arterial walls. EAG significantly increased high-density lipoproteins (HDL), slightly decreased LDL, and potentially reduced the atheroma area to 16.6%. The results indicate that EAG attenuates atherosclerosis not only by inhibiting VASC proliferation, but also by increasing blood HDL levels. Therefore, it is suggested that EAG could be an alternative or an adjunct therapy for the improvement of hypercholesterolemia and atherosclerosis.

The inhibitory effects of perilla oil on the platelet aggregation in vitro and thrombosis in vivo were investigated in comparison with aspirin, a well-known blood flow enhancer. Rabbit platelet-rich plasma was incubated with perilla oil and aggregation inducers collagen or thrombin, and the platelet aggregation rate was analyzed. Perilla oil significantly inhibited both the collagen- and thrombin-induced platelet aggregations, in which the thromboxane B2 formation from collagen-activated platelets were reduced in a concentration-dependent manner. Rats were administered once daily by gavage with perilla oil for 1 week, carotid arterial thrombosis was induced by applying 35% FeCl3-soaked filter paper for 10 min, and the blood flow was monitored with a laser Doppler probe. Perilla oil delayed the FeCl3-induced arterial occlusion in a dose-dependent manner, doubling the occlusion time at 0.5 mL/kg. In addition, a high dose (2 mL/kg) of perilla oil greatly prevented the occlusion, comparable to the effect of aspirin (30 mg/kg). The results indicate that perilla oil inhibit platelet aggregation by blocking thromboxane formation, and thereby delay thrombosis following oxidative arterial wall injury. Therefore, it is proposed that perilla oil could be a good candidate without adverse effects for the improvement of blood flow.

The effects of nattokinase on the in vitro platelet aggregation and in vivo thrombosis were investigated in comparison with aspirin. Rabbit platelet-rich plasma was incubated with nattokinase and aggregation inducers collagen and thrombin, and the platelet aggregation rate was analyzed. Nattokinase significantly inhibited both the collagen- and thrombin-induced platelet aggregations. Nattokinase also reduced thromboxane B2 formation from collagen-activated platelets in a concentration-dependent manner. Rats were orally administered with nattokinase for 1 week, and their carotid arteries were exposed. Arterial thrombosis was induced by applying 35% FeCl3-soaked filter paper for 10 min, and the blood flow was monitored with a laser Doppler probe. Nattokinase delayed the FeCl3-induced arterial occlusion in a dose-dependent manner, doubling the occlusion time at 160 mg/kg. In addition, a high dose (500 mg/kg) of nattokinase fully prevented the occlusion, as achieved with aspirin (30 mg/kg). The results indicate that nattokinase extracted from fermented soybean inhibit platelet aggregation by blocking thromboxane formation, and thereby delay thrombosis following oxidative arterial wall injury. Therefore, it is suggested that nattokinase could be a good candidate without adverse effects for the improvement of blood flow.