Maho Sumiyoshi

Low molecular weight chitosan inhibits obesity induced by feeding a high-fat diet long-term in mice.

Three low molecular weight chitosans (molecular weight: 21, 46 and 130 kDa) obtained by enzymatic hydrolysis of a high molecular weight chitosan (average molecular weight: 650 kDa) had low viscosity and were water‐soluble. The effects of these water‐soluble chitosans on pancreatic lipase (in‐vitro) and the elevation of plasma triacylglycerol concentration after the oral lipid tolerance test were examined in mice. The water‐soluble 46‐kDa chitosan was the most effective at inhibiting pancreatic lipase activity (in‐vitro) and plasma triacylglycerol elevation after the oral lipid tolerance test. Based on this result, the effects of the 46‐kDa chitosan on increases in bodyweight, various white adipose tissue weights, and plasma and liver lipids were examined in mice fed a high‐fat diet for 20 weeks. Water‐soluble 46‐kDa chitosan (300 mg kg−1, twice daily) prevented increases in bodyweight, various white adipose tissue weights and liver lipids (cholesterol and triacylglycerol) in mice fed a high‐fat diet, and further increased the faecal bile acid and fat. The results suggest that the lipid‐lowering effects of the 46‐kDa chitosan may be mediated by increases in faecal fat and/or bile acid excretion resulting from the binding of bile acids, and by a decrease in the absorption of dietary lipids (triacylglycerol and cholesterol) from the small intestine as a result of the inhibition of pancreatic lipase activity. Water‐soluble 46‐kDa chitosan (100 and 300 mg kg−1, twice daily) did not cause liver damage with the elevation of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase, or kidney damage with the elevation of blood nitrogen urea. It was concluded that watersoluble 46‐kDa chitosan is a safe functional food.

Isolation of an anti-angiogenic substance from Agaricus blazei Murill: its antitumor and antimetastatic actions.

We previously found that ergosterol isolated from Agaricus blazei inhibited tumor growth through the inhibition of tumor‐induced neovascularization. In the present study, we isolated further anti‐angiogenic substances (A‐1 and A‐2) from this fungus using an assay system of angiogenesis induced by Matrigel supplemented with vascular endothelial growth factor, and A‐1 was identified as sodium pyroglutamate. Next, we examined the antitumor and antimetastatic actions of A‐1 using Lewis lung carcinoma (LLC)‐bearing mice. A‐1 (30, 100 and 300 mg/kg) inhibited tumor growth and metastasis to the lung. The reduction of the numbers of splenic lymphocytes, CD4+ and CD8+ T cells in LLC‐bearing mice was inhibited by the oral administration of A‐1 (30, 100 and 300 mg/kg). Further, A‐1 increased the number of apoptotic cells of tumors and the numbers of CD8+ T and natural killer cells invading the tumors, and inhibited the increase of von Willebrand factor expression (a measure of angiogenesis) in the tumors. These results suggest that the antitumor and antimetastatic actions of A‐1 (sodium pyroglutamate) may be associated with inhibition of the reduction of immune response caused by the tumor growth and tumor‐induced neovascularization. This is the first report showing that sodium pyroglutamate isolated from A. blazei as an anti‐angiogenic substance has potent antitumor and antimetastatic actions, as well as immune‐modulatory activity, in tumor‐bearing mice.

Effects of ginseng saponins isolated from Red Ginseng roots on burn wound healing in mice.

1 We recently demonstrated that ginsenoside Rb1 (C54H92O23, molecular weight 1108) isolated from ginseng, when intravenously infused into rats with permanent middle cerebral artery occlusion, reduced cerebral infarct volume and ameliorated place navigation disability of the animals, through an anti‐apoptotic action and possibly promotion of vascular regeneration. To investigate the ginsenoside Rb1‐mediated vascular regeneration in vivo in a more easily accessible experimental systems, we made a burn wound on the backs of mice and topically applied either Vaseline (vehicle) alone or Vaseline containing low doses of ginsenoside Rb1 to the wound. 2 Surprisingly, we found that ginsenoside Rb1 at low concentrations (100 pg g−1, 1 pg g−1 and 10 fg g−1 ointment) exhibited the strongest burn wound‐healing action. Furthermore, ginsenoside Rb1 (100 fg–1 ng per wound) increased neovascularization in the surrounding tissue and production of vascular endothelial growth factor (VEGF) and interleukin (IL)‐1β from the burn wound, compared to those mice with burn wounds treated with vehicle alone. 3 In human keratinocyte cultures (HaCaT cells), ginsenoside Rb1 (100 fg ml−1 to 1 ng ml−1) enhanced VEGF production induced by IL‐1β and expression of hypoxia‐inducible factor (HIF)‐1α. 4 These findings suggest that the promotion of burn wound healing by ginsenoside Rb1 might be due to the promotion of angiogenesis during skin wound repair via the stimulation of VEGF production, through the increase of HIF‐1α expression in keratinocytes, and due to the elevation of IL‐1β resulting from the macrophage accumulation in the burn wound.

Facilitating action of asiaticoside at low doses on burn wound repair and its mechanism

Some reports published from 1967 to 1999 describe the use of ointments containing high doses (0.1 to 0.2%, w/w) C. asiataica herb extracts to enhance wound repair. Lower doses at which burn wound repair is enhanced by such topical applications have not been established yet. We found that the application of asiaticoside at low doses of 10(-8) to 10(-12)% (w/w) facilitated burn wound repair. To clarify the accelerating mechanisms of asiaticoside on burn wound repair, we examined the effects of asiaticoside on the levels of various cytokines produced at the site of the burn wound. The topical application of a low dose (10 pg, 1 ng, or 100 ng/wound area) of asiaticoside increased monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), and interleukin (IL)-1beta levels in burn wound exudates. Asiaticoside (10 pg to 100 ng/ml) enhanced MCP-1 production in HaCaT cells, but it had no direct effect on VEGF production. Furthermore, asiaticoside (10 pg to 100 ng/ml) increased the IL-1beta production in THP-1 macrophages with MCP-1, but it had no effect on IL-1beta production without MCP-1 or with lipopolysaccharide (LPS). These findings suggest that the enhancement of burn wound healing by asiaticoside might be due to the promotion of angiogenesis during skin wound repair as a result of the stimulation of VEGF production caused by the increase in MCP-1 expression in keratinocytes and the increase in IL-1beta expression in macrophages induced cooperatively by asiaticoside plus MCP-1.

Olive Leaf Extract and Its Main Component Oleuropein Prevent Chronic Ultraviolet B Radiation-Induced Skin Damage and Carcinogenesis in Hairless Mice

Chronic exposure to solar UV radiation damages skin, increasing its thickness and reducing its elasticity, and causes skin cancer. Our aim in this study was to examine the effects of an olive leaf extract and its component oleuropein on skin damage and the incidence of skin tumors caused by long-term UVB irradiation in hairless mice. Male hairless mice (5 wk old) were divided into 6 groups, including a non-UVB group, a vehicle-treated UVB group (control), 2 olive leaf extract-treated UVB groups, and 2 oleuropein-treated UVB groups. Five groups were UVB irradiated (36-180 mJ/cm(2)) 3 times each week for 30 wk and skin thickness and elasticity after UVB irradiation were measured every week. Olive leaf extract (300 and 1000 mg/kg) and oleuropein (10 and 25 mg/kg) were administered orally twice daily every day for 30 wk. The extract and oleuropein significantly inhibited increases in skin thickness and reductions in skin elasticity, and skin carcinogenesis and tumor growth. Furthermore, they prevented increases in the expression of matrix metalloproteinase (MMP)-2, MMP-9, and MMP-13 as well as in levels of vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2) in the skin. Based on histological evaluation, they prevented increases in the expression of Ki-67 and CD31-positive cells induced by the irradiation. These results suggest that the preventative effects of the olive leaf extract and oleuropein on chronic UVB-induced skin damage and carcinogenesis and tumor growth may be due to inhibition of the expression of VEGF, MMP-2, MMP-9, and MMP-13 through a reduction in COX-2 levels.

Antitumor activities of synthetic and natural stilbenes through antiangiogenic action

We reported that the antitumor and antimetastatic actions of resveratrol might be due to the inhibition of tumor‐induced angiogenesis. To search for anticancer agents with stronger activity than resveratrol, we examined the antiangiogenic effects of 21 synthetic and/or natural stilbenes. Among these 21 stilbenes, 2,3‐, 3,4‐, and 4,4′‐dihydroxystilbene inhibited the pro‐matrix metalloproteinase (pro‐MMP)–9 production in colon 26 cells at 5–25 µM, vascular endothelial growth factor (VEGF)–induced human umbilical vein endothelial cell (HUVEC) migration at 10 and 25 µM, and VEGF‐induced angiogenesis at 5–50 µM. Resvertarol inhibited the pro‐MMP‐9 production and VEGF‐induced angiogenesis at 25 or 50 µM. Thus, the inhibition of pro‐MMP‐9 production in colon 26 cells and VEGF‐induced angiogenesis by three dihydroxystilbenes were greater than those of resveratrol. The three dihydroxystilbenes (8 mg/kg, intraperitoneal injection) inhibited the tumor‐induced neovascularization in colon 26–packed chamber‐bearing mice and the tumor growth in colon 26–bearing mice. Furthermore, the three dihydroxystilbenes inhibited VEGF‐induced VEGFR‐2 phosphorylation. On the other hand, the three dihydroxystilbenes had no effect on VEGFR‐1 and‐2 expression, and VEGF‐induced VEGFR‐1 phosphorylation in HUVECs. These findings suggest that the inhibition of tumor‐induced neovascularization by these three dihydroxystilbenes may be due to the inhibition of VEGF‐induced endothelial cell migration and VEGF‐induced angiogenesis through the inhibition of VEGF‐induced VEGFR‐2 phosphorylation in endothelial cells and pro‐MMP‐9 expression in colon 26 cells. (Cancer Sci 2008; 99: 2083–2096)

Effects of a turmeric extract (Curcuma longa) on chronic ultraviolet B irradiation-induced skin damage in melanin-possessing hairless mice.

Turmeric (the rhizomes of Curcuma longa L., Zingiberacease) is widely used as a dietary pigment and spice, and has been traditionally used for the treatment of inflammation, skin wounds and hepatic disorders in Ayurvedic, Unani and Chinese medicine. Although the topical application or oral administration of turmeric is used to improve skin trouble, there is no evidence to support this effect. The aim of this study was to clarify whether turmeric prevents chronic ultraviolet B (UVB)-irradiated skin damage. We examined the effects of a turmeric extract on skin damage including changes in skin thickness and elasticity, pigmentation and wrinkling caused by long-term, low-dose ultraviolet B irradiation in melanin-possessing hairless mice. The extract (at 300 or 1000 mg/kg, twice daily) prevented an increase in skin thickness and a reduction in skin elasticity induced by chronic UVB exposure. It also prevented the formation of wrinkles and melanin (at 1000 mg/kg, twice daily) as well as increases in the diameter and length of skin blood vessels and in the expression of matrix metalloproteinase-2 (MMP-2). Prevention of UVB-induced skin aging by turmeric may be due to the inhibition of increases in MMP-2 expression caused by chronic irradiation.

Effects of baicalein and wogonin isolated from Scutellaria baicalensis roots on skin damage in acute UVB-irradiated hairless mice

Solar ultraviolet (UV) radiation causes skin damage including increases in skin thickness, edema, and flush. In this study, we examined the effects of two main flavonoids (wogonin and baicalein) isolated from the roots of Scutellaria baicalensis, a traditional remedy for allergic inflammatory diseases long used in China and Japan, on acute UVB irradiation-induced skin damage in hairless mice. Baicalein and wogonin (10 or 50 mg/kg) were administered orally twice daily for 14 consecutive days. The UVB irradiation was performed at a dose of 200 mJ/cm(2) on days 7 and 8 of the treatment with the two main flavonoids. Baicalein, and wogonin prevented the increases in skin thickness and the levels of matrix metalloproteinase (MMP)-9, and vascular endothelial growth factor (VEGF) induced by the irradiation. Wogonin reduced the levels of cyclooxygenase (COX)-2 and hypoxia inducible factor (HIF)-1α in UVB-treated HaCaT cells. These findings suggest that wogonin inhibits irradiation-induced skin damage by suppressing increases in the levels of MMP-9, and VEGF through the inhibition of COX-2 and HIF-1α expression. Baicalein inhibited COX-2 and NF-κB/p65 expression, but stimulated HIF-1α expression. Therefore, its inhibitory action is likely due to the expression of MMP-9 and VEGF through the suppression of COX-2 and NF-κB/p65 expression. Furthermore, the inhibitory effects of baicalein on UVB-irradiated hyperplasia of skin epidermis may be due to the stimulation of HIF-1α expression.

Antitumor and antimetastatic actions of anthrone‐C‐glucoside, cassialoin isolated from Cassia garrettiana heartwood in colon 26‐bearing mice

We examined the antitumor and antimetastatic actions of 10‐hydroxy‐anthrone‐C‐glucoside cassialoin isolated from Cassia garrettiana heartwood in colon 26‐bearing mice. Cassialoin (5 and 10 mg/kg) inhibited tumor growth and metastasis to the abdomen and the expression of CD31 (angiogenesis marker) in the tumors, and it increased the numbers of the γ‐interferon (IFN‐γ)‐positive, CD8+ T and natural killer cells in the small intestine or spleen of colon 26‐bearing mice. Furthermore, cassialoin inhibited tumor‐induced angiogenesis in colon 26‐packed chamber‐bearing mice. We examined the metabolic activities in the blood, stomach and small intestine after p.o. administration of cassialoin to mice. These results suggest that cassialoin might be converted to chrysophanol through chrysophanol‐9‐anthrone and metabolized to aloe‐emodin from chrysophanol. Chrysophanol‐9‐anthrone inhibited vascular endothelial growth factor (VEGF) and matrix metallopeptidase‐9 expression in colon 26 cells at 5 and 10 µM, and it inhibited VEGF‐induced angiogenesis and migration in human umbilical vein endothelial cells (HUVEC) at 0.5–10 µM. Furthermore, chrysophanol‐9‐anthrone inhibited VEGF receptor (VEGFR)‐2 expression and VEGF‐induced VEGFR‐2 phosphorylation. Aloe‐emodin also inhibited the VEGF‐induced angiogenesis by HUVEC at 1–100 µM. Cassialoin, chrysophanol‐9‐anthrone and aloe‐emodin enhanced concanavalin A‐induced IFN‐γ production in splenocytes of colon 26‐bearing mice at a low concentration of 0.1 µM. From these results, it is suggested that the antitumor and antimetastatic actions of p.o. administered cassialoin may be partly due to cassialoin and its metabolites such as chrysophanol‐9‐anthrone and aloe‐emodin through their anti‐angiogenic activities and/or the modulation of the immune systems in the spleen and small intestine in tumor‐bearing mice. (Cancer Sci 2008; 99: 2336–2348)

Effects of an Atractylodes lancea rhizome extract and a volatile component β-eudesmol on gastrointestinal motility in mice

AIM OF THE STUDY The rhizomes of Atractylodes lancea DC (Compositae) are used clinically to treat gastrointestinal symptoms, including functional dyspepsia and gastroparesis, in China and Japan, but their influence and mechanism on gastrointestinal motility are not yet proven in detail. MATERIALS AND METHODS This study examined the effects of an Atractylodes lancea extract, and isolated β-eudesmol, on gastric emptying and small intestinal motility in atropine-, dopamine-, and 5-hydroxytryptamine (5-HT)-treated mice. RESULTS AND CONCLUSIONS The extract (500 or 1000mg/kg) and β-eudesmol (50 or 100mg/kg), as well as itopride hydrochloride (a dopamine D(2) receptor antagonist, 10 or 50mg/kg), stimulated small intestinal motility in normal mice. They inhibited reductions in gastric emptying and gastrointestinal motility induced by dopamine (1mg/kg, intraperitoneal injection, ip). The extract (1000mg/kg) and β-eudesmol (100mg/kg) inhibited the atropine-induced decrease in small intestinal motility, but not gastric emptying. Furthermore, the extract (500 or 1000mg/kg) and β-eudesmol (25, 50, or 100mg/kg) inhibited reductions in gastric emptying and small intestinal motility caused by 5-HT (4mg/kg, ip) or the 5-HT(3) receptor agonist 1-(3-chlorophenyl) biguanide (0.5mg/kg, ip), but not a 5-HT(2C) receptor agonist. These findings suggest that the extract of Atractylodes lancea and β-eudesmol may stimulate gastric emptying or small intestinal motility by inhibiting the dopamine D(2) receptor and 5-HT(3) receptor.