Safety evaluation of proanthocyanidin-rich extract from grape seeds.

Proanthocyanidins, extracted from grape seeds, are widely used mainly as nutritional supplements. However, there has not been a systematic report to investigate toxicological studies on proanthocyanidins, especially in oral administration. In our studies, proanthocyanidin-rich extract from grape seeds was subjected to a series of toxicological tests to document its safety for use in various foods. The grape seed extract (GSE) was examined for acute and subchronic oral toxicity using Fischer 344 rats and for mutagenic potential by the reverse mutation test using Salmonella typhimurium, the chromosomal aberration test using CHL cells, and the micronucleus test using ddY mice. No evidence of acute oral toxicity at dosages of 2 and 4 g/kg, and no evidence of mutagenicity in the above tests was found. Administration of GSE as a dietary admixture at levels of 0.02, 0.2 and 2% (w/w) to the rats for 90 days did not induce noticeable signs of toxicity. The no-observed-adverse-effect level (NOAEL) of GSE in the subchronic toxicity study was 2% in the diet (equal to 1410 mg/kg body weight/day in males and 1501 mg/kg body weight/day in females). The results of our studies indicate a lack of toxicity and support the use of proanthocyanidin-rich extract from grape seeds for various foods.

Isoflavone aglycone-rich extract without soy protein attenuates atherosclerosis development in cholesterol-fed rabbits.

The antiatherogenic effect of soy protein with intact isoflavones is well established, but the effects of isoflavones without soy protein have not been determined. We investigated the antiatherogenic effect of an isoflavone aglycone-rich extract (containing 429.4 mg/g isoflavone aglycones) without soy protein from fermented soy in cholesterol-fed rabbits. We fed 12-wk-old New Zealand white male rabbits diets containing 1 g/100 g cholesterol with 0, 0.33 or 1 g/100 g isoflavone aglycones for 8 wk. We also fed the rabbits a diet containing 1 g/100 g cholesterol with 1.09 g/100 g soy saponin-rich extract, a component other than isoflavone aglycones in the isoflavone aglycone-rich extract. Controls did not consume cholesterol, isoflavone aglycones or saponins. The isoflavone aglycone- and saponin-rich extracts did not affect the serum lipid profile of cholesterol-fed rabbits. The serum concentration of daidzein in its conjugated form was significantly higher in the high isoflavone group than in the low isoflavone group. The level of cholesteryl ester hydroperoxide (ChE-OOH) induced by CuSO(4) in plasma in the high isoflavone group was significantly less than that in the cholesterol group, and the ChE-OOH levels of LDL in the low and high isoflavone groups were significantly less than those in the cholesterol group. The ChE-OOH levels in plasma and LDL in the saponin group did not differ from the cholesterol group. In the aortic arch, the cholesterol concentration was significantly lower in the high isoflavone group, and malondialdehyde concentration was significantly lower in the low and high isoflavone groups compared with the cholesterol group; however these concentrations in the saponin group did not differ from those in the cholesterol group. The atherosclerotic lesion area of the aortic arch was significantly lower in the isoflavone groups (26.3% lower in the low isoflavone group and 36.9% lower in the high isoflavone group) than in the cholesterol group. The lesion areas were not different in the soy saponin and cholesterol groups. Immunohistochemical analysis revealed fewer oxidized LDL-positive macrophage-derived foam cells in atherosclerotic lesions in the aortic arch of isoflavone groups compared with that of the cholesterol group. These results suggest that the antioxidative action of isoflavones and their antioxidative metabolites inhibit the oxidation of LDL, thereby exerting an antiatherosclerotic effect.

Safety evaluation of yeast glutaminase.

The consumption of soy and soy products (including soy sauce) has been increasing in Western countries due to purported health benefits of soy (cancer protective, estrogenic effects). In addition to providing soy proteins and isoflavones, soy sauce also functions as a flavor enhancer and is able to impart a "umami" taste. Glutaminases are used in the production of soy sauce and enzymatically hydrolyzed protein. The glutaminases described herein were produced from the cultured broth of Cryptococcus albidus (ATCC-20293) which is designated as CK, a mutant of C. albidus (ATCC-20293) which is designated as CK-D10 and the newly isolated Cryptococcus sp. NISL-3771 which is designated as TK. All three preparations (CK, CK-D10 and TK) were evaluated for pathogenicity and virulence in mice and were found to be non-pathogenic. The acute LD(50)s for CK in male mice was greater than 4.8 g/kg body weight and for female mice was greater than 6.5 g/kg body weight. Acute LD(50)s for CK and CK-D10 in male and female rats was greater than 7.5 g/kg body weight, and that for TK was greater than 10 g/kg body weight. Subchronic (90-day) feeding studies (wherein the glutaminases were presented as dietary admixtures) were conducted in mice and rats. The NOAEL for CK in mice was 7.5 g/kg body weight/day. The NOAELs in rats were as follows: for CK, 9 g/kg body weight/day; for CK-D10, 1.2 g/kg body weight/day, and for TK, 8 g/kg body weight/day. Mice received CK as a dietary admixture at levels of 0, 1.0 and 10.0% for 1 year. The NOAEL was 13 g/kg body weight/day. The glutaminases from C. albidus described herein demonstrate very low toxicity.

Increase of antioxidative potential of rat plasma by oral administration of proanthocyanidin-rich extract from grape seeds.

The effect of a single oral administration of proanthocyanidins, oligomeric and polymeric polyhydroxyflavan-3-ol units, on the antioxidative potential of blood plasma was studied in rats. Proanthocyanidin-rich extract from grape seeds was administered by intragastric intubation to fasted rats at 250 mg/kg of body weight. The plasma obtained from water- or proanthocyanidin-administered rats was oxidized by incubation with copper sulfate or 2, 2'-azobis(2-amidinopropane) dihydrochloride (AAPH) at 37 degrees C, and the formation of cholesteryl ester hydroperoxides (CE-OOH) was followed. The plasma obtained from proanthocyanidin-administered rats was significantly more resistant against both copper ion-induced and AAPH-induced formation of CE-OOH than that from control rats. The lag phase in the copper ion-induced oxidation of rat plasma was remarkably increased at 15 min after administration of proanthocyanidins and reached a maximum level at 30 min. When the plasma from proanthocyanidin-administered rat was hydrolyzed by sulfatase and beta-glucuronidase following analysis by high-performance liquid chromatography with electrochemical detection, metabolites of proanthocyanidins occurred in rat plasma at 15 min after administration, three peaks of which were identified as gallic acid, (+)-catechin, and (-)-epicatechin. These results suggest that the intake of proanthocyanidins, the major polyphenols in red wine, increases the resistance of blood plasma against oxidative stress and may contribute to physiological functions of plant food including wine through their in vivo antioxidative ability.

Daidzein and genistein but not their glucosides are absorbed from the rat stomach.

Absorption of isoflavone aglycones and glucosides was compared in rats. Daidzein, genistein, daidzin and genistin were orally administered at a dose of 7.9 micromol/kg in 25 mM Na2CO3 and next their metabolite concentration in blood plasma was monitored for 30 min. After isoflavone glucosides administration, their metabolites appeared in plasma with a few minutes delay as compared to aglycones, which suggested that aglycones, but not glucosides, were absorbed already in the rat stomach. This observation was confirmed when absorption site was restricted solely to the stomach and absorption was shown to be independent of the vehicle pH used for administration.

Proanthocyanidin-rich extract from grape seeds attenuates the development of aortic atherosclerosis in cholesterol-fed rabbits.

The aim of this study was to evaluate the antiatherosclerotic effect of proanthocyanidin-rich extracts from grape seeds in cholesterol-fed rabbits. Proanthocyanidin-rich extracts (0.1% and 1% in diets [w/w]) did not appreciably affect the changes in serum lipid profile of cholesterol-fed rabbits. The level of cholesteryl ester hydroperoxides (ChE-OOH) induced by 2,2'-azobis(2-amidinopropane-dihydrochloride (AAPH) were lower in the plasma of rabbits fed proanthocyanidin-rich extract plus cholesterol than in the plasma of rabbits fed cholesterol alone, but not in the low-density lipoprotein (LDL). Aortic malondialdehyde (MDA) content decreased in rabbits fed proanthocyanidin-rich extract. Feeding proanthocyanidin-rich extracts (0.1 and 1% in the diet) to rabbits significantly reduced severe atherosclerosis in the aorta. Immunohistochemical analysis revealed a decrease in the number of oxidized LDL-positive macrophage-derived foam cells in atherosclerotic lesions in the aorta of rabbits fed proanthocyanidin-rich extract. When proanthocyanidin-rich extract was administered orally to rats, proanthocyanidin was detected in the plasma by Porters method but not in the lipoproteins (LDL plus VLDL). In an in vitro experiment using human plasma, proanthocyanidin-rich extract added to the plasma inhibited the oxidation of cholesteryl linoleate in LDL, but not in the LDL isolated after the plasma and the extract were incubated in advance. These results suggested that proanthocyanidins, the major polyphenols in red wine, might trap reactive oxygen species in aqueous series such as plasma and interstitial fluid of the arterial wall, thereby inhibiting oxidation of LDL and showing an antiatherosclerotic activity.

Antioxidative activities of 4-hydroxy-3(2H)-furanones and their anti-cataract effect on spontaneous cataract rat (ICR/f).

We determined the anti-cataract effects and antioxidative activities of four 4-hydroxy-3(2H)-furanones. These four furanones showed similar antioxidative activities in the ferric ion reduction model. 4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) and 2(or 5)-ethyl-4-hydroxy-5(or 2)-methyl-3(2H)-furanone (EHMF) exhibited a higher suppression effect on lipid peroxidation in human plasma than the other furanones did. The effects of hydroxy furanones on the onset of cataract in spontaneous cataract rat (ICR/f rat) were tested, and it was observed that HDMF and EHMF inhibited cataract formation. These results suggest that the antioxidative activity of HDMF and EHMF against superoxide radicals in lens tissue contributed to inhibiting the onset of spontaneous cataract.

Synthesis of 4-hydroxy-3(2H)-furanone acyl derivatives and their anti-cataract effect on spontaneous cataract rats (ICR/f).

4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) and 2(or 5)-ethyl-4-hydroxy-5(or 2)-methyl-3(2H)-furanone (EHMF) are known to inhibit cataract development in spontaneous cataract rats (ICR/f). Forty-five acylated hydroxyfuranone derivatives were designed and synthesized for an anti-cataract test, and their hydrophobic constants were also tested. Among these derivatives, 2,5-dimethyl-4-pivaloyloxy-3(2H)-furanone (HDMF pivalate) exerted a marked protective effect against the development of cataract in a galactose-induced model using cultured rat lens (in vitro). When tested on an ICR/f cataract model (in vivo), HDMF pivalate showed more significant inhibition of cataract development than parent compound HDMF. This derivative is more lipophilic than HDMF, so that HDMF pivalate can penetrate the cornea more easily than HDMF. The inhibition of cataract development by HDMF converted from HDMF pivalate is supported by the fact that HDMF was observed in the lens of ICR/f rats treated with HDMF pivalate.

Safety evaluation of tannase enzyme preparation derived from Aspergillus oryzae.

Tannase is an acylhydrolase enzyme preparation from Aspergillus oryzae that can be used as a processing aid for the manufacture of cold water-soluble tea beverages. A 91-day oral toxicity study in the rat and a gene mutation study in Salmonella typhimurium were performed to establish the safety of the enzyme preparation for the consumer. General toxicity was low, with no adverse effects observed at the highest dose tested, 1% in the diet. There was no evidence of mutagenic potential with or without metabolic activation. These results, together with knowledge of the production organism and the chemical and microbiological characterization of the enzyme preparation, indicate that tannase can be regarded as safe for its intended use in processing tea.

Anti-inflammatory effects and specificity of L-156,602: comparison of effects on concanavalin A and zymosan-induced footpad edema, and contact sensitivity response.

We investigated the in vivo selective anti-inflammatory effect of L-156,602, which was first identified as a preferential delayed-type hypersensitivity-suppressant in our screening program and first reported to be a C5a antagonist. The agent most profoundly suppressed footpad edema 4 h after elicitation by concanavalin A (con A) and also caused a significantly impaired response after a further 20 h. Footpad edema induced by either serotonin, carrageenan or zymosan was not much influenced by the agent. Although the dominant cell population that migrated in response to con A and zymosan 4 h after elicitation was neutrophils, L-156,602 specifically prevented the con-A-induced migration of neutrophils, suggesting a distinct mechanism of neutrophil recruitment between con A and zymosan-induced inflammation. The agent also reduced the contact-sensitivity response, especially in host mice sensitized with a moderate dose of picryl chloride and almost completely suppressed the infiltration of mononuclear leukocytes and neutrophils into the site of inflammation. These selective effects of L-156,602 on inflammatory reactions appeared to be not merely via C5a antagonism.

[Micro-canaliculi in juxtaglomerular cells of two-kidney Goldblatt hypertensive rats].

Juxtaglomerular (JG) cells in two-kidney Goldblatt hypertensive (2KGH) rats were examined ultrastructurally. In JG cells of the clipped kidneys of 2KGH rats, micro-canaliculi branched off from channel-like invaginations of plasma membrane and connected with the secretory renin granules. These findings suggest that micro-canaliculi may play important role in renin secretion from JG cells in 2KGH rats.

Purification and identification of an angiotensin I-converting enzyme inhibitor from soy sauce.

The inhibitory activity of an angiotensin I-converting enzyme (ACE) detected in soy sauce was fractionated into two major fractions of high molecular weight (Hw) and low molecular weight (Lw) by gel filtration chromatography on Bio-gel P-2 after treating with ethanol. The Hw fraction reduced the blood pressure in hypertensive rats after orally administering, while the Lw fraction did not. The ACE inhibitor in the Hw fraction was further purified by Dowex 50W ion-exchange chromatography and four subsequent steps of HPLC. On the basis of the SIMS-mass spectrum, NMR spectrum and other characteristics, the purified ACE inhibitor was identified as nicotianamine (N-[N-(3-amino-3-carboxypropyl)-3-amino-3-carboxypropyl]azetidine-2- carboxylic acid). The IC50 value for this ACE was 0.26 microM.

Immunohistochemical findings of juxtaglomerular cells on acute phase in two-kidney Goldblatt hypertensive rats and spontaneously hypertensive rats.

Juxtaglomerular (JG) cells on the acute phase in two-kidney Goldblatt hypertensive (2KGH) rats and spontaneously hypertensive rats (SHR) were examined immunohistochemically. JG cells in 2KGH rats and SHR were positively stained with anti-renin serum and anti-angiotensin II (A II) serum. In 2KGH rats, the number of renin and AII immunoreactive JG cells in the clipped kidneys increased throughout the observation period. The number of renin and AII immunoreactive JG cells in the unclipped kidneys was almost the same as that in control rats, although immunoreactivity of these cells was weak and they were small in size. These changes in the unclipped kidneys became obvious with the time course after operation. We did not see any changes in these cells in SHR. In 2KGH rats treated with captopril, the number of renin immunoreactive JG cells in the clipped kidneys increased, whereas that of AII immunoreactive JG cells in the bilateral kidney decreased. When captopril was administered to SHR, the number of renin immunoreactive JG cells in the bilateral kidney increased, whereas that of AII immunoreactive JG cells in the bilateral kidney decreased. These results suggested that the JG cell in the bilateral kidney was closely related to the development of hypertension in 2KGH rats, but not in SHR. The increase of renin immunoreactive JG cells in 2KGH rats and SHR treated with captopril was probably due to the removal of negative feedback inhibition of AII on JG cells.

[Effects of captopril on acute phase in two-kidney Goldblatt hypertensive rats and spontaneously hypertensive rats].

Effects of continuous oral administration of captopril were investigated on acute phase in two-kidney Goldblatt hypertensive (2 KGH) rats and spontaneously hypertensive rats (SHR). Systolic blood pressure gradually rose throughout the experimental period of 7, 14, 21 and 28 days in both 2 KGH rats and SHR. These gradual increases of systolic blood pressure were reduced by administration of captopril in both rats. Plasma renin activity were markedly increased throughout the experimental period in both rats treated with captopril, and were modestly increased in 2 KGH rats. In contrast, those changes in plasma renin activity were not obvious in SHR. In 2 KGH rats, juxtaglomerular index (JGI) and juxtaglomerular cell count (JGCC) of the clipped kidneys increased whereas JGI of the opposite kidneys decreased. In contrast, those changes in JGI and JGCC were not obvius in SHR. On the other hand, JGI and JGCC of the clipped kidneys increased in 2 KGH rats treated with captopril and those of the both kidneys increased in SHR treated with captopril. These results suggested that juxtaglomerular cells were related to the development of hypertension in 2 KGH rats, but were not clear in SHR. And these results were found that captopril showed antihypertensive effects, in spite of rises in JGI and JGCC of both 2 KGH rats and SHR.

[Clinical and pathological findings on acute phase in two-kidney Goldblatt hypertensive rats].

Clinical and pathological changes were investigated on acute phase in two-kidney Goldblatt hypertensive rats. Systolic blood pressure, plasma renin activity, plasma aldosterone and juxtaglomerular cell count (JGCC) increased slightly as early as 7 days after operation. Systolic blood pressure and plasma renin activity rose in the course of time after operation. Juxtaglomerular index (JGI) and JGCC of the clipped kidneys increased and JGI of the opposite kidneys decreased with the increase in systolic blood pressure. These results suggested that juxtaglomerular cells had an important role on acute phase in two-kidney Goldblatt hypertensive rats.