The Methanol Extract of Azadirachta indica A. Juss Leaf Protects Mice Against Lethal Endotoxemia and Sepsis.

In the present study, the inhibitory effect of neem leaf extract (NLE) on lipopolysaccaride (LPS)-induced nitric oxide (NO) and tumor necrosis factor-α (TNF-α) production was examined both in vitro and in vivo. In vitro study revealed that NLE treatment (100 µg/ml) inhibits LPS (100 ng/ml)-induced NO production by 96% and TNF-α production by 32%. The reduction in NO production is probably conferred by the complete suppression of inducible nitric oxide synthase (iNOS) expression. Interestingly, in vivo NLE significantly improved the survival rate of mice in an experimental sepsis model. Administration of NLE (100 mg/kg) 24 h before LPS treatment (20 mg/kg) improved the survival rate of mice by 60%. The inhibition of plasma NO and TNF-α production by NLE is likely to account for the improved survival of mice. Our results suggest that NLE may present a promising avenue in the development of therapeutic agents for the treatment of inflammatory diseases.

Matcha, a powdered green tea, ameliorates the progression of renal and hepatic damage in type 2 diabetic OLETF rats.

Matcha, a powdered green tea produced by grinding with a stone mill, has been popularly used in the traditional tea ceremony and foods in Japan. Matcha is well known to be richer in some nutritional elements and epigallocatechin 3-O-gallate than other green teas. In our previous study, epigallocatechin 3-O-gallate exhibited protective effects against renal damage in a rat model of diabetic nephropathy. In the present study, we investigated the preventive effects of Matcha (50, 100, or 200 mg/kg/day) on the progression of hepatic and renal damage in type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. OLETF rats were orally administered Matcha for 16 weeks, and we assessed biochemical parameters in the serum, liver, and kidney and expression levels of major products of advanced glycation end products (AGEs), N(6)-(carboxylmethyl)lysine (CML) and N(6)-(carboxylethyl)lysine (CEL), receptor for AGE (RAGE), and sterol regulatory element binding proteins (SREBPs)-1 and -2. Serum total protein levels were significantly increased by Matcha administration, whereas the serum albumin and glycosylated protein levels as well as the renal glucose and triglyceride levels were only slightly or not at all affected. However, Matcha treatment significantly lowered the glucose, triglyceride, and total cholesterol levels in the serum and liver, renal AGE levels, and the serum thiobarbituric acid-reactive substances levels. In addition, Matcha supplementation resulted in decreases in the renal CML, CEL, and RAGE expressions as well as an increase in hepatic SREBP-2 expression, but not that of SREBP-1. These results suggest that Matcha protects against hepatic and renal damage through the suppression of renal AGE accumulation, by decreases in hepatic glucose, triglyceride, and total cholesterol levels, and by its antioxidant activities.

Protective effect of ursolic acid from Cornus officinalis on the hydrogen peroxide-induced damage of HEI-OC1 auditory cells.

The fruits of Cornus officinalis have been used in traditional oriental medicine for treatment of inner ear diseases, such as tinnitus and hearing loss. In the present study, we investigated the protective effect of C. officinalis on hydrogen peroxide-induced cytotoxicity in HEI-OC1 auditory cells. The results from bioassay-guided fractionation of methanol extract of C. officinalis fruits showed that ursolic acid is a major active component. Ursolic acid (0.05-2 microg/ml) had protective effect against the HEI-OC1 cell damage and reduced lipid peroxidation in a dose-dependent manner. In addition, pre-treatment with ursolic acid significantly attenuated the decrease of activities of catalase (CAT) and glutathione peroxidase (GPX), but superoxide dismutase (SOD) activity was not significantly affected by ursolic acid. These results indicate that ursolic acid protects hydrogen peroxide-induced HEI-OC1 cell damage through inhibition of lipid peroxidation and induction of antioxidant enzymes, CAT and GPX, and may be one of the active components responsible for these effects of C. officinalis fruits.

Beneficial effects of beta-sitosterol on glucose and lipid metabolism in L6 myotube cells are mediated by AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that has been implicated as a key factor for controlling intracellular lipids and glucose metabolism. Beta-sitosterol, a plant sterol known to prevent cardiovascular disease was identified from Schizonepeta tenuifolia to an AMPK activator. In L6 myotube cells, beta-sitosterol significantly increased phosphorylation of the AMPKalpha subunit and acetyl-CoA carboxylase (ACC) with stimulating glucose uptake. In contrast, beta-sitosterol treatment reduced intracellular levels of triglycerides and cholesterol in L6 cells. These effects were all reversed by pretreatment with AMPK inhibitor Compound C or LKB1 destabilizer radicicol. Similarly, beta-sitosterol-induced phosphorylation of AMPK and ACC was not increased in HeLa cells lacking LKB1. These results together suggest that beta-sitosterol-mediated enhancement of glucose uptake and reduction of triglycerides and cholesterol in L6 cells is predominantly accomplished by LKB1-mediated AMPK activation. Our findings further reveal a molecular mechanism underlying the beneficial effects of beta-sitosterol on glucose and lipid metabolism.

Protective effects of morroniside isolated from Corni Fructus against renal damage in streptozotocin-induced diabetic rats.

In our previous study, we reported the renoprotective effect of Hachimi-jio-gan, a Chinese traditional prescription consisting of eight medicinal plants, and also reported the effect of Corni Fructus (Cornus officinalis SIEB. et ZUCC.), a component of Hachimi-jio-gan, on diabetic nephropathy using diabetic rats. In this study, we investigated the effects of morroniside isolated from Corni Fructus on renal damage in streptozotocin-treated diabetic rats. Oral administration of morroniside at a dose of 20 or 100 mg/kg body weight/d for 20 d to diabetic rats resulted in significant decreases in increasing serum glucose and urinary protein levels. Moreover, the decreased levels of serum albumin and total protein in diabetic rats were significantly increased by morroniside administration at a dose of 100 mg/kg body weight/d. In addition, morroniside significantly reduced the elevated serum urea nitrogen level and showed a tendency to reduce creatinine clearance. Morroniside also significantly reduced the enhanced levels of serum glycosylated protein, and serum and renal thiobarbituric acid-reactive substances. Protein expressions related to the advanced glycation endproduct (AGE) level and actions, oxidative stress such as N(epsilon)-(carboxyethyl)lysine, as well as receptors for AGE and heme oxygenase-1 were increased in diabetic rats, but the levels were also significantly decreased by the administration of morroniside. This suggests that morroniside exhibits protective effects against diabetic renal damage by inhibiting hyperglycemia and oxidative stress. These results indicate that morroniside is one component partly responsible for the protective effects of Corni Fructus and Hachimi-jio-gan against diabetic renal damage.

Beta-secretase (BACE1) inhibitors from Perilla frutescens var. acuta.

In the course of screening for anti-dementia agents from natural products, two beta-secretase (BACE1) inhibitors were isolated from the methanolic extract of Perilla frutescens var. acuta and identified as luteolin (1) and rosmarinic acid (2) with IC50 values of 5.0 x 10(-7) M and 2.1 x 10(-5) M, respectively. They inhibited BACE1 in a non-competitive manner with a substrate in Dixon plots, suggesting that they might bind to either beta-secretase subsite or to another regulatory site. Kivalues of 1 and 2 were 6.2 x 10(-5) M and 3.9 x 10(-5) M, respectively. They were less inhibitory against other enzymes such as alpha-secretase (TACE), acetylcholine esterase (AchE), chymotrypsin, and elastase, indicating that they were relatively specific inhibitors of BACE1.

Changes in aurantio-obtusin and glucoaurantio-obtusin content in Cassiae Semen via treatment with a crude enzyme extract from Aspergillus usamii.

Cassiae Semen (seeds of Cassia tora) showed a remarkably different HPLC chromatogram after being treated with a crude enzyme extract from Aspergillus usamii. Increased and decreased compounds were identified as aurantio-obtusin and glucoaurantio-obtusin, respectively. The aurantio-obtusin content reached its maximum level (133.58 +/- 0.39 microg/mg extract) after being incubated for 50 min at 37 degrees C, whereas the inactivated crude enzyme-treated control remained unchanged (54.13 +/- 1.33 microg/mg). On the other hand, the glucoaurantio-obtusin content decreased by less than one-third (51.09 +/- 1.63 microg/ mg) of the untreated control (143.19 +/- 2.12 microg/mg), suggesting that an increase in aurantio-obtusin content originated from the enzymatic cleavage of its glucoside glucoaurantio-obtusin.

Plant phenolics as prolyl endopeptidase inhibitors.

Prolyl endopeptidase (PEP, EC 3.4.21.26), a serine protease, is widely distributed in various organs, particularly in the brains of Alzheimer's disease patients. The expression of PEP in Alzheimer's patients has been found to be significantly higher than that of the normal person, suggesting that a specific PEP inhibitor can be a good candidate for an anti-amnestic drug. In the current study, thirty-nine plant phenolics were investigated to determine their roles as prolyl endopeptidase (PEP) inhibitors. Nineteen compounds such as 1,2,3-trigalloyl glucopyranoside, 1,2,6-trigalloyl glucopyranoside, 1,2,3,4,6-pentagalloyl gluco-pyranoside, 1,2,6-trigalloyl alloside, 1,3,6-trigalloyl alloside, 1,2,3,6-tetragalloyl alloside, acetonyl geraniin, corilagin, elaeocarpusin, euphorscopin, geraniin, helioscopin B, helioscopinin A, helioscopinin B, jolkinin, macranganin, rugosin E, supinanin, and teracatain exhibited strong inhibition against PEP (IC50 26.7 - 443.7 x 10(-9) M). Rugosin E (IC50 26.7 x 10(-9) M) showed the most effective inhibition followed by 1,2,6-trigalloyl glucopyranoside (IC5031.4 x 10(-9) M) and macranganin (IC5042.6 x 10(-9) M). No significant structure-activity relationship was found; however, at least, three pyrogallol groups seem to be a minimal requirement for stronger activity against PEP All 19 active compounds inhibited PEP in a non-competitive mode with a substrate in Dixon plots. They did not show significant effects against other serine proteases such as trypsin, chymotrypsin and elastase, indicating that they were relatively specific PEP inhibitors.

Effects of methanol extract of Alisma orientale rhizome and its major component, alisol B 23-acetate, on hepatic drug metabolizing enzymes in rats treated with bromobenzene.

In the course of screening for hepatoprotective agents from natural products, the effects of the methanol extract (ME) of the rhizome of Alisma orientale (Alismataceae) and its major component, alisol B 23-acetate (ALB) on hepatic lipid peroxidation and drug-metabolizing enzymes were evaluated in rats intoxicated with bromobenzene (BB). Pretreatment with ME and ALB had no effect on hepatic antioxidant enzymes such as glutathione reductase and a-glutamylcysteine synthetase. ME and ALB had also no effect on the reduction in glutathione content caused by BB. In contrast, ME recovered the BB-induced decrease in epoxide hydrolase and glutathione S-transferase, enzymes that remove toxic epoxides. ME also attenuated the BB-induced increase in aminopyrine N-demethylase and aniline hydroxylase, enzymes that produce toxic intermediates. This effect was greater than that seen with ascorbic acid, which was used as a positive control. ALB had similar effects on the activities of antioxidant enzymes to ME, and may be partly responsible for the effects of ME.

Effects of the aerial parts of Orostachys japonicus and its bioactive component on hepatic alcohol-metabolizing enzyme system.

In the course of screening medicinal plants that modulate hepatic alcohol-metabolizing enzymes and lipid peroxidation, effects of the methanol extract (ME) of Orostachys japonicus and its major bioactive compound, gallic acid (GA), were investigated in rats treated with 10% ethanol solution for 6 weeks. The ME and GA greatly enhanced the activities of hepatic alcohol dehydrogenase (ADH), the microsomal ethanol-oxidizing system (MEOS), and aldehyde dehydrogenase (ALDH) in a dose-dependent manner, but had no effect on catalase. The hepatic lipid peroxide level increased by ethanol administration was moderately reduced by treatment with ME or GA. The results suggest that the detoxification of hepatic alcohol by O. japonicus ME under our experimental conditions was due to the enhanced activities of the alcohol-oxidizing enzymes, ADH, MEOS, and ALDH. In addition, GA may be partly responsible for the effects.

Asarum sieboldii extracts attenuate growth, acid production, adhesion, and water-insoluble glucan synthesis of Streptococcus mutans.

Asarum sieboldii has been used in traditional folk medicine to treat dental caries and periodontal disease. In the present study, we investigated the inhibitory effect of the ethanol and aqueous extracts of A. sieboldii on the growth, acid production, adhesion, and water-insoluble glucan synthesis of Streptococcus mutans. The ethanol and aqueous extracts of A. sieboldii inhibited the growth and acid production of S. mutans. In the bacterial adherence assay, the ethanol and aqueous extracts of A. sieboldii significantly lowered the adherence of S. mutans. We also found that the ethanol and aqueous extracts of A. sieboldii significantly inhibited the synthesis of water-insoluble glucan by crude glucosyltransferase. These results suggest that A. sieboldii extracts may inhibit the caries-inducing properties of S. mutans. Further studies are necessary to clarify the active constituents of A. sieboldii extracts responsible for such biomolecular activities.

Beta-secretase (BACE1) inhibitors from Sanguisorbae Radix.

In the course of screening anti-dementia agents from natural products, two beta-secretase (BACE1) inhibitors were isolated from the ethyl acetate soluble fraction of Sanguisorbae Radix by the activity-guided purification using silica gel, Sephadex LH-20, and RP-HPLC. They were identified as 1,2,3-trigalloyl-4,6-hexahydroxydiphenoyl-beta-D-glucopyranoside (Tellimagrandin II, 1) and 1,2,3,4,6-pentagalloyl-beta-D-glucopyranoside (2) and were shown to non-competitively inhibit beta-secretase (BACE1) with the IC50 values of 3.10x10(-6) M and 3.76x10(-6) M, respectively. The Ki values of 1 and 2 were 6.84x10(-6) M and 5.13x10(-6) M. They were less inhibitory to alphasecretase (TACE) and other serine proteases such as chymotrypsin, trypsin, and elastase, suggesting that they were relatively specific inhibitors of BACE1.

Cryptotanshinone but not tanshinone IIA inhibits angiogenesisin vitro.

In the course of screening of angiogenesis inhibitor from natural products, cryptotanshinone from Salvia miltiorrhiza was isolated as a potent small molecule inhibitor of angiogenesis. Cryptotanshinone inhibits bFGF-induced angiogenesis of BAECs at ten micromolar ranges in vitro without cytotoxicity. Tanshinone IIA, another tanshinone isolated from S. miltiorrhiza, which is structurally very similar to cryptotanshinone except C-15 position of dihydrofuran ring does not inhibit angiogenesis induced by bFGF. These results demonstrate that cryptotanshinone is a new anti-angiogenic agent and double bond at C-15 position of the dihydrofuran ring plays a crucial role in the activity.

beta-Secretase (BACE1) inhibitors from pomegranate (Punica granatum) husk.

In the course of screening for anti-dementia agents from natural products, two beta-secretase (BACE1) inhibitors were isolated from the husk of pomegranate (Punica granatum) by activity-guided purification. They were identified as ellagic acid and punicalagin with IC50 values of 3.9 x10(-6) and 4.1x10(-7) M and Ki values of 2.4x10(-5) and 5.9x10(-7) M, respectively. The compounds were non-competitive inhibitors with a substrate in the Dixon plot. Ellagic acid and punicalagin were less inhibitory to alpha-secretase (TACE) and other serine proteases such as chymotrypsin, trypsin, and elastase, thus indicating that they were relatively specific inhibitors of BACE1.

Antibacterial effect of Phellinus linteus against methicillin-resistant Staphylococcus aureus.

Methanol extract and its fractions (CHCl3, n-BuOH and H2O) of the fruit body of Phellinus linteus mushroom were investigated for antibacterial activity against methicillin-resistant Staphylococcus aureus. The n-BuOH fraction showed a good antibacterial activity (MIC, 63-125 microg/ml) against all tested strains.

Effects of methanol extract of Cirsium japonicum var. ussuriense and its principle, hispidulin-7-O-neohesperidoside on hepatic alcohol-metabolizing enzymes and lipid peroxidation in ethanol-treated rats.

Effects of the methanol extract of Cirsium japonicum var. ussuriense and hispidulin 7-O-neohesperidoside isolated from the plant on hepatic alcohol-metabolizing enzymes and lipid peroxidation were studied in rats treated with ethanol. Rats treated with 10% alcohol solution for 6 weeks were orally administered with 250 or 500 mg of methanol extract or 10 or 20 mg of hispidulin 7-O-neohesperidoside per kg body weight daily during the last week of ethanol treatment. The administration of the methanol extract of herbal plant and hispidulin 7-O-neohesperidoside in ethanol-treated rats significantly enhanced the activities of hepatic alcohol dehydrogenase, microsomal ethanol-oxidizing system and aldehyde dehydrogenase in a dose-dependent manner. The extract and the compound decreased hepatic lipid peroxidation along with an increase in hepatic content of reduced glutathione. The methanol extract and hispidulin 7-O-neohesperidoside of C. japonicum var. ussuriense also increased the activity of glutathione reductase, but had no effect on gamma-glutamylcysteine synthase. The results suggest that C. japonicum var. ussuriense may alleviate alcoholic toxicity by enhancing ethanol oxidation as well as inhibiting lipid peroxidation, and hispidulin 7-O-neohesperidoside is one of the active substances responsible for the protective effects of this plant.

Effect of methanol extract of Zanthoxylum piperitum leaves and of its compound, protocatechuic acid, on hepatic drug metabolizing enzymes and lipid peroxidation in rats.

The effect of methanol extract and protocatechuic acid from the leaves of Zanthoxylum piperitum on lipid peroxidation and drug metabolizing enzymes were investigated in the liver of bromobenzene-treated rats. The methanol extract and protocatechuic acid reduced the level of lipid peroxide induced by bromobenzene. The methanol extract and protocatechuic acid reduced the activity of aniline hydroxylase that had been increased by bromobenzene, while did not affect the activities of aminopyrine N-demethylase and glutathione S-transferase. The methanol extract and compound effectively restored the activity of epoxide hydrolase which had been decreased by bromobenzene. These results may suggest that the methanol extract of Z. piperitum and protocatechuic acid prevented lipid peroxidation by reducing the activity of aniline hydroxylase, an epoxide-producing enzyme, and by enhancing the activity of epoxide hydrolase, an epoxide-removing enzyme, in rats that had been intoxicated with bromobenzene.

Inhibitory effects of Korean medicinal plants and camelliatannin H from Camellia japonica on human immunodeficiency virus type 1 protease.

To identify substances with anti-human immunodeficiency virus (HIV) activity in traditional medicines, 101 extracts of Korean medicinal plants were screened for their inhibitory effects on HIV type 1 protease (PR). The enzyme activity was determined by HPLC. Of the extracts tested, strong inhibitory effects were observed in the acetone extracts of the pericarp and leaves of Camellia japonica, the water extract of the leaves of Sageretia theezans and the methanol extract of the aerial part of Sophora flavescens. Camelliatannin H from the pericarp of C. japonica, showed a potent inhibitory activity on HIV-1 PR with IC(50) of 0.9 microM.

Effects of extract from Angelica keiskei and its component, cynaroside, on the hepatic bromobenzene-metabolizing enzyme system in rats.

The effects of Angelica keiskei Koidz. on hepatic lipid peroxide and the activities of free radical generating and scavenging enzymes were investigated in bromobenzene-induced hepatic lipid peroxidation in rats. The level of lipid peroxide elevated by bromobenzene was significantly reduced by the methanol extract from the aerial parts of A. keiskei and its component, cynaroside. Epoxide hydrolase activity was decreased significantly by the treatment of bromobenzene. However, the enzyme activity was restored in the liver of rats given the methanol extract and cynaroside. The results suggest that the reduction of bromobenzene-induced hepatic lipid peroxidation by the extract of A. keiskei and cynaroside under our experimental conditions is thought to be through enhancing the activity of epoxide hydrolase, an enzyme removing bromobenzene epoxide.