Sonchus asper extract inhibits LPS-induced oxidative stress and pro-inflammatory cytokine production in RAW264.7 macrophages

BACKGROUND/OBJECTIVESSonchus asper is used extensively as an herbal anti-inflammatory for treatment of bronchitis, asthma, wounds, burns, and cough; however, further investigation is needed in order to understand the underlying mechanism. To determine its mechanism of action, we examined the effects of an ethyl acetate fraction (EAF) of S. asper on nitric oxide (NO) production and prostaglandin-E2 levels in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages.MATERIALS/METHODSAn in vitro culture of RAW264.7 macrophages was treated with LPS to induce inflammation.RESULTSTreatment with EAF resulted in significant suppression of oxidative stress in RAW264.7 macrophages as demonstrated by increased endogenous superoxide dismutase (SOD) activity and intracellular glutathione levels, decreased generation of reactive oxygen species and lipid peroxidation, and restoration of the mitochondrial membrane potential. To confirm its anti-inflammatory effects, analysis of expression of inducible NO synthase, cyclooxygenase-2, tumor necrosis factor-α, and the anti-inflammatory cytokines IL-1β and IL-6 was performed using semi-quantitative RT-PCR. EAF treatment resulted in significantly reduced dose-dependent expression of all of these factors, and enhanced expression of the antioxidants MnSOD and heme oxygenase-1. In addition, HPLC fingerprint results suggest that rutin, caffeic acid, and quercetin may be the active ingredients in EAF.CONCLUSIONSTaken together, findings of this study imply that the anti-inflammatory effect of EAF on LPS-stimulated RAW264.7 cells is mediated by suppression of oxidative stress.     

Anti-inflammatory effect of enzymatic hydrolysates from Styela clava flesh tissue in lipopolysaccharide-stimulated RAW 264.7 macrophages and in vivo zebrafish model

BACKGROUND/OBJECTIVES

In this study, potential anti-inflammatory effect of enzymatic hydrolysates from Styela clava flesh tissue was assessed via nitric oxide (NO) production in lipopolysaccahride (LPS) induced RAW 264.7 macrophages and in vivo zebrafish model.

MATERIALS/METHODS

We investigated the ability of enzymatic hydrolysates from Styela clava flesh tissue to inhibit LPS-induced expression of pro-inflammatory mediators in RAW 264.7 macrophages, and the molecular mechanism through which this inhibition occurred. In addition, we evaluated anti-inflammatory effect of enzymatic hydrolysates against a LPS-exposed in in vivo zebrafish model.

RESULTS

Among the enzymatic hydrolysates, Protamex-proteolytic hydrolysate exhibited the highest NO inhibitory effect and was fractionated into three ranges of molecular weight by using ultrafiltration (UF) membranes (MWCO 5 kDa and 10 kDa). The above 10 kDa fraction down-regulated LPS-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), thereby reducing production of NO and prostaglandin E₂ (PGE₂) in LPS-activated RAW 264.7 macrophages. The above 10 kDa fraction suppressed LPS-induced production of pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. In addition, the above 10 kDa fraction inhibited LPS-induced phosphorylation of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase (JNK), and p38. Furthermore, NO production in live zebrafish induced by LPS was reduced by addition of the above 10 kDa fraction from S. clava enzymatic hydrolysate.

CONCLUSION

The results of this study suggested that hydrolysates derived from S. clava flesh tissue would be new anti-inflammation materials in functional resources.     

Antioxidant activity and anti-inflammatory activity of ethanol extract and fractions of Doenjang in LPS-stimulated RAW 264.7 macrophages

BACKGROUND/OBJECTIVESFermentation can increase functional compounds in fermented soybean products, thereby improving antioxidant and/or anti-inflammatory activities. We investigated the changes in the contents of phenolics and isoflavones, antioxidant activity and anti-inflammatory activity of Doenjang during fermentation and aging.MATERIALS/METHODSDoenjang was made by inoculating Aspergillus oryzae and Bacillus licheniformis in soybeans, fermenting and aging for 1, 3, 6, 8, and 12 months (D1, D3, D6, D8, and D12). Doenjang was extracted using ethanol, and sequentially fractioned by hexane, dichloromethane (DM), ethylacetate (EA), n-butanol, and water. The contents of total phenolics, flavonoids and isoflavones, 2,2-diphenyl-1 picryl hydrazyl (DPPH) radical scavenging activity, and ferric reducing antioxidant power (FRAP) were measured. Anti-inflammatory effects in terms of nitric oxide (NO), prostaglandin (PG) E2 and pro-inflammatory cytokine production and inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expressions were also measured using LPS-treated RAW 264.7 macrophages.RESULTSTotal phenolic and flavonoid contents showed a gradual increase during fermentation and 6 months of aging and were sustained thereafter. DPPH radical scavenging activity and FRAP were increased by fermentation. FRAP was further increased by aging, but DPPH radical scavenging activity was not. Total isoflavone and glycoside contents decreased during fermentation and the aging process, while aglycone content and its proportion increased up to 3 or 6 months of aging and then showed a slow decrease. DM and EA fractions of Doenjang showed much higher total phenolic and flavonoid contents, and DPPH radical scavenging activity than the others. At 100 µg/mL, DM and EA fractions of D12 showed strongly suppressed NO production to 55.6% and 52.5% of control, respectively, and PGE2 production to 25.0% and 28.3% of control with inhibition of iNOS or COX-2 protein expression in macrophages.CONCLUSIONSTwelve month-aged Doenjang has potent antioxidant and anti-inflammatory activities with high levels of phenolics and isoflavone aglycones, and can be used as a beneficial food for human health.     

Repression of proinflammatory gene expression by lipid extract of Nostoc commune var sphaeroides Kützing,a blue-green alga,via inhibition of nuclear factor-κB in RAW 264.7 macrophages

We investigated whether lipid extract from a blue-green alga, N commune, modulates proinflammatory gene expression in RAW 264.7 macrophages. The cells were incubated with N commune lipid extract (0-100 μg/mL) and subsequently activated by LPS (100 ng/mL). Quantitative real-time PCR analysis showed that mRNA abundance of proinflammatory mediators, including TNF-α, COX-2, IL-1β, IL-6, and iNOS, was significantly reduced by N commune lipid extract in a dose-dependent manner. Secretion of TNF-α and IL-1β into cell culture medium was also significantly decreased by N commune lipid extract. Thin-layer chromatography–densitometry analysis showed that N commune lipid extract contained approximately 15% of fatty acids. To determine whether the inhibition of proinflammatory mediator production by N commune lipid extract is primarily conferred by fatty acids in the lipid extract, macrophages were incubated with 100 μg/mL of N commune lipid extract or 15 μg/mL of a fatty acid mixture, which was formulated to reflect the fatty acid composition of N commune lipid extract. The fatty acid mixture significantly reduced RNA abundance of TNF-α and COX-2, but to a lesser extent than did the N commune lipid extract, suggesting the presence of additional bioactive compounds with an antiinflammatory property in the lipid extract. As NF-κB is a major regulator for the proinflammatory gene expression, we measured its DNA-binding activity. DNA-binding activity of NF-κB was significantly reduced by N commune lipid extract. In conclusion, our study suggests that N commune lipid extract represses the expression of proinflammatory genes in RAW 264.7 macrophages, at least in part, by inhibiting the activation of NF-κB pathway.     

Acanthopanax koreanum Nakai modulates the immune response by inhibiting TLR 4-dependent cytokine production in rat model of endotoxic shock

The hepatoprotective activity of Acanthopanax koreanum Nakai extract (AE) was investigated against D-Galactosamine/Lipopolysaccharide (D-GalN/LPS)-induced liver failure rats compared with that of acanthoic acid (AA) isolated from AE. Although D-GalN/LPS (250 mg/kg body weight/10 µg/kg body weight, i.p.) induced hepatic damage, pretreatments with AE (1 and 3% AE/g day) and AA (0.037% AA, equivalent to 3% AE/g day) alleviated the hepatic damage. This effect was the result of a significant decrease in the activity of alanine transaminase. Concomitantly, both the nitric oxide and IL-6 levels in the plasma were significantly decreased by high-dose AE (AE3) treatment compared to the GalN/LPS control (AE0). This response resulted from the regulation of pro-inflammatory signaling via a decrease in TLR4 and CD14 mRNA levels in the liver. While a high degree of necrosis and hemorrhage were observed in the AE0, pretreatment with AE3 and AA reduced the extent of hepatocyte degeneration, necrosis, hemorrhage and inflammatory cell infiltrates compared to the AE0. In conclusion, these results suggest that especially high-dose AE are capable of alleviating D-GalN/LPS-induced hepatic injury by decreasing hepatic toxicity, thereby mitigating the TLR 4-dependent cytokine release. The anti-inflammatory effect of AE could be contributing to that of AA and AE is better than AA.     

The anti-inflammatory effect of Indonesian Areca catechu leaf extract in vitro and in vivo

BACKGROUND/OBJECTIVES

Overproduction of nitric oxide (NO) by the inducible nitric oxide synthase (iNOS) enzyme can cause inflammation. Cyclooxygenase-2 (COX-2) is also involved in the inflammatory response through regulation of nuclear factor-kappa B (NF-κB). Areca catechu is one of the known fruit plants of the Palmaceae family. It has been used for a long time as a source of herbal medicine in Indonesia. In this study, we explored the effect of Indonesian Areca catechu leaf ethanol extract (ACE) in lipopolysaccharide (LPS)-induced inflammation and carrageenan-induced paw edema models. Recently, this natural extract has been in the spotlight because of its efficacy and limited or no toxic side effects. However, the mechanism underlying its anti-inflammatory effect remains to be elucidated.

MATERIALS/METHODS

We measured NO production by using the Griess reagent, and determined the expression levels of inflammation-related proteins, such as iNOS, COX2, and NF-κB, by western blot. To confirm the effect of ACE in vivo, we used the carrageenan-induced paw edema model.

RESULTS

Compared to untreated cells, LPS-stimulated RAW 264.7 cells treated with ACE showed reduced NO generation and reduced iNOS and COX-2 expression. We found that the acute inflammatory response was significantly reduced by ACE in the carrageenan-induced paw edema model.

CONCLUSION

Taken together, these results suggest that ACE can inhibit inflammation and modulate NO generation via downregulation of iNOS levels and NF-κB signaling in vitro and in vivo. ACE may have a potential medical benefit as an anti-inflammation agent.     

Ethanol extract of Innotus obliquus (Chaga mushroom) induces G1 cell cycle arrest in HT-29 human colon cancer cells

BACKGROUND/OBJECTIVES

Inonotus obliquus (I. obliquus, Chaga mushroom) has long been used as a folk medicine to treat cancer. In the present study, we examined whether or not ethanol extract of I. obliquus (EEIO) inhibits cell cycle progression in HT-29 human colon cancer cells, in addition to its mechanism of action.

MATERIALS/METHODS

To examine the effects of Inonotus obliquus on the cell cycle progression and the molecular mechanism in colon cancer cells, HT-29 human colon cancer cells were cultured in the presence of 2.5 - 10 µg/mL of EEIO, and analyzed the cell cycle arrest by flow cytometry and the cell cycle controlling protein expression by Western blotting.

RESULTS

Treatment cells with 2.5 - 10 µg/mL of EEIO reduced viable HT-29 cell numbers and DNA synthesis, increased the percentage of cells in G₁ phase, decreased protein expression of CDK2, CDK4, and cyclin D1, increased expression of p21, p27, and p53, and inhibited phosphorylation of Rb and E2F1 expression. Among I. obliquus fractions, fraction 2 (fractionated by dichloromethane from EEIO) showed the same effect as EEIO treatment on cell proliferation and cell cycle-related protein levels.

CONCLUSIONS

These results demonstrate that fraction 2 is the major fraction that induces G₁ arrest and inhibits cell proliferation, suggesting I. obliquus could be used as a natural anti-cancer ingredient in the food and/or pharmaceutical industry.     

A mixture of Salacia oblonga extract and IP-PA1 reduces fasting plasma glucose (FPG) and low-density lipoprotein (LDL) cholesterol levels

At present, lifestyle-related diseases are one of the most critical health issues worldwide. It has been reported that lipopolysaccharide derived from a Gram-negative bacteria (IP-PA1) symbiotic with wheat exhibited several advantageous biological effects, such as the reduction of plasma glucose levels in NOD mice and low-density lipoprotein (LDL) levels in WHHL rabbits. In this study, the beneficial effects on plasma glucose and lipids of a tea (SI tea) consisting of IP-PA1 and Salacia (which contains an inhibitor of α-glucosidase) were investigated in the KK-Ay/TaJcl type 2 diabetic model mice and in human subjects with premetabolic syndrome in a double-blind, randomized study. SI tea significantly decreased plasma glucose levels in KK-Ay/TaJcl mice. A clinical trial of SI tea was performed with 41 subjects between the ages of 40 and 69, who belonged either to a high plasma glucose group (HG: FPG 100-125 mg/dl) or to a hyperlipidemia group (HL: TG ≥ 150 mg/dl, or LDL ≥ 120 mg/dl, or HDL < 40 mg/dl). These subjects ingested either Salacia without IP-PA1 (the control) or SI tea. Blood samples were collected at 0, 30, and 60 days after initiating SI tea treatment, and were measured for FPG, HbA1c, TG, LDL, and HDL. These results showed that SI tea reduced FPG and HbA1c more rapidly than the control in the HL group, and also significantly improved LDL and HDL levels in the HG group. Thus, SI tea may be helpful in preventing lifestyle-related diseases.     

Pancreatic lipase inhibitory activity of taraxacum officinale in vitro and in vivo

Obesity has become a worldwide health problem. Orlistat, an inhibitor of pancreatic lipase, is currently approved as an anti-obesity drug. However, gastrointestinal side effects caused by Orlistat may limit its use. In this study the inhibitory activities of dandelion (Taraxacum officinale) against pancreatic lipase in vitro and in vivo were measured to determine its possible use as a natural anti-obesity agent. The inhibitory activities of the 95% ethanol extract of T. officinale and Orlistat were measured using 4-methylumbelliferyl oleate (4-MU oleate) as a substrate at concentrations of 250, 125, 100, 25, 12.5 and 4 µg/ml. To determine pancreatic lipase inhibitory activity in vivo, mice (n=16) were orally administered with corn oil emulsion (5 ml/kg) alone or with the 95% ethanol extract of T. officinale (400 mg/kg) following an overnight fast. Plasma triglyceride levels were measured at 0, 90, 180, and 240 min after treatment and incremental areas under the response curves (AUC) were calculated. The 95% ethanol extract of T. officinale and Orlistat, inhibited, porcine pancreatic lipase activity by 86.3% and 95.7% at a concentration of 250 µg/ml, respectively. T. officinale extract showed dose-dependent inhibition with the IC₅₀ of 78.2 µg/ml. A single oral dose of the extract significantly inhibited increases in plasma triglyceride levels at 90 and 180 min and reduced AUC of plasma triglyceride response curve (p<0.05). The results indicate that T. officinale exhibits inhibitory activities against pancreatic lipase in vitro and in vivo. Further studies to elucidate anti-obesity effects of chronic consumption of T. officinale and to identify the active components responsible for inhibitory activity against pancreatic lipase are necessary.     

Suppressive effects of Schizandra chinensis Baillon water extract on allergy-related cytokine generation and degranulation in IgE-antigen complex-stimulated RBL-2H3 cells

Schizandra chinensis Baillon is a traditional folk medicine plant that is used to treat and prevent several inflammatory diseases and cancer in Korea, but the underlying mechanisms involved in its anti-allergic activity are not fully understood. This study was designed to investigate mechanisms of anti-allergic activity of a Schizandra chinensis Baillon water extract (SCWE) in immunoglobulin E (IgE)-antigen complex-stimulated RBL2H3 cells and to assess whether gastric and intestinal digestion affects the anti-allergic properties of SCWE. Oxidative stress is an important consequence of the allergic inflammatory response. The antioxidant activities of SCWE increased in a concentration-dependent manner. RBL-2H3 cells were sensitized with monoclonal anti-dinitrophenol (DNP) specific IgE, treated with SCWE, and challenged with the antigen DNP-human serum albumin. SCWE inhibited β-hexosaminidase release and expression of interleukin (IL)-4, IL-13, and tumor necrosis factor-alpha (TNF-α) mRNA and protein in IgE-antigen complex-stimulated RBL2H3 cells. We found that digested SCWE fully maintained its antioxidant activity and anti-allergic activity against the IgE-antigen complex-induced activation of RBL-2H3 cells. SCWE may be useful for preventing allergic diseases, such as asthma. Thus, SCWE could be used as a natural functional ingredient for allergic diseases in the food and/or pharmaceutical industries.     

Anti-inflammatory and carbonic anhydrase restoring actions of yam powder (Dioscorea spp) contribute to the prevention of cysteamine-induced duodenal ulcer in a rat model

Increased acid output, accompanied with a defective defense system, is considered a fundamental pathogenesis of duodenal ulcer (DU). However, relapse of DU occurs despite proton pump inhibitors and H₂ receptor antagonists, hence imposing the enforcement of the defense system. Dried powder of the yam tuber (Dioscorea spp) has been used in traditional folk medicine as a nutritional fortification. We hypothesized that dried-yam powder would prevent DU through improvement of anti-inflammatory actions and carbonic anhydrase (CA) activity. Therefore, we investigated the preventive effects of dried-yam powder against the cysteamine-induced DU and elucidated the underlying mechanisms. Duodenal ulcers were induced in Sprague-Dawley rats by intragastric administration of 500 mg/kg cysteamine-HCl. The dried-yam powder was used as a pretreatment before the cysteamine-HCl. The number and size of DU were measured. The expressions of inflammation mediators were checked in duodenal tissues, and the expressions of CAs and malondialdehyde levels were also examined. Cysteamine provoked perforated DU, whereas dried-yam powder significantly prevented DU as much as pantoprazole and significantly reduced the incidence of perforation. The messenger RNA expressions of cyclooxygenase-2 and inducible nitric oxide synthase were remarkably decreased in the yam group compared with the cysteamine group, and the serum levels of proinflammatory cytokines including interleukin-1β, interleukin-6, and tumor necrosis factor were significantly attenuated in the yam group. Cysteamine significantly decreased the expression of CAs, whereas yam treatment significantly preserved the expressions of CA IX, XII, and XIV. In conclusion, dried-yam powder exerts a significant protective effect against cysteamine-induced DU by lowering the activity of inflammatory cytokines and free radicals and restoring the activity of CAs, except in CA IV.     

Capsaicin attenuates palmitate-induced expression of macrophage inflammatory protein 1 and interleukin 8 by increasing palmitate oxidation and reducing c-Jun activation in THP-1 (human acute monocytic leukemia cell) cells

Capsaicin, a spicy component of hot peppers, has been shown to improve inflammatory disease and obesity. In this study, we tested the hypothesis that the anti-inflammatory activity of capsaicin can be used to improve free fatty acid (FFA)-induced inflammation by reducing gene expression of macrophage inflammatory protein 1 (MIP-1) and interleukin 8 (IL-8) in THP-1 (human acute monocytic leukemia cell) macrophages. To investigate whether capsaicin ameliorates palmitate-induced MIP-1 and IL-8 gene expressions, we treated THP-1 cells with palmitate in the presence or absence of capsaicin and measured MIP-1 and IL-8 by real-time polymerase chain reaction. To elucidate the mechanism by which capsaicin effects on palmitate-induced MIP-1 and IL-8 gene expressions, we performed immunoblotting with stress kinase-related antibodies and measured palmitate oxidation and palmitate oxidation-related gene expression. Palmitate and stearate but not the unsaturated FFA oleate significantly increased MIP-1 and IL-8 expressions in THP-1 macrophages. Treatment with capsaicin or FFA oxidation stimulators inhibited palmitate-induced MIP-1 and IL-8 expressions in THP-1 macrophages. Capsaicin increased the gene expression of carnitine palmitoyltransferase 1 and the β-oxidation of palmitate. Furthermore, capsaicin significantly reduced palmitate-stimulated activation of c-Jun N-terminal kinase, c-Jun, and p38. Our data suggest that the attenuation of palmitate-induced MIP-1 and IL-8 gene expressions by capsaicin is associated with reduced activation of c-Jun N-terminal kinase, c-Jun, and p38 and preserved β-oxidation activity.     

Hypoglycemic effect of Rehmannie Radix Preparata (Sookjihwang) extract in streptozotocin-induced diabetic rats

Rhemannie Radix Preparata (RRP) has been previously employed in traditional oriental medicine as a treatment for diabetic thirst and improving blood flow. The aim of this study was to evaluate its hypoglycemic control by assaying the activities of key enzymes of carbohydrate metabolism in streptozotocin-(STZ)-induced diabetic rats. Further, RRP extracts were prepared in water (RRPW), in 50% ethanol (RRP50), and in 100% ethanol (RRP100), respectively, and compared for their actions in diabetic rats. The oral treatment of RRP (5 mg/kg b.w./d) to diabetic rats for 21 days resulted in a significant decline in blood glucose by 67% compared to diabetic control rats (P < 0.05). The altered activities of glucokinase, glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), and acetyl CoA carboxylase (ACC) in the livers of diabetic rats were reversed significantly to near-normal levels by the administration of RRP (P < 0.05). Among the three RRP extracts, RRP100 was the most effective in terms of hypoglycemic action. However, the administration of RRP to diabetic rats did not improve insulin production. The modulatory effects of RRP100 on the attenuation of carbohydrate enzyme activities appear to hold promise for widespread use for the treatment of diabetes in the future.     

Yacon (Smallanthus sonchifolius)-derived fructooligosaccharides improves the immune parameters in the mouse

Owing to its high contents of fructooligosaccharides (FOSs), the yacon (Smallanthus sonchifolius) root is used in traditional Andean medicine as a substitute for cane sugar in diabetes and for obesity prevention. This study was designed to test the hypothesis that regular consumption of yacon works to improve the immune system. BALB/c mice were fed with the AIN-93 diet supplemented with 5% commercial FOS or either 3% or 5% yacon FOS for 30 consecutive days. Animals in the control group were fed with nonsupplemented ration. Food intake; weight gain; serum levels of IgA, IgM, and IgG; levels of fecal IgA, production of nitric oxide by peritoneal macrophages, frequencies of T and B lymphocytes in the spleen and peripheral blood, T-cell proliferation, and cytokine production were evaluated in all groups. No significant differences were observed in food intake and weight gain when the experimental and control groups were compared. Also, serum levels of IgA, IgM, and IgG; nitric oxide production in peritoneal macrophages; frequencies of T and B lymphocytes in the spleen and peripheral blood; T-cell proliferation; and production of interleukin (IL)-4, interferon-γ, IL-10, and tumor necrosis factor α did not differ in the different groups. The intake of FOS, however, led to a significant reduction of the proinflammatory cytokine IL-1β in macrophage cultures and elevation of the levels of fecal IgA. Together, these results indicate that the daily consumption of yacon does not exert negative effects on the immune system, helps to preserve an anti-inflammatory state in phagocytic cells, and improves mucosal immunity, possibly preventing the risks associated with autoimmune and metabolic diseases.     

Inhibitory activity of Euonymus alatus against alpha-glucosidase in vitro and in vivo

The major goal in the treatment of diabetes mellitus is to achieve near-normal glycemic control. To optimize both fasting blood glucose and postprandial glucose levels is important in keeping blood glucose levels as close to normal as possible. α-Glucosidase is the enzyme that digests dietary carbohydrate, and inhibition of this enzyme could suppress postprandial hyperglycemia. The purpose of this study was to test the inhibitory activity of methanol extract of Euonymus alatus on α-glucosidase in vitro and in vivo to evaluate its possible use as an anti-diabetic agent. Yeast α-glucosidase inhibitory activities of methanol extract of E. alatus were measured at concentrations of 0.50, 0.25, 0.10, and 0.05 mg/ml. The ability of E. alatus to lower postprandial glucose was studied in streptozotocin-induced diabetic rats. A starch solution (1 g/kg) with and without E. alatus extract (500 mg/kg) was administered to diabetic rats by gastric intubation after an overnight fast. Plasma glucose levels were measured at 30, 60, 90, 120, 180, and 240 min. Plasma glucose levels were expressed in increments from baseline, and incremental areas under the response curve were calculated. Extract of E. alatus,which had an IC₅₀ value of 0.272 mg/ml, inhibited yeast α-glucosidase activity in a concentration-dependent manner. A single oral dose of E. alatus extract significantly inhibited increases in blood glucose levels at 60 and 90 min (p<0.05) and significantly decreased incremental response areas under the glycemic response curve (p<0.05). These results suggest that E. alatus has an antihyperglycemic effect by inhibiting α-glucosidase activity in this animal model of diabetes mellitus.     

In vitro and in vivo studies on radiobiological effects of prolonged fraction delivery time in A549 cells

Intensity-modulated radiation therapy, when used in the clinic, prolongs fraction delivery time. Here we investigated both the in vivoand in vitroradiobiological effects on the A549 cell line, including the effect of different delivery times with the same dose on A549 tumor growth in nude mice. The in vitroeffects were studied with clonogenic assays, using linear-quadratic and incomplete repair models to fit the dose-survival curves. Fractionated irradiation of different doses was given at one fraction per day, simulating a clinical dose-time-fractionation pattern. The longer the interval between the exposures, the more cells survived. To investigate the in vivoeffect, we used sixty-four nude mice implanted with A549 cells in the back legs, randomly assigned into eight groups. A 15 Gy radiation dose was divided into different subfractions. The maximum and minimum tumor diameters were recorded to determine tumor growth. Tumor growth was delayed for groups with prolonged delivery time (40 min) compared to the group receiving a single dose of 15 Gy (P< 0.05), and tumors with a 20 min delivery time had delayed growth compared to those with a 40 min delivery time [20′ (7.5 Gy × 2 F) vs 40′ (7.5 Gy × 2 F), P= 0.035; 20′ (3 Gy × 5 F) vs 40′ (3 Gy × 5 F); P= 0.054; 20′ (1.67 Gy × 9 F) vs 40′ (1.67 Gy × 9 F), P= 0.028]. A prolonged delivery time decreased the radiobiological effects, so we strongly recommend keeping the delivery time as short as possible.     

Sequence Analysis of the Second Internal Transcribed Spacer (ITS2) Region of rDNA for Species Identification of Trichostrongylus Nematodes Isolated From Domestic Livestock in Iran

Background

Infectivity of herbivores with Trichostrongylus nematodes is widespread in many countries, having a major economic impact on breeding, survivability, and productivity of domestic livestock. This study was carried out on Trichostrongylus species isolated from domestic livestock in order to develop an easy-to-perform method for species identification.

Methods

Trichostrongylus isolates were collected from sheep, goat, cattle, and buffaloes in Khuzestan Province, southwest Iran. Primary species identification was carried out based on morphological characterization of male worms. PCR amplification of ITS2-rDNA region was performed on genomic DNA and the products were sequenced. Phylogenetic analysis of the nucleotide sequence data was conducted employing Bayesian Inference approach. Consequently, a restriction fragment length polymorphism (RFLP) profile was designed to differentiate Trichostrongylus species.

Results

A consensus sequence of 238 nucleotides was deposited in the GenBank for Iranian isolates of Trichostrongylus species including T. colubriformis, T. capricola, T. probolurus and T. vitrinus. The designated RFLP using restriction enzyme TasI could readily differentiate among species having different ITS2 sequence. The molecular analysis was in concordance with morphological findings.

Conclusion

Phylogenetic analysis indicated a close relationship among the sequences obtained in this study and reference sequence of relevant species. ITS2-RFLP with TasI is recommended for molecular differentiation of common Trichostrongylus species.