Antioxidant properties of date seeds extract (Phoenix dactylifera L.) in alloxan induced damage in rats.

The study was conducted to examine the antioxidant activity and evaluate the protective effects of the date seeds powder kentichi against alloxan-induced damage in the liver, kidney, and pancreas in diabetic's rats. Group 1: control group, that did not receive any treatment, Group 2: alloxan was injected intraperitoneally (120 mg/kg body weight) for two days (Diab), Group 3: treated only by date seeds powder added in the diet (300 g/kg) for 6 weeks (DSPK), Group 4: alloxan-diabetic rats treated with date seeds powder (300 g/kg) (DSPK + Diab). Estimations of biochemical parameters in blood were determined. TBARS, SOD, CAT, and GPx activities were determined. A histopathological study was done by immersing pieces of both organs in a fixative solution followed by paraffin hematoxylin-eosin staining. In addition, the antioxidant activities of DSPK were evaluated by DPPH radical scavenging activity, reducing power, and ABTS free radical scavenging. The results revealed that date seeds significantly decreased serum levels of glucose, cholesterol, triglycerides, urea, creatinine, T-protein, ALP, D-bili and T-bili levels. In addition, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities that had been reduced in liver, kidney, and pancreas of the treated group were restored by DSPK treatments and, therefore, the lipid peroxidation level was reduced in the liver, kidney and pancreas tissue compared to the control group. Additionally, the histological structure in these organs was restored after treatment with date seeds powder.

Anti-osteoporosis effects of mammalian lignans and their precursors from flaxseed and safflower seed using zebrafish model.

Secoisolariciresinol diglucoside (SDG) and tracheloside (TCL) are the main lignan components of flaxseed cake and safflower seed cake, which are by-products of oil extraction. Both SDG and TCL are metabolized into mammalian lignan enterolactone (EL) with the involvement of intestinal bacteria. In this research, we evaluated the anti-osteoporosis effects of SDG and the in vivo metabolites EL and enterodiol (ED) prepared in our previous work, as well as the newly isolated chemical constituents from safflower seed, including TCL, the lactone ring opening product of TCL (OTCL) and two alkaloids on the alloxan-induced zebrafish model. All the compounds showed significant anti-osteoporosis effects at 80 µM, with p < 0.05 for EL and p < 0.001 for other compounds compared with the model. SDG and TCL showed the most significant and concentration-dependent effects, with p < 0.001 compared with model at 20 µM. The alkaloids, N-coumaroylserotonin glucoside and N-feruloylserotonin glucoside, also showed anti-osteoporosis at 20 µM with p < 0.01, whereas EL, ED, and OTCL showed no significant effects. Quantitative real-time polymerase chain reaction revealed that SDG and TCL upregulated the expression of osteogenic genes Runx2, SP7, OPG, Col1a1a, Alp, ON, OPN, and OCN in alloxan-treated zebrafish. The in vivo metabolite of lignans, EL, showed significant anti-inflammatory effect (p < 0.01) at 20 µM, which might also help to combat osteoporosis and other complications caused by excessive immune response in the body. The results provided scientific data for using the oil extraction by-products as sources of anti-osteoporosis compounds. PRACTICAL APPLICATION: This study found that lignans in flaxseed cake and safflower seed cake exhibited anti-osteoporosis effects by upregulating the expression of osteogenic genes, making the oil extraction by-products sources of anti-osteoporosis compounds.

Ameliorative Effects of Flavonoids from Platycodon grandiflorus Aerial Parts on Alloxan-Induced Pancreatic Islet Damage in Zebrafish.

Platycodon grandiflorus (balloon flower), used as a food reserve as well as in traditional herbal medicine, is known for its multiple beneficial effects. In particular, this plant is widely used as a vegetable in Republic of Korea. We examined the ameliorative effects of P. grandiflorus on alloxan-induced pancreatic islet damage in zebrafish. The aerial part treatment led to a significant recovery in pancreatic islet size and glucose uptake. The efficacy of the aerial part was more potent than that of the root. Eight flavonoids (1-8) were isolated from the aerial part. Structures of two new flavone glycosides, designated dorajiside I (1) and II (2), were elucidated to be luteolin 7-O-α-L-rhamno-pyranosyl (1 → 2)-(6-O-acetyl)-ß-D-glucopyranoside and apigenin 7-O-α-L-rhamnopyranosyl (1 → 2)-(6-O-acetyl)-ß-D-glucopyranoside, respectively, by spectroscopic analysis. Compounds 1, 3, 4 and 6-8 yielded the recovery of injured pancreatic islets in zebrafish. Among them, compound 7 blocked KATP channels in pancreatic ß-cells. Furthermore, compounds 3, 4, 6 and 7 showed significant changes with respect to the mRNA expression of GCK, GCKR, GLIS3 and CDKN2B compared to alloxan-induced zebrafish. In conclusion, the aerial part of P. grandiflorus and its constituents conferred a regenerative effect on injured pancreatic islets.

Protective effect of mulberry flavonoids on sciatic nerve in alloxan-induced diabetic rats

Mulberry leaves (Morus alba L.) are a traditional Chinese medicine for blood serum glucose reduction. This study evaluated the protective effects of mulberry flavonoids on sciatic nerve in alloxan-induced diabetic rats. In this study, 80 Sprague-Dawley rats were divided into five groups: A (control), B (diabetic treated with saline), C-D (diabetic treated with 0.3, 0.1 g/kg mulberry flavonoids once a day for 8 weeks) and E (diabetic treated with 0.3 mg/kg methycobal). The diabetic condition was induced by intraperitoneal injection of 200 mg/kg alloxan dissolved in saline. At the end of the experimental period, blood, and tissue samples were obtained for biochemical and histopathological investigation. Treatment with 0.3 g/kg mulberry flavonoids significantly inhibited the elevated serum glucose (P< 0.01). The increased myelin sheath area (P< 0.01), myelinated fiber cross-sectional area and extramedullary fiber number (P< 0.05) were also reduced in alloxan-induced rats treated with 0.3 g/kg mulberry flavonoids. 0.3 g/kg mulberry flavonoids also markedly decreased onion-bulb type myelin destruction and degenerative changes of mitochondria and Schwann cells. These findings demonstrate that mulberry flavonoids may improve the recovery of a severe peripheral nerve injury in alloxan-induced diabetic rats and is likely to be useful as a potential treatment on peripheral neuropathy (PN) in diabetic rats.

Folhas de amoreira (Morus alba L.) é um medicamento tradicional chinês para a redução da glicose no soro sanguíneo. Avaliaram-se, neste trabalho, os efeitos protetores dos flavonóides de amora no nervo ciático em ratos diabéticos aloxano-induzidos. Dividiram-se 80 ratos Sprague-Dawley em cinco grupos: A (controle), B (diabétidos tratados com solução salina), C-D (diabéticos tratados com 0,3, 0,1 g/kg) e E (diabéticos tratados com 0,3 mg de metilcobal).A diabetes foi induzida por injeção intraperitoneal de 200 mg/kg de aloxana dissolvida em solução salina. No final do período experimental, obtiveram-se amostras de sangue e de tecido para investigação bioquímica e histopatológica. O tratamento com 0,3 g/kg de flavonóides da amoreira inibiu, significativamente, a elevação de glicose no soro (p <0,01). O aumento da área da bainha de mielina (p <0,01), da área de fibra da seção transversal e do número de fibras mielinizadas extramedulares (p <0,05) foi também reduzido em ratos aloxânicos, tratados com 0,3 g/kg flavonóides de amora. Flavonóides da amoreira na dose de 0,3 g/kg também diminuiram, acentuadamente, a destruição da mielina do tipo bulbo de cebola e as alterações degenerativas das células mitocôndrias e das células de Schwann. Estes resultados demonstram que os flavonóides da amoreira podem melhorar a recuperação de uma lesão nervosa periférica grave em ratos com diabetes, induzida por aloxana, e parece ser útil como tratamento potencial para a neuropatia periférica (PN) em ratos diabéticos.

Efeito do composto "mais vida" na ativação de macrófagos de ratos diabéticos / Effects of "mais vida", a commercial natural mix, on the activation of macrophages from diabetic rats

O objetivo deste trabalho foi avaliar a atividade funcional de macrófagos de ratos diabéticos, através da liberação do ânion superóxido, na presença do composto "mais vida". Os animais foram divididos em dois grupos, controle (N=20) e diabético (N=20). Avaliou-se a glicemia, massa corpórea e a liberação de superóxido pelos macrófagos de baço de ratos. O composto "mais vida" foi obtido através da mistura de extratos de sete plantas, sendo Orbignia martiana Rodr., Tabebuia avellanedae L.G., Arctium lappa L., Rosa centifolia L., Maytenus ilicifolia Mart., Vernonia condensata Baker e Thuja occidentalis L. Observou-se que glicemia foi maior no grupo diabético. A liberação espontânea do ânion superóxido pelos macrófagos foi menor no grupo diabético. O composto "mais vida", independente dos níveis glicêmicos, aumentou a liberação de superóxido dos macrófagos. Quando as células foram estimuladas pelos extratos vegetais isolados, também houve aumento na liberação do ânion superóxido pelos macrófagos em ambos os grupos. As maiores liberações de superóxido ocorreram quando os macrófagos foram estimulados pela Thuja occidentalis L., Rosa centifolia L., Tabebuia avellanedae L.G. e Maytenus ilicifolia Mart. Estes dados sugerem que a ativação de macrófagos pelo composto "mais vida" pode representar um mecanismo alternativo de defesa para infecções em indivíduos diabéticos.

This study investigated the effects of "mais vida", a commercial natural mix, on macrophages functional activity as evaluated by the superoxide release in diabetic rats. The animals were divided into two groups, control (N = 20) and diabetic (N = 20). This was achieved by determining blood glucose weight and the superoxide released by spleen macrophages. The "mais vida" mix was obtained by the combination of extracts from seven medicinal species, which were: Orbignia martiana Rodr., Tabebuia avellanedae L.G., Arctium lappa L., Rosa centifolia L., Maytenus ilicifolia Mart., Vernonia condensata Baker and Thuja occidentalis L. Blood glucose levels were significantly higher (p<0.05) in the diabetic group, as compared to blood glucose levels in the control group. Superoxide levels in macrophages isolated from normoglycemic rats were higher than those obtained from diabetic animals. The "mais vida" mix, independently of glycemic status, increased significantly the superoxide release in the macrophages. Each extract by itself also increased the superoxide release by phagocytes in the macrophages in both groups. The largest superoxide release occurred when the phagocytes were stimulated by Thuja occidentalis L., Rosa centifolia L., Tabebuia avellanedae L.G. and Maytenus ilicifolia Mart. In addition, the activation of macrophages by the "mais vida" mix may represent an additional protection mechanism for diabetic individuals against infections.

Determination of alloxan by fluorometric high-performance liquid chromatography.

A fluorometric, reversed-phase high-performance liquid chromatography (RP-HPLC) method that allows quantitation of low levels of alloxan has been described. The method involved derivatization of alloxan with 500-200,000-fold excess of 1, 2-phenylenediamine (PD) in 0.1 M acetate buffer, pH 4.5 for 15 min at room temperature. The fluorescent product alloxazine (excitation: 382 nm; emission: 435 nm) was then analyzed by RP-HPLC using an Eclipse XDB-C18 (4.6 x 150 mm) column and a mobile phase consisting of 0.1% trifluoroacetic acid in 15/85 (v/v) acetonitrile/water at a flow of 1 mL/min (injection volume: 20 microL). The method is robust, and as low as 0.1 pmol of the analyte could be successfully detected and quantified. Following a minimal pre-treatment such as ultrafiltration (molecular weight cut-off 5000 Da) or protein precipitation using perchloric acid, acetonitrile, or phosphotungstic acid, the method is suitable for analysis of alloxan in complex physiological fluids (e.g. fetal bovine serum) and tissue homogenates (e.g. heart and kidney). The method has been rigorously evaluated and adapted in the laboratory for routine analysis and determination of alloxan added to cell cultures.

Simultaneous quantitative determination of alloxan, GSH and GSSG by HPlc. Estimation of the frequency of redox cycling between alloxan and dialuric acid.

This in vitro study compares the frequency of redox cycling between alloxan and dialuric acid at different initial ratios of glutathione and alloxan. Alloxan oxidizes GSH to GSSG. The rate of GSH oxidation at a given initial GSH concentration of 2.0 mmol/L depends on the initial concentration of alloxan added. The higher the concentration of alloxan in relation to the initial concentration of GSH, the faster GSH oxidation proceeds, as well as oxygen consumption, and therefore, formation of reactive oxygen species. The highest rates of GSH oxidation, i.e. GSSG formation, were found at concentration ratios of between 2.0 mmol/L GSH and 0.2 and 0.04 mmol/L alloxan, respectively. Because 0.04 mmol/L alloxan oxidizes 2.0 mmol/L GSH completely, a frequency of at least 25 cycles between alloxan and dialuric acid within 3 hours can be assumed. During each redox cycle, two molecules of GSH are oxidized to one molecule of GSSG, and during each cycle one molecule of oxygen is reduced simultaneously to one molecule of hydrogen peroxide. In total, therefore, one molecule of alloxan oxidizes at least 50 molecules of GSH and forms about 25 molecules of hydrogen peroxide.

Studies on the 'linear pattern' in renal glomeruli demonstrated with immunofluorescence.

We have experimentally induced the 'linear pattern' in immunofluorescence: the linear deposition of endogenous immunoglobulin (Ig) along the glomerular basement membrane (GBM) in rats injected with both protamine and nephrotoxic serum. This Ig was demonstrated to have no specific antibody activity against GBM or rabbit serum. Our findings could be of value in the analysis of the mechanism and pathological meaning of the 'linear pattern' of endogenous Ig in immunofluorescence.

Diabetogenic action of alloxan-like compounds: the effect of dehydrouramil hydrate hydrochloride on isolated islets of Langerhans of the rat.

Dehydrouramil hydrate hydrochloride (DHU) is an analogue of alloxan which retains the in vivo diabetogenic activity of alloxan but, in contrast to alloxan, is stable in aqueous media at physiological pH. Using rat islets of Langerhans, we have studied the acute effects of DHU on B cell function. Glucose-stimulated insulin release was markedly inhibited by DHU, the concentration of DHU giving 50% inhibition (I50) was 1 mmol/l; this was lowered to 0.5 mmol/l when the islets were exposed to DHU for 5 min before elevation of glucose concentration. The basis for this change appeared to be a protective effect of glucose, since the inclusion of 3-0-methylglucose during re-incubation with DHU also attenuated the subsequent inhibition of glucose-stimulated insulin release. The inhibitory effect on glucose-stimulated insulin release of a 5-min exposure to DHU persisted throughout a subsequent 120-min period in the absence of DHU. DHU also inhibited insulin release stimulated by mannose (20 mmol/l) or by 2-ketoisocaproate (20 mmol/l) with I50 of 1 and 0.5 mmol/l respectively. Concentrations of DHU up to 1 mmol/l had no significant effect on islet glucose oxidation or ATP content; 5 mmol/l DHU did not affect the rate of glucose oxidation, but lowered the ATP content by 30% without pre-incubation and by 60% in islets pre-incubated for 5 min with DHU before addition of glucose.(ABSTRACT TRUNCATED AT 250 WORDS)