Acellular Porcine Cornea Produced by Supercritical Carbon Dioxide Extraction: A Potential Substitute for Human Corneal Regeneration.

PURPOSE: The aim of this study was to develop a non-cytotoxic, biocompatible innovative acellular porcine cornea (APC) for corneal wound healing and corneal blindness treatment. METHODS: APC was produced by using supercritical carbon dioxide (SCCO2) to decellularize the porcine cornea. Decellularization of the porcine cornea was examined by hematoxylin and eosin staining and 4,6-diamidino-2-phenylindole, dihydrochloride staining. The residual DNA content of APC was analyzed in comparison with the native porcine cornea. Virus inactivation up to at least 6 log10 was confirmed for the stepwise process of APC for 4 different model viruses. In addition, a series of in vitro and in vivo tests in accordance with ISO-10993 biocompatibility assay and animal performance tests were performed. RESULTS: APC produced by the SCCO2 process revealed complete decellularization, without any residual non-collagenous proteins. The scanning electron microscopy structural features of the decellularized cornea were similar to those of human. APC was found to be nontoxic and exhibited excellent biocompatibility in both in vitro and in vivo studies. The animal performance test proved that APC exerted excellent adaptability on the cornea and no sign of irritation and good compatibility in lamellar corneal transplantation. CONCLUSIONS: APC manufactured by SCCO2 technology revealed complete cells and non-collagenous protein removal compared with the Triton-sodium dodecyl sulfate decellularization process. APC showed excellent biocompatibility in rabbit lamellar corneal transplantation with a follow-up to 1 year. APC can be a potential substitute for human-donated cornea for corneal transplantation in the near future.

Reconstruction of the orbital floor using supercritical CO2 decellularized porcine bone graft.

Orbital floor fractures subsequently lead to consequences such as diplopia and enophthalmos. The graft materials used in orbital floor fractures varied from autografts to alloplastic grafts, which possess certain limitations. In the present study, a novel porcine bone matrix decellularized by supercritical CO2 (scCO2), ABCcolla® Collagen Bone Graft, was used for the reconstruction of the orbital framework. The study was approved by the institutional review board (IRB) of Kaohsiung Medical University Chung-Ho Memorial Hospital (KMUH). Ten cases underwent orbital floor reconstruction in KMUH in 2019. The orbital defects were fixed by the implantation of the ABCcolla® Collagen Bone Graft. Nine out of ten cases used 1 piece of customized ABCcolla® Collagen Bone Graft in each defect. The other case used 2 pieces of customized ABCcolla® Collagen Bone Graft in one defect area due to the curved outline of the defect. In the outpatient clinic, all 10 cases showed improvement of enophthalmos on CT (computerized tomography) at week 8 follow-up. No replacement of implants was needed during follow-ups. To conclude, ABCcolla® Collagen Bone Graft proved to be safe and effective in the reconstruction of the orbital floor with high accessibility, high stability, good biocompatibility, low infection rate and low complication rate.

Supercritical carbon dioxide decellularized porcine cartilage graft with PRP attenuated OA progression and regenerated articular cartilage in ACLT-induced OA rats.

Knee osteoarthritis (OA) is a common degenerative articular disorder and considered one of the primary causes of pain and functional disability. Knee OA is prevalent in 10% of men and 13% of women aged 60 years above. The study aims to use cartilage tissue engineering that combines the triads of decellularized porcine cartilage graft as "scaffold," plasma rich platelet (PRP) as "signal" and chondrocytes from rat as "cell" to attenuate ACLT-induced OA progression and regenerate the knee cartilage in rats. Decellularization of the porcine cartilage was characterized by hematoxylin and eosin, 4,6-Diamidino-2-phenylindole staining, scanning electron microscopy and residual DNA quantification. The protective effect of decellularized porcine cartilage graft (dPCG) was evaluated by intra-articular administration in surgically induced anterior cruciate ligament transection (ACLT) rat osteoarthritis (OA) model. Supercritical carbon dioxide technology completely decellularized the porcine cartilage. Intra-articular administration of dPCG with or without PRP significantly reduced the ACLT-induced OA symptoms and attenuated the OA progression. Pain-relief by dPCG with or without PRP was assessed by capacitance meter and improved articular cartilage damage in the rat knee was characterized by X-ray and micro-CT. Besides, the histological analysis depicted cartilage protection by dPCG with or without PRP. The repairation and attenuation effect by dPCG with or without PRP in the articular knee cartilage damage were also explored by safranin-O, type II collagen, aggrecan and SOX-9 immuno-staining. To conclude, intra-articular administration of dPCG with or without PRP is efficient in repairing the damaged cartilage in the experimental OA model.

A novel 3D histotypic cartilage construct engineered by supercritical carbon dioxide decellularized porcine nasal cartilage graft and chondrocytes exhibited chondrogenic capability in vitro.

Augmentative and reconstructive rhinoplasty surgical procedures use autologous tissue grafts or synthetic grafts to repair the nasal defect and aesthetic reconstruction. Donor site trauma and morbidity are common in autologous grafts. The desperate need for the production of grafted 3D cartilage tissues as rhinoplasty grafts without the adverse effect is the need of the hour. In the present study, we developed a bioactive 3D histotypic construct engineered with the various ratio of adipose-derived stem cells (ADSC) and chondrocytes together with decellularized porcine nasal cartilage graft (dPNCG). We decellularized porcine nasal cartilage using supercritical carbon dioxide (SCCO2) extraction technology. dPNCG was characterized by H&E, DAPI, alcian blue staining, scanning electron microscopy and residual DNA content, which demonstrated complete decellularization. 3D histotypic constructs were engineered using dPNCG, rat ADSC and chondrocytes with different percentage of cells and cultured for 21 days. dPNCG together with 100% chondrocytes produced a solid mass of 3D histotypic cartilage with significant production of glycosaminoglycans. H&E and alcian blue staining showed an intact mass, with cartilage granules bound to one another by extracellular matrix and proteoglycan, to form a 3D structure. Besides, the expression of chondrogenic markers, type II collagen, aggrecan and SOX-9 were elevated indicating chondrocytes cultured on dPNCG substrate facilitates the synthesis of type II collagen along with extracellular matrix to produce 3D histotypic cartilage. To conclude, dPNCG is an excellent substrate scaffold that might offer a suitable environment for chondrocytes to produce 3D histotypic cartilage. This engineered 3D construct might serve as a promising future candidate for cartilage tissue engineering in rhinoplasty.

Protocols for the preparation and characterization of decellularized tissue and organ scaffolds for tissue engineering.

Extracellular matrix (ECM) scaffolds are extensively used in tissue engineering studies and numerous clinical applications for tissue and organ reconstructions. Due to the global severe shortage of human tissues and organs, xenogeneic biomaterials are a common source for human tissue engineering and regenerative medicine applications. Traditional methods for decellularization often disrupt the 3D architecture and damage the structural integrity of the ECM scaffold. To efficiently obtain natural ECM scaffolds from animal tissues and organs with intact architecture, we have developed a platform decellularization process using supercritical CO2 and tested its potential application in tissue engineering. A combination of human mesenchymal stem cells with a decellularized dermal matrix scaffold allowed complete regeneration of skin structure in a porcine full-thickness wound model.

Protocols for accelerated production and purification of collagen scaffold and atelocollagen from animal tissues.

Traditional purification of atelocollagen involves a harsh extraction process with environment-polluting chemicals and costly and/or time-consuming procedures which include salting-out, alkali, acid and enzymatic treatment and ion-exchange chromatography. The atelocollagen market is growing exponentially, with demand in the skincare industry and for various medical applications. As a result, there is an urgent need for an eco-friendly production process with minimal manipulation. We developed a novel technique involving supercritical carbon dioxide extraction technology to remove the cells and noncollagenous substances from the porcine hide. Subsequent processes allow the production of several products, including decellularized dermal membrane, high-purity collagen particles and atelocollagen. The advantages of our process are its faster speed and lower environmental impact and its generation of multiple products, including high purity atelocollagen with complete removal of telopeptides.

Supercritical Carbon Dioxide-decellularized Porcine Acellular Dermal Matrix combined with Autologous Adipose-derived Stem Cells: Its Role in Accelerated Diabetic Wound Healing.

Diabetes mellitus (DM) causes impaired wound healing by affecting one or more of the biological mechanisms of hemostasis, inflammation, proliferation, and remodeling and a large number of cell types, extracellular components, growth factors, and cytokines. Interventions targeted toward these mechanisms might accelerate the wound healing process. To evaluate the wound healing efficacy of supercritical carbon dioxide (scCO2)-decellularized porcine acellular dermal matrix (ADM) combined with autologous adipose-derived stem cells (ASCs) in streptozotocin (STZ)-induced DM rats. DM was induced by injecting rats with STZ; dorsal full-thickness skin (5 × 5 cm2) was created and treated with and without ASCs-scCO2-treated ADM to evaluate the wound healing rate through histological examination, fluorescence microscopic observation, and immunohistochemical analysis. In the present study, complete decellularization of the porcine dermal matrix was achieved through scCO2. Isolation of ASCs was conducted and evaluated using CD29+/CD31-/CD45-/CD90+ markers in flow cytometry, which indicated that more than 90% of cells were ASCs. The percentage of cells labeled with CD29+ and CD90+ was found to be 97.50% and 99.69%, respectively. The wound healing rate increased in all groups relative to the group with the DM wound without treatment. DM wound treated with ADM-ASCs showed significantly higher (p < 0.01) wound healing rate than DM wound without treatment. ADM-ASC-treated rats showed significantly increased epidermal growth factor, Ki67, and prolyl 4-hydroxylase and significantly decreased CD45 compared with the group with the DM wound without treatment. The intervention comprising ADM decellularized from porcine skin by using scCO2 and ASCs was proven to improve diabetic wound healing. ADM-ASCs had a positive effect on epidermal regeneration, anti-inflammation, collagen production and processing, and cell proliferation; thus, it accelerated wound healing.

Immunomodulatory activity of fucoidan against aspirin-induced gastric mucosal damage in rats.

Gastric ulcers and related complications associated with the use of non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, represent a major global health problem. In the present study, we investigate the immunological activity of fucoidan against aspirin-induced gastric mucosal damage in rats. Thirty-six rats were randomly divided into the following, normal (Carboxy methyl cellulose 0.05 %), aspirin (Asp-400mg/kg) treated, fucoidan alone (Fu-0.02 g/kg, daily for 14 days) and Fu+Asp. Cytokines, total nitrite and nitrate (NOx) analysis and tissue localization of Cyclooxygenase 1, 2 and epidermal growth factor receptor (EGFR) were done using Elisa and immunohistochemistry respectively. Histopathology of gastric tissue, collagen deposition was performed using Hematoxylin and Eosin and Masson's trichrome were performed. Treatment of rats with a single dose of aspirin (400mg/kg, orally) led to significant alterations in the levels of total nitrite and nitrate (NOx), interleukins (IL-4, 6, 10, 12), tumor necrosis factor (TNF-α), and interferon gamma (IFN-γ). Notably, collagen deposition in glandular tissue and localization of cyclooxygenase 1, 2, and epidermal growth factor were considerably affected in aspirin-treated rats. These severities were prevented to a significant extent in rats pretreated with fucoidan (0.02 g/kg/day for two weeks orally). Our findings collectively indicate that the gastro-protective effect of fucoidan against aspirin-induced ulceration in rats is mediated through its immunomodulatory properties.

Ostensibly ineffectual doses of cadmium and lipopolysaccharide causes liver damage in rats.

Various hepatotoxicants co-treated with lipopolysaccharide (LPS) have the potential to cause severe hepatic damage. Whether co-treatment with ostensibly ineffectual (without effect on customary clinical liver function tests, such as aspartate aminotransferase and alanine aminotransferase) doses of cadmium (Cd) and LPS cause liver damage is still unknown. We examined the effects of treating ostensibly ineffectual doses of Cd and LPS on liver dysfunction as well as on liver histopathology. We injected rats with saline only, Cd only, LPS only, or a single ostensibly ineffectual dose of Cd (100 µg/kg body weight) plus LPS (0.1 mg/kg body weight). After 6 h, the rats were killed and their liver damage was assessed. Co-treated with ostensibly ineffectual doses of Cd and LPS had higher levels of aspartate aminotransferase and alanine aminotransferase, hepatic lipid peroxidation, peroxynitrite, nitrite, and interleukin-1ß (IL-1ß), but lower levels of hepatic metallothionein (MT) than did that treated with saline only, Cd only, and LPS only. Histopathological analysis of Cd only and LPS only showed apparent liver damage, but Cd plus LPS showed marked hepatic damage. We conclude that co-treating the rats with ostensibly ineffectual doses of Cd and LPS is hepatotoxic. Cd promotes LPS-initiated oxidative-stress-associated liver damage by increasing IL-1ß and decreasing MT levels in rats.

Gamma-irradiation improves the color and antioxidant properties of Chaga mushroom (Inonotus obliquus) extract.

The objective of this study was to evaluate the effect of ionizing radiation on color and antioxidative properties of Chaga mushroom (Inonotus obliquus) extract (CME). CME (10 mg/mL) was gamma-irradiated at 0, 3, 5, 7, and 10 kGy, and color, antioxidant activity, and total phenolic compound levels were then determined. The lightness and yellowness were increased (P < .05), and the redness was decreased (P < .05), as irradiation dose increased. The antioxidant parameters such as the 2-diphenyl-1-picrylhydrazyl, superoxide, and hydroxyl radical scavenging activities, ferric reducing/antioxidant power, and inhibition of lipid peroxidation increased as the irradiation dose increased. Also, the total phenolic compound levels of CME were increased (P < .05) by gamma-irradiation. These results suggest that gamma-irradiation could be considered a means for improving the antioxidant properties and the color of CME.

Effect of crude sulphated polysaccharide from brown algae against acetaminophen-induced toxicity in rats.

This study was conducted to examine the protective role of crude polysaccharide from brown seaweed Sargassum polycystum against acetaminophen-induced abnormality in blood glucose, serum albumin/globulin ratio, and liver glycogen, lactate, and pyruvate. Liver and renal tissue histology was performed to confirm the efficacy of Sargassum polysaccharide. A toxic dose of acetaminophen (800 mg/kg body weight intraperitoneally) induced severe abnormality in all basic parameters with apparent toxicity in liver (enlargement of hepatocytes, loss of cytoplasmic content with disruption in the hepatic plates and sinusoidal dilation) and renal tissue (glomerular damage with congestion of tubules). The isolated liver cells were stained with acridine orange and examined under fluorescence microscope, which revealed that the acetaminophen induced significant damage. In contrast, the rats pretreated with Sargassum polysaccharide (200 mg/kg body weight) daily for 3 weeks did not show liver and renal tissue with these severe aberrations induced by acetaminophen. Histology results were also consistent with analyzed basic biochemical parameters, which confirmed the effectiveness of the crude polysaccharide against acetaminophen-induced abnormality in rats.

Anti-peroxidative and anti-hyperlipidemic nature of Ulva lactuca crude polysaccharide on D-galactosamine induced hepatitis in rats.

To find whether pretreatment of Ulva lactuca polysaccharide (ULP) extract could be effective against D-Galactosamine (500 mg/kg body weight, i.p.) induced anomaly in rat. Serum total cholesterol (TC), triglycerides (TG), free fatty acid (FFA), phospholipids (PL), high density lipoprotein (HDL), low density lipoprotein (LDL), very low density lipoprotein (VLDL), tissue lipoperoxides (LPO), hepatic protein thiols, non-enzymatic anti-oxidants glutathione (GSH) and vitamins (E and C) were examined using spectrophotometer. The ultra structural changes of liver during D-Galactosamine and protection offered by ULP were examined by electron microscopy. Seaweed histology and chemical composition of polysaccharides in seaweed were examined. Alcian blue staining showed the presence of sulphated polysaccharide with total sugar (65.4%), sulphate (17.4%), and uronic acid (17.2%) content. D-Galactosamine intoxicated rats showed significant (p<0.01) liver damage with acute aberration in serum lipid profile, hepatic protein thiols and tissue non-enzymatic anti-oxidants. Assorted deposits of lipid droplets and abnormal appearance of mitochondria was observed in electron microscopy study. Rats pretreated with ULP (30 mg/kg body weight/day/for 21 days) showed a significant inhibition (p<0.05) against abnormality induced by d-Galactosamine. U.lactuca exhibit anti-peroxidative and anti-hyperlipidemic property.

Comparison of hypolipidemic activity of synthetic gallic acid-linoleic acid ester with mixture of gallic acid and linoleic acid, gallic acid, and linoleic acid on high-fat diet induced obesity in C57BL/6 Cr Slc mice.

Hyperlipidemia is the major risk factors of heart disease such as atherosclerosis, stroke, and death. In the present study, we studied the effect of gallic acid (GA), linoleic acid (LA), mixture of GA and LA (MGL), and chemically synthesized gallic acid-linoleic acid ester (octadeca-9,12-dienyl-3,4,5-trihydroxybenzoate, GLE) on the ability to ameliorate hyperlipidemia in C57BL/6 mice fed a high-fat diet (HFD). GLE, GA, LA, and MGL were mixed with HFD and the composition of the test compounds were 1% of the diet for 7 weeks. After 7 weeks, the average body weight of ND and GLE groups was lower than that of HFD group (P<0.05). The liver weight of mice decreased (P<0.05) in all treatment groups relative to HFD fed group. The plasma lipids such as triglyceride and LDL-cholesterol were found to be decreased (P<0.05) in GLE, GA, LA, and MGL fed mice when compared to that of HFD fed mice. But high-density lipoprotein (HDL) cholesterol increased (P<0.05) in HFD and GLE fed mice when compared to that of ND fed mice. The hepatic accumulation of fat droplets of GA, LA, GLE, and MGL group showed considerably lower than that of HFD group. Adipose histology showed that GLE supplementation was found to be more effective in decreasing the size of adipocyte relative to those of other treatment groups. In conclusion, the supplementation of synthetic GLE from gallic acid and linoleic acid ester may have a potential hypolipidemic effect on mice fed high-fat diet. Further studies are required to prove GLE as a hypolipidemic agent.

Chemopreventive and therapeutic modulation of green tea polyphenols on drug metabolizing enzymes in 4-Nitroquinoline 1-oxide induced oral cancer.

Oral cancer is one of the most common cancers in the world. Drugs can modulate the expression of drug metabolizing enzymes and are useful in chemoprevention as well as therapy in cancer. 4-Nitroquinoline 1-oxide (4-NQO) is used to induce oral cancer in the present study. In the present investigation, the effect of green tea polyphenols (GTP) on the activities of cytochrome b5, cytochrome P450, cytochrome b5 reductase (cyt b5 R), cytochrome P450 reductase (cyt P450 R), arryl hydrocarbon hydroxylase (AHH), DT-diaphorase (DTD)(Phase I enzymes) and glutathione-S-transferase (GST) and UDP-glucuronyl transferase (UDP-GT) (Phase II enzymes) were assessed in tongue and oral cavity. In induced rats, there was a decrease in the activity of Phase II enzymes and an increase in the activity of Phase I enzymes. On supplementation of GTP by both simultaneous and post treatment mode (200mg/kg) there was a significant increase in the activity of GST and UDP-GT and a significant decrease in the activity of Phase I enzymes. There was a significant decline in the number of tumors, tumor volume and oral squamous cell carcinoma in both simultaneous and post GTP treated animals relative to 4-NQO induced animals; on comparing simultaneous and post GTP treated animals the number of tumors, tumor volume and oral squamous cell carcinoma was significantly reduced in post treated animals. Thus inhibition of Phase I enzymes could be attributed to the protective efficacy of GTP which deactivates carcinogen and GTP induced the expression of Phase II enzymes that detoxifies the 4-NQO. It can be proposed that GTP plays role as a detoxifying agent by which its modulating role prevented/inhibited the formation of tumor.

Effect of gamma irradiated hyaluronic acid on acetaminophen induced acute hepatotoxicity.

The hepatoprotective efficacy of irradiated hyaluronic acid (HA) on acetaminophen (APAP) induced acute hepatotoxicity was investigated. BALB/c mice (4-6 weeks of age) were pretreated with unirradiated HA (UIHA), 5 and 50 kGy gamma irradiated HA (GIHA) for 14 days and were dosed APAP (500 mg/kg b.wt). After 9h of APAP dosing animals were euthanized. The degree of acute hepatotoxicity was measured by aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The expression of interferon-gamma (IFN-gamma) in serum and alpha-and mu-class of gluthathione-S-transferase (GSTs), CYP 2E1 class of cytochrome monooxygenase and glutathione (GSH) in liver were quantified. Histological evaluation was done by Hematoxiylin and Eiosin staining, Periodic acid schiffs staining, Manson trichrome staining and histological scorings were done. The degree of acute hepatotoxicity was markedly lower in UIHA and 5 kGy than in 50 kGy GIHA pretreated group and there was negligible difference between 5 and 50 kGy GIHA pretreated group. The expression of interferon-gamma (IFN-gamma) was significantly (P<0.05) suppressed in 5 and 50 kGy GIHA pretreated group. Histological scorings showed a significant protection of liver in UIHA and 5 kGy GIHA pretreated mice. Expression of alpha class GSTs was significantly increased in 5 and 50 kGy GIHA pretreated group. To conclude suppression of IFN-gamma and increase in alpha-class GSTs expression may exert a protective role in acute hepatotoxicity of APAP and 5 kGy GIHA showed comparable protective effect to that of UIHA.

Structural and antioxidant properties of gamma irradiated hyaluronic acid.

Hyaluronic acid (Hyaluronan, HA) was depolymerised by gamma irradiation and its structural changes and antioxidant activities were investigated. The structural changes of gamma irradiated HA were studied by gel-permeation chromatography (GPC), viscosity, pH, Hunter colour measurement, UV spectrophotometry, and FT-IR spectroscopy. The results demonstrated that gamma irradiation decreased molecular weight size, viscosity and pH of the hyaluronic acid and its colour turned to intense yellow. UV spectra of the irradiated HA showed a change at 265nm, which indicates the formation of double bonds. Differences in the height and shape of certain absorption bonds of FT-IR spectra in the range 1700-1750cm(-1) were also observed, which is associated with the formation of carboxylic acid. From these structural changes of the HA, gamma irradiation may have a role in the formation of pyrancarboxylic acid rings. DPPH radical scavenging ability and the reducing power of gamma irradiated HA were significantly higher than that of non-irradiated HA. However, non-irradiated and irradiated HA did not show significant differences in the Rancimat test.

Effect of T. chebula on mitochondrial alterations in experimental myocardial injury.

Mitochondria play a central role in molecular events leading to tissue damage in ischemia. The present study examines the role of the alcoholic extract of T. chebula (TCE) pretreatment (50 mg/100 g body weight) to attenuate the isoproterenol (ISO) (20mg/100g body wt, sc) induced alterations on heart mitochondrial ultrastucture and function in experimental rats. ISO induced cardiotoxicity was evidenced by a significant rise in the level of lactate, decrease in enzyme activities of tricarboxylic acid cycle (TCA), mitochondrial respiration, levels of adenosine triphosphate (ATP) and oxidative phosphorylation. TCE intervention significantly attenuated the above alterations by ISO and retained near normal function of the mitochondria. Electron microscopic studies of the mitochondria further support the isoproterenol induced deleterious changes and accredit the protective effect of TCE on mitochondrial structure and energy metabolism.

Green tea attenuates diabetes induced Maillard-type fluorescence and collagen cross-linking in the heart of streptozotocin diabetic rats.

The enhanced myocardial collagen content, collagen glycation and the resulting advanced glycation end products (AGE) which exhibit the characteristics of increased cross-linking are proposed for the stiffness of myocardium in diabetes. To explore the cardioprotective effect of green tea in diabetes, we study the effect of green tea extract on myocardial collagen characteristics in streptozotocin diabetic rats. The effect of green tea on marker enzymes in serum and cardiac tissues were also assayed to understand the extent of protection. Six weeks after the diabetes induction, diabetic rats were treated with green tea extract [300 mg (kg body weight)(-1)day(-1)] for 4 weeks. AGE were determined by fluorescence assay and cross-linking of collagen by solubility measurement while collagen content was measured by biochemical assay. The activities of aspartate transaminase (AST), lactate dehydrogenase (LDH) and creatine kinase (CPK) were measured by biochemical assay. The increase in blood glucose, glycated hemoglobin and systolic blood pressure in diabetic rats were reduced upon green tea treatment. The activities of AST, LDH and CPK were significantly increased in serum whereas decreased in cardiac tissues in diabetic rats representing the cardiac damage. Administration of green tea to diabetic rats significantly ameliorates these enzyme activities. There was no significant difference in the myocardial collagen content among the experimental rats. A significant (P<0.05) increase in collagen linked Maillard-type fluorescence and decrease in collagen solubility in the myocardium of diabetic rats as compared to control rats (0.955+/-0.02 versus 0.683+/-0.04 and 30+/-1.41 versus 45.17+/-1.17, respectively) indicates the increase in advanced glycation end products formation and degree of collagen cross-linking. Green tea administration to diabetic rats significantly (P<0.05) decreased the fluorescence (0.73+/-0.02) whereas increased the solubility of collagen (41.5+/-1.04) indicating the reduction in advanced glycation end products and collagen cross-linking. The present study reveals that green tea by ameliorating myocardial collagen characteristics may provide a therapeutic option in the treatment of cardiovascular complications of diabetes.

Attenuation of 4-nitroquinoline 1-oxide induced in vitro lipid peroxidation by green tea polyphenols.

Lipid peroxidation is believed to play an important role in pathogenesis of diseases. 4-Nitroquiunoline 1-oxide (4-NQO) a potent oral carcinogen, widely used for induction of oral carcinogenesis, was found to induce lipid peroxidation in vivo and in vitro. Green tea contains high content of polyphenols, which are potent antioxidants. Thus green tea polyphenols (GP) can play a protective role in 4-NQO induced in vitro lipid peroxidation. 4-NQO at the concentration of 1.5 mM was found to induce lipid peroxidation in 5% liver homogenate in phosphate buffered saline and extent of lipid peroxidation at the different time intervals 0, 15, 30 and 45 min where studied by assessing parameters such as hydroxyl radical production (OH), thiobarbituric acid reactants (TBARS), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT). It was found that addition of 4-NQO caused an increase in OH and TBARS level and a decrease in activity of SOD, CAT and the levels of GSH. Simultaneous addition of GP 10 mg/ml significantly decreased lipid peroxidation and increased in antioxidant status. Thus, we conclude that GP, a potent antioxidant, was found to nullify 4-NQO induced lipid peroxidation in vitro and 4-NQO acts initially by causing oxidative stress and leads to carcinogenesis.

Modulatory efficacy of green tea polyphenols on glycoconjugates and immunological markers in 4-Nitroquinoline 1-oxide-induced oral carcinogenesis-A therapeutic approach.

Green tea polyphenols (GTP) has been used as a chemopreventive agent world wide against chemically induced cancer. The present study is aimed to understand the therapeutic action of GTP on glycoconjugates and immunological markers in 4-Nitroquinoline 1-oxide (4-NQO)-induced oral cancer over a period of 30 days at 200mg/kg, p.o., Oral cancer was induced by painting 4-NQO for 8 weeks followed by administration of GTP after 22 weeks, for 30 days. Glycoconjugates such as hexose, hexosamine, sialicacid, fucose and mucoprotein were analysed. Expression of glycoconjugates was examined through histology and SDS-PAGE. Immunological markers such as circulating immune complex and mast cell density were studied. Oral cancer-induced animals showed a significant increase in levels of glycoconjugates and its expression, similar to that observed for immunological markers. Treatment with GTP altered the expression of glycoconjugates as well as immunological markers. The results suggest that GTP modulates both the expression of glycoconjugates and immunological markers resulting in regression of oral cancer.