Inflammation and oxidative stress in corneal tissue in experimental keratitis due to Fusarium solani: Amelioration following topical therapy with voriconazole and epigallocatechin gallate.

Combined antifungal and antioxidant therapy may help to reduce oxidative stress in fungal keratitis. Experimental Fusarium solani keratitis was induced by application of F. solani conidia to scarified cornea (right eye) of 16 rabbits (another four rabbits were negative controls [Group I]). Five days later, F. solani-infected animals began receiving hourly topical saline alone (Group II), voriconazole (10 mg/mL) alone (Group III), epigallocatechin gallate (EGCG, 10 mg/mL) alone (Group IV) or voriconazole and EGCG (Group V). Twenty days post-inoculation, corneal lesions were graded. After animal sacrifice, excised corneas underwent histopathological and microbiological investigations. Corneal tissue levels/activities of interleukin 1 beta (IL-1ß) and tumour necrosis factor alpha (TNF-α) gene mRNA transcripts, matrix metalloproteinase (MMP) 2 and 9 proteins, malondialdehyde (MDA) and reduced glutathione (GSH), and superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), were also measured. Clinical and histopathological scores (severity of corneal lesions; [P < .05]) and mean levels (P < .05) of IL-1ß and TNF-α mRNA transcripts, MMP 2, MMP 9 and MDA were Group II > Groups IV and III > Groups V and I. Mean SOD, CAT, GPx and GSH levels (P < .05) were Group II < Groups IV and III < Groups V and I. Topical voriconazole with EGCG apparently reduces inflammation in experimental F. solani keratitis, as manifested by improved clinical, histological, microbiological and molecular parameters.

Identification of phytoconstituents and in-vitro evaluation of the putative anticataractogenic effect of an ethanolic root extract of Leucas aspera.

Modern herbal medicine has played a significant role in treating oxidative stress and related complications. In the present investigation, gas chromatography-mass spectrometric analysis of ethanolic extracts of the leaf and of the root of Leucas aspera (L. aspera) (Willd.) Link separately showed the presence of various phytoconstituents; major components have already been reported to possess various biological, including antioxidant, activities. Of the two extracts analyzed, the root extract exhibited more potential antioxidant activity than did the leaf extract. Since this finding correlated with more perceptible amounts of antioxidant components being detected in the ethanolic extract of L. aspera root, the root extract was evaluated for possible anticataractogenic potential in cultured Wistar rat lenses. Following incubation of Wistar rat lenses for 24h at 37°C in Dulbecco's modified Eagle's medium (DMEM), gross morphological examination revealed that none of the eight lenses incubated in DMEM alone (Group I) exhibited any opacification (Grade 0), whereas all eight lenses incubated in DMEM that contained sodium selenite (100µM selenite/ml of DMEM) (Group II) exhibited thick opacification (Grade +++). In contrast, only one out of eight lenses incubated in DMEM containing sodium selenite (100µM selenite/ml of DMEM) and simultaneously exposed to the L. aspera root extract (300µg/ml of DMEM) (Group III) exhibited a slight degree of opacification (Grade +) after 24h incubation, while the remaining seven lenses did not show any opacification (Grade 0). The mean activities of catalase, superoxide dismutase, glutathione peroxidase and glutathione-S-transferase and the mean level of reduced glutathione were all significantly (p<0.05) higher in Group III lenses than the mean values in Group II lenses. The mean concentration of malondialdehyde in Group III lenses was significantly (p<0.05) lower than that in Group II lenses. Further, significantly (p<0.05) lower mean mRNA transcript levels of the genes encoding αA- and ßB1-crystallins, as well as significantly lower mean levels of the αA- and ßB1-crystallin proteins themselves, were observed in Group II lenses. However, in Group III lenses, the mean mRNA transcript levels of the crystallin genes, and the mean protein levels, were essentially similar to those noted in normal control (Group I) lenses. The results of the present study suggest that in selenite-challenged Wistar rat lenses simultaneously exposed to an ethanolic extract of L. aspera root, lenticular opacification was prevented by mean activities of enzymatic antioxidants, mean levels of reduced glutathione and malondialdehyde mean expression levels of genes encoding αA- and ßB1-crystallins, and mean levels of the crystallin proteins themselves, being maintained at near normal levels. Further studies are required to confirm whether the ethanolic extract of the root of L. aspera can be developed for pharmacological management of cataract.

Regulatory effect of chrysin on expression of lenticular calcium transporters, calpains, and apoptotic-cascade components in selenite-induced cataract.

PURPOSE: Selenite-induced cataract is associated with oxidative stress, loss of calcium homeostasis, activation of calpain enzymes, and apoptotic cell death in the lens. An evaluation of naturally occurring antioxidants that also restrict calcium influx into the lens and calpain activation and thus prevent lenticular cell death may lead to the development of safe and effective anticataractogenic drugs. This study focuses on a naturally occurring flavone, chrysin, and its efficacy in preventing cataractogenic changes in in vitro cultured Wistar rat lenses. METHODS: Lenses from Wistar rats incubated for 24 h at 37 °C in Dulbecco's modified Eagle's medium (DMEM) were categorized into four main groups: Group I (control, incubated in DMEM alone); Group II (selenite-challenged and untreated, incubated in DMEM that contained 100 µM/ml of sodium selenite only); Group III (selenite-challenged and chrysin-treated, incubated in DMEM that contained sodium selenite [100 µM/ml of DMEM] and chrysin [200 µM/ml of DMEM]); and Group IV (chrysin-treated, incubated in DMEM that contained chrysin [200 µM/ml of DMEM] only). The Group III (selenite-challenged and chrysin-treated) lenses were further categorized into five sub-groups: Group IIIa (incubated for 24 h in DMEM that contained sodium selenite and chrysin added simultaneously), Group IIIb (first incubated for 2 h in DMEM that contained chrysin only and then for up to 24 h in fresh DMEM that contained sodium selenite only), Group IIIc (first incubated for 30 min in DMEM that contained sodium selenite only and subsequently for up to 24 h in DMEM that contained chrysin only), and Groups IIId and IIIe (first incubated for 1 h and 2 h, respectively, in DMEM that contained sodium selenite only and subsequently for up to 24 h in DMEM that contained chrysin only). RESULTS: Gross morphological assessment revealed dense opacification (Grade +++) in the selenite-challenged, untreated lenses (Group II); however, seven of the eight selenite-challenged and simultaneously chrysin-treated (Group IIIa) lenses showed no opacification (Grade 0) after 24 h incubation, while the remaining single lens exhibited only a slight degree of opacification (Grade +). In the Group IIIa lenses, the reduced glutathione, protein sulfhydryl, and malondialdehyde concentrations appeared to have been maintained at near-normal levels. The mean lenticular concentration of calcium was significantly lower in the Group IIIa lenses than that in the Group II lenses and approximated the values observed in the normal control (Group I) lenses. The Group IIIa lenses also exhibited significantly (p<0.05) higher mean lenticular activity of calpain, significantly higher mean mRNA transcript levels of genes that encode m-calpain and lenticular preferred calpain (Lp82), and significantly higher mean levels of the m-calpain and Lp82 proteins than the corresponding values in the Group II lenses. Casein zymography results suggested that chrysin prevented calpain activation and autolysis. Significantly (p<0.05) lower mean levels of mRNA transcripts of the genes that encode calcium transporter proteins (plasma membrane Ca(2+)-ATPase-1 and sarco/endoplasmic reticulum Ca(2+)-ATPase-2) and lenticular apoptotic-cascade proteins (early growth response protein-1, caspase-3, caspase-8, and caspase-9) and significantly (p<0.05) lower mean concentrations of the proteins themselves were seen in the Group IIIa rat lenses in comparison to the values noted in the Group II rat lenses. CONCLUSIONS: Chrysin appears to prevent selenite-induced cataractogenesis in vitro by maintaining the redox system components at near-normal levels and by preventing the abnormal expression of several lenticular calcium transporters and apoptotic-cascade proteins, thus preventing accumulation of calcium and subsequent calpain activation and lenticular cell death in cultured Wistar rat lenses.

Antihypercholesterolemic and Antioxidative Potential of an Extract of the Plant, Piper betle, and Its Active Constituent, Eugenol, in Triton WR-1339-Induced Hypercholesterolemia in Experimental Rats.

Hypercholesterolemia is a dominant risk factor for atherosclerosis and cardiovascular diseases. In the present study, the putative antihypercholesterolemic and antioxidative properties of an ethanolic extract of Piper betle and of its active constituent, eugenol, were evaluated in experimental hypercholesterolemia induced by a single intraperitoneal injection of Triton WR-1339 (300 mg/kg b.wt) in Wistar rats. Saline-treated hypercholesterolemic rats revealed significantly higher mean blood/serum levels of glucose, total cholesterol, triglycerides, low density and very low density lipoprotein cholesterol, and of serum hepatic marker enzymes; in addition, significantly lower mean serum levels of high density lipoprotein cholesterol and significantly lower mean activities of enzymatic antioxidants and nonenzymatic antioxidants were noted in hepatic tissue samples from saline-treated hypercholesterolemic rats, compared to controls. However, in hypercholesterolemic rats receiving the Piper betle extract (500 mg/kg b.wt) or eugenol (5 mg/kg b.wt) for seven days orally, all these parameters were significantly better than those in saline-treated hypercholesterolemic rats. The hypercholesterolemia-ameliorating effect was better defined in eugenol-treated than in Piper betle extract-treated rats, being as effective as that of the standard lipid-lowering drug, lovastatin (10 mg/kg b.wt). These results suggest that eugenol, an active constituent of the Piper betle extract, possesses antihypercholesterolemic and other activities in experimental hypercholesterolemic Wistar rats.