The bengal gram husk (BegH) was procured from the local market. Analytical grade solvents hexane and methanol were purchased form Merck, Mumbai, India. The culture medium for cultivation of T. purpureogenus CFRM02 such as potato dextrose agar (PDA) was obtained from Hi-Media Laboratories, Mumbai, India. Dihydroethidium (Hydroethidine) from Thermo Scientific. TRIS (hydroxy methyl) amino methane (Trizma base), were obtained from Sigma Chemical Co., St. Louis, MO, USA.
Sample preparation and animal experiment
The red pigment was extracted form T. purpureogenus CFRM02 fermented BegH . The experimental protocol was approved by Institutional Animal Ethical Committee of CSIR-Central Food Technological Research Institute, Mysuru-570020 (IAEC No CFT/IAEC/83/17) as per the guidelines of Committee for Control and Supervision of Animals for Experiments (CPCSEA), Ministry of Environment, Forests, and Climate Change, Government of India. Thirty male Wistar rats (Rattus norvegicus ≈150 g, 4-5 week old) were housed 3 per cage and maintained at a controlled ambient temperature (23 ± 2 oC). The rats were randomly divided into five groups with 6 rat in each. Group A (Control): Normal Diet without alcohol; Group B (ALD): Normal Diet + ethanol group (4 g/kg/day); Group C: Normal Diet + ethanol (4 g/kg/day) + Metadoxine (160mg/kg/day); Group D (Rx1): Normal Diet + ethanol (4 g/kg/day) + red pigment (300mg/kg/day); Group E (Rx2): Normal Diet + ethanol (4 g/kg/day) + red pigment (600mg/kg/day). The control group was given orally an equivalent volume of distilled water. At the end of experimental period (after 6 weeks), the rats were euthanized and blood was collected for further analysis.
Packed Cells Volume (PCV), Hemoglobin (Hb), Platelet (PLT), Total White Blood Cell Count (TWBC) were determined directly using a fully automated haematology analyser (Pentra-XL 80, Horiba ABX, USA) for blood cell profiling.
Fluorescence detection of free radicals
The frozen liver tissue blocks were fixed and 5-8 µm sections were obtained using CM 3050S cryo-microtome. From each group, several successive sections were taken for microscopic observation. For in situ detection of ROS in the liver, dihydroethidium was used. To compare the oxidative stress in liver tissues, ROS levels were imaged using dihydroethidium (Ex=588 nm and Em=615 nm). Nonfluorescent dihydroethidium is oxidized by ROS to yield red fluorescent product. The images were then captured using a fluorescence microscope (Nikon, Tokyo, Japan), 10x objective, equipped with DFC 300 FX digital camera .
The histopathological changes of the liver were observed by light microscopy. Liver tissues were fixed in 10% formalin, and sections at 5μ were stained with hematoxylin and eosin staining.
All experiments were performed in triplicate, the results expressed as the mean ±SEM and statistical differences between samples was determined by Duncan’s multiple range test. The p values <0.05 were regarded as statistically significant. The statistical analyses were performed using Graph Pad Prism 7 (Graph Pad Software, Inc., La Jolla, CA).