Anti-Diabetic Activity of Melothria Heterophylla (Lour.) Cogn. in Streptozotocin Induced Diabetic Rats

ABSTRACT Melothria heterophylla (Lour.) Cogn. (Cucurbitaceae) widely distributed in India and used ethnically by tribal people in India for controlling blood sugar. This promotes us to undertake a study to examine the possible antidiabetic activity of the plant extracts in normal and streptozotocin induced diabetic rats. A single dose study was studied in the normal rats for 12hrs. Oral glucose tolerance test was performed in normal rats after receiving glucose orally (2gm/kg). Diabetes was induced with streptozotocin (50mg/kg, i.p.) and a dose of 300mg/kg of Petroleum ether (PEMH), Methanol (MEMH) and Aqueous (AEMH) extracts were then administered orally to experimental diabetic rats for 21 days. Glibenclamide was used as standard reference. Fasting blood glucose levels, changes in body weight and liver weight, serum albumin, serum urea, total protein, total lipid profile, haemoglobin, SOD, GSH and TBARS were evaluated. Single dose study of extracts on normal rats showed a significant decrease in the fasting blood glucose levels when compared with the normal control rats. Oral glucose tolerance test clearly indicate that MEMH and PEMH extracts shown a significant reduce in the blood glucose levels, AEMH extract showed little effect. In diabetic rats, treatment with the PEMH, MEMH and AEMH showing significant reduction in the fasting blood glucose levels, serum cholesterol, serum triglycerides, LDL-C and VLDL-C levels. A significant escalation is seen in the levels of HDL-C, haemoglobin, body weight and liver weight. Whereas the antioxidant levels of SOD, GSH and TBARS improved than the untreated diabetic rats. The study reveals that the plant extracts of Melothria heterophylla showed significant antidiabetic activity in normal fasted rats, OGTT in normal rats and in STZ induced diabetic rats

A retrospective study on the prevalence of urinary tract infections in a tertiary care hospital in Sangareddy district of South India

Background: Urinary tract infections (UTI) affect the portion of the urinary tract including kidneys, ureters, bladder, and urethra, and are among the commonly acquired nosocomial infections. Diagnosis based on the culture and sensitivity profile of the organism is highly beneficial for appropriate antimicrobial therapy of the individual.Methods: A retrospective study of culture isolates obtained from urine samples from different departments of a tertiary care hospital was performed in the period January 2018 to March 2020 in the district of Sangareddy, Telangana, India.Results: A total 204 samples of 1886 exhibited significant growth of organisms i.e., ≥105 colony-forming units of bacteria per millilitre (CFU mL-1). The most common pathogen isolated was Escherichia coli (47.05%), followed by Klebsiella pneumoniae (15.68%), Staphylococcus aureus (10.78%), Pseudomonas aeruginosa (10.78%), Enterobacter spp. (7.84%) and, Candida albicans (7.84%). The presence of Gram-negative organisms was found to be more than Gram-positive organisms among the samples cultured. Resistance was found to be more towards Amoxicillin (57.14%), followed by oxacillin (34%), cefotaxime (23.62%), clarithromycin (12.08%), erythromycin (12.08%), azithromycin (9.34%), linezolid (3.84%) and vancomycin (2.19%).Conclusions: Increasing levels of antimicrobial resistance by uropathogens emphasize the importance of therapy based on the culture and sensitivity of the organisms. Many uropathogens exhibit multi-drugs resistance. Regular surveillance and monitoring are useful in controlling the increasing resistance

Purification and Characterization of a Mitogenic Lectin from Cephalosporium, a Pathogenic Fungus Causing Mycotic Keratitis

Ophthalmic mycoses caused by infectious fungi are being recognized as a serious concern since they lead to total blindness. Cephalosporium is one amongst several opportunistic fungal species implicated in ophthalmic infections leading to mycotic keratitis. A mitogenic lectin has been purified from the mycelia of fungus Cephalosporium, isolated from the corneal smears of a keratitis patient. Cephalosporium lectin (CSL) is a tetramer with subunit mass of 14 kDa, agglutinates human A, B, and O erythrocytes, and exhibits high affinity for mucin compared to fetuin and asialofetuin but does not bind to simple sugars indicating its complex sugar specificity. CSL showed strong binding to normal human peripheral blood mononuclear cells (PBMCs) to elicit mitogenic activity. The sugar specificity of the lectin and its interaction with PBMCs to exhibit mitogenic effect indicate its possible role in adhesion and infection process of Cephalosporium

Rhizoctonia bataticola lectin (RBL) induces caspase-8-mediated apoptosis in human T-cell leukemia cell lines but not in normal CD3 and CD34 positive cells.

We have previously demonstrated immunostimulatory activity of a fungal lectin, Rhizoctonia bataticola lectin (RBL), towards normal human peripheral blood mononuclear cells. The present study aimed to explore the anticancer activities of RBL using human leukemic T-cell lines, Molt-4, Jurkat and HuT-78. RBL exhibited significant binding (>90%) to the cell membrane that was effectively inhibited by complex glycoproteins such as mucin (97% inhibition) and asialofetuin (94% inhibition) but not simple sugars such as N-acetyl-D-galactosamine, glucose and sucrose. RBL induced a dose and time dependent inhibition of proliferation and induced cytotoxicity in the cell lines. The percentage of apoptotic cells, as determined by hypodiploidy, was 33% and 42% in Molt-4 and Jurkat cells, respectively, compared to 3.11% and 2.92% in controls. This effect was associated with a concomitant decrease in the G0/G1 population. Though initiator caspase-8 and -9 were activated upon exposure to RBL, inhibition of caspase-8 but not caspase-9 rescued cells from RBL-induced apoptosis. Mechanistic studies revealed that RBL induced cleavage of Bid, loss of mitochondrial membrane potential and activation of caspase-3. The expression of the anti-apoptotic proteins Bcl-2 and Bcl-X was down regulated without altering the expression of pro-apoptotic proteins--Bad and Bax. In contrast to leukemic cells, RBL did not induce apoptosis in normal PBMC, isolated CD3+ve cells and undifferentiated CD34+ve hematopoietic stem and progenitor cells (HSPCs). The findings highlight the differential effects of RBL on transformed and normal hematopoietic cells and suggest that RBL may be explored for therapeutic applications in leukemia

Effect of RBL on different phases of cell cycle.

<p>(A) Molt -4 and Jurkat cells, treated with RBL, were stained with PI and acquired on FL2-A channel of flow cytometer equipped with 488nm laser. The X-axis represents the DNA content of the cells and the Y-axis represents the cell number. The graph depicts the percentage of Molt-4 (B) and Jurkat (C) cells in different phases of cell cycle. The data is mean ±SE of three independent experiments. *p<0.05 significant difference between untreated and RBL treated cells.</p

RBL induces apoptosis of Molt-4 and Jurkat cells.

<p>(A) Molt-4 cells were treated with RBL (5 µg/ml) for 6, 12 and 24 h followed by Annexin- V-FITC and PI staining. The X-axis depicts Annexin-V positive cells and Y- axis depicts PI positive cells. The numbers in each quadrant represent percent positive cells. Dot plots are representative of three similar experiments. (B) The graph represents % apoptosis (Annexin-V-positive cells) mean±SE of three independent experiments. *p<0.05 indicates significant difference between treated and untreated cells. (C) Whole cell lysates of Molt-4 and Jurkat cells were exposed to RBL (1.25 to 5 µg/ml) for 24 h and probed with anti-PARP antibody. The blot shows total and cleaved PARP. The data is representative of two similar experiments.</p

Effect of caspase-8 inhibition on RBL-induced apoptosis.

<p>Molt-4 (A) and Jurkat cells (B) were treated with RBL(5 µg/ml) for 12 h in the presence or absence of caspase inhibitors(40 µM) zVAD-FMK (pan caspase inhibitor), zIETD-FMK (caspase-8 inhibitor), or zLEHD-FMK (caspase-9 inhibitor) and viability was assessed by MTT assay. The graphs depict mean±SE values from three independent experiments. *p value <0.05 in comparison with untreated controls. Molt-4 (C) and Jurkat (D) cells were treated with RBL (5 µg/ml) for 12 h in the presence or absence of caspase-8 inhibitor (zIETD-FMK) and caspase-3 activation was assessed using flow cytometry. The overlay depicts profile of untreated cells (black line), cells pretreated with caspase-3 inhibitor followed by RBL exposure (red line) and cells treated with RBL alone (blue line).</p

Binding of RBL and inhibition with sugars.

<p>The binding of FITC- labeled RBL to Molt-4 (A) and Jurkat (B) cells was determined by flow cytometry analysis. The histoplots depict the profiles of unstained cells (shadow) and cells stained with FITC-RBL (green). The binding of RBL to cell membrane of Molt-4 (A inset) and Jurkat (B inset) was visualized by confocal laser scanning microscopy. Original magnification 60×. (C) Molt-4 cells were stained with FITC-labeled RBL alone or RBL pre-incubated with different sugars and flow cytometry analysis was performed. X-axis represents fluorescence intensity, Y-axis represents cell number. The overlay shows profiles of the unstained cells (shadow), RBL stained cells (green) and cells stained with RBL +Mucin (red), +asialofetuin (orange), +GalNAc (blue), +glucose (pink), and sucrose +(yellow). (D) The graph represents mean MFI±SE from three independent experiments. *p<0.05 difference in MFI compared to cells stained with RBL alone.</p

Effect of RBL on proliferation and viability of leukemic cell lines-.

<p>Molt-4 (A) and Jurkat (B) cells were exposed to serial concentrations of RBL for 72 h and proliferation was determined by tritiated thymidine incorporation assay. The percentage cell proliferation was calculated by considering the counts per minute of untreated control cells as 100. Molt-4(C) and Jurkat (D) cell lines were exposed to serial concentrations of RBL for different time periods and cell viability was assessed by MTT assay. The absorbance value of untreated cells was considered as 100 to calculate percent viable cell number. The values presented in the graph are mean±SE of three independent experiments done in triplicates. *p<0.05 difference compared to untreated cells.</p