Michael A. Barletta

α-Glucosidase inhibitory activity of Syzygium cumini (Linn.) Skeels seed kernel in vitro and in Goto–Kakizaki (GK) rats

Syzygium cumini seed kernel extracts were evaluated for the inhibition of alpha-glucosidase from mammalian (rat intestine), bacterial (Bacillus stearothermophilus), and yeast (Saccharomyces cerevisiae, bakers yeast). In vitro studies using the mammalian alpha-glucosidase from rat intestine showed the extracts to be more effective in inhibiting maltase when compared to the acarbose control. Since acarbose is inactive against both the bacterial and the yeast enzymes, the extracts were compared to 1-deoxynojirimycin. We found all extracts to be more potent against alpha-glucosidase derived from B. stearothermophilus than that against the enzymes from either bakers yeast or rat intestine. In an in vivo study using Goto-Kakizaki (GK) rats, the acetone extract was found to be a potent inhibitor of alpha-glucosidase hydrolysis of maltose when compared to untreated control animals. Therefore, these results point to the inhibition of alpha-glucosidase as a possible mechanism by which this herb acts as an anti-diabetic agent.

Growth Pattern of Sirc Rabbit Corneal Cells in Microwell Inserts

AbstractThe SIRC rabbit corneal cell line (ATCC CCL 60) has been used in a number of in vitro studies. However, the growth pattern of SIRC rabbit corneal cells on microwell inserts has not been established. Microwell inserts were seeded with SIRC rabbit corneal cells, suspended in growth medium. Cultures were maintained in a humidified incubator (5% CO237°C) for 21 days, with the growth medium replaced in the wells and inserts every seventh day of culture. Light microscopy was performed on hematoxylin and eosin-stained cross sections. The SIRC rabbit corneal cells formed multiple epithelioid cell layers. Maximum cell layer growth occurred at day 10 (4.54 cell layers ± 0.32). Although decreased slightly, the number of cell layers remained stable with an average of more than 4 cell layers observed for days 12-14. On the 21st day of culture, the average number of cell layers decreased to 3.87 (± 0.09) and exfoliation of the surface cells was evident. Microscopy of the cross sections indicates that new cell l...

An in vitro method for assessing corneal opacification potential using a rabbit corneal cell line

AbstractWe report an in vitro method for identifying materials with the potential to cause corneal opacity. Monolayers obtained from an established line of rabbit corneal cell epithelium were exposed to test materials and the increase in light absorbance at 360 nm was assessed after 10 min. Responses obtained proved to be concentration-dependent and the curves generated were of sufficiently different character to allow differentiation among the test materials. Because trichloroacetic acid (2%) caused a linear response over the entire range of initial monolayer densities used and provided the widest degree of response of any of the materials tested, it was chosen as a reference compound. The effects of all other test materials were expressed in relation to that observed for 2% trichloroacetic acid to provide a basis for comparative effects and to control for the effects of intertrial variation in monolayer density. The increased absorbance caused by 20% acetaldehyde and 2% silver nitrate was 30% and 60%, r...

The use of cell lysis as an index of ocular irritation potential

AbstractWe report an in vitro method for assessing ocular irritation by measuring cell lysis of mouse connective tissue (strain L, Clone 929) and rabbit corneal cell lines (SIRC). Lysis of corneal epithelial cells in vivo leads to swelling and subsequent opacification of the underlying corneal stroma. In vitro lysis was determined by the measurement of changes in absorbance (360 nm) of cell suspensions over a 10-min exposure to test materials. These data were expressed as numbers of cells/ml using linear regression. The regression line obtained was linear and similar for both cell types. Loss of viability and spontaneous cell lysis were minimal over the assay interval. In a separate study, rabbit corneal cells were used to assess alteration of cell membrane integrity following 30-min exposures to test materials by measuring trypan blue dye exclusion in these cells. Activities of test materials were ranked according to the lowest concentration capable of producing statistically significant cell lysis as fo...

Release of Histamine from Rat Peritoneal Cells in Vitro as an Index of Irritation Potential

AbstractUsing mixed rat peritoneal cells, including mast cells, a technique was developed to assess the ability of test materials to cause histamine release. This release was determined fluorimetrically, and noted to be concentration-dependent. This allowed the test materials to be ranked in terms of their potential. An order of potency was observed as follows: triethanolamine lauryl sulfate > triethanolamine > propylene glycol, based upon determinations of the lowest concentration causing significant histamine release. Microscopic evidence supported the observation that histamine release occurred to a varying degree depending upon the test material and its concentration. These observations are in agreement with in vivo studies which have rated the primary irritation potential of these materials. Moreover, these findings agree with data assessing the effects of these materials upon membrane integrity of cultured rabbit corneal cells. This similarity of findings suggests that these irritants may act as mem...

Bioassay-guided Isolation of the Antidiabetic Active Principle from Salviamiltiorrhiza and its Stimulatory Effects on Glucose Uptake Using 3T3-L1Adipocytes

Natural products, which reduce hyperglycemia by enhancing the glucose uptake in peripheral tissues, have been considered to be effective for treatment of Type-2 Diabetes Mellitus. Salvia miltiorrhiza (Labiatae), danshen, has been widely used traditional Chinese medicine for the treatment of various cardiovascular and cerebrovascular diseases. In the present study, different extracts of Salvia miltiorrhiza root were investigated for their ability to enhance glucose uptake in differentiated 3T3-L1 adipocytes. An in vitro bioassay guided fractionation approach was adapted to isolate the active principle of Salvia miltiorrhiza using extensive column chromatographic techniques. The structure of active compound was elucidated using various spectroscopic methods (ESI-MS, MALDI-ToF, 1H-NMR, 13C-NMR, COSY, TOCSY, HETCOR) and determined to be magnesium salt of salvianolic acid B (SAB). SAB showed concentration dependent increase in glucose uptake in 3T3-L1 adipocytes. The efficacy of the active principle was also evaluated for its antidiabetic activity in streptozotocin-induced diabetic rats. SAB (25 mg/kg) significantly improved the glucose tolerance in diabetic rats (*p<0.05, ** p<0.01). The SAB treatment group showed significantly lower (*p<0.05) blood glucose levels over 120 min as compared to diabetic control group. Thus, these results suggested that SAB has the potential to be developed as a potential glucose-lowering agent by increasing glucose uptake in peripheral tissues in the treatment of diabetes mellitus.

Effect of Wrightia tinctoria and Parthenocissus quinquefolia on blood glucose and insulin levels in the Zucker diabetic rat model.

The aim of this study is to evaluate the antidiabetic activity of two Indian Ayurvedic herbs using an oral glucose tolerance test and blood insulin levels to understand the mechanism of action using the Zucker diabetic rat model. Herbal extracts of Wrightia tinctoria and Parthenocissus quinquefolia at a dose of (250 mg/kg body weight) were used throughout the study. Following a glucose challenge of 2 gm/kg using oral gavage, a timed glucose tolerance test was used to determine the ability of these extracts to alter glucose levels in diabetic animal model. The glucose lowering activities of these extracts were then compared to the controls. Both tested herbal extracts have shown to exhibit significant (P < 0.05) hypoglycemic activity compared to the control. W. tinctoria and P. quinquefolia have an antidiabetic activity which reduced the blood glucose level in oral glucose tolerance test significantly compared with the control. To further understand their mechanism of action, blood insulin levels were also studied using an insulin Elisa assay. These studies revealed that the herbal extract of P. quinquefolia has direct correlation between glucose and insulin levels. However, W. tinctoria significantly lowered blood glucose levels (P< 0.05), while it did not show any correlation between blood glucose and insulin levels. Based on these findings, it can be concluded that hypoglycemic effects of W. tinctoria are more complicated than P. quinquefolia, and may involve other possible mechanism of action.

Maternal-fetal electrocardiographic effects and pharmacokinetics after an acute IV administration of caffeine to the pregnant rat

The relationship between fetal exposure and cardiovascular functional effects in the caffeine-treated pregnant rat was investigated. Caffeine (100 mg/kg) was administered intravenously to dams on day 21 of gestation. The transplacental transport of caffeine was studied by obtaining maternal and fetal blood (umbilical vein) samples at designated times after drug administration. Concurrent maternal-fetal electrocardiograms (ECGs) were measured and evaluated for caffeine-induced changes. Maternal and fetal plasma caffeine levels as well as area under the curve values were proportionally related throughout the experiment, indicative of equal exposure to caffeine. The fetal ECG exhibited more extensive changes associated with caffeine than did the dams, but the effects were not detected in the first 30 min, suggesting a lag period for the action of caffeine on the fetal heart. The frequency of fetal ectopic beats and abnormal T waves were directly related to fetal plasma caffeine levels. Fetal ECG combined with the fetal blood microsampling technique was a practical method of testing for prefunctional effects of caffeine in the rat fetus.

A method to obtain maternal-fetal plasma samples using a microsampling technique in the rat: Transplacental passage of cefoxitin☆

A microsampling technique that allows taking blood samples from the umbilical vein of the pregnant rat is described. Such techniques are needed in order to allow pharmacokinetic and embryo exposure to be correlated with teratogenic endpoints. Cefoxitin was administered intravenously (300 mg/kg) into tracheotomized, pentobarbital anesthetized dams on day 21 in gestation. Blood samples were collected via the carotid artery from the dam and the umbilical vein of the fetus at designated times. Up to three samples of 20 to 30 microliters each, were taken from individual fetuses at 20-min intervals. With few exceptions, fetal cefoxitin concentrations were homogeneous at each sampling period. Fetal concentrations were low compared to maternal concentrations as seen by the small fetal/maternal area under the curve ratio (0.053 +/- 0.006).

Structure elucidation and inhibitory effects on human platelet aggregation of chlorogenic acid from Wrightia tinctoria.

Abstract Background: Interest in natural compounds as sources of potentially new treatment options is growing rapidly. Preliminary screening of many different plant extracts showed that Wrightia tinctoria acts as a potent human platelet aggregation inhibitor. The aim of the present study was to isolate and characterize the active compound responsible for potent inhibition of human platelet aggregation in vitro. Methods: A 70% ethanolic extract derived from W. tinctoria seeds was fractionated with chloroform followed by ethyl acetate. The ethyl acetate fraction was further fractionated and purified through a series of three successive column chromatographic separations using silica gel, Sephadex LH‐20, and C‐18 columns. Liquid chromatography coupled to negative electrospray ionization tandem mass spectrometry (LC‐MS/MS) and nuclear magnetic resonance (NMR) studies were performed in the structure determination of the active phenolic compound present in the ethyl acetate fraction of W. tinctoria seeds. Results: A phenolic compound has been isolated and identified as chlorogenic acid by LC‐MS/MS and NMR studies. Chlorogenic acid showed concentration-dependent inhibitory effect on collagen-induced platelet aggregation in vitro with an IC50 of 0.2363 μg/μl. Conclusion: The present data suggest that chlorogenic acid can be developed as potential antiplatelet agent in the treatment of cardiovascular diseases in diabetes mellitus.