Application of modified in vitro screening procedure for identifying herbals possessing sulfonylurea-like activity
Introduction
Diabetes mellitus is a serious chronic metabolic disorder characterized by a decrease or cessation of insulin secretion in response to normal physiological stimuli as well as a reduced responsiveness of peripheral tissues to insulin. If untreated, diabetes leads to severe complications such as atherosclerosis, retinopathy, neuropathy, ulceration and gangrene of the extremities. Diabetes has significant impact on the health, quality of life and life expectancy of patients. In 1999, it was the sixth leading mortality cause in the United States (Anderson, 2001).
More than 1200 species of organisms have been used ethnopharmocologically or experimentally to treat symptoms of diabetes mellitus. They represent more than 725 genera in 183 families ranging from marine algae to higher plants. The phylogenetic distance between families is a strong indication of the varied nature of the active constituents and mechanisms of actions. Therefore, the study of traditional remedies for diabetes might yield an excellent return in potential new sources of anti-diabetic drugs (Marles and Farnsworth, 1995).
Traditionally, various in vivo models (e.g. alloxan or streptozotocin-induced diabetic rats) are used as a main tool in studying hypoglycemic herbs. The in vivo models, however, have many drawbacks. The studies take considerable time to complete and often require use of a relatively large number of animals. In vivo research requires a large amount of experimental compound to dose the animals. This is often a very difficult task in the case of active components of herbals because their isolation and purification is frequently a tedious and low-yielding process. In addition, the precise mechanism of action of the experimental chemical is difficult to determine from an in vivo experiment since the measured end-point is mediated by multiple complementary mechanisms.
The various in vitro models designed to simulate specific events occurring at the molecular, sub-cellular and cellular levels offer an attractive alternative to in vivo studies. They are especially useful during the initial screening steps when large numbers of compounds are to be tested for selected pharmacological activities. In vitro models usually require very small amounts of experimental compounds and utilize efficient liquid handling automation techniques. Therefore, hundreds of compounds can be screened within a single assay run. Those compounds satisfying the criteria of the in vitro screens, can then be tested in relevant, low-throughput in vivo models. This concept has been successfully applied by pharmaceutical companies to the screening of extensive combinatorial libraries. The same approach was recently advocated for application in herbal research (Gebhardt, 2000).
Sulfonylureas and their analogues are clinically proven therapeutic agents in managing Type II diabetes. They exert their pharmacological activity by interacting with ATP-sensitive K+ channels (SUR1 receptors) in pancreatic β-cells. Closing of the channel by sulfonylureas results in a decrease in K+ efflux that depolarizes the cells. This leads to Ca2+ influx via opening of voltage-gated Ca2+ channels. Increase in intracellular Ca2+ stimulates insulin release from β-cell secretory granules (Panten et al., 1996). The development of new, safer and more efficacious agents is still ongoing. In fact, recently one such drug, Nateglinide, has been approved for sale in the US by the FDA.Therefore, we decided to modify an in vitro screening procedure successfully used in development of sulfonylurea-related drugs (Onhota et al., 1994, Fujita et al., 1996) and apply it for identifying herbals possessing sulfonylurea-like activity. The procedure consists of a combination of the SUR1 binding assay and an insulin secretion assay in cultures of HIT-T15 cells. The experimental protocols were optimized to enable screening of the crude herbal extracts. Once the details of the procedure were developed, it was applied to a selected set of hypoglycemic and non-hypoglycemic herbs.
Section snippets
Materials and methods
Powdered American ginseng (Panax quinquefolium L.) roots were purchased from Sigma (St. Louis, MO, USA). Powdered valerian roots (Valeriana sp.) were obtained by emptying “Valerian” capsules (Nature’s Way Products, Inc, Springville, UT, USA). The capsule material offered the characteristic odor of Valerian and demonstrated a positive identification assay according to the method in the German Commission E monograph (Blumenthal, 1998). The fresh leaves of bitter melon (Momordica charantia L.) and
Preparation of herbal extracts
Valerian, American ginseng and fenugreek were extracted with 70% aqueous ethanol. Five grams of the powdered material of each herbal product was agitated with 200 ml of the solvent at 50 °C for 4 h. Following centrifugation, the clear supernatant was evaporated on a rotary evaporator at 40 °C resulting in concentrated aqueous solutions that were lyophilized to yield the dry products.
Fresh bitter melon and cerasse leaves were macerated with a limited volume of water (1 ml/1 gm of fresh leaves),
Cell culture conditions and preparation of cell membranes
HIT-T15 cells (passage 65–80) were cultured in 300 cm2 vented cap tissue culture flasks using Hams F-12K modified nutrient mixture supplemented with 10% dialyzed horse serum and 2.5% bovine foetal serum. The cells were maintained at 37 °C in an atmosphere of humidified air and CO2 (95:5). The cells were harvested by scraping in ice-cold 40 mM Tris, 250 mM sucrose buffer (pH 7.4). The resulting suspension was homogenized in a Polytron and centrifuged at 900×g to remove large debris. The supernatant
Receptor binding assay
In saturation binding experiments, HIT-T15 cell membranes (100 μg/ml) were incubated with various concentrations of -glibenclamide (from 0.025 to 10 nM) in 1 ml per tube of the assay buffer (40 mM Tris, pH 7.4) for 2 h at room temperature. Non-specific binding was determined in the presence of 1 μM unlabeled glibenclamide. Binding was terminated by rapid filtration through Whatman GF/B filters followed by two washes with 5 ml of ice-cold assay buffer. The filters were transferred into scintillation
Insulin secretion in cultures of HIT-T15 cells
HIT-T15 cells were seeded into collagen-coated sterile 96-well plates at a density of 5×104 cells per well. Following a 24-h recovery period, the spent media was removed and the cells were washed with 200 μl of Hanks balanced salt solution (HBSS). After pre-incubation in the same buffer for 30 min at 37 °C, the buffer was removed. Solutions of various concentrations of glibenclamide or crude herbal extracts in HBSS were added to the wells and the cells were further incubated for 1 h at 37 °C. The
Herbal extracts
The yields of dried crude extracts of herbs and herbal products used in this research are listed in Table 1.
Receptor binding
To confirm the presence of high affinity sulfonylurea receptors in HIT-T15 cell membranes, a value for the dissociation constant (Kd) of -glibenclamide was determined. Total and non-specific bindings were examined in the presence and absence of 1 μM unlabeled glibenclamide. The results are depicted in Fig. 1. The Kd for glibenclamide was 0.71 nM and the maximum number of binding sites was
Receptor binding assay
Receptor binding is a well-established technique for studying the interactions of various ligands with receptors (Weiner, 1992). The HIT-T15 cell membranes were successfully used in elucidating the mechanism of therapeutic activity of sulfonylureas (Gaines et al., 1988) as well as in the course of discovery of the new therapeutic agents (Onhota et al., 1994, Fujita et al., 1996). The Kd values for -glibenclamide binding as well as the Ki for glipizide that were obtained using our membrane
Conclusion
The in vitro procedure described in this paper for identifying herbals possessing sulfonylurea-like activity appears to be well suited for screening a large number of crude herbal extracts. A non-hypoglycemic herb and two hypoglycemic herbs with mechanisms of activity different than that of the sulfonylureas were correctly identified as inactive by the combined results of the receptor binding and insulin secretion assays. In addition, the collected data suggests that the hypoglycemic properties
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