A Kinetic Study for In-vitro Intestinal Uptake of Monosaccharide across Rat Everted Gut Sacs In the Presence of Some Antidiabetic Medicinal Plants
M Patel, S Mishra
diabetes mellitus, enicostemma hyssopifolium, eugenia jumbolana, gymnema sylvestre, tinospora cordifolia, trigonella foenum
M Patel, S Mishra. A Kinetic Study for In-vitro Intestinal Uptake of Monosaccharide across Rat Everted Gut Sacs In the Presence of Some Antidiabetic Medicinal Plants. The Internet Journal of Alternative Medicine. 2008 Volume 7 Number 1.
Diabetes mellitus (DM) is a debilitating and often life threatening disease with increasing incidence throughout the world. It was postulated that DM is the most common chronic disorder affected more than 176 million people worldwide, and this global figure has been set to double by the year 2030, Tiwari \& Madhusudaan . The treatment of DM relied heavily on dietary measures, which included the use of traditional plant therapies. Several reviews on plants with known antidiabetic activity or with traditional use as antidiabetic remedies have been published, Ajganonkar , Oliver-Bever , Bailey \& Day . Traditional antidiabetic plants might provide a useful source for developing new oral hypoglycemic compounds as pharmaceutical entities or simple dietary adjuncts to the exiting therapies. Studying such traditional medicines might offer an alternative and natural key to unlock diabetologists’ pharmacy. On the other hand, suggested mechanisms describing therapeutic effects of several traditional medicinal plant systems are holistic, Handa et al . Most of the reported hypoglycemic plants are anecdotal, and only few have received adequate scientific evaluation. The fundamental mechanisms of these medicinal systems are still unexplainable using modern tools, Rahman \& Zaman . It is claimed that most medicinal preparations in traditional medicines contain a variety of synergistically acting phytochemicals that are thought to act on a variety of targets by various modes and mechanisms, Tiwari \& Madhusudaan . In this study, we investigated the effect of
Materials and methods
Preparation of the aqueous extracts
Dried aerial parts of EH, seeds of EJ, stem of TC, leaves of GS and seeds of TF were purchased form local market of Ahmedabad, Gujarat. Air dried plant materials were groung to powder, 100g of each was extracted with 500 mL water by maceration. The water was evaporated under vacuum at 50°C. Extracts were diluted in distilled water to use in experiments.
Adult male Swiss albino rats weighing 150-180 g and housed at temperature 25 ± 2 °C were used in this study. Animals were maintained on commercial feed and tap water
Preparation of everted gut sacs
Intestinal segments (5 ± 1 cm) were everted according to the method described by Wilson & Wiseman. After being everted, segments of guts were blotted with a piece of whatman filter paper no. 40 and weighted. A 1 g glass weight was fixed and tied to the end of the everted gut segment to make an empty gut sac. This was important to prevent peristaltic muscular contractions, which may otherwise alter the shape and internal volume of the sac. The 1 g glass weight was the minimum weight to secure the above-mentioned conditions. After weighing, the empty sac was filled with 0.5 mL of Krebs-Henseleit bicarbonate buffer (KHB). The composition of the buffer was: NaHCO3 25 mM/L; NaCl 118 mM/L; KCl 4.7 mM/L; MgSO4 1.2 mM/L; NaH2PO4 1.2 mM/L; CaCl2 1.2 mM/L; and Na4EDTA 9.7 mg/L. Glucose (2g/L) was added to the medium just before the start of the appropriate experiment. The pH was maintained at 7.4. The sac was filled with a blunted-ended syringe and then the needle was slipped off carefully, and the proximal end of the sac was tightly tied with the thread. The compartment containing the buffer in the sac was named serosal fluid compartment. The distended sac was placed inside a 40 mL KHB bath (mucosal fluid compartment) and mounted. This gut sac bath was placed in a carbon dioxide incubator adjusted at 5 % CO2 and 37°C. For studying the effect of the plant extracts on the uptake of glucose (substrates), glucose at varying concentrations was added into mucosal compartment fluid. The plant extracts were also added in the same compartment after digestion in 2 mL of simulated gastric fluid (5 mg/mL). At the end of the incubation period (60 min), the sacs were removed from the gut sac bath, blotted by a standardized procedure as described above and weighted. The serosal fluid was drained through a small incision into a test tube. The emptied sac was shaken gently to remove the adhered fluid and the tissue was weighted. The final serosal volume was determined by subtracting (after incubation) the weight of the empty sac from that of the filled sac. The gut fluid uptake was determined by measuring an increase in the volume of fluid in the gut wall. Glucose concentrations in both the compartment were measured using a commercially available glucose oxidase kit (Span Diagnostics Ltd. Sachin, India). The amount D (+)-glucose transported from the mucosal compartment was characterized as ‘uptake’ while the serosal gain of the substances is treated as ‘release’. Uptake and release of glucose was expressed as mM/g tissue wet weight/h.
In each series of experiments, control everted gut sacs derived from the same rat in a buffer containing no substrate were run in parallel. The controls were run either with or without acid digested plant extracts and results were corrected accordingly.
All experiments were carried out in triplicate. Comparison of D (+)-glucose uptake difference between the controls and experimental groups were examined using paired t - test for mean ± SEM. In terms of enzyme kinetics, glucose transported per hour was analogue to the velocity of transfer, in other words, to the concentration difference of glucose between compartments at the beginning and end of an experiment. The Michaelis – Menten constant (Km), which is the affinity of the transferring enzyme for the substrate, and maximal velocity (Vmax), which is the rate of transfer reaction, in the presence as well as in the absence of studied plant extracts were determined from the differences of uptake and release values using the Michaelis-Menten and Lineweaver-Burk Plots in Microsoft Excel. Any difference with p values less than 0.05 were considered as statistically significant. Mean ± SEM of Km and Vmax values were presented Table 1.
Biochemical parameters of D (+)-glucose transport across rat everted small intestines
The everted gut sacs were incubated in Krebs- Henseleit buffer (pH= 7.4) at 37°C.
The most challenging goal in the management of patients with DM is to achieve blood glucose level as close to normal as possible. Unfortunately, postprandial hyperglycaemia (PPHG) or hyperinsulinaemia are independent risk factors for the development of vascular complications in DM patients Tiwari \& Madhusudaan . Mechanisms playing role in release and transport of glucose across the intestinal brush border membrane down to the blood stream have attracted much attention recently as potential targets to control PPHG. In this category, majority of recent studies reported the potential use of antidiabetic medicinal plants on inhibition of glucose transport. Drugs that reduce PPHG by suppressing the absorption of carbohydrate are effective in prevention and treatment of non-insulin dependent DM. Alpha-glucosidase inhibitors are used to establish greater glycemic control over hyperglycemia in diabetes mellitus type 2, particularly with regard to PPHG. They may be used as monotherapy (acarbose, miglitol, voglibose) in conjunction with an appropriate diabetic diet and exercise, or they may be used in conjunction with other anti-diabetic drugs. They may also be useful in patients with diabetes mellitus type 1; however, this use has not been officially approved by the Food and Drug Administration.
Our findings would tend to indicate that glucose transport was significantly decreased in the presence of the aqueous extract of EJ seeds, TC stem, GS leaf, and TF seeds which caused a decrease in the Vmax. Only the aqueous extract of aerial parts of EH did not decreased the glucose absorption
Although our findings are promising, uncertainties remain. Some investigators suggested that flavonoids decreased glucose uptake by a sodium-dependent pathway
In conclusions, our study provides evidence for a biochemical mechanism which carries blood glucose lowering effect of EJ seeds, TC stem, GS leaf and TF seeds in intestine via non-competitive inhibition. However, further kinetic data on carrier-mediated transport of D (+)-glucose is needed.
One of the authors, Mr. Mayurkumar Patel, would like to thank All India Council for Technical Education (AICTE), New Delhi, for providing the financial assistance, in the form of National Doctoral Fellowship (NDF) to carry out this work.