Screening of Plants Constituents for Effect on Glucose Transport Activity in Ehrlich Masticates Tumour Cells
Sadahiko Ishibashi, Fabian Dayrit, William G. Padolina, and Kazuo Yamasaki. Chikage, Murakami, Keiko Myoga, Ryoji Kasai, Kazuhiro Ohttani, Tomonori Kurokawa
Institute of Pharmaceutical Sciences, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-Ku, Hiroshima, Japan Department of Chemistry. Received May 10, 1993
The effect of plant extracts on D-glucose uptake by Ehrlich ascot tumour cells was examined. Among the 23 extracts of medicinal plants, five samples inhibited, and six samples activate, the uptake significantly. From one of the active plants, Lagerstroemia specious, two tritepenoids, colosolic acid and maslinic acid were isolated. Colosolic acid was shown to be a glucose transport activator. Since the compound was known to have hypoglycemic activity, our simple in vitro bioassay method can at least be used for anti-diabetic activity
Glucose transport is one of the most important functions of all cells to acquire energy. Several types of a glucose transporter are known in cell membranes of mammalian tissues. Glucose transporter is important in regulating the level of intracellular glucose.
Modification of the activity of glucose transport would cause several physiological effects, i.e., lowering blood glucose level, etc. Up to now, only a few compounds have been known to affect glucose transport activity. The compounds, which inhibit glucose transporter activity, are forskolin, diterpene isolated from Labiatac plant, phorezin, and dihydrochalcone of Rosaceae and cylochalasm B, one of the mycoloxins. On the other hand, no other agent able to increase glucose transport activity is known except insulin, a pancreatic hormone, which regulates sugar increasingly.
Systematic research in the pursuit of an agent to modify glucose transport actively has been carried out in search for a new type of agent for the treatment of diabetes, a tonic for the aged, etc. In particular, finding of activator is more important.
In this report we describe the establishment of a screening method for measuring glucose transport activity which can be used for rapidly evaluating many types of sample, ranging from crude extracts to pure compounds.
Ehrlich ascites tumour cells were used to measure glucose transport activity because this cell are known to contain a glucose transporter, and they can be easily propagated and used as an experimental system without the need for a complex procedure to separate cells, which might injure the cellular membrane.
The finding of a glucose transport activator in plant extracts and the isolation of an active principle from one of these active plant extracts are also described.
Result and Discussion
The time course of 2-deoxy-D-glucose (2DG) uptake by Ehrlich cells was measured (Fig.1). The rate of uptake was linear up to 2 min at concentration of 0.2 – 1 mM. Accordingly, experiments with test solutions were carried out using an incubation time of 1min. Under these experimental conditions, the Km and Vmax values were 1.7 mM and 1.4 mnol/min/10^6 cells, respectively, calculated from Lineweaver- Bark plots. The Km value obtained was consistent with reported value with reported value for type 1 glucose transporter.
The effect on glucose transport activity of forskolin, a known glucose transport inhibitor, was measured in this system, it inhibited 2-DG uptake at a concentration of 20mm by 51% (Fig. 2). Forskolin can accordingly be used as control in our system.
Then the effect of 23 methanolic extracts of medicinal plants on glucose transport activity was measured at three different concentrations. The plants were randomly chosen from Southeast Asia herbal medicine (Table 1), and Japanese medicinal plants used mainly for the treatment of diabetes (Table II). Among the 23 samples, 6 samples accelerated 2-DG uptake and 5 reduced it, while the others were ineffective. (Fig. 3)
Although both effects were interesting, we focused on stimulation in this report. Since, as mentioned above, no stimulating agent of glucose transport has been reported except insulin. In addition, among the plants exhibiting a positive effect, Lagerstroemia speciosa and Alomordica charantia were used as antidiabetic agents in the Southeast Asia, and the hypoglycemic effect of Tinospora cordifolia (syn. T. namphii) has recently been reported. Preliminary results of the effect of ginseng extract have also been reported. These screening results prompted us to study the active principle of this glucose transport stimulating plants.
The target plant, Lagerstroemia speciosa L. is distributed all over the Southeast Asia, as well in India, South China and tropical Australia. The leaves of this plant are called “Banaba” in the Philippines, and used as an anti diabetic, the decoction has been clinically tested and found to reduce blood sugar.
The bioactive McO11 extract of banaba was fractionated and subjected to column chromatography. The bioactivity of each fraction was monitored at each stage of the isolation process. From the active McO11 fraction eluded from a Diaion HP-20 chromatography column, compounds 1 and 2 were isolated by silica-gel column chromatography in yields of 0.01 and 0.0016%, respectively. Compounds 1 and 2 were identified by means of NMR as known tritepenes, colosolic acid (2a-hydroxyursoloic acid) and maslinic acid (2a-hydroxyoleanolic acid), respectively.
The bioactivity of 1 and 2 was measured by the above method. Colosolic acid (1) showed a significant glucose transport-stimulating activity at a concentration of 1mm, while 2 were inactive (Table III).
The hypoglycemic effect of 1 has recently been reported in normoglycemic rats following oral administration. This evidence strongly suggest that our in vitro bioassay is closely related to the hypoglycemmic effect and maybe used as a first screening method for anti-diabetic substances without the need for any animals, as in an in vivo assay. Examinations of the correlation of both activities and a further search for active substances in other plants are in progress.
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