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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