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.
