Note: Descriptions are shown in the official language in which they were submitted.
CA 02441656 2004-07-13
Electrolytic Reduced Water, Anti-Cancer Drug, and Producing Method and
Apparatus Thereof.
This application is a division of Canadian Patent Application Serial No.
2,316,656, filed on August 25, 2000.
Field of the Invention
The present invention generally relates to water obtained from
reduction by electrolysis (hereinafter, referred to as "electrolytic reduced
water"), and more particularly, to electrolytic reduced water having an effect
to inhibit metastasis of cancer cells. The present invention also relates to
the
method and apparatus of producing such electrolytic reduced water.
Description of the Background Art
In recent years, worldwide mortality from cancer has been on the rise. A
major factor of death caused by cancer is remote metastasis to other organs,
which in many cases has already occurred when a person is diagnosed with
1~ cancer.
Curing a cancer once it has metastasized is often difficult using current
conventional cancer therapies. Solving this problem will likely be a key to
curing cancer.
Metastasis of a cancer cell involves three steps: adhesion;
decomposition; and invasion of the cancer cell to a basement membrane that is
formed of collagen, laminin, fibronectin or the like. Activation of a group of
metal catalysts, called matrix metalloprotease, by the cancer cell is known to
play an important role in metastasis. At present, chemotherapy for cancer
focuses on the treatment of cancer cells with abnormal pathophysiology. Such
2o therapy often exhibits insufficient effects due to the problems of
selectivity,
side effects and resistance with respect to the cancer. Thus, as a new means
for
cancer treatment, an anti-cancer drug that can suppress metastasis with fewer
side effects has been under development.
It is known that intracellular oxidation in various cancer cell lines
is considerably greater than in normal cell lines. It has also been reported
that the superoxide anion radical (hereinafter, referred to as "SAR")
promotes the metastasis of cancer cells. The applicant has already
proposed that high concentration hydrogen dissolved water obtained by
-1-
CA 02441656 2004-07-13
electrolysis has the potency to prevent or repair damage to DNA caused by
[SAR Japanese Patent Laying-Open No. 10-118653 (U.S. Serial No.
08/917,336)].
To produce such high concentration hydrogen dissolved water (i.e.,
electrolytic reduced water) applicable to cancer treatment, tap water has
been electrolyzed with NaCI dissolved therein as an electrolysis promoting
catalyst. This method has the advantage that it is possible to obtain not only
the
electrolytic reduced water (at the cathode side), but also bactericidal water
having an oxidizing property at the anode side. However, the method also
poses a problem in that, at the time of electrolysis of the NaCI solution,
hypochlorus acid and chlorine gas are produced in large amounts and dissolve
into the electrolytic reduced water. Water containing hypochlorous acid and
chlorine gas is unsuitable for drinking and is considered to be carcinogenic.
Thus, the conventional method is unable to produce electrolytic reduced water
which is highly effective for cancer treatment.
SUMMARY OF THE INVENTION
The present invention is directed to solve the above-mentioned
problems. A main object of the present invention is to provide electrolytic
reduced water completely free of hypochlorous acid and chlorine gas that is
applicable to cancer treatment.
Another object of the present invention is to provide a method of
producing such electrolytic reduced water.
Yet another object of the present invention is to provide an
apparatus for producing such electrolytic reduced water.
In general, electrolytic water is obtained by electrolyzation of water,
both in the cathode and anode chambers. The electrolytic reduced water
described herein, however, does not refer to all such electrolytic water.
Reduction only takes place in the cathode chamber, and therefore, the
reduced water is obtained only in the cathode chamber. Thus, the
electrolytic reduced water disclosed in the present invention can be defined
as water which has been reduced by electrolysis in the cathode chamber and
has an oxidation-reduction potential of a negative value.
The electrolytic reduced water according to a first aspect of the
-2-
CA 02441656 2003-09-24
present invention is obtained by electrolyzing water including NaOH, wherein
the
concentration of the NaOH is within a range from O.OOO1N to 0.02N, as claimed
in
Canadian Patent Application Serial No. 2,316,656.
As such NaOH solution is completely free of chlorine, the electrolysis of the
solution produces neither hypochlorous acid nor chlorine gas.
If bubbles are vigorously produced during electrolysis, atomic hydrogen or
hydrogen radicals themselves are also coupled to form hydrogen gas and escape
from
within the water. Thus, in the electrolysis accompanied by such intense
bubbling, the
amount of hydrogen radicals dissolved in the electrolytic reduced water
(refined liquid
to at the cathode side) is unlikely to increase from a fixed amount.
Therefore, it is
desirable that the least possible amount of bubbles is produced during the
electrolysis
to attain a larger amount of dissolved hydrogen. By selecting the NaOH
concentration
within the range from 0.0001 N to 0.02N, substantially no bubbles are
generated during
the electrolysis, and thus, it is possible to obtain stable electrolytic
reduced water.
t5 In addition, by selecting the NaOH concentration within this range, it is
possible to cause electrolytic reaction approximately at the same level as in
the case of
tap water.
In the electrolytic reduced water of the present invention, the concentration
of
NaOH is preferably within the range from O.OOO1N to 0.002N.
2o By selecting the NaOH concentration within this range, electrolytic reduced
water with an increased amount of hydrogen dissolved therein can be obtained.
According to a second aspect of the present invention, there is provided
electrolytic reduced water including a hydrogen radical that is obtained by
electrolyzing
water containing NaOH, wherein the concentration of said NaOH is within a
range from
25 O.OOO1N to 0.02N, as claimed in Canadian Patent Application Serial No.
2,316,656.
The electrolytic reduced water of the present invention is obtained by
electrolyzing water containing NaOH, which has an oxidation-reduction
potential of at
most -50 mV, a dissolved oxygen amount of at most 9.5 ppm and a dissolved
hydrogen
amount of at least 300 ppb.
_3_
CA 02441656 2003-09-24
According to a third aspect of the present invention, there is provided a
method
of producing electrolytic reduced water, which comprises introducing a water
solution
including NaOH to both a cathode chamber and an anode chamber separated by a
diaphragm, wherein the concentration of NaOH is within a range from O.OOO1N to
0.02N; applying electricity between a cathode electrode immersed in the
cathode
chamber and an anode electrode immersed in the anode chamber to perform
electrolysis
of the water solution including NaOH; and drawing out electrolytic reduced
water
obtained at the cathode chamber, as claimed in Canadian Patent Application
Serial No.
2,316,656.
l0 According to the method, the NaOH solution is used as electrolyte, which is
free of chlorine. Therefore, it is possible to obtain electrolytic reduced
water
completely free of hypochlorous acid and chlorine gas.
In the method of producing electrolytic reduced water of the present
invention,
the electrolysis is effected with the cathode and anode chambers both being
sealed.
Accordingly, it is possible to suppress generation of hydrogen gas while
conducting the electrolysis, thereby increasing the amount of dissolved
hydrogen.
In the method of producing electrolytic reduced water of the present
invention,
the electrolysis is conducted with voltage, current and time that are selected
such that
no hydrogen gas is generated from the cathode chamber.
2o Accordingly, cathode water having a large amount of dissolved hydrogen can
be obtained in the cathode chamber.
According to a fourth aspect of the present invention, there is provided an
apparatus for producing electrolytic reduced water using the method of the
present
invention, comprising: filter means for filtrating raw water to produce clear
water;
NaOH add means for adding a NaOH solution to the clear water filtered by the
filter
means; and an electrolysis tank having cathode and anode chambers separated by
a
diaphragm, to which the clear water having the NaOH solution added therein is
introduced, as claimed in Canadian Patent Application Serial No. 2,316,656.
-4-
CA 02441656 2004-07-13
As the apparatus is provided with the NaOH add means, it is possible to
electrolyze the water solution including NaOH, producing neither hypochlorous
acid
nor chlorine gas.
The apparatus for producing electrolytic reduced water of the present
invention, may further comprise; a first conduit provided between the NaOH add
means
and the electrolysis tank to introduce the clear water with the NaOH solution
to the
electrolysis tank; a second conduit connected to the electrolysis tank, which
draws
cathode water discharged from the cathode chamber outwards; a third conduit
connected to the electrolysis tank to draw anode water discharged from the
anode
l0 chamber outwards; first, second and third valves provided in the first,
second and third
conduits, respectively, to open/close the relevant conduits; and means for
controlling
the opening/closing of the first, second and third valves.
According to the apparatus, it is possible to perform electrolysis with the
cathode and anode chambers being sealed by closing the first, second and third
valves.
This enables production of the cathode water containing a large amount of
dissolved
hydrogen.
The electrolytic reduced water of the present invention is obtained in the
cathode chamber.
According to a fifth aspect of the present invention, there is provided
electrolytic reduced water including a hydrogen radical.
According to a sixth aspect of the present invention, there is provided an
anti-
cancer drug, comprising a water solution including a hydrogen radical.
The foregoing and other objects, features, aspects and advantages of the
present invention will become more apparent from the following detailed
description
of the present invention when taken in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a diagram showing a NaOH solution electrolyzing system
according to the present invention.
Figure 2 is a diagram showing an electrolysis tank according to the
-5-
CA 02441656 2004-07-13
present invention.
Figure 3 illustrates a cancer cell metastasis inhibiting effect exhibited by
NaOH electrolytic reduced water.
Figure 4 illustrates the result of a cytotoxicity test of the NaOH
electrolytic reduced water.
Figure 5 illustrates the result of analysis of gelatinase/IV type
collagenase activity by zymography.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Hereinafter, embodiments of the present invention will be described.
Figure 1 schematically shows an apparatus for producing electrolytic
reduced water free of hypochlorous acid and chlorine gas that is effective for
cancer treatment. Specifically, Figure 1 illustrates a NaOH solution
electrolyzing system.
Referring to Figure 1, raw water (tap water) is pressurized by a pump
and filtrated by a reverse osmosis membrane to obtain purified water. A
NaOH solution is added to the purified water via a metering pump. The
metering pump is controlled to achieve a prescribed concentration by
measuring electrical conductivity of the solution. The NaOH solution is
supplied to an electrolysis tank through a flow rate sensor and an
electromagnetic valve 1. Once the electrolysis tank is filled with the NaOH
solution, the flow rate becomes 0, and a stop signal is supplied from the flow
rate sensor to a control circuit. In response to the stop signal supplied, the
pump and the metering pump stop, and electromagnetic valve 1 is closed.
A timer is activated, and a direct current for use in electrolysis is supplied
to
the electrolysis tank for a prescribed time period. When the time is up,
electromagnetic valves 2 and 3 are opened to draw out reduced water and
acidic water produced respectively. After the produced water is drawn out,
each electromagnetic valve attains its initial state, and the NaOH solution
is supplied for next electrolysis.
Figure 2 schematically shows the electrolysis tank. The electrolysis tank
includes a cathode chamber 2 containing a cathode electrode 1, and an anode
chamber 4 containing an anode electrode 3. Chambers 2 and 4 are
separated by a diaphragm 5. A cathode water outlet pipe 6 is connected to
-6-
CA 02441656 2003-09-24
cathode chamber 2 to draw out the cathode water (the electrolytic reduced
water). A drain pipe 7 is connected to anode chamber 4 to discharge the
anode water (the acidic water) outwards. Feed pipes 8 are connected to
cathode chamber 2 and anode chamber 4, respectively, to supply them with
purified water having NaOH of a prescribed amount added therein.
Example 1
Using purified water filtered through a reverse osmosis membrane
or the like, 0.01% NaOH solution was prepared and subjected to electrolysis.
Electrolytic reduced water was obtained completely free of hypochlorous
acid and chlorine gas, since the NaOH solution was used.
'Vhen electrolyzing water, oxygen gas is generated at the anode side
and hydrogen gas at the cathode side. The hydrogen gas is generated
because a hydrogen ion produced by the electrolysis and an electron supplied
from the cathode electrode are coupled to form atomic hydrogen, and two
hydrogen atoms are then coupled to form by drogen gas. As w ill be
described later, the electrolytic reduced water thus obtained has potency to
prevent or suppress growth and metastasis of cancer cells, which is
considered attributable to strong antioxidation property of the atomic
hydrogen. Therefore, it is desirable that a large amount of hydrogen in its
atomic state is dissolved in water. In the cathode water obtained from high
current electrolytic reaction utilizing high voltage, hydrogen tends to be
gasified for the most part, thereby reducing the dissolved amount of atomic
hydrogen. To avoid such phenomena, it is preferable to perform the
electrolysis over a long period of time under the condition that can prevent
generation of hydrogen gas. In other words, the electrolysis is preferably
conducted under low voltage and low current over a long period of time. It
was found that the electrolytic reduced water could be obtained without
generating hydrogen gas if the NaOH solution was electrolyzed under the
conditions of a voltage from 5V to 100V, a current from 5mA to 2 A, and a
time period from 5 to 120 minutes. The obtained electrolytic reduced water
exhibited pH of 11.5 and ORP (oxidation-reduction potential) of -850 mv.
Here, the oxidation-reduction potential was measured at room
temperature employing an "oxidation-reduction potential meter" available
_7_
CA 02441656 2004-07-13
from Toa Electronics (Toa Denpa Kogyo), by immersing its electrodes for
measurement into the test water.
The characteristics of the obtained electrolytic reduced water are
shown in Tables 1 and 2.
The results shown in Tables x and 2 are similar to each other, but
they were measured on different days taking different water samples.
Comparison of the tables show that it is possible to obtain data
exhibiting good reproducibility.
Table 3 shows conditions of electrolysis, i.e., current density values,
corresponding to electrolyzed degrees 1-5. The current density of tap water
is expressed as 0.0 mA/cm2, because it has undergone no electrolysis. The
current density, which is controlled by a microcomputer, is one of the most
important conditions of electrolysis. Once the current density is
determined, the voltage and the NaOH concentration are consequently
determined.
In Tables 1 and 2, the amounts of dissolved oxygen were measured
using a dissolved oxygen meter of DO-14P type available from Toa
Electronics (Toa Denpa Kogyo). The amounts of dissolved hydrogen were
measured using a dissolved hydrogen meter of DHD1-1 type also available
from Toa Electronics.
Table 1
oxidation-dissolveddissolved
water presence/
pH reductionoxygen hydrogen
temperature absence
of
(aC) potentialamount amount H radical
(m~ (PPm) (PPb)
tap 13.1 7.5 +652 10.0 2.3 - x
water 2.6
ED: 12.7 9.8 - 94 9.4 400 - 0
1 450
ED: 13.2 10.3 -247 8.6 690 - 0
2 ?20
reduced
water ED: 13.2 10.4 -494 8.2 880 - 0
3 900
ED: 13.7 10.7 -729 ?.2 1030 - 0
4 1060
ED: 14.0 11.5 -850 6.8 1090 - 0
5 1130
ED: electrolyzed degree
_g_
CA 02441656 2004-07-13
Table 2
oxidation-dissolveddissolved
water presence/
pH reductionoxygen h dro
en
y g
temperature absence
of
(aC) potentialamount amount H radical
(m~ (PPm) (PPb)
tap 12.5 6.8 +681 10.2 2.4 - x
water 2.6
ED: 12.6 9.6 - ?0 9.5 360 - 0
1 410
reducedED: 12.8 9.9 -211 8.7 660 - 0
2 700
water ED: 13.1 10.2-452 8.4 840 - 0
3 890
ED: 13.4 10.4-676 7.4 980 - 0
4 1040
ED: electrolyzed degree
Table 3
tap water 0.0 mA/cm2
electrolyzed degree: 1 3.2 mA/cm2
electrolyzed degree: 2 6.4 mA/cm2
electrolyzed degree: 3 12.9 mA/cm2
electrolyzed degree: 4 16.2 mA/cmz
electrolyzed degree: 5 25.8 mA/cmz
Tables 1 and 2 also include the results showing presence/absence
of hydrogen radicals. x represents that hydrogen radicals were not
included; O represents that hydrogen radicals were included. The
presence/absence of hydrogen radicals (atomic hydrogen) was confirmed
utilizing a characteristic of tungsten oxide (in the form of plate). Tungsten
oxide has potency to adsorb hydrogen radicals in a specific manner, and it
turns
blue when adsorbing the hydrogen radicals. The obtained electrolytic reduced
water was contacted with tungsten oxide to qualitatively determine the
presence/absence of hydrogen radicals.
Example 2
The evaluation results of cancer cell metastasis inhibiting effects of
the obtained electrolytic reduced water (with electrolyzed degree of 5 in
Table 1) will be described.
Figure 3 shows the inhibiting effects of the electrolytic reduced water
-9-
1 450
ED: 13.2 10.3 -24
CA 02441656 2004-07-13
against highly metastatic human fiber sarcoma cell lines HT1080 in an in vitro
metastasis model system. Here, HT1080 cells available from a cell bank [e.g.,
JCRB Cell Bank or ATCC (in U.S.A.)] were employed.
The HT1080 cells were cultured in 10% fetal bovine serum
supplemented MEM (Minimum Essential Medium; medium including
the least possible amount of nutritious ingredients) medium at a temperature
of
37°C under 5% COZ/95% air environment. A chemotaxel filter (pore size:
8,um) was coated with matrigel at 25 ~cg/filter. Sub-confluent HT1080 cells
were suspended in the MEM medium containing 0.1 % bovine serum albumin
(BSA) and the number of cells was adjusted to 4x105/ml. 200 ,ul of the
resultant cell suspension was added to a chamber in its upper room.
Immediately after addition of the cells, 700 ~cl of the MEM containing 10
,uglml
of fibronectin was added to the chamber in its lower room (having a
24 holes plate) (a 24 holes plate side), and cultured in a C02 incubator.
After six hours, the chamber was taken out. Cells were removed from
the upper surface of the filter with a cotton bud, and moved to the 24 holes
plate containing WST-1 (an indicator that changes its color depending on
metabolic ability specific to living cells, or the number of living cells).
After
culture for 16 hours, absorbance at 450 nm was measured. Referring to Figure
3, "ctrl" represents the result when purified water was used, and "NaOH mix"
represents the result when the electrolytic reduced water obtained with
electrolyzed degree of 5 in Table 1 was used. As seen from Figure 3, invasive
metastasis of HT1080 cells is dramatically reduced in the case of NaOH mix
compared to ctrl. This means that the electrolytic reduced water has
suppressed
2~ the invasive metastasis of the human fiber sarcoma cells.
Figure 4 illustrates the result of a one-week cytotoxicity test.
HT1080 cells were cultured in 10% fetal bovine serum supplemented
MEM medium that had been prepared using purified water or the
electrolytic reduced water (with electrolyzed degree of 5). After culture
for one week, WST-1 was added, and the number of living cells was
measured with absorbance at 450 nm. No significant difference was found
between the result obtained using the electrolytic reduced
-10-
CA 02441656 2004-07-13
water (NaOH mix). This means that the electrolytic reduced water has no
adverse effects on growth of healthy cells. Thus, from the results shown in
Figures 3 and 4, it has become clear that the NaOH electrolytic reduced water
is
capable of suppressing the invasive metastasis activity without introducing
cytotoxicity.
Referring to Figure 5, matrix metalloprotease (MMP), which plays an
important role in the metastasis of cancer cells, was analyzed focusing on
MMP2 and MMP9 among others, which are known as being particularly
deeply involved in the cancer metastasis.
Figure 5 illustrates the analysis result of gelatinase/IV type
collagenase activity in zymography. Specifically, HT1080 cells were
cultured on a chemotaxel chamber for 48 hours, and then the supernatant of
the cultured cells was purified using a centrifugal device before recovery.
12.1 of the supernatant was added to 10% polyacrylamide gel containing 1
mg/ml of gelatin. After gel electrophoresis, the gel was washed with 2%
TritonT"s X-100 for one hour and kept at 37 ° C for 60 hours.
Thereafter, the
gel was dyed with 0.1% Ponceau S to detect gelatinise activity, which was
represented as a white band on a colored background. In Figure S, the wider
the white band, the greater the activity of MMP promoting the metastasis of
cancel cells.
The results of the analysis showed that the NaOH electrolytic
reduced water had no effect on manifestation of MMP2 and MMP9, but it
significantly suppressed activation of MMP2.
From the results described above, it has become clear that the
NaOH electrolytic reduced water has cancer cell metastasis inhibiting
effects by suppressing the activation of MMP2.
Inhibiting the metastasis mechanism of cancer cells is important not
only for preventing the metastasis itself, but also for suppressing
vascularization due to the invasive activity of the cancer cells as well as
for
preventing the cancer cells from becoming malignant. Further, a drug for
inhibiting the cancer metastasis must maintain its effects for a long period
of time and have the least number of side effects. In the present invention,
it
has been proved that the NaOH electrolytic reduced water can suppress the
-11-
CA 02441656 2004-07-13
metastasis of cancer cells without damaging the cells. This suggests that
utilizing such water daily as drinking water may prevent progress of the
cancer,
and thus, it is considered to have a great significance for cancer treatment
in the
future.
As explained above, the electrolytic reduced water obtained by the
present invention is a kind of water ehibiting an antioxidation property and
contains no oxides such as hypochlorous acid and chlorine gas, which is not
only applicable to medical treatment but also suitable for drinking and other
uses.
Although the present invention has been described and illustrated in
detail, it is clearly understood that this is by way of illustration and and
example
only and is not to be taken by way of limitation, the spirit and scope of the
present invention being limited only by the terms of the appended claims.
-12-
