Scientific and Educational Reports of the Facultyof Science and Technology, Kochi University
Vol. 2 (2019), No. 10

Life-prolonging effect of deep sea drinking water in the hypotrichousciliated protozoan Euplotes woodruffi
Mikihiko Arikawa* and Tatsuomi Matsuoka
Department of Biological Sciences, Faculty of Science and Technology, Kochi University, Akebono, Kochi, Japan.

Keywords:
deep sea watercell lifespanlife-prolongation
magnesium
Euplotes

Abstract
Deep sea drinking water (DSDW), which is produced by processes ofdesalination and mineralization of deep sea water, contains various
minerals and is drunk to replenish the balance of minerals, particularly Mg.
Although the effect of DSDW on living organisms has been well studied,few studies investigating the effect of DSDW on cell lifespan have so far
been reported. Therefore, to evaluate the life-prolonging properties of
DSDW at a cellular level, we cultured ciliated protozoan Euplotes
woodruffi in natural mineral water (NMW) and DSDW, and compared thenumber of cells surviving during the experimental period. Results showedthat cells cultured in DSDW lived longer than those cultured in
NMW—possibly by consuming a substance richly available in DSDW. To
isolate this key element in DSDW, we investigated the effect of various
ions on the cell lifespan and found that only cells in the Mg-containing
culture water survived as long as cells in DSDW. Judging from the results
obtained in the study, we concluded that DSDW can prolong the cell
lifespan and that Mg plays a key role in the life-prolonging effect of
DSDW.

Introduction

Due to its balance of essential minerals, DSW isthought to have potential as a good source ofhigh-mineral content products ranging fromhealth and beauty—such as cosmetics andbathing
preparations—to
foods
and
beverages—such as alcohol and drinking water.
Deep sea drinking water (DSDW) has been
produced exclusively by processes ofdesalination and mineralization of DSW tomake it suitable for drinking. DSDW contains
various minerals and is drunk for the
replenishment of the balance of minerals,particularly Mg. Therefore, the effect of DSDW
on living organisms has been well studied inhuman and experimental animals, and recent
reports provide accumulating evidence of thetherapeutic or preventive effects of DSDW. For

In recent years, deep sea water (DSW) has
attracted considerable attention along with therise of health-consciousness worldwide. DSW isgenerally defined as sea water that is pumped
up from a depth of more than 200 meters and ischaracterized by a low and uniform temperature,high purity, and a rich mixture of inorganic
nutrients [1]. In Japan, the aquamarine research
project focusing on the effective utilization of
DSW was started in 1985 by the Science andTechnology Agency (presently the Ministry of
Education, Culture, Sports, Science andTechnology) at Muroto City, Kochi Prefecture.
*Corresponding author.
E-mail address: marikawa@kochi-u.ac.jp
1
Received: August 21, 2019
Reviewed by anonymous referee(s), and accepted: September 28, 2019
Published: November 29, 2019

example, DSDW has the ability to 1) improve
cardiovascular hemodynamics in Kurosawaand Kusanagi-Hypercholesterolemic rabbits [2],2) improve skin symptoms and mineral
imbalances in human atopic eczema/dermatitis
syndrome [3,4], 3) prevent hyperlipidemia andatherosclerosis in cholesterol-fed rabbits [5,6],
and 4) reduce allergic skin responses and serum
levels of cytokines in patients with allergic
rhinitis [7]. Moreover, Liao et al. reported that
DSDW enhances the longevity of diabetic rats
by enhancing cardiac function and that theunderlying molecular mechanism includes the
suppression of pro-apoptotic proteins and theenhancement of survival proteins [8]. However,few studies investigating the life-prolonging
effect of DSDW at the cellular level have so far
been reported—possibly due to the fact that an
appropriate experimental model for research on
the cell lifespan has not yet been established.
In the present study, we investigated theeffect of DSDW on the cell lifespan using afree-living
unicellular
eukaryotic
microorganism, Euplotes woodruffi, classified
into the Phylum Ciliophora and the Kingdom
Protista. Usually, Euplotes lives in freshwater
environments, such as ponds and swamps, but it
can be cultured in a laboratory with naturalmineral water (NMW), which is normallyavailable on the commercial market [9,10]. The
advantages in using the ciliate E. woodruffi inthe study are that 1) they are large enough to be
counted individually under a conventional
stereoscopic microscope, 2) dead cells are easily
distinguished from live cells because healthy
cells move continuously, while dead cells
eventually disappear, and 3) they do not rely on
feeding on bacteria in order to grow. This third
criterion is thought to be the key advantage inthe study. The majority of ciliates areheterotrophs and feed on smaller organisms
such as bacteria and algae. However, somespeciesof
Euplotes—includingE.
woodruffi—are unable to survive by feeding
only on bacteria. Therefore, in our laboratory,
we feed them with separately-cultured algal
cells (Chlorogonium elongatum or C.
capillatum) twice a week [9,11]. In other words,

if E. woodruffi are left in culture water without
the addition of food sources, they stop growing
and dividing, and finally die and disappear
within a few weeks. This property of E.
woodruffi is convenient for our experiments
because it is not necessary to consider the effectof feeding on bacteria on the life-prolonging
properties of DSDW during the experimental
periods.
We cultured Euplotes cells with both NMW
and DSDW, and compared the lifespan of thesecells. Our results indicated that DSDW
possesses life-prolonging properties and thatMg plays a key role in the beneficial effect ofDSDW.

Materials and Methods

The ciliated protozoan Euplotes woodruffi
was obtained from the Iwakuni City Micro-life
Museum (Yamaguchi, Japan), and was cultured
in NMW (Delicious water Rokko, Asahi Soft
Drinks Co., Ltd., Tokyo, Japan) at 25°C with
Chlorogonium capillatum added as a foodsource. C. capillatum was cultured axenically ina medium (1 mg/ml sodium acetate and 5
mg/ml yeast extract) at room temperature undercontinuous lighting conditions. In the present
study, to investigate the life-prolonging
properties of DSDW, we used desalinatedMuroto Deep-Seawater (Amami’s Water, AKO
KASEI Co., Ltd., Kochi, Japan). The water
hardness of NMW and DSDW was calculated to
be 48.4 and 1001, respectively, by the following
equation: hardness (mg/l) = Ca (mg/l) × 2.497 +Mg (mg/l) × 4.118. Therefore, NMW isconsidered to be soft water and DSDW is
considered to be hard water. The elements andhardness of these waters are listed in Table 1.
For cell survival experiments, the culture of E.
woodruffi in NMW was first starved for morethan 3 days after its last feeding to obtain cells
in a uniform condition. Cells were thencollected by centrifugation, resuspended in
fresh culture water (NMW, DSDW, or
conditioned water), seeded into wells of aplastic
plate,
andculturedinatemperature-controlled incubator at 25°C. The
2

was shown. For nonparametric comparison
between two groups, as shown in Fig. 1, a
Mann–Whitney U test was performed. P values< 0.05 were considered statistically significant.

Table 1. Levels of elements and hardness ofnatural mineral water (NMW) and deep sea
drinking water (DSDW).
Elements

Content in water (mg/l)
NMW

DSDW

Na
Ca
Mg
K

29
9.5
6
1

74
71
200
69

Hardness

48.4

1001

Results and Discussion

Experiment 1
The objective of the present study was toevaluate the life-prolonging effects of DSDW.
As shown in Fig. 1, the number of cells cultured
in NMW (open circles) drastically decreased at
4 days, and all cells had died at 6 days after the
onset of the experiment. On the contrary, thenumber of cells cultured in DSDW (filled
circles) remained unchanged up to 8 days, andonly after 11 days all cells had died. Cells
cultured in DSDW survived significantly longer
than those cultured in NMW, indicating that
DSDW possesses life-prolonging properties.

Water hardness was calculated by the following
equation: hardness (mg/l) = Ca (mg/l) × 2.497 +Mg (mg/l) × 4.118.

number of surviving cells was counted daily
under a stereoscopic microscope andcontinuously recorded until all cells had died.
No food was supplied during the experimental
period. The detailed procedures of eachexperiment are as follows.
Experiment 1: To see the difference in
culturing cells in NMW and DSDW, cells were
cultured in both water types, and counted thenumber of surviving cells daily.
Experiment 2: To investigate in detail the
life-prolonging effect of DSDW on the cell
lifespan, cells were cultured in DSDW diluted
with deionized water at different dilution rates
or in NMW and DSDW with different cellconcentrations.
Experiment 3: To characterize thelife-prolonging effect of DSDW, the culture
water was exchanged at the middle of theculture period.
Experiment 4: To investigate the effect ofions contained in DSDW on the cell lifespan,
the conditioned water was prepared by theaddition of Na, Ca, Mg, and K—each alone andin various combinations—to control NMW. The
concentration of each ion in the conditioned
water was identical to that in DSDW or adjusted
to between 1 mM and 5 mM.
The experiment was performed in duplicate
or triplicate and only the representative result

Experiment 2
Next, we cultured cells under various
conditions. As shown in Fig. 2a, cells cultured
in NMW survived for 9 days (open bar). On theFig. 1. Survival curves of Euplotes woodruffi
cultured in natural mineral water (NMW, open
circles) and deep sea drinking water (DSDW, filled
circles). Cells cultured in DSDW survived
significantly longer than those cultured in NMW.
Bars represent standard errors. Asterisks indicate
significant difference at p < 0.05 between NMW and
DSDW (Mann–Whitney U test).
3

a

b

Fig. 2. Survival days of cells cultured in NMW and DSDW under various culture conditions. (a) Cells were
cultured in NMW (open bar) and DSDW diluted at different dilution rate (filled bars). The life-prolonging effectof DSDW was decreased with an increase in the dilution rate. (b) Cells were cultured in NMW (open bars) and
DSDW (filled bars) with different cell concentrations. The life-prolonging effect of DSDW was observed under
all culture conditions, independently of cell concentrations. Moreover, the survival days of cells increased with adecrease in the cell concentration regardless of the water types.

contrary, cells cultured in non-diluted DSDW
survived for 15 days (filled bar labeled as 1/1),
indicating the life-prolonging effect of DSDW.
When cells were cultured in DSDW diluted
with deionized water at different dilution rates,
survival time was decreased with an increase in
the dilution rate (filled bars labeled as 1/2, 1/4,
1/8, and 1/16). Next, we cultured cells in NMW
and DSDW with different cell concentrations.
As shown in Fig. 2b, cells cultured in NMW
survived for 7 days (extreme left open bar),whereas cells cultured in DSDW survived for
14 days (extreme left filled bar), indicating
again the life-prolonging effect of DSDW. The
effect was observed under all culture conditions
independentlyofcell
concentrations.
Furthermore, the lower the cell concentration in
culture was, the longer cells survived,
regardless of the water types. These results
suggest the possibility that cells consume asubstance contained in the culture water during
the survival period and DSDW contains thesubstance to a greater degree than does NMW,
so that cells cultured in DSDW lived longer
than those cultured in NMW.

Experiment 3
Then, we conducted an experiment inwhich the culture water was exchanged 4 days
after the onset of the experiment. As shown in
Fig. 3, cells cultured in NMW (open bar labeled
as NMW–NMW) and in DSDW (filled barlabeled as DSDW–DSDW) survived for 8 days
and 17 days, respectively. In both cases, theculture water was exchanged with a new sampleof the same water. The life-prolonging effect of
DSDW was confirmed in the experiment. Cells
cultured in NMW followed by DSDW survived
for 15 days (gradient bar labeled as NMW–
DSDW), indicating that cells which would
otherwise have died at 8 days survived for
additional 7 days. It was therefore clearly
demonstrated that DSDW prolonged the cell
lifespan. In contrast, cells cultured in DSDW,
which survive for 17 days, survived for only 12days when cultured in DSDW followed by
NMW (gradient bar labeled as DSDW–NMW);
thus, their lifespan was shortened by 5 days.
The cell lifespan was demonstrably shortenedby the exchange of culture water from DSDW
to NMW. The period when cells were cultured
in DSDW was 0 days in NMW–NMW, 4 days4

indicate the dependence of the life-prolonging
effect of DSDW on the period over which cells
are cultured in DSDW.
In the present study, the survival days ofcells were different among the experiments. For
example, cells cultured in NMW survived for 6
days (Fig. 1) and for 9 days (Fig. 2a). Similarly,
cells cultured in DSDW survived for 11 days
(Fig. 1) and for 15 days (Fig. 2a). This is due tothe difference between the periods in whichcells were cultured in the control NMW before
the onset of the experiments. Whether theperiod in which cells were cultured in NMW
before the onset of the experiments affects thelife-prolonging effect of DSDW is unclear.
However, cells cultured in DSDW neverthelesssurvived longer than those cultured in NMW,
indicating the life-prolonging effects of DSDW.
Moreover, the experiment in which culture
water was exchanged in the middle of theculture period revealed that the longer cells
were cultured in DSDW, the longer theysurvived (Fig. 3). This indicates that thelife-prolonging effect of DSDW depends on
how long cells are cultured in this medium.

Fig. 3. The effect of exchange of the culture water onsurvival days of cells. At the middle of the culture
period, the culture water was exchanged from NMW
to NMW (NMW–NMW), from NMW to DSDW(NMW–DSDW), from DSDW to NMW (DSDW–NMW), and from DSDW to DSDW (DSDW–
DSDW). The survival days increased with anincrease of the period in which cells were cultured in
DSDW.

in DSDW–NMW, 11 days in NMW–DSDW,
and 17 days in DSDW–DSDW, and the cell
lifespan was 8 days in NMW–NMW, 12 days in
DSDW–NMW, 15 days in NMW–DSDW, and17 days in DSDW–DSDW. These resultsabFig. 4. The effect of elements in the culture water on the cell survival. (a) Cells were cultured in the conditioned
water (gray bars) containing various ions. Compared with the control NMW (open bar), only cells cultured in the
Mg-containing conditioned water survived as long as did cells cultured in DSDW (filled bar). (b) Cells were
cultured in the conditioned water (gray bars) containing high concentrations of Na, Ca, Mg, or K ions. Althoughhigh concentrations of Ca are lethal, cells cultured in the conditioned water survived longer than those cultured in
the control NMW (open bar) in a concentration-dependent manner. Among the ions tested, Mg showed thegreatest life-prolonging effect.
5Experiment 4
To identify the key element that provides
cells with an additional lifespan, cells were
cultured in the conditioned water which was
prepared based on the composition and theconcentration of DSDW listed in Table 1. In theexperiment, as shown in Fig. 4a, cells cultured
in NMW (open bar) and DSDW (filled bar)survived for 10 days and 13 days, respectively.
Among cells cultured in the conditioned water(gray bars), on the other hand, only cells
cultured in the Mg-containing conditioned water
survived as long as cells cultured in DSDW.
Next, we investigated the effect of highconcentrations of ions on the cell lifespan (Fig.
4b). Cells cultured in NMW survived for 9 days
(open bar). Although high concentrations of Ca
are lethal, cells cultured in the conditioned
water containing high concentrations of Na, Mg,
or K nevertheless survived for longer periods
compared with cells in the control NMW.
Moreover, the high concentrations of ionsprolongedthecelllifespaninaconcentration-dependent manner. Among theions tested, Mg showed the greatest effect on
the cell lifespan. Cells cultured in theconditioned water containing the highest
concentration of Mg (5 mM) survived over twotimes longer than the control cells after the
onset of the experiment. These results indicatethat Mg plays the most important role in thelife-prolonging effect of DSDW. Moreover,these results indicate two possibilities: either
that not only Mg but also high Na and high K
have life-prolonging properties, or that cells
survive long in high ionic strength conditions.
Judging from the fact, as shown in Fig. 4a, that
only cells cultured in the conditioned watercontaining Mg survived longer than thosecultured in NMW, only Mg, and not Na or K,might play a key role in the life-prolonging
effect of DSDW. A high ionic strength condition
might also have a life-prolonging effect, but it
cannot be compared with the effect of Mg alone.
Since the concentration of Mg in DSDW iscalculated to be approximately 2 mM, higher
concentrations of Mg are expected to have astrongerlife-prolonging
effect.
Furtherexperiments to investigate whether the cell
lifespan is affected by the ionic strength of theculture medium are needed.
As mentioned above, among ions containedin DSDW, Mg is the most important element in
the life-prolonging effect of DSDW. However,
the mechanism by which Mg affects cells is still
unknown. Mg has so far been reported tosupport a number of significant functions in thehuman body. For example, Mg inhibits platelet
aggregation and suppresses thrombus formation[12,13]. Mg lowers blood pressure by
improving blood fluidity and decreasing
peripheral vessel resistance [14]. Mg is also
found to have a significant influence on bonemetabolism [15-17]. The fact that a shortage ofMg causes various illnesses—including
ischemic heart diseases [18,19]—indicates that
Mg has important biological functions and is
involved in the physiological regulation oftissues and organs. On the other hand, studies atthe cellular and molecular levels have revealed
that Mg acts as an important modulator of cellfunctions. For example, in Mg-deficient rats,
various inflammatory cytokines and excessive
amounts of free radicals are released byleukocytes and macrophages [20], and reactive
oxygen species are produced by neutrophils[21]. On the contrary, a high concentration of
Mg reduces the production of superoxide anion
by eosinophils, of sulphidoleukotrienes byleukocytes, and of proinflammatory cytokines
by human blood cells [22,23]. These studies
suggest that Mg plays an essential role in a wide
range of fundamental cellular reactions. We
demonstrated that cells cultured in DSDW
survived longer, possibly through their effective
utilization of Mg. Mg is quickly captured andsmoothly accumulated in cells [24]. However,in terms of the life-prolonging properties of
DSDW, how Mg works after being taken into
cells is still unknown. Further studies arenecessary to clarify whether 1) Mg attenuates
the activity of cellular metabolism, 2) Mg
improves the efficiency of ATP synthesis, 3) Mg
downregulates ATP consumption, or 4) Mg
increases the number of mitochondria. Resultsfrom such experiments would guide the6understanding of how Mg provides cells withtolerance against unfavorable environmental
conditions. Judging from the findings of thepresent study, it can also be said that Mg
inhibits cell aging. In this case, we must
consider carefully the term “aging” separatelyfrom the technical term “clonal aging” because,in the present study, Euplotes cells cultured in
the mineral water showed neither division nor
proliferation during the experimental period. We
reported that DSDW prolonged the cell lifespan,or to be exact, prolonged the survival period ofcells cultured without food supply. Although
what occurs intracellularly in cells cultured in
DSDW is still unknown, culturing the ciliatedprotozoan E. woodruffi in conditioned mineralwater can be a useful experimental model forthe study of the possible anti-“aging” effect of
Mg or of DSDW.

5.

6.

7.

8.

9.

Acknowledgments

The authors are deeply grateful to Dr.
Yoshihiro Hataguchi (Ako Kasei Co., Ltd.) for
his generous support of this study. This research
did not receive any specific grant from funding
agencies.

10.

Conflict of Interest

11.

The authors declare no conflict of interest.

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