Experiment On Rats

Abstract
This study was performed to obtain the scientific evidence for biological effect of the DB-functional water. Total seventy nine clinically healthy Sprague-Dawley rats were used in this study. One, two and three weeks after the exposure to the Daemyung Bio-functional water (DB-functional water), liver grossly showed bright coloration and turgor, compared to control which was exposed to conventional purified water. Microscopically hepatic parenchyma showed bright field, especially in low magnification, that turned to be due to the bright cellular cytoplasm of each liver cells. Boundary among the hepatic cells was more obvious and sinusoidal area including Disse space was very well defined. From these results it is suggested that the DB-functional water have obvious improving effect on histological structure of rat liver for functioning. So the DB-functional water is highly recommendable as drinking water for improving and keeping hepatic health.

Introduction
The social and economical development of the world gives rise to the �ell-being� code, which means more upgraded life. Recently people prefer foods and drinks that keep healthy rather than simple food satisfying hunger and thirst. In addition, the style of enjoying food is going to change with time. Food materials have been regarded as substitute medical supplies rather than simple food source. Mushroom, milk and rice which have pharmacological effect are commercially available.
Recently people have had special interest in drinking water that they are consuming daily because water can keep their health. Various kinds of specified water were commercially available in the market of Korea. However there are no scientific or medical evidence that can verify their beneficial effect of their so-called specified water. So we need to find out real specified water that can be effective in health care.
Recently we became to know specified water produced by a company called Damyung Bio. Co., Ltd.. This water is called functional water by producer. It has been proved to be effective in treating the various kinds of so called incurable diseases. The diseases include B and C hepatitis, diabetes mellitus, hypertension etc. which we can exemplify in human cases. In this study we were trying to obtain the effectiveness of the functional water on the liver of clinically healthy rat by supplying the functional water.
In this study we focused on the effectiveness of gross and microscopic findings on the rat liver because introduced water into gastrointestinal tract is expected to be most effective in hepatic circulation. These experimental results can be expected partly to support the effectiveness of the functional water in treating and relieving the incurable diseases, especially hepatitis type B and C.

Materials and MethodsAnimals
Total seventy nine clinically healthy, Sprague-Dawley rat(Hyochang Science, Co, Ltd.) were used as experimental animals in total two times of experiment (Table 1). In the 1st and 2nd trials of experiment, body weight of rats ranged 50~120g (4 weeks of age), 35~74g(3 weeks of age), respectively. They maintained at room temperature of 22~23C, under humidity of 50~55%. During experimentation standard pellet food(Superfeed, Co. Ltd.) and drinking water(purified conventional and functional water obtained as below) was supplied ad libitum.

Preparation of drinking water
Functional water was directly obtained from tap water using a functional water purifier made by Daemyung Bio Co., Ltd. Purified tap water was also directly obtained from tap water using a water purifier also made by the same company.

Clinical examination
During the experiment, rats were clinically observed at least 2 times daily with a focus on their probable behavioral change.

Gross and histopathological examination of liver
In the 1st and 2nd trials of experiment, animals were autopsied and grossly examined liver 7 days after the exposure to functional water with the control exposed to conventional water.
For autopsy all rats were anesthetized with ether(J. T. Baker) and weighed. Abdominal area with livers exposed was photographed to visualize the gross findings of liver including the coloration by a digital camera(COOLPIX 8400, Nikon, Japan). After gross examination of the other internal organs, whole livers of each rat were carefully removed from the body and weighed. A part of liver tissue was dissected and pre-fixed in Bouin� fixative solution. Following post-fixation the liver tissue blocks were washed with tap water and then dehydrated in graded series of 70~100% alcohol. After cleaning and paraffination, about 4㎛-thickness tissue slices were obtained with use of microtome (Reichert-jung 820, Leica, Germany). All preparation of the slices was stained with Hematoxylin and Eosin (HE) for examining the histologic structure of liver with a light microscope (U-MDOB, Olympus optical Co. Ltd., Japan) As needed, photographs were taken using digital camera (Polaroid DMC-3, Polaroid, USA).

Results
1. Clinical findings
Clinically rats supplied by the functional water were more active but gentle, and more rapid in food recognition. They had a larger radius of action, compared to the control supplied by the conventional purified water.

2. Gross findings
There were no statistical difference in hepatosomatic index(HSI) between two experimental groups (Table 2). The coloration of liver surface was light brown and taut, in contrast to that of the control rat which was blackish brown and drooped (Fig. 1-1, 2). Other peritoneal organs and tissues had excsellent resiliency, compared to that of the control supplied by the conventional purified water.

3. Histopathological findings
Microscopically hepatic parenchyma showed bright field, especially in low magnification, that turned to be due to the bright cellular cytoplasm of each liver cells (Fig. 1-1, 2). The bright area of cytoplasm presumed to be areas occupied by lipid and glycogen, which remains to be proved. Boundary among the hepatic cells was more obvious and sinusoidal area including Disse space was very well defined, compared to those of the control supplied by conventional purified water (Fig. 1-3, 4). Two weeks after the supply, hepatic cells had more clean and homogeneous cytoplasm in contrast to those of the control which had vacuolated cytoplasmic areas probably representing secondary lysosomes or lipid vacuoles.

Discussion
From this study, livers grossly and microscopically showed obvious changes by the supply of the functional water, which meant to exert the obvious effectiveness of the water to the rat liver. Functional water is made by letting conventional tap water pass a series of filters. By passing a series of filters water is purified, energized by upbringing ray(4~14㎛ of wave length), and weakly alkalized. This energized functional water is known to have smaller molecular clusters so that water is well driven into cellular interior with Ca2+ to activate various cellular biochemical events.
The DB-functional water is considered to be effective in improvement of hepatic health even in clinically healthy individuals. The exact mechanism for the improvement can not be fully explained from this study. However this histopathological data using clinically healthy rats is sufficiently supportive to the clinical results that were proven to be effective in treating hepatic B and C hepatitis. Gross and histological findings strongly suggest that hepatic cells of rat could be activated by letting lipid and glycogen introduce into the cell interior.
This data is also expected to be fundamentals to obtain the experimental evidences of functional water effectiveness for other various curable and incurable diseases. Furthermore hepatic health is critically important because liver is well known to be responsible for above 90% of total body health.
Accordingly functional water made by the DB-functional water purifier is highly recommendable as daily drinking water for keeping people of worldwide healthy.

Table 1. Experimental groups of rats, Sprague-Dawley

Experiment	Drinking water	Duration of drinking	Numbers of rats used
1st trial	CW	One week	10
	FW	One week	9
	CW	Two weeks	10
	FW	Twe weeks	10
2nd trial	CW	One week	10
	FW	One week	10
	CW	Two weeks	5
3rd trial	FW	Two weeks	5
	CW	Three weeks	5
	FW	Three weeks	5

CW : conventional drinking water, FW: functional drinking water

Table 2. Hepatosomatic index(HIS) of rats, Sprague-Dawley

	Duration of drinking	Average body weight(g)		Liver weight(g)		HSI(%)
Experiment		CW	FW	CW	FW	CW	FW
1st trial	One week	81.94	83.96	3.60	4.07	4.37	4.85
	Two weeks	79.05	87.75	3.17	3.30	4.05	3.79
2nd trial	One week	73.42	70.15	2.96	2.71	4.03	3.87
	Two	76.98	75.89	3.26	2.52	4.26	3.33
	Three weeks	83.24	82.90	3.03	3.06	3.64	3.71

HIS : hepatosomatic index, CW: conventional drinking water, FW: functional drinking water

Fig. 1. Gross and microscopic findings of rat liver one week after the supply of drinking water. The livers of functional water group grossly shows coloration of light brown(2) in contrast to deep brown coloration of conventional purified water group(1). Microscopically more well defined histological structure of parenchyma including Disse space and sinusoidal area can be noted in the liver of functional water group(4) in contrast to the control which is ambiguous even in outline of hepatic cells (3). H&E, x400