U.S. patent application number 09/755020 was filed with the patent office on 2002-07-25 for composition of multipurpose high functional alkaline solution composition, preparation thereof, and for the use of nonspecific immunostimulator.
Invention is credited to Choi, Hyun-Suk, Choi, Soo-IL, Jeon, Kyung-Soo, Park, Yong-Ho, Yoo, Byung-Woo.
Application Number | 20020098980 09/755020 |
Document ID | / |
Family ID | 36747572 |
Filed Date | 2002-07-25 |
United States Patent
Application |
20020098980 |
Kind Code |
A1 |
Choi, Soo-IL ; et
al. |
July 25, 2002 |
Composition of multipurpose high functional alkaline solution
composition, preparation thereof, and for the use of nonspecific
immunostimulator
Abstract
Disclosed are a multipurpose, high-functional, alkaline solution
composition, preparation therefor and use thereof as a nonspecific
immunostimulator. The composition comprises 1-25 parts by weight of
borax (Na.sub.2B.sub.4O.sub.7.10H.sub.2O), 10.sup.-5-10.sup.-4
parts by weight of sodium thiosulfate
(Na.sub.2S.sub.2O.sub.3.5H.sub.2O), 30-150 parts by weight of
potassium carbonate, 30-200 parts by weight of refined sugar
(C.sub.12H.sub.22O.sub.11), and 100-200 parts by weight of water,
based on 100 parts by weight of sodium metasilicate
(Na.sub.2SiO.sub.3.5H.sub.2- O). In addition to bringing about an
improvement in disease resistance, weight gain rate, crop yield,
crop quality, harvest time, the composition shows nonspecific
immunostimulating activities, including antibody production and
immune enhancement, by activating immune cells, thereby maximizing
vaccination effects on malignant viral diseases.
Inventors: |
Choi, Soo-IL; (Ansung,
KR) ; Choi, Hyun-Suk; (Ansung, KR) ; Jeon,
Kyung-Soo; (Ansung, KR) ; Yoo, Byung-Woo;
(Seoul, KR) ; Park, Yong-Ho; (Seoul, KR) |
Correspondence
Address: |
JACOBSON HOLMAN PLLC
400 SEVENTH STREET N.W.
SUITE 600
WASHINGTON
DC
20004
US
|
Family ID: |
36747572 |
Appl. No.: |
09/755020 |
Filed: |
January 8, 2001 |
Current U.S.
Class: |
504/187 ;
424/184.1; 504/362 |
Current CPC
Class: |
A61P 31/04 20180101;
Y10S 514/885 20130101; Y10S 426/807 20130101; A61P 31/12 20180101;
A61K 33/22 20130101; A61P 35/00 20180101; A61K 31/7016 20130101;
A61K 33/10 20130101; A61P 37/02 20180101; Y10S 514/908 20130101;
A61K 33/38 20130101; A61P 15/00 20180101; A61P 37/04 20180101; Y10S
514/874 20130101; A61K 31/7016 20130101; A61K 2300/00 20130101;
A61K 33/10 20130101; A61K 2300/00 20130101; A61K 33/22 20130101;
A61K 2300/00 20130101; A61K 33/38 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
504/187 ;
424/184.1; 504/362 |
International
Class: |
A01N 059/06; A61K
039/00; A01N 059/16 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 23, 2000 |
KR |
2000-70054 |
Claims
What is claimed is:
1. A multipurpose, high-functional, alkaline solution composition,
comprising 100 parts by weight of sodium metasilicate
(Na.sub.2SiO.sub.3.ident.5H.sub.2O), 1-15 parts by weight of borax
(Na.sub.2B.sub.4O.sub.7.ident.10H.sub.2O), 10.sup.-5-10.sup.-4
parts by weight of sodium thiosulfate
(Na.sub.2S.sub.2O.sub.3.ident.5H.sub.2O), 30-150 parts by weight of
potassium carbonate, 30-200 parts by weight of refined sugar
(C.sub.12H.sub.22O.sub.11), and 100-200 parts by weight of
water.
2. The multipurpose, high-functional, alkaline solution composition
as set forth in claim 1, further comprising at least one compound
selected from the group consisting of sodium chloride, silver
thiosulfate and sodium molybdate at an amount of 10.sup.-3 parts by
weight or less based on 100 parts by weight of sodium
metasilicate.
3. A method for preparing a multipurpose, high-functional, alkaline
solution composition, comprising the steps of: adding 100 parts by
weight of anhydrous sodium metasilicate, 1-15 parts by weight of
borax, 10.sup.-5-10.sup.-4 parts by weight of sodium thiosulfate,
30-150 parts by weight of potassium carbonate, 30-200 parts by
weight of refined sugar to 100-200 parts by weight of water
maintained at 60-80.degree. C. with stirring for 2-3 hours to
complete dissolution; and admixing 30-200 parts by weight of
refined sugar to the solution with stirring for 2-4 hours.
4. The method as set forth in claim 3, further comprising the step
of admixing to the solution at least one compound selected from the
group consisting of sodium chloride, silver thiosulfate and sodium
molybdate at an amount of 10.sup.-1 parts by weight or less.
5. Multipurpose, high-functional feedstuff, in which the
composition of claim 1 or 2 or a dilution thereof is added by
spraying.
6. Multipurpose, high-functional feedstuff and a feedstuff
additive, in which the composition of claim 1 or 2 or a dilution
thereof is fermented.
7. A high-functional fertilizer, in which the composition of claim
1 or 2 or a dilution thereof is added by spraying.
8. A high-functional fertilizer and a fertilizer additive, in which
the composition of claim 1 or 2 or a dilution thereof is
fermented.
9. A method for enhancing non-specific immunostimulating activity
in the immune system of an animal or a plant, in which the
composition of claim 1 or 2 or a dilution thereof is added in food,
fertilizer, feedstuff, drug or water, and fed to the animal or
fertilized to the plant.
10. A method for keeping foods fresh, in which the foods are
treated with the composition of claim 1 or 2 or a dilution
thereof.
11. A method for treating human or animal tumors, in which the
composition of claim 1 or 2 or a dilution thereof is administered
to humans or animals to remove the tumor cells or inactivate
pathogens.
12. A method for promoting the differentiation of human or animal
oosperms, in which the composition of claim 1 or 2 or a dilution
thereof is administered to humans or animals.
13. A method for enhancing human or animal immune defense against
bacteria and viruses, in which the composition of claim 1 or 2 or a
dilution thereof is administered to humans or animals.
14. A method for promoting the germination and growth of a plant,
in which seeds of the plant are treated with the composition of
claim 1 or 2 or a dilution thereof.
15. A method for improving a livestock weight gain rate, in which
the feedstuff of claim 5 or 6 or a dilution thereof is fed to
livestock.
16. A method for improving the yield of crop, in which the
fertilizer of claim 7 or 8 is applied to the crop.
17. A method for improving a livestock weight gain rate, in which
the composition of claim 1 or 2 is diluted in water and fed to
livestock.
18. A method for improving the yield of crop, in which the
composition of claim 1 or 2 is diluted in water and sprayed over
the crop.
19. A method for activating water, in which the composition of
claim 1 or 2 is added in the water to minimize the number of bound
water molecules.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the invention
[0002] The present invention relates to a multi-purpose,
high-functional, alkaline solution composition, a preparation
method therefor, and use thereof as an nonspecific
immunostimulator. More particularly, the present invention relates
to an alkaline solution composition which mostly comprises sodium
metasilicate (pentahydrate) and shows nonspecific immunostimulating
activities, including antibody production and immune enhancement,
by activating immune cells, thereby maximizing vaccination effects
versus malignant virus diseases, preparation therefor, and use
thereof as an immunostimulator.
[0003] 2. Description of the Prior Art
[0004] The functionality of alkaline mass in the body has been of
great interest since the early 20.sup.th century. Extensive
research has recently revealed that alkaline mass in the body
increases ionization ratios of potassium and sodium to heighten the
purification capability of blood, resulting in blood clearance,
fatigue-reduction, and aging retardation.
[0005] One alkaline solution composition is disclosed in Korean
Pat. No. 128,110, yielded to the present inventor, which comprises
10-18 parts by weight of sodium silicate, 0.1-0.5 parts by weight
of sodium hyperoxide, 5-10 parts by weight of potassium carbonate,
1 part by weight of sodium carbonate, 10-18 parts by weight of
refined sugar and 0.1-3.0 parts by weight of silver thiosulfate in
water. The said composition is now used for the post-treatment of
fiber products and the fermentation of feedstuff and in the
agricultural industry by virtue of its high far infrared radiation
efficiency, antibacterial activity and deodorizing activity. The
composition, however, is disadvantageous in that its preparation is
complicated and it is difficult to store for a long period of
time.
[0006] Meanwhile, the amount of antibiotics used each year in the
world is increasing. However, the more antibiotics are used, the
greater the side effects are. For example, higher dosages of
antibiotics are needed to treat patients who have overused
prescription antibiotics because they have become resistant to
antibiotics. Furthermore, misuse and abuse of antibiotics has
resulted in the appearance of super-bacteria which are completely
immune to existing antibiotics. In an effort to curtail the use of
antibiotics, consideration has been taken of the general
enhancement of the immune system in the body, which leads to an
improvement in vaccination effects. For example, nonspecific
immunostimulators (hereinafter, referred to as "NIS"), which induce
the body to increase its immune response to external pathogens, are
now of great interest to medical personnel and extensive and
intensive research has been focused on the development of NIS over
the world.
[0007] In Japan, an ingredient extracted from edible mushroom
(Lentinus edoddes) was reported to show an anti-cancer effect. In
addition, NIS was observed in bacteria one hundred years ago. Along
with the effects of forming antibodies and inducing cytokines, the
immune enhancement of NIS has recently been under active study. For
instance, a cell wall extract from Norcardia opaca is reported to
induce the activation of the macrophages derived from murine
peritoneal cavities (Barot-Ciobaru et al., 1987). RU41740, derived
from Klebsiella pneumoniae, KP-40, derived from Propionibacterium
avidum, and QH-B, derived from Quillaja saponaria, are found to
have useful functions in association with the induction of
cytokines and the stimulation of immune cells (Bessler et al,
1997); Nimier et al., 1999; Ronnberg et al., 1997; Siwiki et al.,
19988; Tewari et al., 1996). Recently, bacterial DNA-derived CPG
motifs, called immunostimulatory motifs, have been revealed to
effectively induce the expression of IL-6, IL-12, IL-18 and
IFN-.gamma. in immune cells (Bohle et al., 1999; Klinman et al.,
1999; Krieg, 1999).
[0008] These and other NIS developed thus far suffer from the
problems of being produced in complicated processes, inconvenient
for long-term storage, and expensive.
DISCLOSURE OF THE INVENTION
[0009] With problems encountered in prior arts in mind, the present
invention has an objective of providing an alkaline solution
composition, which is easy to produce and store for a long period
of time, has inhibitory activity against bacteria and virus
proliferation, and can serve as a nonspecific immunostimulator.
[0010] It is another object of the present invention to provide a
method for preparing such an alkaline solution composition.
[0011] It is a further object of the present invention to provide
uses of the alkaline solution composition.
[0012] It is still a further object of the present invention to
provide methods for improving the weight gain rate of livestock and
the yield of crops.
[0013] It is still another object of the present invention to
provide a method for keeping agricultural products, fishes or meats
fresh for a long period of time.
[0014] It is still yet another object of the present invention to
provide a nontoxic, nonspecific immunostimulator with an anticancer
effect.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The above and other objects, features and other advantages
of the present invention will be more clearly understood from the
following detailed description taken in conjunction with the
accompanying drawings, in which:
[0016] FIG. 1 is a grayph in which changes in the proportions of
porcine CD4.sup.+ T lymphocytes in the BARODON.RTM.-fed groups and
in the control group are plotted against time;
[0017] FIG. 2 is a graph in which changes in the proportions of
porcine MHC class II-presenting cells in the BARODON.RTM.-fed
groups and in the control group are plotted against time;
[0018] FIG. 3 is a graph in which changes in the proportions of
porcine Non T/Non B lymphocytes (N lymphocytes) are plotted against
time;
[0019] FIG. 4 is a graph in which changes in the proportions of
porcine CD8.sup.+ T lymphocytes in the BARODON.RTM.-fed groups and
in the control group are plotted against time;
[0020] FIG. 5 is a histogram showing the lymphoproliferation
activities of porcine lymphocytes isolated from the peripheral
blood of the BARODON.RTM.-fed groups and the control group in
response to stimulation with Con A, PHA, PWM and LPS;
[0021] FIG. 5 is a histogram showing the lymphoproliferation
activities of porcine lymphocytes isolated from the mesenteric
lymph nodes of the BARODON.RTM.-fed groups and the control group in
response to stimulation with Con A, PHA, PWM and LPS;
[0022] FIG. 7 is a graph showing anti-cholera antibody
productivities of the BARODON.RTM.-fed groups and the control
group;
[0023] FIG. 8 is a graph in which changes in muscle Ca-ATPase
activity of cods in the BARODON.RTM.-treated groups and the control
group are plotted against storage time;
[0024] FIG. 9 is an NMR spectrum showing an absorption wavelength
band for .sup.17O of tap water; and
[0025] FIGS. 10 and 11 are NMR spectra showing absorption
wavelength bands for .sup.17O of water molecules in an aqueous
solution containing the composition of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention contemplates an NIS based on an
alkaline solution composition. This composition comprises sodium
metasilicate, borax, sodium thiosulfate, potassium carbonate,
refined sugar, and water, optionally sodium chloride, silver
thiosulfate and/or sodium molybdate.
[0027] Sodium metasilicate, useful in the present invention, has
five crystalline water molecules and comprises silicon dioxide
(SiO.sub.2) at an amount of 27.5-29.0% and sodium oxide (Na.sub.2O)
at an amount of 28.5-30.0% with a content error margin less than
2%. This compound is quite stable compared with commercially
available liquid sodium silicate. Existing as a white powder or
grain, sodium metasilicate is easy to weigh accurately and
convenient for long-term storage and transportation. When being
dissolved in water, sodium metasilicate is of strong alkalinity.
Silicon, like the other constituents of sodium metasilicate, is an
essential element for the growth of animals and plants.
[0028] Borax having with ten crystal water molecules has a specific
density of 1.715. As a constituent of borax, boron (B) is a trace
element in which animals and plants are likely to be deficient.
Fruit trees, when soils are insufficient in boron, do not bear well
and, if produced, many of their fruits are defective. Generally,
borax is used as a bacterium repellent, an insect repellent, a
styptic, and a glaze material. When melted together with metals,
borax has the property of dissolving the metals. A solution of
borax dissolved in water is alkaline with approximately pH 9.5. In
the composition of the present invention, the amount of borax is
preferably adjusted within the range of 1-15 parts by weight based
on 100 parts by weight of sodium metasilicate. For instance, if
borax is used at an amount less than 1 part by weight, no effects
thereof are exhibited. On the other hand, if the borax content
exceeds 15 parts by weight, toxicity may be generated.
[0029] With five crystal water molecules, sodium thiosulfate is not
dissolved in alcohol, but dissolved in water, giving a
characteristic salty taste. Its aqueous solution is neutral with a
pH in the range of 6.5-8.0. With a chemical property of dissolving
silver halide or other silver salts, sodium thiosulfate is
generally used to extract silver from ores. Additionally, sodium
thiosulfate finds applications in the removal of chlorine and heavy
metals. In the present invention, sodium thiosulfate is used at an
amount of approximately 10.sup.-5-10.sup.-4 parts by weight based
on 100 parts by weight of sodium metasilicate. For instance, no
additional effects can be obtained when the amount of sodium
thiosulfate is less than the lower limit. On the other hand, an
amount exceeding the upper limit of sodium thiosulfate, when being
applied to animals, acts to sediment tissular Ca, causing the
animals to be excited and to have loose bowels.
[0030] Potassium carbonate is dissolved well in water and its
solution is of strong alkalinity with pH 11.6. Like sodium,
potassium is an essential element for the body, playing an
important role in metabolism and blood circulation. For example, an
appropriate balance between potassium and sodium in vivo is needed
to prevent hypertension and diabetes mellitus. In the present
invention, a preferable amount of potassium carbonate falls within
the range of 30-150 parts by weight based on 100 parts by weight of
sodium metasilicate. When being applied to animals or plants, for
example, an amount outside this range of potassium carbonate may
upset the in vivo balance of sodium/potassium.
[0031] In the composition of the present invention, refined sugar
prevents ionized inorganic materials from recombining so as to
stabilize the composition. In addition, refined sugar converts
inorganic materials to organo-type materials in sugar-associated
forms, as well as acting to improve the adhesiveness or adsorption
of the composition. Within the amount range of 30-200 parts by
weight based on 100 parts by weight of sodium metasilicate, refined
sugar can perform these roles.
[0032] Sodium chloride, an optional compound in the composition of
the present invention, is used as a source of sodium to control the
ratio of sodium/potassium. As for silver thiosulfate, it is often
used to suppress the ethylene synthesis of plants or to facilitate
the differentiation of plants. In an aqueous solution, silver
thiosulfate forms [Ag(S.sub.2O.sub.3.sup.2-).sub.2].sup.3- at a low
concentration of S.sub.2O.sub.3.sup.2- while forming
[Ag.sub.2(S.sub.2O.sub.3.sup.2-).sub.- 6].sup.10- at a high
concentration of S.sub.2O.sub.3.sup.2-. In the present invention,
silver thiosulfate, existing in a polyvalent anionic form like
sodium thiosulfate, acts to facilitate cell differentiation. In the
present invention, sodium molybdate serves as a source of
molybdenum, which is an element which animals and plants are likely
to lack. The optional compounds are each used at an amount of
10.sup.-1 parts by weight or less based on 100 parts by weight of
sodium metasilicate.
[0033] After complete dissolution of the constituents, the
composition of the present invention is tinged with ivory. The
composition is odorless and nontoxic in addition to being very
stable with a specific viscosity of 1.43-1.50, maintaining pH 13
and ranging in viscosity from 61.0-239.0. Particularly, even when
being treated with HCl, the composition of the present invention
hardly undergoes solidification or pH changes.
[0034] Various experimental results obtained over a long period of
time demonstrate that the composition of the present invention
increases weight-gaining rates in livestock and the yield from
crops, as well as serving as an excellent NIS in both plants and
animals.
[0035] The composition of the present invention may be fed to
livestock, along with feedstuff, for example, in a mixture with
feedstuff or after being fermented in feedstuff, or as a dilution
with water. After feeding, the composition brings about excellent
immune enhancement in the livestock. For example, the composition
of the present invention is effectively preventive of porcine
epidemic diarrhea (PED) and hog cholera, which cause serious
problems for the hog raising industry. Also, the composition was
found to be effective for the prophylaxis and treatment of fowl
typhoid, which shows a high mortality, causing an enormous loss in
the poultry industry. In the case of milk cows fed with the
composition, the somatic cell count per volume of milk, which is an
index determining milk quality, was decreased. Other effects of the
composition of the present invention on livestock include growth
facilitation and improvement in flesh quality. Sheds in which the
livestock lived while being fed with the composition of the present
invention were found to have less offensive odors than those in
which the livestock lived while being fed with ordinary
feedstuff.
[0036] For plants, the composition of the present invention, which
is applied in mixtures of fertilizers or as dilutions in water,
exhibits useful effects, including facilitation of germination and
growth, improvement in disease resistance, increased crop yields,
improvement in crop quality, etc.
[0037] A better understanding of the present invention may be
obtained in light of the following examples which are set forth to
illustrate, but are not to be construed to limit the present
invention.
PREPARATION EXAMPLE 1
[0038] In purified water (500 kg) maintained at 60-80.degree. C.,
sodium metasilicate (pentahydrate, 300 kg), borax (decarbohydrate,
35 kg), sodium thiosulfate (0.01 kg), sodium chloride (1 kg), and
potassium carbonate (150 kg) were sequentially added, and stirred
for 3 hours for dissolution. To the homogeneous solution was added
refined sugar (450 kg), followed by stirring for 4 hours to give an
alkaline solution with pH 13 (hereinafter referred to as
"BARODON.RTM.-1").
PREPARATION EXAMPLE 2
[0039] BARODON.RTM.-1 (1,436 kg), prepared in Preparation Example
1, was dropwise added with a solution of silver thiosulfate (0.02
kg) in water (1 L), stirred and maintained at about 50.degree.C.
for 4 hours in an incubator. The resulting solution was designated
"BARODON .RTM.-2".
PREPARATION EXAMPLE 3
[0040] In purified water (5 L) maintained at 100.degree. C., sodium
molybdate (Na.sub.2MoO.sub.4A2H.sub.2O, 0.3 kg) was added with
stirring to give a colorless, odorless solution, which was then
dropwise added to BARODON.RTM.-1, stirred, and maintained at
50.degree. C. for 4 hours in an incubator. The resulting solution
was designated "BARODON.RTM.-3".
PREPARATION EXAMPLE 4
[0041] Using 6 kg of borax and 300 kg of potassium carbonate, an
alkaline solution was obtained in a manner similar to that of
Preparation Example 1. To the alkaline solution (1,557 kg), a
solution of silver thiosulfate (0.02 kg) in water (1 L) was
dropwise added, followed by stirring and standing at 50.degree. C.
for 4 hours in an incubator. The resulting alkaline solution was
designated "BARODON.RTM.-4".
PREPARATION EXAMPLE 5
[0042] Using 150 kg of refined sugar, the procedure of Preparation
Example 4 was repeated to give an alkaline solution with a
relatively low viscosity (hereinafter referred to as
"BARODON.RTM.-5").
EXAMPLE 1
Effect on Rice Cultivation
[0043] BARODON.RTM.-1 to -5 were tested for immune enhancement and
growth facilitation effects on the cultivation of rice plants on
paddy fields.
[0044] BARODON.RTM.-1 to -5 were each diluted in 10 volumes of
water and 500-fold diluted again with water. Just after being
immersed for 24 hours in the diluted solution, rice seeds were sown
on seedbeds. A couple of days before the transplantation of rice
plants from the seedbeds to paddy fields, the diluted solutions
were sprayed over the nursery rice plants in an ordinary manner.
The spraying of the diluted solution was conducted again a couple
of weeks before rice booting (rice earing).
[0045] On seedbeds, the nursery rice plants treated with the
composition of the present invention grew more uniformly and
strongly and suffered almost no damages due to cold weather,
compared with a control group which was not treated with the
composition. Also, the nursery rice plants treated with the
composition were observed to completely root only three days after
the transplantation. No diseases, including damping-off, leaf
blast, and sheath blight, effected the treated rice plants during
their growth on field. In addition, the treated rice plants were
improved in lodging-resistance and were found to suffer no lodging
following a typhoon, while 25% of the control was lodged.
[0046] Amounts of produced rice were measured and the results are
given in Table 1, below.
1 TABLE 1 Composition Crop yield per km.sup.2 (Chuchung .RTM. Rice)
Control 575 kg BARODON .RTM.-1 655 kg BARODON .RTM.-2 690 kg
BARODON .RTM.-3 710 kg BARODON .RTM.-4 680 kg BARODON .RTM.-5 670
kg
EXAMPLE 2
Effect on Pear Tree Cultivation
[0047] BARODON.RTM.-3 was mixed with 9 volumes of water and then
re-diluted 500 times. After applying dung-manure and organic
fertilizers on a pear orchard, the dilution was sprayed thereon.
Thereafter, a roundabout was installed along the circumference of
the orchard to prevent the interference of animals. About a couple
of weeks before the blooming of pear blossoms, BARODON.RTM.-2 was
sprayed on the land. The pear trees fertilized with the
compositions of the present invention burst into bloom about 4-5
days earlier and produced harvestable fruits about 15 days earlier
than did a control, which was not fertilized with the compositions.
The pears harvested from the treated trees were graded the highest
in appearance, size and Brix degree. The pears harvested from the
trees fertilized with the compositions were 60% reduced in black
spot occurrence and smoother in appearance, compared with the
control. No soggy pears were found in the treated trees. The pear
trees treated with the compositions of the present invention showed
a fruit drop rate due to typhoon 20% less than that of the control.
The pears of the treated trees were about 15% greater in size
compared with those of the control. The Brix degree of the pears of
the treated trees was measured to range from 13.0 to 15.0, which
was about 12% greater than that of the control. The storage period
was also improved.
EXAMPLE 3
Effect on Plant Growth
[0048] A 16-fold dilution of BARODON.RTM.-3 in water was further
diluted by the dilution factors given in Table 2, below and then
sprayed over leaves of specimen plants. The leaves were found to be
greater by 15.6% in length, 8.7% in width, and 7-47% in foliage
fresh weight, compared with a control group which was not treated
with the solution.
2 TABLE 2 Leave Width at Maximum (cm) Dilution Factor Non- Pot No.
200-Fold 300-Fold 400-Fold Spraying 9th day after 1 6.1 6.1 6.2 5.4
Spraying 2 6.0 6.3 6.2 5.9 (10, July) Avg. 6.1 6.2 6.2 5.7 Foliage
Fresh Weight (g/pot) Dilution Factor Non- Pot No. 200-Fold 300-Fold
400-Fold Spraying 9th Day After 1 57.8 59.2 44.6 39.9 2nd Spraying
2 56.3 60.5 42.8 41.5 (10, July) Avg. 57.1 59.9 43.7 40.7 (140%)
(147%) (107%) (100%)
PREPARATION EXAMPLE 6
[0049] BARODON.RTM.-2, prepared in Preparation Example 2, was
diluted by a factor of 10 and 500 g of the resulting dilution was
sprayed over 1 ton of combined feedstuff to give a high-functional
feedstuff (hereinafter referred to as "BARODON.RTM.-6").
PREPARATION EXAMPLE 7
[0050] A 10-fold dilution of BARODON.RTM.-2, prepared in
Preparation Example 2, in water (10 L) was added, together with
refined sugar (3 Kg), sodium chloride (1 Kg) and water (75 L), to
combined feedstuff (1 ton), after which the resulting formulation
was fermented for 24 hours with stirring to give high-functional
feedstuff (hereinafter referred to as "BARODON.RTM.-7").
EXAMPLE 4
[0051] The BARODON.RTM. series was tested for weight gain and
immune enhancement effects on animals as follows.
[0052] (1) Weight-Gaining Effect on Hog
[0053] 30 heads of three-way cross hybrid
(Yorkshire.times.Landrace.times.- Durroc), fattening hogs, each of
which was 15 weeks (104.+-.4 days) old, were selected. They were
all admitted to 3 hog pens, each with a size of 4 M.times.4.2 M, at
a population of 10 heads per hog pen and adapted to the new
circumstance for one week before testing.
[0054] BARODON.RTM.-6, prepared in Preparation Example 6, was fed
to a group of 10 hogs (designated "Tx-1 group") in one hog pen for
9 weeks while the feedstuff containing 3% of BARODON.RTM.-7,
prepared in Preparation Example 7, was given to a group of 10 hogs
(designated "Tx-2 group") in another hog pen. For a control group,
the same feedstuff, but lacking "BARODON.RTM." was supplied.
Thereafter, all the hogs were fed with common feedstuff. During the
testing period, the hogs were all allowed free access to
feedstuff.
[0055] A measurement was made of weight gain and feedstuff intake
for 6 weeks for each group and, from the measured values, the feed
efficiency (feedstuff intake/weight gain) was calculated. The
average daily weight gain was measured to be 842.86 g for the
control group, 890.48 g for the Tx-1 group and 880.95 g for the
Tx-2 group, the latter two groups being 5.65% and 4.52% improved,
respectively, compared with the control group. As for average daily
feedstuff intake, 2.71 kg of feedstuff was consumed by the control
group, 2.77 kg of feedstuff by the Tx-1 group and 2.65 kg of
feedstuff by the Tx-2 group. Therefore, the control group showed a
feed conversion rate of 3.22 while the Tx-1 group was calculated to
be 3.11 in feed conversion rate with an improvement of 3.54% and
the Tx-2 group grew at a feed conversion rate of 3.01, which was
6.98% improved.
[0056] (2) Perception of Pork
[0057] The pork of each of the test groups was tasted by healthy
men and women and they were interviewed regarding taste and flesh
quality.
3 TABLE 3 Softness of Flesh Taste Soft Tough Good Moderate Control
0 8 0 3 Tx-1 6 1 8 2 Tx-2 11 0 10 0 *numbers of the persons who
responded to the questions after a sampling party.
[0058] (3) Distribution of Immune Cells in Peripheral Blood of
Hog
[0059] Using pig leukocyte surface-specific monoclonal antibodies
and a flow cytometry such as that manufactured by Dickinson
Immunocytometry System, San Jose, Calif., U.S.A., identified as
FACSCalibur, proportions of major histocompatibility complex (MHC)
expressing cells and lymphocyte subpopulations were examined in the
peripheral bloods of the Tx-1 and the Tx-2 group as well as the
control group.
[0060] (3-1) Isolation of Leukocyte from Peripheral Blood
[0061] The blood taken from the swine fore vena cava was well mixed
with acid citrate dextrose (ACD)-ethylenediamine tetraacetic acid
(EDTA) and laid over a layer of Hypaque Ficoll (Histopaque, Sigma,
St. Louis, Mo., U.S.A.). After centrifugation at 1,500 rpm for 30
min, leukocytes were taken, washed three times with phosphate
buffered saline (PBS, pH 7.2), and suspended in an RPMI-1640 medium
(GibcoBRL, Grand Island, N.Y., U.S.A.). For examination, leukocyte
cell counts were adjusted to 1.times.10.sup.7 cells/ml while viable
cells were counted according to the tryphan blue exclusion
technique. (3-2) Monoclonal Antibodies for Examination of Leukocyte
Subpopulation
[0062] Effects on porcine immunological properties, including
immune cell populations, were determined using monoclonal
antibodies specifically reactive with cell surface molecules of
porcine leukocytes, i.e., MHC class I antigens, MHC class II
antigens, Po(porcine)CD2, PoCD4, PoCD8, surface IgM (sIgM), Non
T/Non B (.gamma..delta. TCR), and granulocyte and monocyte (G+M),
as shown in Table 4, below.
4TABLE 4 Isotype MAb* of MAb Molecules** Cell type*** Reference
PT85A IgG.sub.2a MHC class I All nucleated Davis et al. cells
(1987) H42A IgG.sub.2a MHC class II Ag-presenting Davis et al.
cells (1987) TH81A5 IgG.sub.2a MHC class II Ag-presenting Davis et
al. cells (1987) MSA4 IgG.sub.2a PoCD2 T cells Davis et al. (1987)
PT90A IgG.sub.2a PoCD4 T h/i cells Davis et al. (1987) PT81B
IgG.sub.2a PoCD8 T c/s cells Davis et al. (1987) P1g45A IgG.sub.2b
slgM B cells Davis et al. (1987) PT79A IgG.sub.2a .gamma..delta.TCR
N cells Davis et al. (1987) DH59B IgG.sub.1 Granulocyte +
Granulocyte + Davis et al. Monocyte Monocyte (1987) MAB*:
Monoclonal antibodies specifically reactive with leukocyte
differentiation. Molecules**: Porcine leukocyte differentiation
molecules. Cell type***: Cells expressing molecules.
[0063] (3-3) Flow Cytometry
[0064] Using a flow cytometry CellQuest program, proportions of
leukocyte subpopulations were analyzed according to the method of
Davis et al (1990). In order to take advantage of a flow cytometry
using a laser beam, cells were labeled with one or two fluorescent
dyes such as fluorescein isothiocyanate (FITC) and phycoerythrin
(PE) in an indirect manner. In each well of a V-bottom 96-well
microplate, 100 .mu.l of the leukocytes isolated from blood (cell
density 1.times.10.sup.7 cells/ml) was added, along with 50 .mu.l
of a monoclonal antibody (concentration 15 .mu.g/ml) and sensitized
for 30 min at 4.degree. C., followed by washing three times with a
first washing buffer [PBS 450 ml, ACD 50 ml, 20% NaN.sub.3 5 ml,
gamma globulin free horse serum (GibcoBRL) 10 ml, 250 mM EDTA 20
ml, 0.5% phenol red 1 ml] through centrifugation. After decanting
the supernatant, the cell pellet on the bottom was suspended using
a plate mixer or a vortex mixer (Scientific Industries, Bohemia,
N.Y., U.S.A.).
[0065] In a single dyeing test, an FITC-conjugated goat anti-mouse
IgG+IgM antibody (Caltag Lab, U.S.A.), serving as a secondary
antibody, was diluted by a factor of 200 and added at an amount of
100 .mu.l to each well in which the suspended leukocytes were
contained. After sensitization for 30 min at 4.degree. C., washing
was conducted three times with a second washing buffer, which was
the same as the first washing buffer, but being free of the horse
serum, by centrifugation. A 2% PBS-formalin (38% formalin 20 ml,
PBS 980 ml) solution was added at an amount of 200 .mu.l to each
well to fix the cells.
[0066] In a double dyeing test, PoCD4 (FITC) was coupled with PoCD8
(PE), PoCD4 (FITC) with MHC class II (PE), and PoCD8(FITC) with MHC
class II (PE) to dye cells. In detail, leukocyte cells in one well
were mixed with a pair of monoclonal antibodies and primarily
sensitized according to the result of the single dyeing test,
followed by washing three times with the first washing buffer at
4.degree. C. Thereafter, a goat antibody specific for each
monoclonal antibody isotype was used at a concentration of 1.0
.mu.g per well for FITC conjugates and at a concentration of 0.1
.mu.g per well for the PE conjugate, followed by secondary
sensitization for 30 min at 4.degree. C. Washing and fixing
procedures were conducted in the same manner as in the single
dyeing test.
[0067] After completion of the dyeing, cells were stored at
4.degree.C. in a dark, cold space until examination. Using a flow
cytometry, 2,000 or more dyed cells were analyzed to count
positive-response cells. In regard to the measurement and data
analysis, FACScalibur and CellQuest program (Becton Dickinson) was
employed.
[0068] The results showed that the proportion of CD4.sup.+ T
lymphocytes in porcine peripheral blood started to increase after
three weeks from the feeding with BARODON.RTM.-containing
feedstuff, as shown in FIG. 1 and, on the 8.sup.th week after the
feeding, considerably higher levels of the CD4.sup.+ T lymphocytes
were maintained by the Tx-1 and the Tx-2 groups than by the control
group (p<0.05). Particularly, the Tx-1 group maintained
CD4.sup.+ T lymphocytes at high levels over a period from the
8.sup.th week to the 13.sup.th week post-application
(p<0.05).
[0069] As for CD8.sup.+ T lymphocytes, a high level was detected in
the Tx-2 group on the third week after the feeding (p<0.01), but
no noticeable differences were found from the 8.sup.th week after
the feeding, compared with the control group (p<0.05), as shown
in FIG. 4.
[0070] Cells expressing MHC class II antigens, mainly macrophages,
were counted at a considerable high level for the Tx-1 group at the
11.sup.th week after the feeding (p<0.05) and for the Tx-2 group
at the 8.sup.th week after the feeding, compared with the control
group, as shown in FIG. 2.
[0071] The count of Non T/Non B lymphocytes (N lymphocytes) in the
Tx-2 group was maintained higher than that in the control group
(p<0.01) from the 3.sup.rd week after the feeding, as shown in
FIG. 3, showing the possibility of enhancing both nonspecific and
specific immune defense responses. Over a period from the 11.sup.th
week to the 13.sup.th week after the feeding, the Non T/Non B
lymphocytes were maintained at considerably higher levels in the
Tx-2 group than in the control group (p<0.01).
[0072] Comparing the two BARODON.RTM.-fed groups, the Tx-2 group
stayed distinctively predominant over the Tx-1 group in MHC-class
II antigen-expressing cell level during a period from the 3.sup.rd
week to the 8.sup.th week after the feeding (p<0.05) as shown in
FIG. 2. A little more CD4 and CD8 expressing cells were counted in
the Tx-2 group than in the Tx-1 group on the 3.sup.rd week after
the feeding (p<0.1), as shown in FIGS. 1 and 4. Over the Tx-1
group, the Tx-2 group attained noticeable superiority in Non T/Non
B lymphocytes on the 13.sup.th week after the feeding, as shown in
FIG. 3.
[0073] (4) Effect on Activity of Blood and Lymph Nodal
Lymphocytes
[0074] In order to examine activities of blood and lymph nodal
lymphocytes, their proliferative responses were determined by
measuring [.sup.3H]-thymidine incorporation of porcine lymphocytes
obtained from peripheral blood and mesenteric lymph nodes after
stimulation with Concanavalin A (Con A), Phytohemagglutinin (PHA),
Pokeweed mitogen (PWM) and Lipopolysaccharide (LPS).
[0075] When being stimulated with PWM, the lymphocytes obtained
from the peripheral blood of the Tx-2 group after 8 weeks of the
feeding with the BARODON.RTM.-containing feedstuff were observed to
be highly proliferative as proven by a significantly larger
stimulation index for the Tx-group than for the control group
(p<0.05). At 11.sup.th week post-feeding, the lymphocytes
isolated from the peripheral blood of both the Tx-1 group and the
Tx-2 group gave greater proliferative responses to the stimulation
with each of PHA, PWM and LPS than those isolated form the control
group, showing higher SI values (p<0.01), as depicted in FIG.
5.
[0076] As for lymphocytes isolated from mesenteric lymph nodes,
significantly higher SI values were observed in both the Tx-1 group
and the Tx-2 group in response to the stimulation with PHA
(p<0.05) and PWM (p<0.01) at the 8.sup.th week after the
feeding than in the control group. At 11 weeks post-application,
the Tx-1 group showed higher Con A- and PHA-stimulated
lymphoproliferative responses than the control group (p<0.01)
while the Tx-2 group was measured to have significantly high SI
values in response to the stimulation with Con A, PHA and PWM
(p<0.05), as depicted in FIG. 6.
[0077] (5) Effect on Proportions of Splenic and Lymph Nodal
CD4.sup.+CD8.sup.+ T Lymphocyte Subpopulations
[0078] Immunohistochemistry was used to analyze proportions of T
lymphocytes. After being immunologically dyed by use of the ABC
method, CD4.sup.+, CD8.sup.+, and CD4.sup.+CD8.sup.+ dpp T
lymphocyte cell counts in mesenteric lymph nodes and spleens were
measured with the aid of an image analyzer (Olympus, U.S.A.). The
immunohistochemical analysis results are given in Tables 5 and 6,
below. In spleens, as apparent from Table 5, an increase was seen
in the proportion of CD4.sup.+ T lymphocytes for the Tx-2 group
only while CD8.sup.+and CD4.sup.+CD8.sup.+ T lymphocyte counts were
significantly increased for the Tx-1 and the Tx-2 group, both
(p<0.001). In mesenteric lymph nodes, on the other hand, both
the Tx-1 and the Tx2 groups were significantly increased in all
CD4.sup.+, CD8.sup.+ and CD4.sup.+CD8.sup.+ dpp T lymphocyte
proportions (p<0.01), as specified in Table 6, below.
Particularly, such higher distributions of T lymphocytes
subpopulations were more evident in the Tx-2 group (p<0.01).
5 TABLE 5 Group CD4.sup.+ CD8.sup.+ CD4.sup.+CD8.sup.+dpp Control
11 .+-. 1 8 .+-. 1 3 .+-. 1 Tx-1 11 .+-. 1 11 .+-. 1 6 .+-. 1 Tx-2
14 .+-. 1 17 .+-. 1 11 .+-. 1
[0079]
6 TABLE 6 Group CD4.sup.+ CD8.sup.+ CD4.sup.+CD8.sup.+dpp Control
32 .+-. 5 29 .+-. 2 10 .+-. 1 Tx-1 35 .+-. 4 39 .+-. 4 32 .+-. 3
Tx-2 40 .+-. 4 47 .+-. 5 35 .+-. 4
[0080] (6) Effect on Antibody Production After Vaccination with Hog
Cholera Vaccine
[0081] Anti-hog cholera virus IFA titers were determined after the
vaccination with a hog cholera vaccine in BARODON.RTM.-fed groups
and a non-BARODON.RTM. fed group. Significantly higher antibody
titers were measured after three weeks of the feeding of
BARODON.RTM. in the BARODON.RTM.-fed groups (Tx-1 and Tx-2) than in
the control group and these titers were maintained till the
11.sup.th week after the feeding (p<0.01), as depicted in FIG.
7.
[0082] (7) Effect on Typhoid-Resident Fowl Treated with No
Antibiotics
[0083] 10,000 heads of chickens infected with typhoid were fed for
10 weeks with the feedstuff containing 5wt % of BARODON.RTM.-7,
prepared in Preparation Example 7. Their mortalities by week were
measured and the results are given in Table 7, below. During the
testing term, no antibiotics were administered to the
typhoid-infected chickens. As specified in Table 7, the
BARODON.RTM.-fed group showed a mortality of as low as 0.3% on
average until the 15.sup.th week after the testing whereas
additional typhoid-infected 10,000 heads in a control group, which
were not fed with BARODON.RTM., were, for the most part, dead only
4 days after the testing.
7 TABLE 7 Mortality with Lapse of Time (%) Age Week Fowl@ (week) 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 BARODON .RTM.-fed 27 .34 .36 .31
.37 .30 .28 .3 .27 .3 .4 .35 .33 .33 .32 .31 Control 28 1.12 1.45
1.73 7.19 almost all dead
[0084] (8) Effect on Somatic Cell Count in Milk Cow Suffering From
Mastitis
[0085] In order to examine the effect of BARODON.RTM. on the
somatic cell counts of milk cows suffering form mastitis, they were
fed with the feedstuff containing 5 weight % of BARODON.RTM.-7,
prepared in Example 7, for a couple of months. Measurement was made
of somatic cell counts. The results are given in Table 8, below, in
which the term "liquid medicine" means breasts of the cows were
rubbed with towels cleaned with a 100 fold dilution of
BARODON.RTM.-2, the term "diluted in water" means that the cows
were allowed to drink a 200-fold dilution of BARODON.RTM.-2, and
the character "q" stands for the number of breasts
8 TABLE 8 No. of Somatic Cells (.times.1,000) Farm A Farm B Week
Feed + Liquid Diluted Feed + Liquid After medicine Feed in Water
Control medicine Feed Control Feeding (q = 10) (q = 34) (q = 6) (q
= 37) (q = 16) (q = 12) (q = 36) Before 2,457 356 1,471 168 967 619
155 2 1,846 205 1,999 276 1,214 886 197 (75) (57) (135) (164) (125)
(150) (127) 4 1,543 144 1,683 371 1,017 772 305 (62) (40) (114)
(220) (105) (124) (196) 6 2,001 228 2,367 517 1,137 681 353 (81)
(64) (160) (307) (117) (115) (147) 8 1,788 272 845 423 936 606 210
(72) (76) (57) (251) (96) (97) (135)
[0086] Main pathogens causing mastitis were also counted and the
results are given in Table 9, below.
9 TABLE 9 No. of Infected BARODON .RTM.-fed Group Control Pathogen
Pre-Feed 4 weeks 8 weeks Pre-Fee 4 weeks 8 weeks Staphylo- 11 7 6
12 14 15 cocci sp. Streptococci 2 2 1 2 3 3 sp. Gram (-) 3 2 2 3 4
4 bacilli Fungi/Bacili 0 0 0 1 2 2 Total 16 11 9 18 23 24
EXAMPLE 5
[0087] BARODON.RTM. was also investigated for its immune
enhancement of the human body. In this regard, texts for children
were manufactured with sheets of paper, over which a 100-fold
dilution of BARODON.RTM.-5, prepared in Preparation Example 5, had
been sprayed, and measured for QRS waves in Japanese Far InfraRed
Ray Application Institute, located in Osaka, Japan. The results are
given in Table 10, below. For reference, QRS wave numbers can give
physically beneficial effects to the body when being within the
range of 10,000 to 20,000.
10 TABLE 10 Wave No. BARODON .RTM.-S.F Paper Non-treated Paper
Allergy 1,700 11 Ocular Nerve 1,300 13 Autonomic Nerve 1,500 11
EXAMPLE 6
[0088] Toxicity Test The toxicity of BARODON.RTM. was determined
through in acute toxicity tests using rats at Screening and
Toxicology Center of the Korea Research Institute of Chemical
Technology (Test No. S-700). The acute tests were conducted with a
10-fold dilution of BARODON.RTM.-4, according to the National
Institute of Health's Notification No. 94-3 `Toxicity Testing
Practice for Medicines` (Apr. 14, 1994) and the Ministry of Health
and Welfare's Regulation No. 87-80 `Korean Good Laboratory
Practice` (Oct. 29,1987).
[0089] As a result of the test, toxic symptoms, including death,
eight change, and other side effects, were not observed in males
and females injected with the testing material while the LD.sub.50
value was reported to be over 5,000 mg/kg for male and female,
both. This means that the composition of the present invention is
non-toxic to human bodies.
EXAMPLE 7
Mutation Test
[0090] To determine whether BARODON.RTM. acts as a mutagen, a
returning mutation test was carried out at Screening and Toxicology
Center of the Korea Research Institute of Chemical Technology (Test
No. S-694) in accordance with the National Institute of Health's
Notification No. 94-3 `Toxicity Testing Practice for Medicines`
(Apr. 14, 1994) and the Ministry of Health and Welfare's Regulation
No. 87-80 `Korean Good Laboratory Practice` (Oct. 29, 1987),
treating four salmonella strains (His-less mutants of Salmonella
typhimurium TA100/TA1535 (base pair substitution type) and
TA98/TA1537 (frame shift type) with a 10-fold dilution of
BARODON.RTM.-4.
[0091] From all the four bacterial strains, negative records were
observed, indicating that the test material did not cause the
return mutation from His.sup.31 strains into His.sup.+ ones. This
result means that the composition of the present invention is safe
to the human body.
EXAMPLE 8
Effect on Cell Proliferation of Oosperm
[0092] Immature ova recovered from cow ovaries were subjected to in
vitro maturation for 24 hours and then to in vitro fertilization
for 20 hours. The resulting oosperms were developed for 9 days in
media in which a 10-fold dilution of BARODON.RTM.-4 was further
diluted by factors of 500, 200, 100 and 50, respectively and in
control media. Bull spermiducts, obtained from a local slaughter
house, and bovine oviduct epithelial cells (BOEC) and granulosa
cells (GC), both taken from cow ovaries, were cultured to the third
generation in the same media. Viable cells were counted using a
hemocytometer.
[0093] After the development, the externally fertilized oosperms
underwent cleavage at a rate of 60.0% in the control media, at a
rate of 62.4% in the 500-fold diluted media, at a rate of 66.3% in
the 200-fold diluted media, and at a rate of 73.7% in the 50-fold
diluted media. Development rates of the blastocyst embryos were
measured to be 13.3% in the control media, 20.0% in the 500-fold
diluted media, 21.3% in the 200-fold diluted media, 18.4% in the
100-fold diluted media, and 11.0% in the 50-fold diluted solution.
After culturing, viable cells numbered 2.0.times.10.sup.5 cells for
BOEC and 3.2.times.10.sup.5 cells for GC in the control media,
2.4.times.10.sup.5 cells for BOEC and 3.9.times.10.sup.5 cells for
GC in the 500-fold diluted media, 2.5.times.10.sup.5 cells for BOEC
and 3.7.times.10.sup.5 cells for GC in the 200-fold diluted media,
2.6.times.10.sup.5 cells for BOEC and 2.7.times.10.sup.5 cells for
GC in the 100-fold diluted media, and 2.5.times.10.sup.5 cells for
BOEC and 2.8.times.10.sup.5 cells for GC in the 50-fold diluted
media.
[0094] As demonstrated in the above data, the composition of the
present invention has a positive influence on both the cell
proliferation of BOEC and GC and the proliferation and development
of oosperms and blastocyst embryos. Particularly, blastocyst
embryos were improved in proliferation rate by 50.4-60.2% when
being cultured in the 500-fold and 200-fold diluted media, compared
with when being cultured in the control media.
EXAMPLE 9
Anti-Cancer Activity
[0095] After being diluted by factors of 600-900, BARODON.RTM.-4
was tested for inhibitory activity against human tumor (cancer)
cells of four kinds.
[0096] Human leukemic cells (Jurkat), human lung carcinoma cells
(NCl-H69), human stroma cells (SW579), and human osteogenic sarcoma
cells (U-2 OS) were obtained from the American Type Culture
Collection, Rockvile, Md., U.S.A. Each of these cells was cultured
in media in which BARODON.RTM.-4 was diluted by factors of 600,
700, 800 and 900. Cells were examined and measured for their
proliferation rates at each sub-culturing while viable cells were
counted using a tryphan blue method.
[0097] Prior to the examination of anticancer activity, normal
cells were cultured in the same diluted media to determine whether
BARODON.RTM. has cytotoxicity. The results are given in Table 11,
below, demonstrating that the diluted media did not inhibit the
growth rate of human fibroblast cells nor prevented the
proliferation of hamster kidney cells.
11TABLE 11 Condition Culture Dilution of No. of of Cell lines Media
BARODON .RTM.-4 passages Cells* CCD-27SK DMEM + Control P8
Excellent (Normal skin 10% FCS 1/900 P8 Excellent fibroblasts 1/800
P8 Excellent of human 1/700 P8 Very good 1/600 P7 Poor BHK-21
Normal DMEM + Control P17 Excellent hamster 10% FCS 1/900 P17
Excellent kidney cells 1/800 P17 Very good 1/700 P17 Good 1/600 P11
Poor *dyed with 0.4% tryphan blue and counted under a
microscope
[0098] Culture results of the cancer cells of four kinds are given
in Table 12, below.
12TABLE 12 Condition Dilution of No. of of Cell Lines Culture Media
BARODON .RTM.-4 passages cells* Jurkat RPMI - 1640 + Control P7
Excellent leukemic 10% FCS 1/900 P7 97% Died leukocytes in P7 of
human 1/800 P7 99% Died in P2 1/700 P2 99% Died 1/600 P2 100% Died
in P2 NCI-H69 RPMI - 1640 + Control P6 Excellent Small cell 10% FCS
1/900 P6 81% Died carcinoma in P6 of human 1/800 P5 98% Died lung
in P5 1/700 P2 100% Died in P2 1/600 P2 100% Died in P2 SW579 RPMI
- 1640 + Control P10 Excellent Thyroid 10% FCS 1/900 P10 Excellent
carcinoma 1/800 P10 Excellent of human 1/700 P10 Very good 1/600 P9
Good U-20S Mccoy`s 5A + Control P10 Excellent Osteo- 15% FCS 1/900
P10 Excellent genic 1/800 P10 Very good Sarcoma 1/700 P9 39% of
Died in human P2** 1/600 P9 47% Died in P2** *dyed with 0.4%
tryphan blue and counted under a microscope **100% dead after
P3
[0099] As apparent from Tables 11 and 12, the compostitions of the
present invention have inhibitory activity against human tumor
cells while giving no damages to normal cells. Therefore, the
present invention can be used for the effective treatment of human
tumors of some kinds even though the efficacy may be different
depending on kinds of tumors.
EXAMPLE 10
Effect on Freshness Retention
[0100] Fresh cods were flash-frozen with dry ice before being
transported to a laboratory. After being immersed in one of a
BARODON.RTM.-free solution, a 0.05%, 0.1% or 0.5% BARODON.RTM.-2
solution for 10 min, they were stored at 0.degree. C. for 7 days
during which their freshness was examined.
[0101] As a fresh index for fishes, the activity of muscular
Ca-ATPase, which is connected with protein denaturation, was
measured in the subjects. The best results were observed in the
cods immersed in the 0.5% BARODON.RTM.-2 solution, indicating the
retention of the original freshness for 5 days, as shown in FIG.
8.
EXAMPLE 11
Effect on Water Activation
[0102] Aqueous solutions in which BARODON.RTM.-5 was diluted at
dilution ratios of 1:2,800 and 1:5,600 with tap water were measured
for .sup.17O-NMR of H.sub.2O. The spectroscopic examination was
conducted at 20.degree. C. with the aid of a spectrophotometer
JNMEX-270 at the Japanese Water Science Research Meeting, Japan,
using as a control tap water. .sup.17O absorption wavelength bands
were measured at 149.6 Hz (full width at half maximum) for the
control (FIG. 9), 53.6 Hz for the 2,800-fold diluted solution (FIG.
10), and 54.7 Hz for the 5,600-fold diluted solution (FIG. 11).
[0103] These spectroscopic analysis results indicated that the tap
water is of a heterologous phase, resulting from the aggregation of
various water molecule combinations which differ from one to
another in the number of bound water molecules whereas the
BARODON.RTM.-containing solutions are of near crystalline phases in
which water molecule combinations are composed of constant, minimal
numbers of bound water molecules. With smaller numbers of bound
water molecules, water is in a more active state, showing better
fluidity and biopermeability. Because activated water can be useful
for the growth of animals and plants, the composition of the
present invention can be used as a beverage additive or a
wastewater-treating agent.
EXAMPLE 12
Behavior of BARODON.RTM. Upon Neutralization
[0104] BARODON.RTM.-4, prepared in Preparation Example 4, was of
strong alkalinity with pH 13.20. When BARODON.RTM.-4 was
neutralized with 0.1 N HCl, a precipitate formed at the contact
area. Further, the composition did not show detectable pH change
even after addition of a considerable amount of HCl. This behavior
indicates that the composition of the present invention is highly
reactive because of being sufficiently ionized.
[0105] While a 1,000-fold dilution of the composition was
neutralized with 0.1 N HCl, neither coagulates nor bubbles were
observed under a microscope. In this case, a change occurred in the
pH.
[0106] From this behavior of BARODON.RTM. a upon neutralization, it
can be recognized that, when being used at an appropriate amount,
the composition of the present invention does not produce
coagulates after the reaction with HCl within human and animal
stomach. In addition, no gas is produced by the introduction of the
present invention in the stomach, so that the composition is free
of the problems associated with gas production.
[0107] When being applied to animals and plants, as described
hereinbefore, the composition of the present invention can bring
about an improvement in disease resistance, weight gain rate, crop
yield, crop quality, harvest time. Also, the composition of the
present invention shows nonspecific immunostimulating activities,
including antibody production and immune enhancement, by activating
immune cells, thereby maximizing vaccination effects versus
malignant virus diseases.
[0108] With remarkable inhibitory activity against some kinds of
human tumors, the composition of the present invention can be used
as a therapeutic or a preventive agent.
[0109] In addition, when the composition of the present invention
is fed in mixture with feedstock to livestock, the sheds in which
the livestock lived were found to have less offensive odors and the
feedstuff is little infested with noxious insects. Further, the
composition of the present invention is highly useful as a
preservative for keeping foods fresh.
[0110] The present invention has been described in an illustrative
manner, and it is to be understood that the terminology used is
intended to be in the nature of description rather than of
limitation. Many modifications and variations of the present
invention are possible in light of the above teachings. Therefore,
it is to be understood that within the scope of the appended
claims, the invention may be practiced otherwise than as
specifically described.
* * * * *