Penjelasan IMSdanPATEN

Berikut LINK MIMS untuk produk:
Utsukushhii: https://www.mims.com/indonesia/drug/info/utsukushhii
Sop Subarashi: https://www.mims.com/indonesia/drug/info/utsukushhii
UTSUKUSHHII
BPOM RI ML 867031004482
Patent Manfaat :
• JP-3040711B2 : Anti Tumor Agent…………………………………………………….. Hal. 03
• JP-3272023B2 : Menaikkan System Immun Tubuh……………………………………. Hal. 07
• JP-2712000 : Membantu Terapi Hepatitis C……………………………………………. Hal. 11
SOP SUBARASHI (Triple Peptide)*

BPOM ML 830531001482
Patent Manfaat :
• JP-3946238 : Efek regenerasi sel…………………………………………………….…. Hal. 15
• JP-3946239 : Efek dalam perbaikan sirkulasi pembuluh darah………………………… Hal. 19
• JPW O2003055901A1 : Anti hipertensi agent………………………………………….. Hal. 24
• JP 2009538895A : Mencegah terjadinya pembekuan darah……………………………. Hal. 47
PATEN FUNGSI artinya MUTLAK HASILNYA, tapi cepat atau lambat hasilnya di tiap orang
berbeda-beda (tergantung dari banyak faktor, misalkan tingkat kerusakan sel, pola makan, pola
hidup, dll).
LINK CHECK PATENT :
UTSUKUSHII:
https://patents.google.com/patent/JP3040711B2/en?oq=JP-3040711B2
https://patents.google.com/patent/JP3272023B2/en?oq=JP-3272023B2
https://patents.google.com/patent/JP2712000B2/en?oq=JP-2712000
SUBARASHI:
https://patents.google.com/patent/JP3946239B1/en?oq=JP3946239
https://patents.google.com/patent/JP3946238B1/en?oq=JP3946238
https://patents.google.com/patent/JPWO2003055901A1/en?oq=JPWO2003055901A1
https://patents.google.com/patent/JP2009538895A/en?oq=JP2009538895A
UTSUKUSHHII
Petent pertama: JP-3040711B2 : Anti Tumor Agent
Description
DETAILED DESCRIPTION OF THE INVENTION

[0001]
[0001] The present invention relates to an antitumor agent and a method for producing the same.
[0002]
2. Description of the Related Art Most of anti-tumor agents which are currently mainly used
have a function of acting as a blocking agent at the time of cell proliferation. Alkylating agents
and platinum preparations that have a DNA synthesis inhibitory action, antimetabolites that are
taken up during cell metabolism and inhibit the action of enzymes, and antitumor drugs that act
on metabolic enzymes and nucleic acids derived from natural products to exert antitumor effects
Examples include cancer antibiotics and plant alkaloids.
Most of these drugs act specifically on cells that are actively undergoing cell division, so that
both normal cells and bone marrow cells and intestinal epithelial cells, which have a relatively
fast cell division cycle, can be treated similarly to cancer cells. The drug works. For this reason,
serious side effects such as a decrease in immunity due to a decrease in leukocyte function and
gastrointestinal tract disorders are observed.
[0003]
It is known that immune cells such as lymphocytes have an action of recognizing and damaging
tumor cells. Therapy using it is BRM (Biological ResponseMod)
ifiers) therapy, in which immune cells in the body, particularly effector cells such as T cells, are
activated by administering a bacterial-derived substance, a polysaccharide, or the like, thereby
increasing the rate of damage to tumor cells. Until now, Lentinan, OK-432, P
Immunostimulants such as SK and cytokines such as interferon and interleukin have been used.
However, these drugs also have side effects such as rash, drug hypersensitivity, and fever, and in
particular, injection preparations tend to have more side effects.
[0004]
The chemotherapeutic agent currently used as an antitumor agent always has side effects due to
its mechanism of action. On the other hand, BRM preparations are used for the purpose of
restoring the immunity, which is reduced due to cancer, or suppressing the growth rate of tumors.
Therefore, there is a need for a drug that has no side effects, has higher immunity in the body,
and is highly effective in suppressing tumor growth.
[0005]
It is known that the genus Enterococcus, which is a kind of lactic acid bacterium, has a function
of a BRM preparation, that is, an antitumor effect by enhancing immunity (Ohashi et al.,
Pharmaceutical Journal, 113). , 396-399 (1
993)). The present inventors have conducted various studies in order to obtain such cells having
a stronger antitumor action or a processed product thereof. As a result, the processed cells
obtained by treating the cells of lactic acid bacteria with an enzyme were found to be non-
enzymatically. The present inventors have found that they have a much stronger antitumor effect
than the treated cells, and have completed the present invention.
[0006]
BEST MODE FOR CARRYING OUT THE INVENTION The present invention is
characterized in that a lactic acid bacterium belonging to the genus Enterococcus is acted on by
an enzyme, and further subjected to a heat treatment to obtain a treated product of the bacterium.
The enzymes used in the present invention include lysozyme, chitinase, N-
Any enzyme can be used as long as it is a lytic enzyme such as acetylglucosaminidase or a
converting or degrading enzyme for a cell surface structure or an antibiotic, and the antitumor
effect of the cell is enhanced by the enzyme treatment. There are lysozymes, however, that give
the most effective results. Further, as an example of the cell treatment conditions, treatment at a
final concentration of lysozyme of 10 to 3000 μg / ml at 37 ° C. for 1 to 3 hours is preferable.
[0007]
To prepare the cells or a processed product thereof, carriers such as starch, lactose and soybean
protein, additives such as excipients, binders, disintegrants, lubricants, stabilizers and corrigents
are used. It is formulated into tablets and granules by well-known methods.
[0008]
The amount used varies depending on symptoms, age, etc.

As an active ingredient, 0.002 to 0.1 g / kg body weight per day can be usually administered to
an adult once or several times a day.
[0009]
EXAMPLES Examples will be shown below, but the present invention is not limited by the
descriptions of these examples.
Embodiment 1 FIG. (Culture of lactic acid bacteria) Enterococcus faecali
s) Inoculation of NF-1011 (Microtechnical Laboratories No. 12564) into Rogosa liquid medium
having the following composition, and the number of bacteria was 10:
6
/ ml), and cultured at 37 ° C for 10 to 16 hours.
A culture solution of 09 cells / ml was obtained. The obtained culture solution is 12,000 rpm
The cells were collected by centrifugation for 20 minutes and washed twice with distilled water
to obtain cells.
The composition of Rogosa liquid medium is shown. Trypticase 10 g Yeast extract 5 g Tryptose
3 g Monopotassium phosphate 3 g Dipotassium phosphate 3 g Triammonium citrate 2 g Tween
80 (surfactant) 1 g Glucose 20 g Cysteine hydrochloride 0.2 g Salt solution (as per 1) 5 ml
Distilled water 1000 ml (adjusted to pH 7.0, 121 ° C. 15 minutes heat-sterilized in) (1) saline: MgSO 4
· 7H 2 O 11.5g FeSO 4 · 7H 2 O 0.68g MnSO 4 · 2H 2 O 2.4g distilled 100 ml of water
Embodiment 2 FIG. (Enzyme treatment and heat treatment) The cells obtained in Example 1:
sterilized distilled water = 1: 10 (v /
Resuspended to the ratio of v). To this was added filter-sterilized egg white lysozyme for feeding
(manufactured by Taiyo Kagaku Co., Ltd.) to a final concentration of 100 μg / ml.
Incubated at 2 ° C. for 2 hours Next, this enzyme-treated cell was
Heat treatment in an autoclave at 10 ° C. for 10 minutes,
After the bacterial solution was cooled, it was dried with a freeze dryer to obtain a powder
sample.
Embodiment 3 FIG. (Heat treatment) The cells obtained in Example 1: sterilized distilled water =
1: 10 (v /
It was suspended to the ratio of v). This at 110 ° C for 10
After performing heat treatment by autoclave for 110 minutes,
The lysozyme used in Example 2, which had been inactivated by heating for 10 minutes, was
added in an amount corresponding to a final concentration of 100 μg / ml. Next, freeze-drying
was performed to obtain a powder sample.
Embodiment 4 FIG. (Anti-Tumor Effect) C3H / HeN mice (male, 7 weeks old) purchased from
SLC Japan were administered according to the body weight according to the standard
administration group obtained in Example 2 (group A) and the standard obtained in Example 3.
Product group (group B) and control group (group C) were divided into three groups (10 animals
each).
MM4 was intraperitoneally injected into C3H / HeN mice.

Six weeks after transplantation of breast cancer, one week later, cancer cells were collected and
CaCl
2 and MgCl 2 .6H 2 O were suspended in a phosphate buffered saline (PBS (−)), and the cells
were washed three times by centrifugation at 700 rpm for 5 minutes.
The cell concentration was adjusted with (-). The cell suspension was transplanted into the
abdominal skin of the mice of all the above groups so that the number of viable cells was 1 ×
10 6 cells / animal.
Each of the preparations obtained in Examples 2 and 3 was mixed with a powdered feed (CE-2:
CLEA Japan) in an amount of 5% to prepare a mixed feed. From the day after the transplantation
of the MM46 breast cancer cells, the mouse groups (Group A and Group B)
Had free intake daily. Group C was fed a powdered feed without the standard.
After transplantation of the cancer, the size of the formed tumor was measured. From the fourth
day after cancer transplantation, the major axis (a) and the minor axis (b) were measured using
calipers every three days, and the size (diameter) of the tumor was determined by the following
formula. The results are shown in FIG. Tumor diameter (mm) = (a x b) 1/2
The group A and the group B have significantly smaller tumor diameters from day 18 after
cancer transplantation (risk ratio 1% to 5%) as compared with the group C.
This effect persisted up to 38 days after cancer transplantation. When group A and group B were
compared, the tumor diameter of group A tended to be smaller than 25 days after cancer
transplantation.
Forty-one days after transplantation of the cancer, the tumor portion of all mice was excised and
weighed. The results are shown in FIG. Compared to group C, group B showed significant
weight suppression at a 5% risk factor. On the other hand, group A also showed significant
suppression of tumor weight with a 1% risk factor,
The suppression effect was higher than that of group B.
[0020]
EFFECT OF THE INVENTION The enzyme-treated product of lactic acid bacteria obtained in
the present invention has no side effects and has a high immunostimulatory effect, so that tumor
growth can be more efficiently suppressed. In addition, it is also possible to use this treated
product with an existing anticancer agent.
[Brief description of the drawings]
FIG. 1 is a graph showing changes in tumor diameter of MM46 breast cancer.
FIG. 2 is a diagram showing the average of tumor weight per mouse.
────────────────────────────────────────────────── (5)
Continuation of the front page (51) Int.Cl. 7 Identification code FI C12R 1:01) (56) References
JP-A-8-27010 (JP, A) JP-A-56-158717 (JP, A) Pharmaceutical Magazine, Vol. 113, No. 5,
(1993), p. 396-399 Journal of the Japanese Society of Agricultural Chemistry, Vol. 69, No. 4,
(1995), p. 443-446 Okayama Magazine, Vol. 14, (1995), p. 89-105 Osaka University Dental
Journal, Vol. 39, No. 2, (1994), p. 356-377 (58) Field surveyed (Int. Cl. 7 , DB name) A61K
35/74 A61P 35/00 C12N 1/20 CA (STN) REGISTRY (STN)
Petent kedua: JP-3272023B2 : Menaikkan System Immun Tubuh
Description
[0001]
BACKGROUND OF THE INVENTION This invention is, Enterococcus (En

The present invention relates to a preventive / therapeutic agent for leukopenia containing cells of a
microorganism belonging to the genus terococcus ) or a processed product thereof.
[0002]
2. Description of the Related Art There are 100 kinds, 100 trillion intestinal bacteria inhabiting the human
intestinal tract. The presence of intestinal bacteria can be grasped as one ecosystem. In other words, the
types and amounts of the constituent microorganisms of the intestinal flora (flora) exist while maintaining
a complicated relationship between the microorganisms and the host organism, such as interdependence
and antagonism. This intestinal flora can be regarded as a single organ, and the importance of its role in
the host organism is increasingly recognized. Its role is directly related to development, differentiation,
immune activity, nutrition (digestion and absorption), cancer, heart disease, brain disease and other
diseases such as adult diseases and digestive diseases, as well as aging and longevity. Or it seems to be
indirectly related.
[0003]
Some chemotherapeutic agents (cancer drugs) for cancer have already been used for treatment, but not
only destruction of cancer cells but also side effects due to administration of chemotherapeutic agents,
especially leukocytes (neutral neutrophils) The number of spheres is remarkably reduced, and more
serious cases are often caused by immunodeficiency or bacterial replacement. Therefore,
Leukopenia, which is the greatest problem of chemotherapeutic agents, is also serious in diseases such
as bone marrow transplantation and aplastic anemia, and there is a need for a drug that prevents and
treats such leukopenia without side effects.
[0004]
As a result of the research, the present inventors have found that leukopenia is prevented by
microorganisms belonging to the genus Enterococcus, which are enterococci derived from the human
intestinal tract (resident in the human intestine). And found a therapeutic effect.
Furthermore, since the present bacterium is a kind of resident bacterium in the human intestinal tract,
virtually no toxicity by oral ingestion is recognized, which has led to the present invention.
[0005] As a microorganism belonging to the genus Enterococcus usable in the present invention is
Enterococcus faecalis (Enterococcus faecalis) and Enterococcus faecium (Enterococcus faecium) and
the like, particularly useful strains are healthy person feces by the present inventors Enterococcus
faecalis, a new strain isolated from Enterococc
us faecalis ) NF-1011. The strain has been deposited with the Institute of Microbial Industry and
Technology of the National Institute of Advanced Industrial Science and Technology under No. 12564 of
Microbial Laboratory Bacteria.
[0006] Enterococcus faecalis NF

1 shows the isolation means of -1011 and the mycological and physiological properties of the strain.
(1) Separation means A 10-fold dilution of feces of a healthy subject with heat-sterilized water is spread
on a suitable selective medium (KMN agar plate and SF agar plate), and is subjected to aerobic
conditions at 37 ° C. for 48 to 48 hours. After culturing for 72 hours, bacterial colonies appeared. The
bacterial colonies were streaked on another homogeneous plate medium and cultured in the same
manner to allow bacterial colonies to reappear. The same operation was repeated several times to
separate a single colony consisting of only a single strain. The microbial (morphological, biochemical and
serological) properties of this new isolate were examined and classified and identified as belonging
to Enterococcus faecalis .
[0008] (2) Bacteriological and physiological properties {Properties determination} ───────────────

Gram staining + bacterial morphology Spherical catalase-hemolytic α serogroup D Proliferative 10 ° C +
45 ° C + 50 ° C + heat tolerance 60 ° C for 30 minutes + bile esculin Growth in supplemented medium +
Growth in pH 9.6 medium + Growth in medium supplemented with 6.5% sodium chloride + Methylene
blue reducing + Gelatin liquefaction-Growth in medium supplemented with 0.01% TTC + Growth in
medium supplemented with tellurite + Presence or absence of acid generation Glycerol + L-arabinose-D-
ribose + D-xylose-D-glucose + D-galactose + D-fructose + D-mannose + maltose + mannitol + sucrose +
L-sorbo -D-sorbitol + L-rhamnose-lactose + amygdalin + esculin + cellobiose + melibiose-inulin-
melezitose + ＋ + Positive,-; negative TTC; 2,3,5-triphenyltetrazolium chloride
(Medium) As a typical medium for culturing microorganisms used in the present invention, a Rogosa liquid
medium having the following composition can be used. Trypticase 10 g Yeast extract 5 g Tryptose 3 g
Monopotassium phosphate 3 g Dipotassium phosphate 3 g Triammonium citrate 2 g Tween 80
(surfactant) 1 g Glucose 20 g Cysteine hydrochloride 0.2 g Salts Solution (1) 5 ml Distilled water 1,000 ml
(pH adjusted to 7.0 with sodium hydroxide solution, sterilized by heating at 121 ° C. for 15 minutes) (1)
Salt solution: MgSO 4 .7H 2 O 11.5 g FeSO 4 · 7H 2 O 0.68g MnSO 4 · 2H 2 O 2.4 g 100 ml distilled water
(Culture method) A microorganism belonging to the genus Enterococcus is inoculated into 10 ml of

Rogosa liquid medium and incubated at 37 ° C. for 10 minutes.
Aerobic stationary culture (pre-culture) is performed for about 16 hours to obtain a culture solution (seed)
of about 10 9 cells / ml. This is added to 10 liters of a sterilized Rogosa liquid medium, and the cells are
similarly statically cultured.
(Bacterial collection, washing and drying methods) The culture solution obtained as described above is
centrifuged at 8,000 to 12,000 rpm to obtain viable cells (precipitate). The viable cells are washed twice
with a physiological saline solution (0.85% NaCl aqueous solution) twice (similarly to the centrifugation
operation), and then suspended in distilled water to obtain 100 ml of a bacterial solution (about 10 11 cells
/ ml).
The obtained viable cell suspension is heated at 110 ° C. for 10 minutes to obtain a dead cell suspension.
Next, drying is performed by an appropriate method such as a hot air drying method or a freeze drying
method to obtain dried dead cells.
As the agent of the present invention, the dead cells obtained as described above, or their crushed
products and aqueous extracts can be used. In order to formulate these, starch, lactose, carriers such as
soy protein, excipients, binders, disintegrants, lubricants, stabilizers, tablets and tablets by known
methods using additives such as flavoring agents Formulated in granules.

As an active ingredient, 0.002 to 0.1 g / kg body weight per day can be usually administered to an adult
once or several times a day.
[0015]
EFFECT OF THE INVENTION Since the Enterococcus genus of the present invention is an intestinal
lactococci, it has no toxicity and no side effects,
It has a prophylactic or therapeutic effect on leukopenia due to administration of chemotherapeutic agents
such as immunosuppressants.
[0016]
【Example】
Embodiment 1 FIG. (Preparation of bacterial cells) Enterococcus faecalis NF-1011 was inoculated into 10
liters of Rogosa liquid medium (the number of bacteria: 10 6 cells / ml), and 37
The culture was allowed to stand at 16 ° C. for 16 hours to obtain a culture solution with about 10 9 viable
cells / ml. The obtained culture solution was centrifuged at 12,000 rpm to collect the cells, and this was
collected in a physiological saline (0.85% NaC).
aqueous solution), suspended in 100 ml of distilled water,
A bacterial solution was obtained. This bacterial solution was heated at 110 ° C. for 10 minutes, and then
freeze-dried to obtain dead bacterial cell powder.
Embodiment 2 FIG. An ICR mouse (male, 6 weeks old, body weight 23 ± 2 g) was treated with mouse
solid feed (CE-
2) and sterilized tap water, and the room temperature (25
(± 2 ° C.) and 60 ± 10% humidity for one week. The mice were divided into two groups (5 mice per group),
and one group was orally administered with a feed containing 0.25% of the bacterial cell preparation
obtained in Example 1 and containing 5% ethanol from the 7th day. They had free access to drinking
water. The other group was orally administered a feed containing no cell preparation, and similarly, on
day 7, they were allowed to freely ingest 5% ethanol-containing drinking water. Both groups were bred for
28 days after administration of ethanol-containing drinking water.
Blood was collected from the orbital vein on the 7th and 28th days after administration of ethanol-
containing drinking water, and the white blood cell count was measured using a hemocytometer. Table 1
shows the white blood cell count. In the above-mentioned group treated with the bacterial sample, an
improvement and a therapeutic effect on leukopenia due to oral ingestion of alcohol were observed.
Table 1 エタノール Days of ethanol administration ───────────────── {728} Control group

5100 ± 591 4030 ± 760 Bacterial group 4933 ± 262 5830 ± 83 ─────────────────────
Numerical values are mean ± SD (per μl)
Embodiment 3 FIG. Healthy dogs (cross-breeds, 1 to 3 years old, male or female, weighing about 10 kg)
were fed dog food (dry and canned) manufactured by AGF and drinking water, and bred in a normal
breeding environment.
Each dog was intravenously administered 10 mg / kg of cyclophosphamide (Endoxane, Shionogi

Pharmaceutical Co., Ltd.) for three consecutive days, and experimentally caused leukopenia. 1 group 15
At the same time as cyclophosphamide administration, the bacterial sample obtained in Example 1 was
dissolved or suspended in physiological saline (0.85% NaCl aqueous solution) in one group.
0mg / kg body weight, mix with feed
Oral administration once a day for 14 consecutive days. The other group was similarly fed with a diet
containing no cell preparation. All animals of both groups were observed for general health during the
experiment, and examined for blood properties and bone marrow.
(1) Effect on White Blood Cell Count In the control group to which a diet containing no cell preparation
was administered, the white blood cell count became the lowest on day 8 after cyclophosphamide
administration, and then increased. On the other hand, the white blood cell count was the lowest on day 7
in the group treated with the above-mentioned cell preparation, and the recovery was faster than that in
the control group. There was a tendency for many (FIG. 1).
(2) Bone marrow image The bone marrow component collected by performing a bone marrow puncture
by an ordinary method is smeared,
Observation was made for a bone marrow image. Table 2 shows the ratio (M / E ratio) of the total number
of granulocytic cells (M) to the total number of erythroid cells (E) on days 5, 11, and 14 from
cyclophosphamide administration.
It was shown to. In particular, the M / E ratio of the control group on the fifth day showed a remarkably low
value, whereas the M / E ratio in the group treated with the bacterial cell standard
An increase in the E ratio was observed, and administration of the bacterial cell preparation improved the
suppression of bone marrow function associated with the use of chemotherapeutic agents, neutrophils
(white blood cells)
Enhancement of production was observed.
Table 2 ───────────────────────── Days ５ 5 11 14 ─────────────────────────

Control group 0.67 1.28 0.95 Bacterial cell administration group 2.34 2.44 1. 73
─────────────────────────
(3) Side effects and deaths The general health condition of each dog after administration of
cyclophosphamide (fever, activity, appetite, vomiting, cough, dehydration, purpura, hematuria,
Cystitis or the like) was observed. In the group treated with the bacterial cell, the side effect (general
health condition) due to cyclophosphamide treatment was mild throughout the entire experimental period.
In the control group, there were deaths, whereas in the group treated with the bacterial cell, there were no
deaths (Table 3).
[0026]
Embodiment 4 FIG. (Formulation example) (1) 150 mg of the dead bacterial cell powder obtained in
Example 1 is mixed with 150 mg of purified starch powder and 700 mg of lactose to prepare tablets or
granules.
(2) 300 mg of dead bacterial cell powder obtained in Example 1

Is mixed with 300 mg of soy protein and 400 mg of lactose to make tablets or granules.
As shown in Examples 2 and 3 above, it was confirmed that the dead bacterial cell powder of the present
invention has remarkable preventive and therapeutic effects on leukopenia, and has little or no side
effects. Was done.
FIG. 1 is a graph showing a white blood cell count recovery effect.
──────────────────────────────────────────────────続き Continuation of the

front page (72) Inventor Hideyo Yamaguchi 2-15-5 Kuriya, Tama-ku, Kawasaki City, Kanagawa
Prefecture (56) References JP-A-62-103023 (JP, A) International Publication 89/425 (WO , A1) (58) Field
surveyed (Int. Cl. 7 , DB name) A61K 35/74 CA (STN) BIOSIS (DIALOG) MEDLINE (STN)
Claims (1)
Hide Dependent
(57) [Claims]
1. 1. A preventive agent for leukopenia, comprising a heat-killed cell of a microorganism belonging

to the genus Enterococcus or a processed product thereof as an active ingredient.
Petent ketiga: JP-2712000 : Membantu Terapi Hepatitis C
Description
[0001]
BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a
therapeutic agent for hepatitis C, comprising a lactic acid bacterium or a processed product thereof as an
active ingredient.
[0002]
2. Description of the Related Art In 1989, hepatitis C virus (HC)

V) A method for measuring gene antibodies has been developed,
95% of about 200,000 patients annually who are said to have non-B hepatitis
Was found to have hepatitis C, and about 70,000 C
Hepatitis patients are increasing.
[0003]
Hepatitis C is most commonly transmitted by blood, such as transfusion of blood or blood products, or
infection of patients or HCV carriers by instruments contaminated with the blood of HCV, but unlike
hepatitis B, it is caused by sexual activity. Infection, family transmission, and mother-to-child transmission
are low.
[0004]
The natural cure rate of hepatitis C is low, and after the onset of acute hepatitis, persistent infection
occurs, and about 60% of cases shift to chronic hepatitis, and some progress to cirrhosis and
hepatocellular carcinoma. In addition, even in the case of primary infection in adults, about 70% of the
people become carriers and shift to chronic hepatitis. Therefore, the number of chronic patients with
hepatitis C is currently 1
It is estimated that over half a million people and careers will exceed two million.
[0005]
At present, as a drug for treating chronic active hepatitis C, an antiviral agent, interferon (IF) is used.
N) and two types of strong neo-minophagen C (SNMC), which is a glycyrrhizin preparation, are mainly
used.
[0006]
INF has an action of destroying HCV-RNA, but requires long-term administration, and about half of the
group with normal GPT levels have recurred hepatitis after discontinuing INF administration. In addition,
side effects are large, such as influenza-like symptoms (headache, fever, joint pain, muscle pain,
anorexia, general malaise, nausea, vomiting, etc.), loss of effect due to induction of INF antibodies,
autoimmune diseases, depression symptoms ( Insomnia, irritability, etc.), hair loss, thyroid dysfunction,
blurring, etc. In particular, the sudden increase in suicides due to depression is a major problem.
[0007]
SNMC has an anti-inflammatory effect, and a lower transamylase effect has been confirmed. In chronic
hepatitis, 20
４０40 ml / day intravenous injection or 5% glucose 200-50
0 ml and 4 weeks or more by intravenous drip. If liver damage is strong and GPT value is more than 200
IU / dl, 100 ml / day is mixed with 200-500 ml of 5% glucose and 4 to 8 weeks by intravenous drip. Long-
term large dose administration. SNM
Glycyrrhetinic acid, which is produced by the conversion of glycyrrhizin, which is the main component of
C, has the effect of suppressing the inactivation of corticosteroids in the liver, so no side effects have
been reported with small doses of SNMC. It has been reported that glycyrrhetinic acid causes
pseudoaldosterone symptoms such as hypertension and edema, and hypokalemia during the
administration period has also been observed.
[0008]
Although there are oral preparations containing glycyrrhizin, the active ingredient glycyrrhizin is converted
to glycyrrhetinic acid in the stomach and loses its original activity. The usage is low because it can be
seen.
Although currently limited to hepatitis B, immunomodulators have also been used. This drug has the
effect of stimulating immune cells and enhancing immunity against chronic hepatitis whose immunity is
mildly reduced. Examples of immunomodulators include Sho-saiko-to, OK-432 (Picibanil), Lentinus
edodes
mycelia (LEM), recombinant interleukin 2 (r
IL-2).
When liver damage occurs, GOT (glutamate oxaloacetate transaminase) in the blood
The abnormalities of the liver can be known by remarkably increasing the values and GPT (glutamate
pyruvate transamylase) values. Γ-G for alcohol overdose
TP (gamma glutamyl transpeptidase) shows a high value. In acute hepatitis, GOT value is several
hundred to several thousand IU / dl
To go to chronic hepatitis.
The T value is less than 1000 IU / dl, and the GPT value is rather high. Also, with appropriate treatment,
these values can be reduced to the normal range. Thus, these test values are important as indices
indicating liver damage or liver function status.
[0011]
At present, most of the above-mentioned drugs used for treatment are injections. Moreover, in the case of
long-term parenteral administration, antibodies to the administered drug are likely to be generated, in
which case the effects are diminished and sometimes serious side effects are caused. Therefore, for
chronic hepatitis that requires long-term treatment, it is impossible to treat only outpatients.
Or they are forced to stay inside and outside the hospital for long periods of time with daily visits. Such
treatments are particularly time consuming, physically and mentally and economically burdensome for
patients, limiting their range of activity and quality of life (QOL). It will take time to reintegrate into society
by imposing a decline.
In order to achieve early rehabilitation of patients, it is preferable to use internal medicine that can be
used easily even at home. Moreover, there are no side effects as described above,
There is a need for drugs that have strong pharmacological effects.
[0013]
Means for Solving the Problems In order to solve this problem, the present inventors have found that a
method of enhancing immunity in the body and promoting the elimination of virus is effective in treating
hepatitis and preventing recurrence of viral antigens. As a result of repeated studies based on the fact
that there is, it is found that oral administration of killed cells of lactic acid bacteria having
immunostimulatory effects has a remarkable therapeutic effect on chronic active hepatitis C or an effect
that completely cures it. Thus, the present invention has been completed.
Since the agent of the present invention is a lactic acid bacterium cell or a processed product thereof, it
has no toxicity and has no concern about side effects. It can also be used in combination with other
drugs, and is effective even when administered to patients who are ineffective with IFN therapy.
The lactic acid bacterium cells or processed products thereof used in the present invention are formulated
by using a carrier such as starch, lactose, soy protein, etc.
Tablets and granules can be prepared by known methods using additives such as excipients, binders,
disintegrants, lubricants, stabilizers, and corrigents.

As an active ingredient, 2 to 100 mg / kg body weight per day can be usually administered to an adult
once or several times a day.
[0017]
Industrial Applicability The therapeutic agent for hepatitis C of the present invention has no side effects
and toxicity and can promptly treat chronic active hepatitis C. Since this drug can be used regardless of
the subtype of hepatitis virus, it can be used in patients who are not effective by IFN therapy. In addition,
since it is not necessary to go to the hospital every day for oral administration and can be used easily
even at home, it is possible for the patient to return to society early.
[0018]
EXAMPLES Examples will be shown below, but the present invention is not limited by the descriptions of
these examples.
Embodiment 1 FIG. (Culture of Enterococcus) Enterococcus faecali

s) Inoculation of NF-1011 (Microtechnical Laboratories No. 12564) into Rogosa liquid medium having the
following composition, followed by the number of bacteria: 10 6
Cells / ml) at 37 ° C. for 10 to 16 hours to obtain a culture solution with about 10 9 viable cells / ml. The
obtained culture solution is
The cells were collected by centrifugation at 2,000 × g for 20 minutes and washed twice with distilled
water to obtain cells. The cells were suspended in distilled water and heated at 110 ° C. for 10 minutes to
obtain a dead cell suspension.
Next, the cells were dried by a suitable method such as hot air drying or freeze drying to obtain dried
dead cells.
The composition of Rogosa liquid medium is shown below. Trypticase 10 g Yeast extract 5 g Tryptose 3 g
Monopotassium phosphate 3 g Dipotassium phosphate 3 g Triammonium citrate 2 g Tween 80
(surfactant) 1 g Glucose 20 g Cysteine hydrochloride 0.2 g Salt solution (as per 1) 5 ml Distilled water
1000ml (pH
Adjusted to 7.0, 15 minutes heat sterilized at 121 ℃) (1) saline: MgSO 4 · 7H 2 O 11.5
g FeSO 4 · 7H 2 O 0.68g MnSO 4 · 2H 2 O 2.4g distilled water 100ml
Clinical Example 1 54-year-old man Hepatitis (Hepatitis) was pointed out in 1979, and he was diagnosed
with chronic active hepatitis C. He had been treated with SNMC, but had a GOT value of 100 IU / dl and a
GPT value of 120 IU / dl.
Since the effect of the present invention containing the dried Enterococcus faecalis cells obtained in
Example 1 was obtained from June 1993, the amount of the present invention was reduced to 10 mg /
day from June 1993 because there was no effect because the concentration of the cells did not decrease
to the normal value. The patient was taken once a day at a daily weight of kg. About half a year later, the
blood test value in January 1994 was a GOT value of 45 IU / d.
1. The GPT value was only 61 IU / dl, a slight increase from the normal value, the γ-GPT value was 28 IU
/ dl, and the serum amylase value was 104 IU / dl, each of which fell to the normal range.
When a liver biopsy was performed in February of the same year, there was almost no tissue wetting and
no tendency to reconstruct lobules was observed. Therefore, IF
There was no need for N adaptation, and periodic follow-up was diagnosed as good.
Clinical Example 2 50-year-old male In November 1990, hepatitis C was diagnosed, and Sho-saiko-to
and SM
Although the combination therapy of NC was continued and the administration of IFN was continued for
one year (3 million units / day) at the same time, when the administration was stopped with remission of
the symptoms, the GOT and GPT values were increased again. Dosing (10 million units / day) was
started. Because the GOT and GPT values decreased after 3 months of administration, when the
treatment was interrupted again, both values increased again, and deterioration of liver function was
observed. As described above, since the blood test values are not good and the therapeutic effect cannot
be obtained, the agent of the present invention including the dried Enterococcus faecalis cells obtained in
Example 1 obtained in November 1993 as a cell mass of 45 mg from November 1993. / Kg
Body weight was taken once daily. Four months later, the blood test values in March 1994 were as
follows: GOT value was 130 IU / dl to 80 IU / dl.
GPT value decreases from 265 IU / dl to 139 IU / dl,
The γ-GTP value dropped to the normal value of 56 IU / dl.
Clinical Example 3 A 48-year-old male was found to have high GOT and GPT levels at a health checkup
in 1990, and became HCV antibody-positive in September 1991, and was diagnosed with hepatitis C.
After that, IFN administration (3 million units /
Day), and after one year, the GOT value and the GPT value both decreased to a normal range of 25 to 45
IU / dl and became stable. When the administration was stopped, the GOT value was 90 IU / dl.
The GPT value rose again to 145 IU / dl.
In the month, she was hospitalized and re-administered IFN (10 million units / day). GOT value 30 IU / dl,
GPT 2 months after re-administration
Since the value was 45 IU / dl, when the operation was stopped again, the GOT value was 70 IU / dl and
the GPT value was 140 IU / dl again.
Repeated attempts at IFN therapy showed a similar course.
As described above, despite improvement by IFN therapy, relapse occurred after discontinuation of
administration, and side effects during administration were strong. Therefore, IFN therapy was
discontinued in January 1994.
From February 1994, the above-mentioned patient was orally administered once daily with 45 mg / kg
body weight of the agent of the present invention containing the dried cells of Enterococcus faecalis
obtained in Example 1 as the amount of cells. Four months later, the blood test value in July 1994 was G
OT value from 80 IU / dl to 41 IU / dl, GPT value 180
It decreased from IU / dl to 54 IU / dl. No side effects were observed during administration.
Clinical Example 4 52-year-old woman In 1986, the GOT suddenly became 1500 IU / dl, and she was
discharged one month after emergency hospitalization. Later, another hospital pointed out hepatitis C. He
accidentally inserted a needle to which hepatitis C patient's blood adhered while he was working as a
nurse, which is considered to be the cause. Unable to receive IFN physically, around GOT90IU / dl,
GPT120I
With the figures around U / dl, I didn't make any mistakes. From July 1994, the patient was administered
the agent of the present invention containing the dried cells of Enterococcus faecalis obtained in Example
1 at a dose of 45 mg / kg body weight daily. One month later, the blood test value in August 1994 was
compared to the value tested in January of the same year, and the GOT value was 86 IU / dl to 55 IU / dl.
The PT value dropped from 120 IU / dl to 64 IU / dl.
Clinical Example 5 A 58-year-old female was diagnosed with a disease in 1983 when she was a nurse
and accidentally inserted a needle to which hepatitis C blood had adhered.
FN therapy was given. When the first test was performed, side effects such as fever were strongly
exhibited, but the liver function values on blood biochemistry did not decrease so much. The
administration was repeated with different types of IFN. A few days after administration, visual field loss
due to fundus bleeding occurred, and IFN administration was stopped. From June 1994, the patient was
administered the agent of the present invention containing the dried cells of Enterococcus faecalis
obtained in Example 1 at a dose of 45 mg / kg body weight daily. Table 1 shows the values before and
after drinking the agent of the present invention.
[0027]
The unit is IU / dl for GOT, GPT and LDH.

Embodiment 2 FIG. (Preparation Example) (1) 50 mg of the dried cells obtained in Example 1 were mixed
with 50 mg of purified starch powder and 200 mg of lactose,
Make into tablets or granules.
(2) 10 cells of the dried cells obtained in Example 1
0 mg, soy protein 100 mg and lactose 200 m
g) to make tablets or granules.
Claims (2)
Hide Dependent
(57) [Claims]
1. 1. A therapeutic agent for hepatitis C, which comprises a lactic acid bacterium or a processed
product thereof as an active ingredient.
2. 2. The method according to claim 1, wherein the lactic acid bacterium is of the genus
Enterococcus.
2. The cell of the microorganism belonging to s) or a processed product thereof.
Hepatitis C therapeutic agent as described above
SUBARASHI
Patent pertama: JP-3946238 : Efek regenerasi sel
IGF-1 level increasing agent
Abstract
The present invention provides a new use of components extracted from ovarian membranes of fish.
An anti-aging agent comprising a component extracted from the ovarian membrane of fish, which can
increase again the IGF-1 value decreased by aging, alleviating and improving various symptoms
associated with the aging can do. The component extracted from the ovarian membrane is a component
extracted by treating the ovarian membrane with a proteolytic enzyme. The ovarian membrane is a spider
ovarian membrane.
[Selection] Figure 2
Description
The present invention relates to an agent for increasing IGF-1 level .
Conventionally, a method of extracting amino acids and peptides by treating the ovarian membrane (fish
egg rind) of fish with ozone water in advance and then enzymatically degrading myofibrillar protein, which
is a constituent protein thereof, is known (for example, patent document). 1).
The amino acids and peptides can be used as physiologically active substances or as food fortifiers. More
specifically, the amino acids and peptides have ACE inhibitory activity and act as blood pressure increase
inhibitors (hypertensive agents).
However, it is desired to develop more uses for the components extracted from the ovarian membrane of
fish.
JP 2004-73186 A
In view of such circumstances, an object of the present invention is to provide a new use of a component
extracted from the ovarian membrane of fish.
In order to achieve such an object, the agent for raising IGF-1 level of the present invention is
characterized by containing a component extracted by treating the ovarian membrane of sputum with a
proteolytic enzyme .
In the living body, after the adulthood, various symptoms appear, such as deterioration of metabolism
with aging, becoming lethargic, rough skin texture, no gloss, no stickiness, more spots and freckles. The
various symptoms associated with the aging are grasped as a decrease in IGF-1 value, and it is usually
said that the IGF-1 value once decreased does not increase again.
However, according to the IGF-1 level increasing agent of the present invention, the IGF-1 level can be
increased again, and various symptoms associated with the aging can be alleviated and improved.
Next, embodiments of the present invention will be described in more detail with reference to the
accompanying drawings. FIG. 1 is a graph showing changes over time in IGF-1 values when an IGF-1
level-increasing agent of this embodiment is ingested and when a placebo (placebo) is ingested, and FIG.
2 is a graph showing IGF-1 of this embodiment. FIG. 3 is a graph showing the amount of change in IGF-1
value when taking a value increasing agent and when taking a placebo (placebo), FIG. 3 shows the
physical condition after ingesting the IGF-1 increasing agent of this embodiment for 8 weeks It is a graph
which shows the comparison with the physical condition 2 weeks after an intake stop.
The IGF-1 level increasing agent of this embodiment includes a component extracted from the ovary
membrane of rabbit (hereinafter abbreviated as an ovarian membrane extraction component). The
ovarian membrane extract component can be obtained by, for example, a method of filtering a solution
obtained by performing enzyme treatment on the ovary membrane of salmon and drying the obtained
filtrate.
Specifically, in the method, first, the ovarian membrane of the pupa is used as a raw material, and water
is added to the ovarian membrane in a weight ratio of ovarian membrane: water = 1: 1 to 1: 3, and the
mixture is stirred and mixed. Furthermore, a proteolytic enzyme is added in the range of 1 to 3% by
weight with respect to the total amount of the ovarian membrane, and heated at a temperature of 45 to 55
° C. for 30 minutes to 5 hours, preferably 2 hours. If it does in this way, the component decomposed |
disassembled with the said proteolytic enzyme will elute in water among the components of the said
ovary membrane, and the aqueous solution of this component will be obtained.
Next, the proteolytic enzyme contained in the aqueous solution is inactivated. The deactivation can be
performed, for example, by heating the aqueous solution at a temperature of 90 ° C. for 5 minutes.
Next, the aqueous solution is simply filtered through a wire mesh of about 30 mesh to remove coarse
materials such as undecomposed ovarian membranes. Then, activated carbon is added to the obtained
filtrate to deodorize, decolorize, and degrease the filtrate. The deodorization, decolorization, and
degreasing of the filtrate is performed by adding activated carbon in the range of 2 to 4% by weight to the
total amount of the ovarian membrane as the raw material and heating the filtrate at a temperature of 60 °
C. for 30 minutes, for example. be able to.
After the deodorization, decolorization, and degreasing treatment with the activated carbon, the filtrate is
filtered by, for example, a filter press, and the obtained filtrate is concentrated at a temperature of 60 ° C.
under reduced pressure, for example, and then maintained at a temperature of 80 ° C. for 10 minutes, for
example. And sterilize. Then, the ovarian membrane extract component can be obtained by drying the
sterilized filtrate by spray drying. The ovarian membrane extract component contains amino acids,
peptides, vitamins, minerals, sugars, enzymes, nucleic acids and their metabolites, various growth
factors, cytokines, and the like.
The IGF-1 level increasing agent of the present embodiment is prepared by, for example, formulating the
ovarian membrane extract component in the form of a tablet or the like. 1 value can be raised again, and
various symptoms accompanying the aging can be alleviated and improved.
Next, examples of the present invention and comparative examples will be described.
In this example, first, an ovarian membrane extract component of salmon was formulated in the form of a
tablet to produce an IGF-1 increasing agent . The tablet consists of 245 mg of the ovarian membrane
extract and 5 mg of excipient (Loveli wax (registered trademark)) and has a diameter of 8 mm.
Next, 10 healthy female monitors aged 38 to 42 years old took 4 tablets per day for 8 weeks as a health
food. Each monitor has not taken supplements or medicines (including herbal medicines) since one
month ago.
And before the start of ingestion, 4 weeks after the start of ingestion and 8 weeks after the start of
ingestion, a blood test was performed to measure the IGF-1 value. The average value of IGF-1 values of
the 10 monitors is shown in FIG. 1, and the amount of change in the average value of IGF-1 values from
the start of intake to the 4th week and from the start of intake to the 8th week is shown in FIG. . In FIG. 1,
“0 week” is shown before the start of ingestion, “4 weeks” is the 4th week of ingestion, and “8 weeks” is
the 8th week of ingestion. “0-4 weeks” and “8-8 weeks” from the start of intake are described.
Moreover, regarding the physical condition by self-report of each monitor, the state of the 8th week from
the start of intake and the state after 2 weeks from the stop of intake were compared. The results are
shown in FIG.
[Comparative Example]
In this comparative example, first, a placebo (placebo) consisting of 125 mg of corn starch and 125 mg of
lactose was formulated in the form of a tablet with a diameter of 8 mm instead of the IGF-1 level
increasing agent of the above example.
Next, the placebo capsules were administered 4 times a day for 8 weeks to 10 healthy female monitors
aged 38 to 42 years different from the Examples. Each monitor has not taken supplements or medicines
(including herbal medicines) since one month ago.
Then, the IGF-1 value was measured in exactly the same manner as in the above example. The average
value of the IGF-1 values of the 10 monitors is shown in FIG. 1, and the amount of change in the average
value of the IGF-1 values at the 4th week of ingestion and the 8th week of ingestion is shown in FIG.
From FIG. 1, according to the IGF-1 level-increasing agent (Example) of the present embodiment,
the IGF-1 value increased in the 4th week after ingestion compared to before the start of ingestion, and 8
weeks after the start of ingestion. In the eyes, it is further increased than the fourth week after ingestion,
and it is apparent that the number is increasing monotonously. On the other hand, according to the
placebo (comparative example), the IGF-1 value increased in the 8th week after ingestion compared to
before the start of ingestion, but before the start of ingestion in the 4th week after ingestion. In
comparison, it is clear that the IGF-1 value has once decreased and is not a monotonous increase.
Moreover, it is clear from FIG. 2 that the amount of increase in the IGF-1 value is greater than that of the
placebo according to the IGF-1 value increasing agent of the present embodiment.
Furthermore, it is clear from FIG. 3 that the physical condition at 8 weeks after the start of ingestion of
the IGF-1 level-increasing agent of this embodiment is superior to the physical condition at 2 weeks after
ingestion. This is because in 8 weeks after the start of ingestion of the IGF-1 level increasing agent of this
embodiment, the physical condition improved due to the increase in IGF-1 value, but the increase in IGF-
1 value with the discontinuation of intake It is thought that the effect was reduced.
The graph which shows the time-dependent change of the IGF-1 value at the time of ingesting the case
where the IGF-1 value raising agent which concerns on this invention is ingested, and the placebo
(placebo). The graph which shows the variation | change_quantity of the IGF-1 value when the case
where the IGF-1 value raising agent which concerns on this invention is ingested, and the case where a
placebo (placebo) is ingested. The graph which shows the comparison with the physical condition after
ingesting the IGF-1 value raising agent which concerns on this invention for 8 weeks, and the physical
condition after 2 weeks from an ingestion discontinuation.
Explanation of symbols
No sign.
Claims (1)
Hide Dependent
1. An agent for raising IGF-1 level, comprising a component extracted by treating ovarian
membrane of salmon with a proteolytic enzyme .
Patent kedua: JP-3946239 : Efek dalam perbaikan sirkulasi pembuluh darah
Abstract
The present invention provides a new use of components extracted from ovarian membranes of fish.
A peripheral blood flow-improving agent containing a component extracted from the ovarian membrane of
fish, which can alleviate and improve various symptoms caused by a decrease in peripheral blood
circulation. The component extracted from the ovarian membrane is a component extracted by treating
the ovarian membrane with a proteolytic enzyme. The ovarian membrane is a spider ovarian membrane.
[Selection] Figure 1
Description
The present invention relates to an acceleration pulse wave aging index raising agent .
Conventionally, a method of extracting amino acids and peptides by treating the ovarian membrane (fish
egg rind) of fish with ozone water in advance and then enzymatically degrading myofibrillar protein, which
is a constituent protein thereof, is known (for example, patent document). 1).
The amino acids and peptides can be used as physiologically active substances or as food fortifiers. More
specifically, the amino acids and peptides have ACE inhibitory activity and act as blood pressure increase
inhibitors (hypertensive agents).
However, it is desired to develop more uses for the components extracted from the ovarian membrane of
fish.
JP 2004-73186 A
In view of such circumstances, an object of the present invention is to provide a new use of a component
extracted from the ovarian membrane of fish.
In order to achieve this object, the accelerated pulse wave aging index increasing agent of the present
invention is characterized by containing a component extracted by treating the ovarian membrane of
sputum with a proteolytic enzyme .
In the living body, after the adulthood, with aging, various symptoms appear due to a decrease in
peripheral blood circulation such as rough skin, stiff shoulders, joint pain, swelling, coldness and anemia.
The decrease in peripheral blood circulation is caused not only by the above-mentioned aging but also by
low blood pressure, a decrease in heart pump function, muscle shortage, and the like. The decrease in
the peripheral blood circulation is grasped as a decrease in the acceleration pulse wave aging index.
The acceleration pulse wave aging index is an index indicating the flexibility of peripheral blood vessels.
For example, the fingertip is a folded portion where blood pumped from the heart passes through an
artery and reaches a vein. By measuring the change in the volume (volume) of blood flowing in the
peripheral blood vessels of the fingertips, the expansion / contraction state of the peripheral blood vessels
can be obtained as a waveform, and this waveform is called “fingertip pulse wave”. An acceleration pulse
wave showing a waveform of the systole of the heart is obtained by second-order differentiation of the
fingertip pulse wave. For example, the acceleration pulse wave shown in FIG. 6 has four waves, the a
wave being the initial contraction positive wave, the b wave being the initial contraction negative wave,
the c wave being the middle systolic re-rising wave, and the d wave being the late systolic re-falling wave.
. The a wave and the b wave depend on the driving pressure wave generated by the ejection of blood,
and the c wave and the d wave depend on the reflected pressure wave that the driving pressure wave
propagates to the periphery and is reflected back. The acceleration pulse wave aging index is obtained
from the waveform of the acceleration pulse wave by the formula “{(−b + c + d) / a} × 100”. Note that “a”,
“b”, “c”, and “d” in the previous equation are the heights of the a wave, b wave, c wave, and d wave. The
acceleration pulse wave aging index generally takes a value of −120 to 120, and the higher the value, the
more flexible the peripheral blood vessels and the better the peripheral blood circulation.
The inventors of the present application ingested the accelerated pulse wave aging index increasing
agent of the present invention, and examined the peripheral blood circulation state using the accelerated
pulse wave aging index, various symptoms due to a decrease in peripheral blood circulation It was found
that this can be relaxed and improved.
Next, embodiments of the present invention will be described in more detail with reference to the
accompanying drawings. FIG. 1 is a graph showing a comparison between the physical condition after
ingesting the acceleration pulse wave aging index increasing agent of this embodiment for 8 weeks and
the physical condition after 2 weeks from the discontinuation of intake, and FIG. 2 is an acceleration pulse
wave addition of this embodiment. FIG. 3 is a graph showing the change over time of the acceleration
pulse wave aging index when taking an age index increasing agent and when taking a placebo (placebo),
FIG. 3 is taking the acceleration pulse wave aging index increasing agent of this embodiment FIG. 4 is a
graph showing the amount of change in the acceleration pulse wave aging index when taking a placebo
and when taking a placebo, and FIG. 4 shows the case where the acceleration pulse wave aging index
increasing agent of this embodiment is taken and the placebo is taken FIG. 5 is a graph showing the
amount of change in IGF-1 value when the accelerated pulse wave aging index raising agent of this
embodiment is ingested and when the placebo is ingested. is there.
The acceleration pulse wave aging index increasing agent of the present embodiment contains a
component extracted from the ovarian membrane of rabbit (hereinafter abbreviated as an ovarian
membrane extraction component). The ovarian membrane extract component can be obtained by, for
example, a method of filtering a solution obtained by enzymatic treatment of salmon ovarian membrane
and drying the obtained filtrate.
Specifically, in the method, first, the ovarian membrane of the pupa is used as a raw material, and water
is added to the ovarian membrane in a weight ratio of ovarian membrane: water = 1: 1 to 1: 3, and the
mixture is stirred and mixed. Furthermore, a proteolytic enzyme is added in the range of 1 to 3% by
weight with respect to the total amount of the ovarian membrane, and heated at a temperature of 45 to 55
° C. for 30 minutes to 5 hours, preferably 2 hours. If it does in this way, the component decomposed |
disassembled with the said proteolytic enzyme will elute in water among the components of the said
ovary membrane, and the aqueous solution of this component will be obtained.
Next, the proteolytic enzyme contained in the aqueous solution is inactivated. The deactivation can be
performed, for example, by heating the aqueous solution at a temperature of 90 ° C. for 5 minutes.
Next, the aqueous solution is simply filtered through a wire mesh of about 30 mesh to remove coarse
materials such as undecomposed ovarian membranes. Then, activated carbon is added to the obtained
filtrate to deodorize, decolorize, and degrease the filtrate. The deodorization, decolorization, and
degreasing of the filtrate is performed by adding activated carbon in the range of 2 to 4% by weight to the
total amount of the ovarian membrane as the raw material and heating the filtrate at a temperature of 60 °
C. for 30 minutes, for example. be able to.
After the deodorization, decolorization, and degreasing treatment with the activated carbon, the filtrate is
filtered by, for example, a filter press, and the obtained filtrate is concentrated at a temperature of 60 ° C.
under reduced pressure, for example, and then maintained at a temperature of 80 ° C. for 10 minutes, for
example. And sterilize. Then, the ovarian membrane extract component can be obtained by drying the
sterilized filtrate by spray drying. The ovarian membrane extract component contains amino acids,
peptides, vitamins, minerals, sugars, enzymes, nucleic acids and their metabolites, various growth
factors, cytokines, and the like.
The acceleration pulse wave aging index increasing agent of the present embodiment is prepared by
formulating the ovarian membrane extract component, for example, in the form of a tablet or the like. For
example, by taking it as a food such as a health food, the peripheral blood circulation is reduced. Can
alleviate and improve symptoms.
Next, examples of the present invention and comparative examples will be described.
In this example, first, an ovarian membrane extract component of sputum was formulated in the form of a
tablet to produce an acceleration pulse wave aging index raising agent . The tablet consists of 245 mg of
the ovarian membrane extract and 5 mg of excipient (Loveli wax (registered trademark)) and has a
diameter of 8 mm.
Next, 10 healthy female monitors aged 38 to 42 years old took 4 tablets per day for 8 weeks as a health
food. Each monitor has not taken supplements or medicines (including herbal medicines) since one
month ago.
And about the physical condition by the self-report of each monitor, the state of the 8th week after an
intake start and the 2 weeks after an intake stop was compared. The results are shown in FIG.
Further, before the start of ingestion and 8 weeks after the start of ingestion, the peripheral blood
circulation at the tip (fingertip) of the right index finger was examined to obtain an acceleration pulse wave
aging index.
Peripheral blood circulation status was examined as follows. Using an acceleration sphygmograph (BC
Checker (registered trademark)) manufactured by Future Wave Inc., the fingertip of the right index finger
is brought into contact with the center part. The acceleration pulse wave meter irradiates the fingertip with
near-infrared light (wavelength 950 nm), captures the light scattered outside the subcutaneous tissue and
absorbed by the blood and then released outside the body, and generates the waveform of the fingertip
pulse wave The waveform of the acceleration pulse wave obtained by second-order differentiation of the
waveform of the fingertip pulse wave is output. The acceleration pulse wave aging index is obtained from
the waveform of the acceleration pulse wave.
The average value of the acceleration pulse wave aging index of the 10 monitors is shown in FIG. 2, and
the amount of change in the average value of the acceleration pulse wave aging index from the start of
intake to the 8th week is shown in FIG. In FIG. 2, “0 week” is indicated before the start of ingestion, “8
week” is indicated after the 8th week from the start of ingestion, and “0-8 weeks” is indicated from the
start of intake to the 8th week in FIG. 3. To do.
Further, before the start of ingestion, 4 weeks after the start of ingestion and 8 weeks after the start of
ingestion, blood tests were performed to measure insulin growth factor-1 (IGF-1: Insulin-like Growth
Factor-1) values. IGF-1 is a polypeptide having a structure very similar to insulin. It is said that the IGF-1
value decreases with aging, and the once lowered IGF-1 value does not increase again.
The average value of IGF-1 values of the 10 monitors is shown in FIG. 4, and the change amount of the
average value of IGF-1 values from the start of intake to the 4th week and from the start of intake to the
8th week is shown in FIG. .
[Comparative Example]
In this comparative example, first, a placebo (placebo) consisting of 125 mg of corn starch and 125 mg of
lactose was formulated in the form of a tablet having a diameter of 8 mm instead of the acceleration pulse
wave aging index raising agent of the above-mentioned example.
Then, the acceleration pulse wave aging index and the IGF-1 value were measured in exactly the same
manner as in the above example. FIG. 2 shows the average value of the acceleration pulse wave aging
index of the 10 monitors, FIG. 3 shows the amount of change in the average value of the acceleration
pulse wave aging index 8 weeks after the start of intake, and FIG. FIG. 4 shows the average value of 1
values, and FIG. 5 shows the amount of change in the average value of IGF-1 values after 4 weeks from
the start of intake and 8 weeks after the start of intake.
From Figure 1, according to the acceleration pulse wave aging index increasing agent of the present
embodiment (Example), the acceleration pulse wave aging index increasing agent of the present
embodiment from the start of ingestion than physical condition after 2 weeks intake discontinued It is
clear that the physical condition at 8 weeks is superior.
Therefore, it is considered that various symptoms due to the decrease in peripheral blood circulation were
alleviated and improved by taking the acceleration pulse wave aging index increasing agent of this
embodiment. Moreover, it is thought that it is effective to ingest the acceleration pulse wave aging index
raising agent of this embodiment continuously.
In addition, from FIG. 2, according to the acceleration pulse wave aging index increasing agent of the
present embodiment, the acceleration pulse wave aging index is increased in the 8th week from the start
of intake compared to before the start of intake. it is obvious. On the other hand, according to the placebo
(comparative example), it is clear that in the 8th week from the start of intake, it is lower than that before
the start of intake.
Moreover, it is clear from FIG. 3 that according to the acceleration pulse wave aging index increasing
agent of the present embodiment, the acceleration pulse wave aging index increases greatly.
Therefore, it is considered that the flexibility of peripheral blood vessels has been improved by taking
the acceleration pulse wave aging index increasing agent of the present embodiment.
Furthermore, from FIG. 4, according to the acceleration pulse wave aging index increasing agent of this
embodiment, the IGF-1 value increased compared to before the start of intake at 4 weeks from the start
of intake, and from the start of intake. At 8 weeks, it is further increased from 4 weeks after the start of
ingestion, and it is clear that it is increasing monotonously. On the other hand, according to the placebo,
although the IGF-1 value increased in the 8th week from the start of the intake compared to before the
start of the intake, it was compared in the 4th week after the start of the intake before the start of the
intake. It is clear that the IGF-1 value has decreased once, not a monotonous increase.
Moreover, from FIG. 5, according to the acceleration pulse wave aging index raising agent of this
embodiment, it is clear that the increase amount of IGF-1 value is larger than a placebo.
Therefore, it is considered that the IGF-1 value increased by taking the acceleration pulse wave aging
index increasing agent of the present embodiment. In addition, the above-described alleviation and
improvement of various symptoms due to the decrease in peripheral blood circulation is considered to be
caused by an increase in IGF-1 level.
The graph which shows the comparison with the physical condition after ingesting the acceleration pulse
wave aging index raising agent which concerns on this invention for 8 weeks, and the physical condition
after 2 weeks from ingestion. The graph which shows the time-dependent change of the acceleration
pulse wave aging index | exponent when the case where the acceleration pulse wave aging index raising
agent which concerns on this invention is ingested, and the case where a placebo (placebo) is
ingested. The graph which shows the variation | change_quantity of the acceleration pulse wave aging
index of the case where the acceleration pulse wave aging index raising agent which concerns on this
invention is ingested, and the case where a placebo is ingested. The graph which shows the time-
dependent change of the IGF-1 value at the time of ingesting the case where the acceleration pulse wave
aging index raising agent which concerns on this invention is ingested, and the placebo. The graph which
shows the variation | change_quantity of the IGF-1 value when the case where the acceleration pulse
wave aging index raising agent which concerns on this invention is ingested, and the placebo is
ingested. The graph which shows an example of an acceleration pulse wave.
Explanation of symbols
No sign.
Claims (1)
Hide Dependent
1. Accelerating pulse wave aging index increasing agent characterized by containing a

component extracted by treating ovarian ovarian membrane with proteolytic enzyme .
Patent ketiga: JPW O2003055901A1 : Anti hipertensi agent
Abstract
The peptide obtained by treating the proteolytic enzyme of fish meat was applied to a peptide-adsorbing
resin (ODS resin, etc.), and this was eluted with water, then with an 11-18V / V% ethanol aqueous
solution, and further with water, elution with water ▲ 1 Peptide SY is produced by fractionating and
mixing the latter fraction, 11-18 V / V% ethanol-eluted fraction, and water-eluting fraction. Moreover, only
the 11-18V / V% ethanol elution fraction is isolated and peptide SY-MD is manufactured. Peptide SY and
peptide SY-MD are both novel peptide mixtures that not only contain a large amount of newly discovered
antihypertensive peptide Val-Tyr, but also have less bitterness and excellent taste and safety. It can also
be used as a blood pressure-lowering agent or a functional food for the purpose of suppressing blood
pressure increase or preventing blood pressure increase.
Description
TECHNICAL FIELD OF THE INVENTION

The present invention relates to a novel peptide SY and an antihypertensive agent and an
antihypertensive functional food that are used for lowering blood pressure. Furthermore, it relates also to
peptide SY-MD contained in peptide SY and its separation method.
Conventional technology
First, the present inventors have carried out heat denaturation treatment of fish meat, inactivated
autodigestive enzyme, hydrolyzed with proteolytic enzyme, inactivated enzyme, and then separated and
processed by ACE (Angiotensin I-). The inventors have succeeded in developing a novel peptide α-1000
having an inhibitory activity (converting enzyme: angiotensin I converting enzyme), and have already
obtained a patent (Japanese Patent No. 3117777: JP-A-5-271297).
Problems to be solved by the invention

With the increase in lifestyle-related diseases, new development of a component effective for preventing
the onset of hypertension or lowering of blood pressure has been demanded in the art, and the present
invention has been made for the purpose of meeting the demand of the art.
Means for solving the problem

As a result of investigations from various directions to achieve the above object, the present inventors
refocused on the ACE inhibitory peptide α-1000 developed by the present inventors, and as a result of
earnest research on various fraction fractions, For the first time, it was found that the fraction containing
the dipeptide Val-Tyr has a higher ACE inhibitory activity, and this Val-Tyr was identified as one of the
main bodies of the antihypertensive peptide.
Based on the above-mentioned useful new knowledge, we decided to develop a new peptide that not only
has a high Val-Tyr content but also a low bitterness and excellent taste and stability, and treated fish
meat with proteolytic enzymes. The obtained peptide (ie, peptide α-1000) was treated with a peptide
adsorption resin (ODS resin) and subjected to three steps of water elution, ethanol water elution and
water elution. The useful new knowledge that most Val-Tyr in fish peptide was collect | recovered in the -
18V / V% ethanol aqueous solution elution fraction and the last water elution fraction was acquired.
Thus, the mixture after the first water elution fraction, the 11-18 V / V% ethanol aqueous solution elution
fraction and the last water elution fraction has not only a high Val-Tyr content but also a low bitterness
and taste. It was excellent in sex and stability, and was confirmed to be a completely novel
antihypertensive functional peptide. This peptide was identified as a novel peptide and designated herein
as peptide SY.
Furthermore, in the present invention, when only the above 11-18 V / V% ethanol aqueous solution
elution fraction was isolated and examined, the Na content was about 0.1 to 0.2% (in peptide SY, Na was
1 to 3). %) And a new peptide with extremely low Na content was obtained, and this fraction was named
peptide SY-MD.
In the present invention, research was conducted for the purpose of continuously recovering peptides
containing as much Val-Tyr as possible using peptide α-1000 as a raw material.
As a result, after α-1000 was treated with ODS resin, water was added to obtain a part of the fraction
eluted with water (the later fraction), and then an aqueous ethanol solution was continuously added. A
fraction eluted with an aqueous ethanol solution is obtained. At this time, since a part of the water used
for water elution (1) remains, the ethanol concentration is 11 to 18 V / V%, preferably 14 to 16 V / V. It
has been found that about V% is suitable.
Further, in obtaining the peptide SY, setting of the starting point for fractionation of the fraction after the
target water elution (1), its end point (ie, the starting point of ethanol elution), The end point (starting point
of water elution (2)) and the end point are identified or judged by measurement and monitoring of
fractionation time, salinity, concentration, Bx, and UV absorption at a wavelength of 280 nm. A method for
producing peptide SY has also been established, and based on these useful new findings, the present
invention has finally been completed as a result of further research.
The physicochemical properties of the peptide SY of the present invention are shown below.
Physicochemical properties of peptide SY
(A) Molecular weight: 200 to 10,000 (by ASAHIPAK GS-320 high performance liquid chromatography):
FIG.
(B) Melting point: Colored and decomposed at 138 ± 3 ° C.
(C) Solubility in solvent: Easily soluble in water, but hardly soluble in ethanol, acetone and hexane.
(D) Appearance properties: white to light yellow powder
(E) Liquidity (pH): 4.0 to 6.0
(F) Ingredient: Moisture 1 to 5 W / W% (normal pressure heating drying method)
Protein 84-94 W / W% (micro Kjeldahl method)
; Lipid 0.5W / W% or less (Soxhlet extraction method)
; Ash content 4 ± 2W / W% (direct ashing method)
(G) Physiological properties: Contains dipeptide Val-Tyr and has ACE inhibitory activity.
(H) Infrared absorption spectrum: FIG.
(I) UV absorption spectrum: FIG.
(J) Specific rotation: [α]D 20= -40 ° to -51 °
And the peptide SY of this invention is manufactured as follows.
That is, a peptide component obtained by elution fractionation treatment with a peptide adsorption resin
using a peptide stock solution, wherein the elution fractionation treatment uses water, an ethanol aqueous
solution and water in this order as a developing solution, and in the elution pattern illustrated in FIG. The
following fractions of water elution (1) obtained with each developing solution (1) and 11-18V / V%
ethanol elution fractions (showing elution with 15% ethanol in FIG. 1) and water elution (2) ▼ Peptide SY
is produced by fractionating and mixing the fractions.
(1) After fraction of water elution (1): Water was used as a developing solution, and the sodium (Na)
amount in the peptide (peptide SY) of water elution (1) was 1 to 3 g / 100 g. Fraction to the final
fractionation point of the fraction after water elution (1) in which the amount of water becomes
substantially 0 g / 100 g.
(2) Eleven to 18 V / V% ethanol elution fraction: Next, an ethanol aqueous solution with a concentration of
11 to 18 V / V% is used as a developing solution, and the elution amount of the peptide passes the peak
and is reduced to about half of the peak. The fraction to be made. (The peptide SY-MD was obtained by
isolating only this fraction.)
(3) Water elution (2) Fraction: Next, a fraction obtained by using water as a developing solution until the
elution fraction treatment reaches the end point.
Next, peptide α-1000, which is a raw material for peptide SY of the present invention, will be described.
Peptide α-1000 is produced from seafood as a raw material, and can be produced, for example,
according to Japanese Patent No. 31177779. First, the seafood is processed by a meat collector,
deboner or the like to separate the fish meat quality. To do. The raw material is preferably as fresh as
possible. The separated fish meat may be divided into about 10 kg of surimi and used for the next
treatment as it is. However, it is rapidly frozen by blowing cold air at −20 to −50 ° C., for example, about
−30 ° C., and −20 to − You may preserve | save at 25 degreeC and you may decide to use this suitably
as needed.
As seafood, red fish such as sardines, horse mackerel, tuna, skipjack, saury, mackerel; white fish such as
flounder, Thailand, kiss, cono, white cod, herring, yellowtail; cartilage fish such as shark, ray; smelt, carp,
char Freshwater fish such as yamame trout; deep sea fish such as sharks and salamanders, shrimp,
crabs, octopuses, and mites can be used as appropriate.
After the meat is minced, the seafood is pulverized by a pulverizer or the like, ½ amount to 20 times the
amount of the raw material weight, preferably the equivalent amount to 10 times the amount of water, and
then heat-treated, In addition to inactivating the self-digesting enzyme and killing bacteria, the protein is
thermally denatured to increase the efficiency of the subsequent enzyme reaction. As heating conditions,
all conditions can be used as long as such an effect is exerted, but for example, 65 ° C. or higher and 2 to
60 minutes, preferably 80 ° C. or higher and 5 to 30 minutes are preferable.
.
Next, an alkaline agent such as ammonia water or an aqueous solution of sodium hydroxide (potassium
hydroxide) is added to adjust the pH to an appropriate value for the proteolytic enzyme used (for
example, in the case of alkaline protease, pH 7.5 or higher, preferably 8). Above), the temperature is also
set to an appropriate enzyme temperature (depending on the enzyme used, 20 to 65 ° C., 35 to 60 ° C.,
preferably 40 to 55 ° C. in the case of alkaline protease), and 30 minutes after adding the proteolytic
enzyme Treatment is performed for 30 hours (30 minutes to 25 hours, preferably 1 to 17 hours in the
case of alkaline protease).
As the proteolytic enzyme, all enzymes can be used alone or in combination as long as they are capable
of degrading proteins under neutral or alkaline conditions. Its origin can be found in microorganisms in
addition to animals and plants, and in addition to pepsin, renin, trypsin, chymotrypsin, papain, promelain,
bacterial protease, filamentous fungal protease, actinomycete protease, etc. can be widely used. These
enzymes are usually commercially available. However, unpurified enzymes, enzyme-containing culture
fluids, and solid or liquid enzyme-containing materials such as sputum should also be used as required.
Can do. The amount of enzyme added may be about 0.1% to 5.0%.
Next, neutralization treatment is performed if necessary, followed by holding at a temperature of 70 ° C.

(preferably 80 ° C.) or higher for 2 to 60 minutes (preferably 5 to 30 minutes) to inactivate the enzyme and
subsequent separation Make good. After heat deactivation treatment, coarse separation is performed with
a vibe screen or the like, and if necessary, diector treatment is performed, followed by ultracentrifugation
treatment to remove suspended matters and precipitates.
Next, the mixture is filtered using a filter aid such as diatomaceous earth (for example, celite), and the
filtrate is treated with activated carbon (0.05 to 20% W / V, preferably 0.1 to 10% W / V used, 20 to 65).
C., preferably 25 to 60.degree. C., 15 minutes to 4 hours, preferably 30 minutes to 2 hours) to deodorize,
decolorize and purify.
This is concentrated under reduced pressure (0 to 50 ° C.) or other conventional methods (to about 30
Bx), and if necessary, it is centrifuged again (ultra) or filtered to obtain a peptide stock solution. The
peptide stock solution thus obtained is sterilized (by UHTST or other conventional method) and then used
as a product (α-1000 (liquid)) filled in a container. If desired, the product can be further concentrated or
diluted in reverse, or powdered to about 60 mesh by a conventional method such as spray drying or
freeze drying, and filled into a container such as a bag ( α-1000 (powder)). For these products, store
liquids in a refrigerated or frozen state, and store powders in a dry, cool and dark place.
The liquid, pasty or powdery peptide thus obtained is α-1000.
The physicochemical properties of peptide α-1000 (spray dry powder) are as shown below.
Physicochemical properties of peptide α-1000 (powder)
(A) molecular weight;
200-10,000 (by Sephadex G-25 column chromatography)
(B) Melting point: colored at 119 ° C. (decomposition point)
(C) Specific rotation
[Α]D 20= -22 °
(D) solubility in solvents;
Easily soluble in water; hardly soluble in ethanol, acetone and hexane.
(E) distinction between acidic, neutral and basic;
Neutral pH 6.0-8.0 (10% solution)
(F) Ultraviolet absorption spectrum: FIG.
(G) Infrared absorption spectrum: FIG.
(H) Appearance, components;
White powder; moisture 5.14% (heat drying under reduced pressure); protein 87.5% (Kjeldahl method,
nitrogen / protein conversion factor 6.25); lipid 0% (Soxhlet extraction method); ash content 5.0%
(directly) Ashing method).
(I) Characteristics;
It is a peptide derived from fish meat, obtained by inactivating a self-digesting enzyme by heating and
hydrolyzing with a proteolytic enzyme.
(J) amino acid composition;
As shown below.
Analysis method: Amino acid automatic analysis method (however, cystine was measured by hydrolyzing
hydrochloric acid after performing formic acid oxidation treatment. Tryptophan was measured by high
performance liquid chromatography.)
The peptide α-1000 prepared in this manner is directly in the case of a liquid, and after adding water in
the case of a powder, the peptide α-1000 is passed through an ODS resin or other peptide-adsorptive
resin as a peptide stock solution to perform “stock solution loading” in FIG. . As the resin, any type of
resin can be used as long as it is a peptide-adsorbing resin. For example, various ODS resins, YMC
ODS-AQ 120-S50 (trade name), hydrophobic adsorption resin SEPABEADS SP207 (Mitsubishi)
Chemical brand name, etc.) are used as appropriate.
The present invention relates to a peptide having a high Val-Tyr content, low bitterness and excellent
taste and stability, among antihypertensive peptides (for example, α-1000) obtained by proteolytic
enzyme treatment of fish meat. As a result of the examination, it was successful for the first time to
fractionate and mix the desired fraction from the above-mentioned various fractions, and the novel
antihypertensive peptide thus fractionated and mixed was named peptide SY. .
Peptide SY can be fractionated and produced from the elution fraction described above. An example of
elution patterns of various eluates is shown in FIG.
As shown in FIG. 1, the peptide SY according to the present invention is added with, for example, α-1000
(stock solution loading) to the peptide adsorbing resin and then eluted with water (water elution (1)).
Manufacture by mixing the post-fraction, the 11-18 V / V% ethanol-eluted fraction eluted with 11-18 V /
V% ethanol solution, and the water-eluted fraction (2) eluted with water. Can do. Setting of the sampling
start time of peptide SY fraction, setting of eluate switching timing, etc. are based on at least one
measured value of Bx, salinity, UV (280 nm absorption), Na, or based on fractionation time What is
necessary is just to determine suitably, It is also possible to monitor these items suitably, such as real
time, and to implement by a computer.
For example, in the elution pattern of FIG. 1, the water separation elution of peptide SY (1) can be
determined as follows by measuring the salinity value at the start of fractionation of the subsequent
fraction.
i) If it is taken from 0 minutes after the start of water elution, the Na content becomes 4 g / 100 g or more,
so that it becomes a high Na material and is not preferable as an antihypertensive agent.
ii) When the fractionation time is 20 minutes after the start of elution with water, the Na content is within
the allowable range of 1 to 3 g / 100 g.
iii) If the subsequent fractionation is started, the Na content is further reduced, but this time, the salt
content is too low, so that guanine contained in the peptide SY tends to precipitate during concentration,
which is not preferable. .
iv) Therefore, the fractionation start time was 20 minutes after the start of water elution, and the amount
of Na was set to around 1 to 3 g / 100 g.
The final fractionation point of the water elution {circle around (1)} fraction was the point at which the Na
amount was substantially 0 g / 100 g.
Next, 11-18 V / V% ethanol aqueous solution is added instead of water from this point, and the addition of
the ethanol aqueous solution is stopped when the peptide elution amount passes the peak and is reduced
to about half of the peak, and obtained here. Fractions were designated as 11-18 V / V% ethanol elution
fractions. (A peptide SY-MD containing almost no Na is obtained by isolating only the 11-18 V / V%
ethanol elution fraction.)
The time when the addition of the 11-18 V / V% ethanol aqueous solution is stopped and switched to the
water addition is also the start of water elution (2), but the UV absorption value at a wavelength of 280 nm
indicating the total peptide amount rapidly decreases. The end point may be a time when the UV
absorption value becomes zero and a steady state is entered. The fraction obtained here was designated
as a water-eluting (2) fraction.
The following fractions obtained from (1) water elution (1), (2) 11-18 V / V% ethanol elution fraction, and
(3) water elution (2) fractions were separated separately or sequentially. The mixture obtained is the
peptide SY of the present invention.
In this way, the fraction from the water-elution (1) fraction in the middle of the fraction to the water-elution
(2) fraction through the 11-18 V / V% ethanol elution fraction is related to the present invention. It can be
obtained as a peptide SY (indicated as Surden peptide SY in FIG. 1).
In FIG. 1, “15% ethanol extraction” corresponds to peptide SY-MD.
Peptide SY contains a high concentration of a dipeptide (valyl-tyrosine: hereinafter also referred to as Val-
Tyr or VY), which was first confirmed by the present inventors as one of the main bodies of
antihypertensive peptides ( VY recovery rate: When α-1000 is 100%, SY is 90 to 95%, whereas SY-MD is
65 to 75%), and not only exhibits very high ACE inhibitory activity, but also exhibits Taste is also greatly
improved. However, since the peptide SY-MD does not contain the water-elution (1) post-fraction, it
remains bitter, but contains almost no Na, so it is a useful antihypertensive agent for people who cannot
take Na.
That is, the “stock solution loading” portion has a strong taste, but also contains some fish odor derived
from the raw material, and has a high Na content. On the other hand, the post-fraction of water elution
{circle around (1)} has little fishy odor derived from the raw material and has a very good taste.
Therefore, by incorporating the post-fraction of water elution (1), a peptide material “Peptide SY” that can
recover a large amount of VY from peptide SY-MD and has excellent taste and stability can be obtained.
It is.
In addition to the blood pressure suppressing action, peptides SY and peptide SY-MD also have functions
such as calcium and iron absorption promoting action, cholesterol suppressing action, and blood sugar
level suppressing action.
The physicochemical properties of peptide SY are as follows:
Physicochemical properties of peptide SY
(A) Molecular weight: 200 to 10,000 (FIG. 2)
(B) Melting point: coloring and decomposition at 138 ± 3 ° C.
(F) component: moisture 1-5 W / W% (normal pressure heating drying method)
: Protein 84-94 W / W% (micro Kjeldahl method)
: Lipid 0.5W / W% or less (Soxhlet extraction method)
: Ash content 4 ± 2W / W% (Direct ashing method)
: Na 1 to 3 W / W% (atomic absorption photometry)
(J) Specific rotation: [α]D 20= -40 ° to -51 °
(K) ACE inhibitory activity value (IC50): 200 μg / ml or less (according to a modified method of Cushman)
(L) Major amino acid composition
(Analysis method: Amino acid automatic analysis method)
The physicochemical properties of peptide SY-MD are as follows.
Physicochemical properties of peptide SY-MD
(A) Molecular weight: 200 to 10,000
: Ash content 3.0 W / W% or less (direct ashing method)
: Na 0.1-0.2 W / W% (atomic absorption photometry)
(G) Ultraviolet absorption spectrum: FIG.
Peptide SY or peptide SY-MD according to the present invention is a novel peptide mixture that has been
conventionally unknown, exhibits excellent blood pressure lowering activity, and has no safety problem.
As a peptide for foods for specified health use. Therefore, this peptide is used as a food additive such as
a seasoning and a food for fortification or as an animal feed additive, and because of its unique
physiological activity, it is used for the prevention of hypertensive diseases and in some cases for the
treatment. Or widely used as an infusion, health food, clinical nutrition food, or the like.
When used as a food, the peptide can be added as it is, or used in combination with other foods or food
ingredients as appropriate. Moreover, when using as a pharmaceutical, it can administer orally or
parenterally. In the case of oral administration, for example, tablets, granules, powders, capsules,
powders, drinks can be used according to conventional methods. In the case of parenteral administration,
for example, injection preparations, infusions, etc. It can be used as a suppository, suppository, etc.
Hereinafter, the present invention will be described in more detail with reference examples and examples.
Reference example
Fresh sardines were processed with a deboner and minced. Divided fish meat is divided into 10kg surimi,
rapidly frozen at -30 ° C or lower, crushed with a pulverizer, added with an equal amount of water, sent to
a tank, and heated at 100 ° C for 10 minutes Then, the autolytic enzyme was inactivated and heat
denatured. Next, aqueous ammonia was added to adjust the pH to 9.5.
A 0.1% solution of a commercially available alkaline protease product was added thereto, and the mixture
was kept at 50 ° C. for 17.5 hours for enzymatic degradation. The enzyme was then inactivated by boiling
for 15 minutes.
This was passed through a vibrator screen (150 mesh), treated with a diector at 5000 rpm, then treated
with a sharp press centrifuge (15000 rpm), and filtered using diatomaceous earth as a filter aid to obtain a
peptide stock solution.
1% W / V of activated carbon was added to the peptide stock solution obtained above, and the mixture
was stirred at 30 ° C. for 60 minutes and filtered to obtain a filtrate. This was concentrated under reduced
pressure (20 ° C.) according to a conventional method and then UHTST sterilized according to a
conventional method to obtain an α-1000 (liquid) product, which was further spray-dried according to a
conventional method to obtain a particle size of 60 mesh. Α-1000 (powder) products were obtained and
each of these products was stored frozen.
Example 1
Dissolve 5 g of peptide α-1000 (powder) obtained in the Reference Example with 500 ml of deionized
water to make a stock solution, and apply it to a hydrophobic adsorption resin SEPABEADS SP207
(Mitsubishi Chemical Corporation) column (3.5 × 13 cm). The column was filled with the α-1000 solution
(stock solution loading), then, according to the elution pattern of FIG. 1, water, 15% ethanol aqueous
solution, then water and 500 ml of each were added, and the Sarden peptide SY in FIG. All fractions, ie,
the fraction after water elution (1), the 15% ethanol elution fraction, and the water elution (2) fraction were
collected and lyophilized to obtain 2.1 g of peptide SY (powder). It was. The Na content of peptide SY
was 1.45 W / W%. (By atomic absorption photometry)
Example 2
(Mitsubishi Chemical Corporation) column (3.5 × 13 cm). The column is filled with the α-1000 solution
(stock solution loading), and only the 15% ethanol-eluted fraction is isolated and collected from all
fractions of the sadden peptide SY shown in the elution pattern of FIG. Was lyophilized to obtain 1.7 g of
peptide SY-MD (powder). The Na content of peptide SY-MD was 0.124 W / W%. (By atomic absorption
photometry)
Example 3
(Drink production)
The above composition was mixed and dissolved at 60 ° C., and then plate sterilization at 128 ° C. for 10
seconds. Next, 100 ml, 50 ml, and 30 ml brown bottles having been thoroughly washed were filled at 90 °
C., allowed to cool at room temperature, and then rapidly cooled in a running water tank to produce a
drink.
Example 4
(Manufacture of tablets)
Tablets were produced with the following formulation.
500 g of peptide SY (powder) obtained in Example 1, 356 g of reduced maltose starch syrup, 100 g of
crystalline cellulose, 40 g of sucrose fatty acid ester and 4 g of sweetener (stevia) are mixed, and the
mixture is compressed by a compression tablet machine to be uncoated. (250 mg × 4000 pieces) were
prepared. This uncoated tablet was coated with 7.5 mg of shellac solution, and 4000 tablets containing
500 mg of peptide SY (powder) per 4 tablets were produced.
Example 5
The example of taking the drink manufactured in Example 3 is shown.
(1) In case of 30ml drink
Peptide SY was 0.5 g / tube, and clinical trials were conducted for patients with mild hypertension divided
into a peptide administration group and a placebo group by randomized double-blind method. Ingested
one daily, 4 weeks later, the blood pressure on the peptide administration group alone was 10.6 mmHg
on average, and the lower blood pressure was significantly reduced by 5.6 mmHg.
Similarly, clinical trials were conducted for patients with mild hypertension, using a randomized double-
blind method, divided into the peptide administration group and the placebo group. Ingested one daily, 4
weeks later, the blood pressure on the peptide administration group alone was 10.2 mmHg on average,
and the lower blood pressure was significantly reduced by 3.8 mmHg.
In the same manner, 4 weeks later, the blood pressure on the peptide administration group alone was 8.2
mmHg on average, and the lower blood pressure was significantly reduced by 3.0 mmHg.
As described above, in any case, by taking a drink containing 0.5 g / book of peptide SY once a day, the
risk rate is 1 without affecting the blood pressure of patients with mild hypertension. It was confirmed that
the decrease was significantly below%.
Example 6
The administration example of the tablet manufactured in Example 4 is shown.
A placebo-controlled double-blind ingestion test was conducted on normal high blood pressure and 88
patients with mild hypertension.
When the tablets were ingested 4 tablets a day (0.5 g / day as peptide SY), the peptide SY administration
group (44 patients) had an upper blood pressure (systolic blood pressure) of 148 ± 11 mmHg at the start
of the test, The blood pressure (diastolic blood pressure) was 92 ± 14 mmHg, but the upper blood
pressure dropped to 138 ± 12 mmHg after 4 weeks, 134 ± 9 mmHg after 8 weeks, and the lower blood
pressure was 84 ± 11 mmHg after 4 weeks, 83 after 8 weeks. Significantly decreased to ± 10 mmHg. No
significant changes were seen in the placebo group (44 patients), and no side effects such as dry cough
were observed in either group. Therefore, the blood pressure of this tablet containing peptide SY against
normal high blood pressure and mild hypertension The inhibitory effect was confirmed.
Example 7
(Drink production)
This 100 ml drink contained 500 mg / peptide SY-MD, and the components were moisture 96.7 g, protein
0.5 g, carbohydrate 4.5 g, calorie 19 Kcal, sodium 7.4 mg, and sorbitol 0.66 g.
Using this drink SY-MD 0.5g / drink 1 bottle / day, systolic blood pressure 130-140mmHg, diastolic blood
pressure 80-90mmHg normal high blood pressure person and systolic blood pressure 140-160mmHg,
diastolic blood pressure A double-blind controlled study was performed on 90-100 mm Hg volunteers with
mild hypertension.
As a result, there was no significant difference in blood pressure fluctuation in the placebo drink group,
but in the peptide drink group, the systolic blood pressure significantly decreased to 14.7 mmHg and the
diastolic blood pressure to 7.6 mmHg after 4 weeks. , Confirmed the effectiveness.
Example 8
500 g of peptide SY-MD (powder) obtained in Example 2, 356 g of reduced maltose starch syrup, 100 g
of crystalline cellulose, 40 g of sucrose fatty acid ester and 4 g of sweetener (stevia) were mixed, and this
mixture was compressed by a compression tablet machine. Uncoated tablets (250 mg × 4000) were
prepared. This uncoated tablet was coated with 7.5 mg shellac solution per tablet, and 4000 tablets
containing 500 mg of peptide SY-MD (powder) per 4 tablets were produced.
In 100 g, the ingredients were 3.3 g water, 44.2 g protein, 3.5 g carbohydrates, 1.6 g ash, 47.4 g
carbohydrates, 566 mg sodium, and an energy level of 398 Kcal.
This tablet was used 4 times a day (0.5 g / day taken as peptide SY-MD), and a 12-week double-blind
comparative control test was conducted using placebo without peptide SY-MD as a control. In volunteers
with normal high blood pressure and mild hypertension, the peptide SY-MD-containing tablet intake group
showed a significant decrease in blood pressure in both systolic blood pressure (SBP) and diastolic blood
pressure (DBP) compared to before the intake. That is, SBP was 145.4 mmHg and DBP was 86.8 mmHg
before ingestion, while SBP dropped to 134.7 mmHg and DBP to 83.0 mmHg at the end of the test. No
significant blood pressure fluctuation was observed in the placebo group, and no subjective symptoms
such as side effects occurred during 12 weeks.
The invention's effect
Peptide SY and peptide SY-MD according to the present invention are not only excellent in
antihypertensive function because they contain a high concentration of Val-Tyr newly found as a
hypotensive component, that is, the main body of the antihypertensive peptide, Furthermore, peptide SY
has a feature that it has excellent taste and stability without bitterness by incorporating a part of the
water-eluted fraction. Since the novel peptide SY according to the present invention has the above-
mentioned characteristics, it can be used not only as a food or drink or as an additive, but also has
excellent ACE inhibitory activity. It can be used for suppression or prevention, and can be advantageously
used as a medicine by formulating into various dosage forms as an ACE inhibitor or a blood pressure
lowering agent.
As is apparent from the above, the novel peptide SY and peptide SY-MD show excellent blood pressure
lowering activity, and since they are originally derived from fish meat, there is no problem in safety (in
fact, When 500 mg per day was forcibly orally administered to rats, no acute toxicity was observed even
after 10 days.) It is also used as a hypotensive agent or a peptide for specified health foods for the
purpose of lowering blood pressure. be able to.
FIG. 1 is a graph of the peptide SY elution pattern in Example 1.
FIG. 2 shows the molecular weight of peptide SY.
FIG. 3 shows the infrared absorption spectrum of peptide SY.
FIG. 4 shows the ultraviolet absorption spectrum of peptide SY.
FIG. 5 shows the peptide α-1000 ultraviolet absorption spectrum.
FIG. 6 shows an infrared absorption spectrum of peptide α-1000.
FIG. 7 shows the ultraviolet absorption spectrum of peptide SY-MD.
FIG. 8 shows the infrared absorption spectrum of peptide SY-MD.
[Document Name] Description [Claims]
[Claim 1]
Peptide SY having the following physicochemical properties:
(A) Molecular weight: 200 to 10,000 (by ASAHIPAK GS-320 high-speed liquid chromatography)
(C) Solubility in solvents: Easily soluble in water, but hardly soluble in ethanol, acetone and hexane.
(D) Appearance property: White to light yellow powder (E) Liquid property (pH): 4.0 to 6.0
(F) component: moisture 1 to 5 W / W% (normal pressure heating drying method)
: Protein 84-94 W / W% (Micro Kjeldahl method)
[Claim 2]
A peptide component obtained by elution fractionation treatment using a peptide adsorption resin with a
peptide stock solution, wherein the elution fractionation treatment uses water, an aqueous ethanol
solution, and water in this order as the development solution, and in the elution pattern of FIG. The
peptide according to claim 1, which is obtained by preparatively mixing a water-eluted (1) post-fraction,
11-18 V / V% ethanol-eluted fraction and a water-eluted (2) fraction as defined below. SY.
(1) Water fraction after elution (1): Water is used as a developing solution, and the amount of sodium
from the time when the amount of sodium (Na) in the entire eluted peptide fraction (peptide SY) becomes
1 to 3 g / 100 g. Is the fraction up to the final fractionation point of the post-fraction of water elution (1) in
which is substantially 0 g / 100 g.
(2) Ethanol elution fraction: Next, a fraction obtained by using an ethanol aqueous solution having a
concentration of 11 to 18 V / V% as a developing solution until the peptide elution amount passes the
peak and is reduced to about half of the peak.
[Claim 3]
The peptide SY according to claim 1 or 2, wherein the peptide stock solution is produced using an
aqueous solution of peptide α-1000 derived from fish meat and having the following physicochemical
properties as a peptide stock solution.
[α] D 20 = −22 °
Neutral (f) appearance, ingredients:
White powder: moisture 5.14% (heat drying under reduced pressure); protein 87.5% (Kjeldahl method,
(G) characteristics;
[Claim 4]
Using only the aqueous solution of 11-18 V / V% concentration in (2) of claim 2 as a developing solution,
only the fraction obtained until the peptide elution amount passes the peak and is reduced to about half of
the peak is isolated. A method for producing a peptide SY-MD,
[Claim 5]
Peptide SY-MD having the following physicochemical properties obtained in claim 4.
Physicochemical properties of peptide SY-MD (A) Molecular weight; 200-10,000
(F) Ingredient: Moisture 2-6 W / W% (normal pressure heating drying method)
: Ash content 3.0W / W% or less (direct ashing method)
[Claim 6]
An antihypertensive agent comprising the peptide SY according to claim 1 or the peptide SY-MD
according to claim 5 as an active ingredient.
[Claim 7]
An antihypertensive functional food comprising the peptide SY according to claim 1 or the peptide SY-MD
itself according to claim 5, or containing the peptide SY or peptide SY-MD.
[Claim 8]
The food according to claim 7, wherein the food is in a liquid or solid form.
[0001]
BACKGROUND OF THE INVENTION

The present invention relates to a novel peptide SY and an antihypertensive agent and an
antihypertensive functional food that are used for lowering blood pressure. Furthermore, it relates also to
peptide SY-MD contained in peptide SY and its separation method.
[0002]
[Prior art]
First, the present inventors heat-denatured fish meat to inactivate the autolytic enzyme, hydrolyze it with a
proteolytic enzyme, inactivate the enzyme, and then separate the ACE (Angiotensin I-). Converting
enzyme: Angiotensin I converting enzyme) has succeeded in developing a novel peptide α-1000 having
inhibitory activity and has already obtained a patent right (Japanese Patent No. 3117777: JP-A-5-
271297).
[0003]
[Problems to be solved by the invention]

With the increase in lifestyle-related diseases, new development of a component effective for preventing
the onset of hypertension or lowering of blood pressure has been demanded in the art, and the present
invention has been made for the purpose of meeting the demand of the art.
[0004]
[Means for Solving the Problems]

As a result of investigations from various directions to achieve the above object, the present inventors
refocused on the ACE inhibitory peptide α-1000 developed by the present inventors, and as a result of
earnest research on various fraction fractions, For the first time, it was found that the fraction containing
the dipeptide Val-Tyr has a higher ACE inhibitory activity, and this Val-Tyr was identified as one of the
main bodies of the antihypertensive peptide.
Based on the above-mentioned useful new knowledge, we decided to develop a new peptide that not only
has a high Val-Tyr content but also a low bitterness and excellent taste and stability, and treated fish
meat with proteolytic enzymes. The obtained peptide (ie, peptide α-1000) was treated with a peptide
adsorption resin (ODS resin) and subjected to three steps of water elution, ethanol water elution and
water elution. The useful new knowledge that most Val-Tyr in fish peptide was collect | recovered in the -
18V / V% ethanol aqueous solution elution fraction and the last water elution fraction was acquired.
Thus, the mixture after the first water elution, the 11-18 V / V% ethanol aqueous solution elution fraction,
and the last water elution fraction has not only a high Val-Tyr content but also a low bitterness and taste.
It was excellent in sex and stability, and was confirmed to be a completely novel antihypertensive
functional peptide. This peptide was identified as a novel peptide and designated herein as peptide SY.
Furthermore, in the present invention, when only the above 11-18 V / V% ethanol aqueous solution
elution fraction was isolated and examined, the Na content was about 0.1 to 0.2% (in peptide SY, Na is 1
to 3). %) And a new peptide with very low Na content was obtained, and this fraction was named peptide
SY-MD.
[0005]
In the present invention, research was conducted for the purpose of continuously recovering peptides
containing as much Val-Tyr as possible using peptide α-1000 as a raw material.
As a result, after α-1000 was treated with ODS resin, water was added to obtain a part of the fraction
eluted with water (the later fraction), and then an aqueous ethanol solution was continuously added. A
fraction eluted with an ethanol aqueous solution is obtained. At this time, since a part of the water used
for water elution (1) remains, the ethanol concentration is 11 to 18 V / V%, preferably 14 to 16 V / V. We
have found that about V% is suitable.
Furthermore, in obtaining the peptide SY, setting of the fractionation start point when fractionating the
fraction after the target water elution (1), its end point (ie, ethanol elution start point), The end point
(starting point of water elution (2)), the end point is identified or determined by fractionation time, salinity
concentration, Bx, UV absorption measurement at wavelength 280 nm, and monitoring, respectively. The
manufacturing method of SY was also established, and based on these useful new findings, as a result of
further research, the present invention was finally completed.
[0006]
The physicochemical properties of the peptide SY of the present invention are shown below.
Physicochemical properties of peptide SY (A) Molecular weight: 200 to 10,000 (according to ASAHIPAK
GS-320 high-speed liquid chromatography): FIG.
(C) Solubility in solvents: Easily soluble in water, but hardly soluble in ethanol, acetone and hexane.
(F) component: moisture 1-5 W / W% (atmospheric pressure heating drying method)
Protein 84-94 W / W% (micro Kjeldahl method)
; Lipid 0.5W / W% or less (Soxhlet extraction method)
; Ash content 4 ± 2W / W% (direct ashing method)
(J) Specific rotation: [α] D 20 = −40 ° to −51 °
[0007]
And the peptide SY of this invention is manufactured as follows.

That is, a peptide component obtained by elution fractionation treatment with a peptide adsorption resin
using a peptide stock solution, wherein the elution fractionation treatment uses water, an ethanol aqueous
solution and water in this order as a developing solution, and in the elution pattern illustrated in FIG. The
following fractions of water elution (1) obtained by each developing solution (1) and 11-18 V / V% ethanol
elution fractions (showing elution with 15% ethanol in FIG. 1) and water elution (2 ) Peptide SY is
produced by fractionating and mixing the fractions.
(1) Subsequent fraction of water elution (1): using water as a developing solution, sodium from the time
point when the amount of sodium (Na) in the peptide (peptide SY) of water elution (1) is 1 to 3 g / 100 g.
Fraction to the final fractionation point of the fraction after water elution (1) in which the amount of water is
(2) Eleven to 18 V / V% ethanol elution fraction: Next, it is obtained until the elution amount of the peptide
passes the peak and is reduced to about half of the peak using an ethanol aqueous solution with a
concentration of 11 to 18 V / V%. The fraction to be made. (The peptide SY-MD was obtained by isolating
only this fraction.)
[0008]
Next, peptide α-1000, which is a raw material for peptide SY of the present invention, will be described.
Peptide α-1000 is produced from seafood as a raw material, and can be produced according to, for
example, Japanese Patent No. 3117777. First, the seafood is processed by a meat collector, deboner,
etc. to separate the fish meat quality. To do. The raw material is preferably as fresh as possible. The
separated fish meat may be divided into about 10 kg of surimi and used for the next treatment as it is.
However, it is rapidly frozen by blowing cold air at −20 to −50 ° C., for example, about −30 ° C., and −20
to − You may preserve | save at 25 degreeC and you may decide to use this suitably as needed.
As seafood, red fish such as sardines, horse mackerel, tuna, skipjack, saury, mackerel; white fish such as
flounder, Thailand, kiss, cono, white cod, herring, yellowtail; cartilage fish such as shark, ray; smelt, carp,
char Freshwater fish such as yamame trout; deep sea fish such as sharks and salamanders, shrimp,
crabs, octopuses, and mites can be used as appropriate.
After the meat is minced, the seafood is pulverized by a pulverizer or the like, ½ amount to 20 times the
amount of the raw material weight, preferably the equivalent amount to 10 times the amount of water, and
then heat-treated, In addition to inactivating the self-digesting enzyme and killing bacteria, the protein is
thermally denatured to increase the efficiency of the subsequent enzyme reaction. As heating conditions,
all conditions can be used as long as such an effect is exerted, but for example, 65 ° C. or higher and 2 to
60 minutes, preferably 80 ° C. or higher and 5 to 30 minutes are preferable. .
Next, an alkaline agent such as ammonia water or an aqueous solution of sodium hydroxide (potassium
hydroxide) is added to adjust the pH to an appropriate value for the proteolytic enzyme used (for
example, in the case of alkaline protease, pH 7.5 or higher, preferably 8). Above), the temperature is also
set to an appropriate enzyme temperature (depending on the enzyme used, 20 to 65 ° C., 35 to 60 ° C.,
preferably 40 to 55 ° C. in the case of alkaline protease), and 30 minutes after adding the proteolytic
enzyme Treatment is performed for 30 hours (30 minutes to 25 hours, preferably 1 to 17 hours in the
case of alkaline protease).
As the proteolytic enzyme, all enzymes can be used alone or in combination as long as they are capable
of degrading proteins under neutral or alkaline conditions. Its origin can be found in microorganisms in
addition to animals and plants, and in addition to pepsin, renin, trypsin, chymotrypsin, papain, promelain,
bacterial protease, filamentous fungal protease, actinomycete protease, etc. can be widely used. These
enzymes are usually commercially available. However, unpurified enzymes, enzyme-containing culture
fluids, and solid or liquid enzyme-containing materials such as sputum should also be used as required.
Can do. The amount of enzyme added may be about 0.1% to 5.0%.
Next, neutralization treatment is performed if necessary, followed by holding at a temperature of 70 ° C.

(preferably 80 ° C.) or higher for 2 to 60 minutes (preferably 5 to 30 minutes) to inactivate the enzyme and
subsequent separation Make good. After heat deactivation treatment, coarse separation is performed with
a vibe screen or the like, and if necessary, diector treatment is performed, followed by ultracentrifugation
treatment to remove suspended matters and precipitates.
Next, the mixture is filtered using a filter aid such as diatomaceous earth (for example, celite), and the
filtrate is treated with activated carbon (0.05 to 20% W / V, preferably 0.1 to 10% W / V used, 20 to 65).
C., preferably 25 to 60.degree. C., 15 minutes to 4 hours, preferably 30 minutes to 2 hours) to deodorize,
decolorize and purify.
This is concentrated under reduced pressure (0 to 50 ° C.) or other conventional methods (to about 30
Bx), and if necessary, it is centrifuged again (ultra) or filtered to obtain a peptide stock solution. The
peptide stock solution thus obtained is sterilized (by UHTST or other conventional method) and then used
as a product (α-1000 (liquid)) filled in a container. If desired, the product can be further concentrated or
diluted in reverse, or powdered to about 60 mesh by a conventional method such as spray drying or
freeze drying, and filled into a container such as a bag ( α-1000 (powder)). For these products, store
liquids in a refrigerated or frozen state, and store powders in a dry, cool and dark place.
The liquid, pasty or powdery peptide thus obtained is α-1000.
[0009]
The physicochemical properties of peptide α-1000 (spray dry powder) are as shown below.
Physicochemical properties of peptide α-1000 (powder) (A) molecular weight;
[α] D 20 = −22 °
Neutral pH 6.0-8.0 (10% solution)
(F) Ultraviolet absorption spectrum: FIG.
(G) Infrared absorption spectrum: FIG.
(H) Appearance, components;
White powder; moisture 5.14% (heat drying under reduced pressure); protein 87.5% (Kjeldahl method,
(I) Characteristics;
[0010]
(J) amino acid composition;
As shown below.
──────────────────────
Analytical test items Results (%)
──────────────────────
All amino acids arginine 3.34
Lysine 6.86
Histidine 3.34
Phenylalanine 2.33
Tyrosine 2.01
Leucine 6.35
Isoleucine 3.27
Methionine 2.26
Valine 4.16
Alanine 5.17
Glycine 3.59
Proline 2.15
Glutamic acid 12.35
Serine 3.30
Threonine 3.70
Aspartic acid 8.36
Tryptophan 0.32
Cystine 0.47
Total amount 73.33
──────────────────────
Analysis method: Amino acid automatic analysis method (however, cystine was measured by hydrolyzing
hydrochloric acid after performing formic acid oxidation treatment. Tryptophan was measured by high
performance liquid chromatography.)
[0011]
The peptide α-1000 prepared in this manner is directly in the case of a liquid, and after adding water in
the case of a powder, the peptide α-1000 is passed through an ODS resin or other peptide-adsorptive
resin as a peptide stock solution to perform “stock solution loading” in FIG. . As the resin, any type of
resin can be used as long as it is a peptide-adsorbing resin. For example, various ODS resins, YMC
ODS-AQ 120-S50 (trade name), hydrophobic adsorption resin SEPABEADS SP207 (Mitsubishi)
Chemical brand name, etc.) are used as appropriate.
The present invention relates to a peptide having a high Val-Tyr content, low bitterness and excellent
taste and stability, among antihypertensive peptides (for example, α-1000) obtained by proteolytic
enzyme treatment of fish meat. As a result of the examination, it was successful for the first time to
fractionate and mix the desired fraction from the above-mentioned various fractions, and the novel
antihypertensive peptide thus fractionated and mixed was named peptide SY. .
[0012]
Peptide SY can be fractionated and produced from the elution fraction described above. An example of
elution patterns of various eluates is shown in FIG.
As shown in FIG. 1, the peptide SY according to the present invention is added with, for example, α-1000
(stock solution loading) to a peptide adsorbing resin and then eluted with water (water elution (1)).
Manufacture by mixing the post-fraction, 11-18V / V% ethanol elution fraction eluting with 11-18V / V%
ethanol solution, and further water elution (2) fraction eluting with water. Can do. Setting of the sampling
start time of peptide SY fraction, setting of eluate switching timing, etc. are based on at least one
measured value of Bx, salinity, UV (280 nm absorption), Na, or based on fractionation time What is
necessary is just to determine suitably, It is also possible to monitor these items suitably, such as real
time, and to implement by a computer.
For example, in the elution pattern of FIG. 1, the starting point of fractionation of the fraction after the
peptide SY water elution (2) can be determined by measuring the salinity value as follows.
i) If it is taken from 0 minutes after the start of water elution, the Na content becomes 4 g / 100 g or more,
so that it becomes a high Na material and is not preferable as an antihypertensive agent.
ii) When the fractionation time is 20 minutes after the start of elution with water, the Na content is within
the allowable range of 1 to 3 g / 100 g.
iii) If the subsequent fractionation is started, the Na content is further reduced, but this time, the salt
content is too low, so that guanine contained in the peptide SY tends to precipitate during concentration,
which is not preferable. .
[0013]
iv) Therefore, the fractionation start time was 20 minutes after the start of water elution, and the amount
of Na was set to around 1 to 3 g / 100 g.
The final fractionation point of the water elution (1) fraction was the point at which the Na amount was
Next, 11-18 V / V% ethanol aqueous solution is added instead of water from this point, and the addition of
the ethanol aqueous solution is stopped when the peptide elution amount passes the peak and is reduced
to about half of the peak. Fractions were designated as 11-18 V / V% ethanol elution fractions. (Only the
11-18 V / V% ethanol-eluted fraction isolated is a peptide SY-MD containing almost no Na.)
The time when the addition of the 11-18 V / V% ethanol aqueous solution is stopped and switched to the
addition of water is also the start of water elution (2), but the UV absorption value at a wavelength of 280
nm indicating the total peptide amount rapidly decreases. The end point may be a period when the UV
absorption value becomes zero and a steady state is entered. The fraction obtained here was used as the
fraction eluted with water (2).
The obtained (1) water elution (1) post-fraction, (2) 11-18V / V% ethanol elution fraction, (3) water elution
(2) fraction, separately or sequentially. The mixture obtained is the peptide SY of the present invention.
In this way, the fraction from the elution fraction from the middle of the water elution (1) fraction to the
water elution (2) fraction through the 11-18 V / V% ethanol elution fraction is related to the present
invention. It can be obtained as a peptide SY (indicated as Surden peptide SY in FIG. 1).
Furthermore, it was "15% ethanol elute" in FIG. 1 corresponds to peptide SY-MD.
[0014]
Peptide SY contains a high concentration of a dipeptide (valyl-tyrosine: hereinafter sometimes referred to

as Val-Tyr or VY), which was first confirmed by the present inventors as one of the main bodies of the
antihypertensive peptide ( VY recovery rate: When α-1000 is 100%, SY is 90 to 95%, whereas SY-MD is
65 to 75%), and not only exhibits very high ACE inhibitory activity, but also exhibits Taste is also greatly
improved. However, since peptide SY-MD does not contain a fraction after water elution (1), it remains
bitter, but contains almost no Na, so it is a useful antihypertensive agent for people who cannot take Na.
That is, the “stock solution loading” portion has a strong taste, but also contains some fish odor derived
from the raw material, and has a high Na content. On the other hand, the post-fraction of water elution (1)
has little fishy odor derived from the raw material and has a very good taste.
Therefore, by incorporating the post-fraction of water elution (1), a peptide material “Peptide SY” that can
recover a large amount of VY from peptide SY-MD and has excellent taste and stability can be obtained.
It is.
In addition to the blood pressure suppressing action, peptides SY and peptide SY-MD also have functions
such as calcium and iron absorption promoting action, cholesterol suppressing action, and blood sugar
level suppressing action.
[0015]
The physicochemical properties of peptide SY are as follows:

Physicochemical properties of peptide SY (A) Molecular weight; 200-10,000 (FIG. 2)
(F) component: moisture 1 to 5 W / W% (normal pressure heating drying method)
(J) Specific rotation: [α] D 20 = −40 ° to −51 °
(K) ACE inhibitory activity value (IC 50 ): 200 μg / ml or less (Cushman
According to a variant of
[0016]
(L) Major amino acid composition ─────────────────────

Amino acid Analysis value (%)
─────────────────────
Aspartic acid 8.0-9.2
Glutamic acid 9.5 to 12.0
Valine 4.5-5.5
Methionine 2.5-3.8
Isoleucine 4.5-5.2
Leucine 7.3-8.5
Tyrosine 3.4-4.8
Phenylalanine 4.5-5.5
Histidine 3.0-3.8
Lysine 6.5-7.8
Arginine 5.0-6.0
─────────────────────
(Analysis method: Amino acid automatic analysis method)
[0017]
The physicochemical properties of peptide SY-MD are as follows.

(F) Ingredient: Moisture 2-6 W / W% (normal pressure heating drying method)
: Ash content 3.0W / W% or less (direct ashing method)
Peptide SY or peptide SY-MD according to the present invention is a novel peptide mixture that has been
conventionally unknown, exhibits excellent blood pressure lowering activity, and has no safety problem.
As a peptide for foods for specified health use. Therefore, the peptide is used as a food additive such as
a seasoning and a food for fortification or as an animal feed additive, and for the prevention of
hypertensive disease, and in some cases, treatment for hypertension due to its unique physiological
activity. Or widely used as an infusion, health food, clinical nutrition food, or the like.
When used as a food, the peptide can be added as it is, or used in combination with other foods or food
ingredients as appropriate. Moreover, when using as a pharmaceutical, it can administer orally or
parenterally. In the case of oral administration, tablets, granules, powders, capsules, powders, drinks can
be prepared according to conventional methods, and in the case of parenteral administration, for
example, injection preparations, infusions, etc. It can be used as a suppository, suppository, etc.
[0018]
Hereinafter, the present invention will be described in more detail with reference examples and examples.
[0019]
[Reference example]
Fresh sardines were processed with a deboner and minced. Divided fish meat is divided into 10kg surimi,
rapidly frozen at -30 ° C or lower, crushed by a pulverizer, added with an equal amount of water, sent to a
tank, and heated at 100 ° C for 10 minutes Then, the autolytic enzyme was inactivated and heat
denatured. Next, aqueous ammonia was added to adjust the pH to 9.5.
A 0.1% solution of a commercially available alkaline protease product was added thereto, and the mixture
was kept at 50 ° C. for 17.5 hours for enzymatic degradation. The enzyme was then inactivated by boiling
for 15 minutes.
This was passed through a vibrator screen (150 mesh), treated with a diector at 5000 rpm, then treated
with a sharp press centrifuge (15000 rpm), and filtered using diatomaceous earth as a filter aid to obtain a
peptide stock solution.
1% W / V of activated carbon was added to the peptide stock solution obtained above, and the mixture
was stirred at 30 ° C. for 60 minutes and filtered to obtain a filtrate. This was concentrated under reduced
pressure (20 ° C.) according to a conventional method and then UHTST sterilized according to a
conventional method to obtain an α-1000 (liquid) product, which was further spray-dried according to a
conventional method to obtain a particle size of 60 mesh. Α-1000 (powder) products were obtained and
each of these products was stored frozen.
[0020]
[Example 1]
(Mitsubishi Chemical Corporation) column (3.5 × 13 cm). The column was filled with the α-1000 solution
(stock solution loading), then, according to the elution pattern of FIG. 1, water, 15% ethanol aqueous
solution, then water and 500 ml of each were added, and the Sarden peptide SY in FIG. All fractions, that
is, the fraction after elution with water (1), the fraction with 15% ethanol elution, and the fraction with
water elution (2) were collected and lyophilized to obtain 2.1 g of peptide SY (powder). It was. The Na
content of peptide SY was 1.45 W / W%. (By atomic absorption photometry)
[0021]
[Example 2]
(Mitsubishi Chemical Corporation) column (3.5 × 13 cm). The column is filled with the α-1000 solution
(stock solution loading), and only the 15% ethanol-eluted fraction is isolated and collected from all
fractions of the sadden peptide SY shown in the elution pattern of FIG. Was lyophilized to obtain 1.7 g of
peptide SY-MD (powder). The Na content of peptide SY-MD was 0.124 W / W%. (By atomic absorption
photometry)
[0022]
[Example 3]
(Drink production)
――――――――――――――――――――――
100ml drink formula list ――――――――――――――――――――――
Fructose dextrose liquid sugar 4.5g
1g sugar alcohol
Sour seasoning 0.2g
Fragrance 0.13g
Sweetener (stevia) 0.03g
Caramel color 0.02g
Peptide SY (powder) 0.5g
(Obtained in Example 1)
Purified water Constant volume to 100 ml -----------
50ml drink formula list ――――――――――――――――――――――――
Fructose dextrose liquid sugar 10g
Fragrance 0.3g
Acidulant 0.16g
30ml drink formula list ――――――――――――――――――――――――
Fructose dextrose liquid sugar 5g
Fragrance 0.25g
Sour seasoning 0.1g
The above blend was mixed and dissolved at 60 ° C. and then subjected to plate sterilization at 128 ° C.
for 10 seconds. Next, 100 ml, 50 ml, and 30 ml brown bottles having been thoroughly washed were filled
at 90 ° C., allowed to cool at room temperature, and then rapidly cooled in a running water tank to
produce a drink.
[0023]
[Example 4]
500 g of peptide SY (powder) obtained in Example 1, 356 g of reduced maltose starch syrup, 100 g of
crystalline cellulose, 40 g of sucrose fatty acid ester, 4 g of sweetener (stevia) are mixed, and this mixture
is compressed by a compression tablet machine to be uncoated tablet (250 mg × 4000 pieces) were
prepared. This uncoated tablet was coated with 7.5 mg of shellac solution, and 4000 tablets containing
500 mg of peptide SY (powder) per 4 tablets were produced.
[0024]
[Example 5]
The example of taking the drink manufactured in Example 3 is shown.
(1) In the case of 30 ml drink Peptide SY is 0.5 g / bottle, and clinical trials are conducted for patients with
mild hypertension divided into a peptide administration group and a placebo group by a randomized
double-blind method. Ingested one daily, 4 weeks later, the blood pressure on the peptide administration
group alone was 10.6 mmHg on average, and the lower blood pressure was significantly reduced by 5.6
mmHg.
(2) In the case of a 50 ml drink Similarly, clinical trials were conducted for patients with mild hypertension
divided into a peptide administration group and a placebo group by a randomized double-blind method.
Ingested one daily, 4 weeks later, the blood pressure on the peptide administration group alone was 10.2
mmHg on average, and the lower blood pressure was significantly reduced by 3.8 mmHg.
(3) In the case of a 100 ml drink, it was carried out in the same manner, and after 4 weeks, the blood
pressure on the peptide administration group alone was 8.2 mmHg on average, and the lower blood
pressure was significantly reduced by 3.0 mmHg.
As described above, in any case, by taking a drink containing 0.5 g / peptide SY once a day, the blood
pressure of mild hypertensives does not affect the subjective symptoms, and the risk rate is 1%. A
significant decrease was confirmed below.
[0025]
[Example 6]
The administration example of the tablet manufactured in Example 4 is shown.
A placebo-controlled double-blind ingestion test was conducted on normal high blood pressure and 88
patients with mild hypertension.
When the tablets were ingested 4 tablets a day (0.5 g / day as peptide SY), the peptide SY administration
group (44 patients) had an upper blood pressure (systolic blood pressure) of 148 ± 11 mmHg at the start
of the test, The blood pressure (diastolic blood pressure) was 92 ± 14 mmHg, but the upper blood
pressure dropped to 138 ± 12 mmHg after 4 weeks, 134 ± 9 mmHg after 8 weeks, and the lower blood
pressure was 84 ± 11 mmHg after 4 weeks, 83 after 8 weeks. Significantly decreased to ± 10 mmHg. No
significant changes were seen in the placebo group (44 patients), and no side effects such as dry cough
were observed in either group. Therefore, the blood pressure of this tablet containing peptide SY against
normal high blood pressure and mild hypertension The inhibitory effect was confirmed.
[0026]
[Example 7]
(Drink production)
――――――――――――――――――――――
100ml drink formula list ――――――――――――――――――――――
Fructose dextrose liquid sugar 4.5g
1g sugar alcohol
Sour seasoning 0.2g
Fragrance 0.13g
Caramel color 0.02g
Peptide SY-MD (powder) 0.5g
Purified water Constant volume to 100 ml This 100 ml drink contains 500 mg / s of peptide SY-MD, and
the ingredients are water 96.7 g, protein 0.5 g, carbohydrate 4.5 g, calorie 19 Kcal, sodium 7.4 mg, It was
0.66 g of sorbitol.
Using this drink SY-MD 0.5g / drink 1 bottle / day, systolic blood pressure 130-140mmHg, diastolic blood
pressure 80-90mmHg normal high blood pressure person and systolic blood pressure 140-160mmHg,
diastolic blood pressure A double-blind controlled study was performed on 90-100 mm Hg volunteers with
mild hypertension.
As a result, there was no significant difference in blood pressure fluctuation in the placebo drink intake
group, but in the peptide drink intake group, the systolic blood pressure significantly decreased to 14.7
mmHg and the diastolic blood pressure to 7.6 mmHg after 4 weeks. , Confirmed the effectiveness
.
[0027]
[Example 8]
500 g of peptide SY-MD (powder) obtained in Example 2, 356 g of reduced maltose starch syrup, 100 g
of crystalline cellulose, 40 g of sucrose fatty acid ester, 4 g of sweetener (stevia) were mixed, and this
mixture was compressed by a compression tablet machine. Uncoated tablets (250 mg × 4000) were
prepared. This uncoated tablet was coated with 7.5 mg shellac solution per tablet, and 4000 tablets
containing 500 mg of peptide SY-MD (powder) per 4 tablets were produced.
In 100 g, the components were moisture 3.3 g, protein 44.2 g, carbohydrates 3.5 g, ash 1.6 g,
carbohydrates 47.4 g, sodium 566 mg, and the energy amount was 398 Kcal.
This tablet was used 4 times a day (0.5 g / day taken as peptide SY-MD), and a 12-week double-blind
comparative control test was conducted using a placebo not containing peptide SY-MD as a control. In
volunteers with normal high blood pressure and mild hypertension, the peptide SY-MD-containing tablet-
intake group showed a significant decrease in blood pressure in both systolic blood pressure (SBP) and
diastolic blood pressure (DBP) compared to before administration. That is, SBP was 145.4 mmHg and
DBP was 86.8 mmHg before ingestion, while SBP dropped to 134.7 mmHg and DBP to 83.0 mmHg at the
end of the test. No significant blood pressure fluctuation was observed in the placebo group, and no
subjective symptoms such as side effects occurred during 12 weeks.
[0028]
【The invention's effect】

Peptide SY and peptide SY-MD according to the present invention are not only excellent in
antihypertensive function because they contain a high concentration of Val-Tyr newly found as a
hypotensive component, that is, the main body of the antihypertensive peptide, Furthermore, peptide SY
has a feature that it has excellent taste and stability without bitterness by incorporating a part of the
water-eluted fraction. Since the novel peptide SY according to the present invention has the above-
mentioned characteristics, it can be used not only as a food or drink or as an additive, but also has
excellent ACE inhibitory activity. It can be used for suppression or prevention, and can be advantageously
used as a medicine by formulating into various dosage forms as an ACE inhibitor or a blood pressure
lowering agent.
As is apparent from the above, the novel peptide SY and peptide SY-MD show excellent blood pressure
lowering activity, and since they are originally derived from fish meat, there is no problem in safety (in
fact, When 500 mg per day was forcibly administered orally to rats, no acute toxicity was observed even
after 10 days. be able to.
FIG. 1 is a graph of a peptide SY elution pattern in Example 1. FIG.
FIG. 2 shows the molecular weight of peptide SY.
FIG. 3 shows an infrared absorption spectrum of peptide SY.
FIG. 4 shows an ultraviolet absorption spectrum of peptide SY.
FIG. 5 shows a peptide α-1000 ultraviolet absorption spectrum.
FIG. 6 shows an infrared absorption spectrum of peptide α-1000.
FIG. 7 shows an ultraviolet absorption spectrum of peptide SY-MD.
FIG. 8 shows an infrared absorption spectrum of peptide SY-MD.
Claims (8)
Hide Dependent
該食品が液状又は固状の形態であることを特徴とする請求項７に記載の食品。
1. Peptide SY having the following physicochemical properties:

(A) Molecular weight: 200 to 10,000 (by ASAHIPAK GS-320 high performance liquid
chromatography)
(C) Solubility in solvent: Easily soluble in water, but hardly soluble in ethanol, acetone and
hexane.
(F) Ingredient: Moisture 1 to 5 W / W% (normal pressure heating drying method)
2. A peptide component obtained by elution fractionation treatment using a peptide
adsorption resin with a peptide stock solution, wherein the elution fractionation
treatment uses water, an aqueous ethanol solution, and water in this order as the
development solution, and in the elution pattern of FIG. The peptide according to claim
1, which is obtained by preparatively mixing the water-elution (1) after-fraction, 11-18V /
V% ethanol-elution fraction and water-elution (2) fraction, which are obtained by SY.
(1) Water elution (1) Post-fraction: Water is used as a developing solution, and the
amount of sodium from the time when the amount of sodium (Na) in the entire eluted
peptide fraction (peptide SY) becomes 1 to 3 g / 100 g. Fraction up to the final
fractionation point of the fraction after water elution (1) in which becomes substantially
0 g / 100 g.
(2) Ethanol elution fraction: Next, a fraction obtained by using an ethanol aqueous
solution having a concentration of 11 to 18 V / V% as a developing solution until the
peptide elution amount passes the peak and is reduced to about half of the peak.
(3) Water elution (2) Fraction: Next, a fraction obtained by using water as a developing
solution until the elution fraction treatment reaches the end point.
3. The peptide SY according to claim 1 or 2, wherein the peptide stock solution is
produced using an aqueous solution of peptide α-1000 derived from fish meat and
having the following physicochemical properties as a peptide stock solution.
(C) Specific rotation [α] D 20 = −22 °
Neutral (f) appearance, ingredients:
White powder: moisture 5.14% (heat drying under reduced pressure); protein 87.5%
(Kjeldahl method, nitrogen / protein conversion factor 6.25); lipid 0% (Soxhlet extraction
method); ash content 5.0% (directly) Ashing method).
(G) characteristics;
It is a peptide derived from fish meat, obtained by inactivating a self-digesting enzyme
by heating and hydrolyzing with a proteolytic enzyme.
4. Using only the aqueous solution of 11 to 18 V / V% in (2) of claim 2 as a developing
solution, only the fraction obtained until the peptide elution amount passes the peak
and is reduced to about half of the peak is isolated. A method for producing a peptide
SY-MD,
5. Peptide SY-MD having the following physicochemical properties obtained in claim 4.
(C) Solubility in solvent: Easily soluble in water, but hardly soluble in ethanol, acetone
and hexane.
(D) Appearance property: White to light yellow powder (E) Liquid property (pH): 4.0 to
6.0
: Ash content 3.0 W / W% or less (direct ashing method)
6. An antihypertensive agent comprising the peptide SY according to claim 1 or the
peptide SY-MD according to claim 5 as an active ingredient.
7. An antihypertensive functional food comprising the peptide SY according to claim 1 or
the peptide SY-MD itself according to claim 5, or containing the peptide SY or peptide
SY-MD.
8. The food according to claim 7, wherein the food is in a liquid or solid form.
Patent keempat: JP 2009538895A : Mencegah terjadinya pembekuan darah.
Description
The present invention relates to a composition for use in the prevention of venous thrombosis.
Venous thrombosis and thrombosis in the vein cause many deaths annually and are a major health
problem worldwide (Lopez, JA, Kearon, C., Lee, AYY (2004). Deep Venous Thrombosis. Hematology
2004: 439-456).
The main pathologies that occur with venous thrombosis include deep vein thrombosis (DVT) and
pulmonary embolism. DVT is a disease in which a thrombus develops in a deep vein, such as a deep vein
in the leg or arm or a deep vein in the lower abdomen. In the UK, it is estimated that 1 to 3 of every 1000
British people develop DVT each year. In most cases of DVT, the thrombus is small and does not cause
any symptoms. The body can break down the thrombus and there are no long-term problems. However,
in many cases, the thrombus may be larger, resulting in partial or total obstruction of the venous blood
flow, resulting in swelling of the muscles surrounding the vein (eg, calf muscle) and muscle pain. is there.
In some cases, a piece of thrombus peels off and travels through the bloodstream to the lungs, where it
forms a pulmonary embolus that obstructs pulmonary blood flow, causing chest pain and shortness of
breath, or, in severe cases, until death May also cause.
There are risk factors that are well-recognized to make individuals more prone to DVT, including: elderly,
long-term immobilisation, recent surgery, trauma such as fractures, oral contraception Drug use, hormone
replacement therapy, pregnancy, puerperium, cancer and cancer treatment, antiphospholipid syndrome,
various genetic risk factors, and plasma risk factors. Genetic risk factors are primarily associated with the
hemostatic system and include mutations in genes encoding antithrombin, protein C and protein S, as
well as factor V Leiden and factor II G20210 A mutations. Plasma risk factors include
hyperhomocysteinemia and increased factor II, factor VIII, factor IX, factor XI, and fibrinogen
concentrations.
Thrombosis occurs due to dysfunction or inappropriate activation of components of the hemostatic

system. The hemostatic system consists of two separate but related systems; platelets and clotting
proteins. Its main function is to prevent blood loss and at the same time to regulate the network of
molecular signals to ensure blood fluidity. In the event of vascular injury, the integrity of the vasculature is
maintained by the blood, which is converted to an insoluble gel at the site of damage in the process of
platelet initiation and clotting protein reinforcement.
The mechanism by which venous and arterial thrombosis occurs and the structure of the thrombus
formed by the two types of thrombosis are substantially different, so venous thrombosis and arterial
thrombosis are generally clearly distinguished It is recognized as a possible disease type.
In arterial thrombosis such as atherosclerosis or arterial thrombotic events such as stroke or myocardial
infarction, platelets are involved in initiating thrombotic events. However, in diseases that occur with
venous thrombosis, the onset of the disease is caused by the initiation of the coagulation cascade, and
platelet aggregation plays a less important role. Indeed, platelet aggregation inhibitors such as aspirin
have been found to be of little use in preventing venous thrombosis.
Tissue factor (TF) is a transmembrane glycoprotein that is a major trigger of the coagulation cascade.
During vascular injury, blood exposure occurs to the subendothelial TF. Exposed TF is a cofactor for
activation catalyzed by factor IX (FIX) and factor Xa (FX), factor VIIa (FVIIa), which are essential
components of the tenase complex and the prothrombinase complex, respectively. Acts as This leads to
the rapid formation of FXa and thrombin. Thrombin then cleaves fibrinogen into fibrin, which subsequently
polymerizes to form a fibrin thrombus.
The FVIIa / TF complex is involved in the pathogenesis of many thrombosis, and the blood level of TF is a
risk factor for thrombosis. Inappropriate exposure of blood to TF leads to long-term up-regulation of
inflammatory cytokines in the blood, which in turn increases blood levels of acute phase inflammatory
markers such as C-reactive protein . This disruption of the inflammatory system leads to TF expression
on monocytes in the circulating blood, contributing to a persistent imbalance in the coagulation cascade
and spilling over to the broader haemostatic activation.
While TF is released into the bloodstream by vascular injury, DVT can often occur even without any
damage to the vein wall. In recent years, evidence has accumulated to indicate that TF circulates in
normal plasma, associated with cell-derived membrane microvesicles and as a soluble alternatively
spliced form ((1) Giesen et al Blood-borne tissue factor: another view of thrombosis. Proc. Natl. Acad. Sci.
US A. 1999; 96: 2311-2315; (2) Koyama et al. Determination of plasma tissue factor antigen and its
clinical significance. J. Haematol. 1994; 87: 343-347; and (3) Albrecht et al. Detection of circulating tissue
factor and factor VII in a normal population. Thromb. Haemost. 1996; 75: 772-777). Microvesicles with
endogenous TF have been found to contribute to experimental thrombosis in vivo in the microcirculation
of the testicular muscle (Falati et al. J Exp Med. 2003; 197: 1585-1598) It has also been shown to
improve hemostasis in hemophilic mice (Hrachovinova et al. Nat. Med. 2003; 9: 1020-1025). In the
experimental system described in the aforementioned literature, microvesicles with TF bind to platelets or
activated endothelial cells at the site of injury, ie P-selectin glycoprotein ligand-1 (PSGL-1 on the
microvesicles). ) And P-selectin on activated platelets appears to be involved in thrombosis through a
series of actions that depend on the interaction.
International patent application WO99 / 55350 discloses the use of a water-soluble extract of tomato as a
platelet aggregation inhibitor. The platelet aggregation inhibitory properties of the tomato extract (known
under the name Cardioflow® or Fruitflow®) have attracted considerable mass media attention, It has
been suggested that tomato extracts derived from said extracts and other sources can reduce the risk of
DVT (eg (a) Vibrant Life, 1 January 2006, ISSN: 0749-3509; Volume 22; Issue 1; (b) Main Report-Health
and Wealth Letter, Drinking Tomato Juice Protects The Heart, 3 October 2005; (c) Citywire, 18 February
2005; (d) Verna Noel Jones, Chicago Tribune, RESOURCES. Q, 16 January 2005; (e) Lindsay McIntosh,
Aberdeen Press & Journal, 23 September 2004; (f) The Express, 23 September 2004, City And Business
Ed. Stephen Kahn; (g) Citywire, 22 September 2004; (h) The Sunday Mail , 5 September 2004; (i) ANSA-
English Media Service, HEALTH: TOMATOES CAN PREVENT HEART DIS EASES, 28 August 2004;
and similar literature).
While each of these references discusses the underlying rationale for the properties of the extract,
speculations about the possibility of using the extract to reduce the risk of DVT are always based on
known extracts. Based on platelet aggregation inhibitory activity. However, as discussed above, platelet
aggregation has not been associated with the onset of venous thrombosis, and platelet aggregation
inhibitors such as aspirin have been found to be of little use in preventing DVT. A report in the leading
media that tomato extract can prevent DVT by its platelet aggregation inhibitory activity is “GP Clinical-
Behind The Headlines-Can” in the medical journal GP (Haymarket Publications, London, UK, 5 April
2004,) "Tomato drink halt blood clots?" (GP Clinical-Behind Big News-Can Tomato Drinks Stop Clots?) In
the paper, the authors conclude that general practitioners should tell their patients that "tomato beverages
may not help prevent DVT because antiplatelet drugs do not have a significant effect on the venous
system" ing.
Therefore, to the best of the applicant's knowledge, there is no evidence in the literature so far that
suggests that tomato extract has some effect on the treatment of venous thrombosis. Furthermore, there
is no suggestion in the literature that the tomato extract has any effect on the coagulation cascade that
initiates the formation of venous thrombi.
[Summary of Invention]
Now, tissue factor (TF) mediated events are found to be affected by water-soluble extracts of tomatoes,
and the extracts are in vitro clotting times in plasma (with the removal of blood cells containing platelets).
Was found to decrease. These results obtained so far indicate that tomato extracts would be useful in
preventing the onset of venous thrombosis.
Accordingly, in a first aspect, the present invention provides a tomato extract or an active fraction thereof
for use in preventing or inhibiting the onset of venous thrombosis.
In another aspect, the present invention provides a tomato extract or an active fraction thereof for use in
preventing or inhibiting the initiation of fibrin thrombus formation in a vein.
As used herein, the term “active fraction” is a fraction isolated from a tomato extract that has the ability to
prevent the onset of fibrin thrombus formation in the vein or prevent the onset of venous thrombosis.
Refers to the fraction having.
The present invention also provides the following:

The use of a tomato extract or its active fraction for the manufacture of a medicament for preventing or
inhibiting the onset of venous thrombosis;
The use of a tomato extract or an active fraction thereof for the manufacture of a medicament for
preventing or inhibiting the initiation of fibrin thrombus formation in veins;
A composition comprising a tomato extract or an active fraction thereof for use in preventing or inhibiting
the onset of venous thrombosis;
A composition comprising a tomato extract or an active fraction thereof for use in preventing or inhibiting
the initiation of fibrin thrombus formation in a vein;
A method for preventing or inhibiting the onset of venous thrombosis in a mammal such as a human,
comprising administering to the mammal an effective amount of a tomato extract or an active fraction
thereof;
A method for preventing or inhibiting the onset of fibrin thrombus formation in a vein, comprising
administering to a patient an effective amount of a tomato extract or an active fraction thereof.
The tomato extract of the present invention is at risk of experiencing the occurrence of venous thrombosis
by belonging to any one or more (in any combination) of the following potentially dangerous
subpopulations: Prevention or inhibition of the onset of venous thrombosis (or prevention or inhibition of
onset of fibrin thrombosis in the vein) in patients who are more likely than normal:
・ Patients over 50 years of age, eg, over 60 years old, over 70 years old or over 80 years old;
Patients who are stationary for a long period of time, e.g. over 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours, or
over 1, 2, 3, 4 or 5 days;
-Medically obese patients;
・ Patients who have undergone surgery recently (eg, in the past month or in the past 21, 14 or 7 days);
・ Patients with trauma such as fractures;
・ Patients taking oral contraceptives;
・ Patients receiving hormone replacement therapy;
・ Pregnant patients;
・ Mother who gave birth recently (partum);
・ Patients with cancer and patients undergoing treatment for cancer;
・ Patients suffering from antiphospholipid syndrome;
• Patients with genetic risk factors; and • Patients with plasma risk factors.
For patients with genetic risk factors, the risk factors may be any one of the following or two (in any
combination):
-Mutations in the gene encoding antithrombin;
-Mutations in the gene encoding protein C;
-Mutations in the gene encoding protein S;
• Factor V Leiden mutation; and • Factor II G20210 A mutation.
For patients with plasma risk factors, the risk factors can be any one of the following, or two or more (in
any combination):
• Hyperhomocysteinemia • Increase in factor II concentration;
Increase in factor VIII concentration;
Increase in factor IX concentration;
Increased factor XI concentration; and increased fibrinogen levels.
Thus, in another aspect, the invention relates to veins in patients belonging to any one or more than one
(in any combination) of the potentially dangerous subpopulations defined herein. Provided is a tomato
extract or an active fraction thereof for use in the prevention of thrombosis.
The present invention also provides for the prevention of venous thrombosis in patients belonging to any
one or two (or any combination) of potentially dangerous subpopulations as defined herein. There is also
provided the use of a tomato extract or an active fraction thereof to produce a composition (eg, a
medicament).
The invention further provides for patients belonging to any one or more (in any combination) of the
potentially dangerous subpopulations defined herein (eg, human patients) There is also provided a
method for preventing venous thrombosis in a mammalian patient comprising administering to said
patient an effective amount of tomato extract or an active fraction thereof.
In a further aspect, the present invention relates to a use, an extract for use, a use wherein the patient is
a member of a subpopulation of persons suffering from recurrent venous thrombosis, such as recurrent
deep vein thrombosis A method or composition for use is provided.
The term “effective amount” as used herein refers to an amount that provides a therapeutic effect to a
patient. The therapeutic effect may be objective (ie, measurable by some test or marker) or subjective (ie,
the patient suggests or feels an effect).
Further aspects and embodiments of the invention are as set forth herein below and in the claims
appended hereto.
The tomato extract of the present invention has been found to act in several ways to prevent or inhibit the
activity of tissue factor (TF).
First, tomato extracts have been found to affect plasma clotting time (PT, TCT, aPTT), thereby implying
potential mediation of clotting factors.
Second, tomato extract was found to reduce the expression of P-selectin on activated platelets. As
mentioned above, it is believed that the binding of tissue factor-bearing microvesicles to the platelet
surface and endothelial cell surface P-selectin is responsible for the initiation of the coagulation cascade.
Third, tomato extract was found to block the interaction of TF with the PAR2 receptor on the surface of
human umbilical vein endothelial cells (HUVEC cells). The PAR2 receptor is a substrate for the TF / FVIIa
complex and FXa.
Based on the three findings shown above, it is believed that the tomato extract and active fraction of the
present invention would be useful in inhibiting the onset of venous thrombosis and fibrin thrombus
formation.
[Preparation and characteristics of tomato extract]

Whole tomatoes or coarsely chopped or ground but unfractionated tomatoes can be used for the
purposes of the present invention, but it is preferred to use an aqueous extract of tomatoes.
Such an extract may be prepared by homogenizing peeled or unpeeled tomato pulp and then filtering the
homogenate to remove solids. Preferably, substantially all water insoluble solids are removed, for
example by centrifugation and / or filtration.
Alternatively, commercially available tomato paste may be used as a starting material for the preparation
of the extract. Tomato paste is generally diluted with water, so water-insoluble solids are removed, for
example, by centrifugation and / or filtration, to obtain a substantially clear solution.
In any case, the removal of the water-insoluble solids has the effect of removing skin debris containing
lycopene. Thus, the preferred tomato extract of the present invention is a water-soluble extract that is
substantially free of lycopene.
Such aqueous filtrates can be further fractionated to provide an active fraction containing one or more
compounds involved in the biological effects or therapeutic effects described herein. Alternatively, the
aqueous filtrate can be evaporated to yield a dry water-soluble extract.
Filtration of tomato homogenate can be performed in one step or in a series of filtration processes to
remove larger pieces of tomato skin and / or other water-insoluble debris of tomato pulp starting from a
relatively coarse filtration or centrifugation step. A further filtration step can then be performed to obtain a
substantially clear solution, such as a solution that passes through a 0.2 μm (0.2 μ) filter without loss of
solids.
Thus, in one preferred embodiment of the present invention, the tomato extract is a water-soluble extract
that is substantially free of lycopene and that can pass through a 0.2 μm (0.2 μ) filter without loss of
solids.
In some embodiments, native sugars are removed from the tomato extract. The advantages of removing
the native sugars are that the activity of the tomato extract is concentrated and that the tomato extract is
not sticky and easy to process in a solid state.
When the starting material for the preparation of tomato extract is tomato paste, the tomato paste is
preferably produced by the “cold break” method rather than the “hot break” method. The terms “hot break”
and “cold break” are well known in the field of tomato processing and commercially available tomato
pastes are generally sold as either hot break pastes or cold break pastes. Cold break paste can be
prepared by a method that includes a tomato homogenization followed by a heat treatment step in which
the tomato is heated to a temperature not exceeding about 60 ° C., in contrast, a hot break paste is a
homogenized tomato. It is heat treated at a temperature of about 95 ° C. (see, for example, Anthon et al.,
J. Agric. Food Chem. 2002, 50, 6153-6159).
In an alternative method, an aqueous extract of tomato or tomato paste is enzymatically digested with
pectin and starch in the homogenized pulp or paste, then the suspended solids are removed from the
homogenate and microfiltered. Alternatively, it can be produced by ultrafiltration to remove higher
molecular weight proteins and residual polysaccharides. The extract can be purified by removal of
monosaccharides such as glucose, fructose and sucrose, and is concentrated water-soluble containing a
wide variety of low molecular weight (<1000 Da) non-sugar tomato components An extract remains. The
removal of monosaccharides can be done by using crystallization, such as ultrasound-assisted
crystallization, or by ethanol precipitation of crystalline glucose and fructose. Alternatively,
monosaccharides can be separated from other extract components by chromatographic methods, for
example, by selectively adsorbing bioactive extract components from aqueous solutions to polystyrene-
based resin materials, and waste stream glucose Allows selective removal of fructose and sucrose. The
adsorbed non-sugar component is then recovered from the adsorbent resin material by elution with
ethanol and subsequent removal of the ethanol by evaporation. Non-sugar components can be dried to
water-soluble powders by spray drying or drum drying, or can be resuspended in water to produce an
aqueous syrup. The aqueous extract thus prepared represents a further aspect of the present invention.
For example, tomato extracts without sugar, as discussed above, typically contain various compounds
with molecular weights less than 1000 Da and represent preferred extracts for use in the present
invention.
We have also found that the tomato extract is effective when it contains no nucleotides or has a low
nucleotide content. Accordingly, preferred extracts have a nucleotide content of less than 10 nM or do not
contain nucleotides therein.
The tomato extract of the present invention contains several physiologically active ingredients. Preferred
extracts comprise a bioactive ingredient selected from: phenolic compounds; amino acids; amino acid
complexes; and tomato flavor compounds.
Said extract preferably comprises a phenolic compound selected from: flavonoids and flavonoid
derivatives such as quercetin, kaempferol and derivatives of naringenin; hydroxycinnamic acids and
hydroxycinnamic acid derivatives such as ferulic acid, coumaric acid And their complexes; benzoic acids
and benzoic acid derivatives such as benzoic acid, hydroxybenzoic acid, gallic acid, salicylic acid and
complexes thereof.
Said extract preferably comprises an amino acid selected from tyrosine, hydroxytyrosine, phenylalanine,
glutamine and complexes thereof.
The flavor compound may be selected from hexanal derivatives, dimethyl sulfide, b-damacenone, 3-
methylbutyric acid, eugenol and methional.
The tomato extract may be subfractionated by HPLC to obtain three subfractions AF1, AF2 and AF3
based on polarity. In the Examples section below, in vitro examples were performed using unfractionated
extract (tAF) and subfractions AF1-AF3.
[Combination of pharmaceuticals and dietary supplements]

The tomato extract or active fraction thereof may be formulated for oral administration. As such, tomato
extracts or active fractions thereof can be formulated as solvents, suspensions, syrups, tablets, capsules,
troches and snack bars, inserts and patches as examples. Such formulations can be prepared according
to methods known per se.
For example, the extract or active fraction thereof is a syrup or other oral agent in the presence of one or
more excipients selected from sugars, vitamins, flavoring agents, colorants, preservatives and thickeners.
It can be made into a liquid for administration, such as a health drink.
Tonicity modifiers such as sodium chloride or sugars can be added to provide solutions of a particular
osmotic power, for example isotonic solutions. One or more pH adjusters, such as buffers, can be used to
adjust the pH to a specific value, and it is preferable to maintain the pH adjusted value. Examples of
buffering agents include sodium citrate / citrate buffer and phosphate buffer.
Alternatively, the extract or its active fraction can be dried, for example by spray drying or freeze drying,
and the dried product can be in solid or semi-solid dosage forms such as tablets, troches, capsules,
powders, granules Or dispensed as a gel.
Simple dry extracts can be prepared without any additional ingredients. Alternatively, a dry extract can be
prepared by adsorption onto a solid support such as a sugar such as sucrose, lactose, glucose, fructose,
mannose, a sugar alcohol such as xylitol, sorbitol or mannitol, or a cellulose derivative. Other particularly
useful adsorbents include starch-based adsorbents such as flour, such as wheat flour and corn flour. For
tablet form, the dried extract is generally of sugars such as sucrose and lactose, and sugar alcohols such
as xylitol, sorbitol and mannitol, or modified cellulose or cellulose derivatives such as powdered cellulose,
microcrystalline cellulose or carboxymethylcellulose. Mixed with such a diluent. A tablet may generally
also contain one or more excipients selected from granulating agents, binders, lubricants and
disintegrating agents. Examples of disintegrants include starch and starch derivatives, and other
swellable polymers such as cross-linked polymer disintegrants such as cross-linked carboxymethyl
cellulose, cross-linked polyvinyl pyrrolidone and starch glycolate. Examples of lubricants include stearates
such as magnesium stearate and stearic acid. Examples of binders and granulating agents include
polyvinyl pyrrolidone. If the diluent is not inherently very sweet, sweeteners such as ammonium glycyl
lysinate or artificial sweeteners such as aspartame or sodium saccharinate can be added.
The dry extract can also be formulated as a powder, granule or semi-solid for incorporation into capsules.
When used in powder form, the dry extract can be formulated with any one or more of the excipients
described above for tablets, or can be present in undiluted form. For presentation in semi-solid form,
viscous liquids such as polyethylene glycol or semi-solid vehicles, for example glycols such as propylene
glycol or liquid carriers such as glycerol, or vegetable or fish oils such as olive oil, sunflower oil, safflower
oil, The dried extract can be dissolved or suspended in an oil selected from evening primrose oil, soybean
oil, liver oil, herring oil and the like. Such extracts can be filled into hard gelatin type or soft gelatin type
capsules, or capsules made from hard gelatin substitutes or soft gelatin substitutes, where soft gelatin
capsules or soft gelatin substitute capsules are viscous Preferred for liquid or semi-solid packing.
The dried extract can also be provided in powder form for incorporation into bar-shaped snack foods such
as fruit bars, nut bars and cereal bars. For presentation in the form of a bar-shaped snack food, the dried
extract is made of any one or more ingredients selected from dried fruits such as dried tomatoes, raisins
and sultanas, peanuts, and grains such as oats and wheat. Can be mixed.
The dried extract can be provided in powder form for reconstitution as a solution. As such, the dried
extract may contain soluble excipients such as sugars, buffering agents such as citrate buffer and
phosphate buffer, for example bicarbonates such as sodium bicarbonate or ammonium bicarbonate. As
well as solid acids such as citric acid or acid citrate.
In one preferred embodiment, the dry extract is provided in powder form for incorporation into a capsule,
eg, a hard gelatin capsule, optionally with a preferred solid (eg, powder) excipient.
In another embodiment, the dried extract is one from which substantially all native sugars have been
removed.
A solid or semi-solid dosage form of the invention can contain up to about 1000 mg of dried extract, for
example up to about 800 mg.
The extract can be presented as a food supplement or food additive, or can be incorporated into a food,
such as a functional food or a dietary supplement.
The composition of the present invention can be presented in the form of a unit dosage form containing a
defined concentration of extract or an active fraction thereof. Such unit dosage forms can be selected to
obtain a desired level of biological activity. For example, a unit dosage form contains an extract or active
fraction in an amount of up to 1000 mg (dry mass), more commonly up to 800 mg, eg 50 mg to 800 mg,
eg 100 mg to 500 mg be able to. Individual amounts of extract or active fraction that can be included in
the unit dosage form are 50 mg, 100 mg, 150 mg, 200 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg,
550 mg, 600 mg, 650 mg, 700 mg, 750 mg and 800 mg may be selected.
The composition of the invention can be placed in a container, package or dispenser together with
instructions for administration.
[Medical use]
For use in preventing or inhibiting the onset of venous thrombosis, the daily amount of extract or active
fraction administered to a patient depends on the strength of the extract as well as the particular condition
or disease being treated and its severity It will depend on the degree, but will ultimately be at the
discretion of the physician. However, the dosage will typically be an amount that is non-toxic and effective
to produce the desired result.
For example, a typical daily dose for a human patient potentially at risk of suffering from venous
thrombosis is 0.0001-0.1 grams per kilogram body weight, preferably 0.001-0.05 grams per kilogram body
weight obtain. When the active fraction is administered separately, the amount of solid material
administered can be reduced by an amount consistent with an increase in the purity of the active fraction.
Typically, a human patient can be administered at least 100 mg of the extract per day (dry weight or
equivalent dry weight), preferably at least 200 mg, more usually at least 500 mg.
The composition can be administered in single dose units or multiple dose units per day, for example 1 to
4 times per day, preferably 1 or 2 times per day.
The extracts of the invention can be administered in solid, liquid or semi-solid form. For example, the
extract can be administered in the form of tomato juice or a concentrate thereof alone or mixed with other
fruit juices such as orange juice.
[Example 1]
As a starting material for the preparation of tomato extracts commercially having a browning index less
than 0.350 AU (browning index is defined as the absorbance at 420 nm of a solution with a concentration
of 12.5 g / L of soluble solids) A tomato extract for use in the treatment methods of the present invention
was prepared using available 28-30 Brix (ie 28-30% solids, w / w) cold break tomato paste. This paste
was diluted with ultrapure water (˜1: 5) and large particulate matter was removed by clarification following
centrifugal filtration using a Westfalia MSB-14 separator (centrifugal disc clarifier) at room temperature.
The smaller particulate material is then removed by microfiltration at temperatures below 45 ° C. and
passed through a 0.2 μm (0.2 μ) filter that does not contain insoluble (settling) solids and without loss of
soluble solids. A clear pale yellow solution was produced. This solution was concentrated to 62-65 Brix
syrup by evaporation using carefully controlled conditions and temperatures below 80 ° C. to limit the
progress of the non-enzymatic browning reaction. A flash sterilization step (T = 105 ° C. for 3 seconds)
was incorporated at the beginning of the evaporation process. The final product was characterized by a
browning index of less than 0.600 AU and a total microbial plate count of less than 1000.
The concentrated extract may be added to an orange juice matrix for administration to a patient.
[Example 2]
Selective preparation of tomato extract and its sub-fraction Homogenization of fresh tomato
(Lycopersicon esculentum, locally sourced), centrifugation and ultrafiltration of the resulting pale yellow
liquid (ultrafiltration membrane: MW) Aqueous extracts derived from ripe tomato fruits were prepared by
clarification with a cutoff of 1000 Da, Millipore (UK) Ltd., Watford, UK). Analytical results showed that the
majority of the tomato aqueous extract (85-90% of the dry product) consisted of soluble saccharides.
Their constituents were removed using solid phase extraction using a styrene-divinylbenzene (SDVB)
cartridge (JT Baker, Mallinckrodt Baker BV, Deventer, Holland) at pH 2.5. Non-sugar components were
retained on the cartridge and eluted in methanol. The separated non-sugar substance (total active
fraction tAF) accounted for about 4% of the aqueous extract dried product. Using semi-preparative HPLC,
the tAF component was subdivided into three broad groups (Synergy Polar-RP, 4 μm (4 μ), 250 × 10 mm
column and Luna C18 (2), 3 μm (3 μ), 250 x 10 mm column, Phenomenex, Macclesfield, UK: acetonitrile /
0.05% TFA gradient). These three groups are shown in the HPLC chromatogram of FIG. 1 and are
named A1, A2 and A3 in order of decreasing polarity. The isolated tAF and subfractions AF1-AF3 are
reconstituted to a known concentration in phosphate buffered saline (PBS, Sigma-Aldrich, Poole, UK) and
the pH of the solution before use in in vitro examples Was adjusted to 7.4.
[Example 3]
Inhibition of clotting time by components in tomato extract It has previously been shown that tomato
extract components inhibit platelet aggregation in vitro and ex vivo (see applicant's earlier application
WO99 / 55350). Platelets are part of the hemostatic system and work in conjunction with the coagulation
cascade to balance blood fluidity and blood coagulation. This experiment was designed to investigate
whether tomato extract can also act on the coagulation cascade independent of its known effects on
platelets. Plasma clotting time (measured in plasma depleted of platelets) was used to provide a
measurement that reflects the state of the coagulation cascade independent of platelet function.
Incorporates the clotting ability of Factor I (Fibrinogen), Factor II (Prothrombin), Factor V, Factor VII and
Factor X to provide specific criteria for the validity of the extrinsic system and Thrombin clotting times (PT
and TCT, respectively) were used. Tissue factor must be added from an external source to allow the
exogenous system to function. Activated partial thromboplastin time (aPTT) is used to characterize the
intrinsic system and also regulates the clotting ability of factor XII, factor XI, factor IX, factor V and factor
VIII, all of which are normally present in plasma. test.
Experimental details: Coagulation time was measured according to the procedure specified by the
manufacturer on a CoaData 4001 coagulometer (Helena Biosciences, UK). Briefly, PT, TCT and aPTT
reagents were obtained from Helena Biosciences with Norm-Trol quality control plasma, and
coagulometers were made to a percentage of PT / TCT / aPTT using a calibrator kit from the company.
Calibrated against. Citrated plasma warmed to 37 ° C was incubated for 15 minutes with either control
solution (saline) or treatment solution (see below). The treated plasma was then incubated with PT / TCT
/ aPTT reagent with agitation and the time taken to clot formation was recorded in duplicate by a
coagulometer. Duplicate measurements with a coefficient of variation of less than 5% were accepted.
Norm-Trol QC plasma was used as a control.
Treatment: Tomato extracts AF1, AF2 and AF3 described in Example 2 were used as treatment agents.
All treatment solutions were made to give a final concentration of 80 μg per mL of plasma. Saline (0.9%
NaCl) was used as a control. All control and treatment solutions were adjusted to pH 7.4 and warmed to
37 ° C. before use.
Results: The data obtained showed an increase in the time at which clot formation occurred in the treated
plasma compared to the control plasma. FIG. 2 shows the results obtained as percent inhibition of clotting
time compared to the control value. Tomato extract fractions AF1 and AF2 had the most significant effect
on the clotting time parameter. The extrinsic pathway (assessed by PT and TCT) was affected more
strongly than the intrinsic pathway.
Conclusion: The results obtained indicate that any component of tomato extract can interact in vitro with
blood factors that together make up the coagulation cascade, and this effect takes into account the
overall function of the hemostatic system Interesting in case. Increased effects on the extrinsic system
suggest that the tissue factor-mediated pathway of clotting, known to be of particular importance in
venous thrombosis, could be beneficially suppressed by using the tomato extract of the present invention
.
[Example 4]
Investigation of the effect of tomato extract on ADP-induced platelet P-selectin expression The following
experiment was designed to further explore the mechanisms by which tomato extract components can
alter blood coagulation. Activation of platelets results in the release of procoagulant signaling molecules
from the surface of activated platelets. Subsequent interaction with the vessel wall results in activation of
the coagulation cascade. One of the most prominent of these procoagulant platelet-derived signaling
molecules is P-selectin. In this experiment, the expression of P-selectin on the surface of activated
platelets was measured using a fluorescently labeled antibody against P-selectin. The level of P-selectin
induced fluorescence was quantified by flow cytometry. Next, the effect of tomato extract components on
P-selectin expression was quantified.
Experimental details: Freshly collected whole blood was diluted 1:10 with HEPES-Mg buffer (pH 7.4) and
pre-incubated with treatment solution (see below) or control solution (HEPES-Mg buffer) for 10 minutes .
To induce P-selectin expression on platelets, aliquots (40 μL) of these mixtures are then combined with
ADP (final concentration 3 μmol / L) in Falcon polystyrene tubes (BD Biosciences, Cowley, UK) or
Incubated at room temperature for 5 minutes without ADP. Next, 10 μL of a saturating concentration of
the two fluorescently labeled antibody was added to the incubation tube. Fluorescein isothiocyanate
labeled anti-CD61 (anti-CD61-FITC) was added to clearly identify all platelets in the test sample (CD61 is
a platelet-specific protein that is not expressed by other blood cells). Phychoerythrin labeled anti-P-
selectin (anti-CD62P-PE) was added and allowed to bind to P-selectin expressed on the platelet surface.
FITC and PE labeled mouse IgG antibodies were used as isotype controls. Incubation was performed in
the dark at room temperature for 20 minutes. Next, ice-cold phosphate buffered saline (2 mL) was added
and analyzed on a FACS caliber flow cytometer using CellQuest software (BD Biosciences, Cowley, UK).
Activated platelets were defined as the percentage of CD61 positive results co-expressing the CD62P
receptor.
Treatment: Tomato extracts tAF and AF1 to AF3 described in Example 2 were used as treatment agents.
All treatment solutions were made to give a final concentration of 50 μg / mL. HEPES-Mg buffer was used
as a control. All control and treatment solutions were adjusted to pH 7.4 and warmed to 37 ° C prior to
use. N = 5 for each measurement.
Results: 41.2-68.2% P-selectin positive platelets were recorded in the control sample for stimulation with
3 μmol / L ADP, with a median of 51.1%. The dispersion obtained from the experiment was below 5%.
Preincubation of diluted whole blood with tAF or AF1-AF3 resulted in significant inhibition of activation-
induced P-selectin expression compared to control values (P <0.001, FIG. 3). The effect of tAF on P-
selectin expression was significantly different from the effect of each of the subfractions AF1-AF3, but no
difference was detected between the individual subfractions. Dose responsiveness was observed in the
inhibition of P-selectin by tAF (final concentration range 0-100 g / mL, FIG. 4).
Conclusion: The results obtained indicate that tomato extract reduces platelet activation in response to
ADP agonists in vitro and inhibits the expression of P-selectin on the platelet surface. This effect is very
important for the coagulation cascade. Expression of P-selectin in activated platelets results in the
release of P-selectin into the bloodstream (soluble P-selectin or sP-selectin), resulting in a protein source
that can be sustained after platelet activation is inhibited . A higher level of soluble P-selectin is a soluble
P-selectin tissue factor that functions as a mediator of soluble P-selectin binding between platelets and
monocytes and provides a link between platelet activation and the coagulation cascade And is associated
with venous thrombosis. Our results show that this link to the coagulation cascade can be broken by the
positive effect of tomato extract on P-selectin expression; thus tomato extract reduces both bound
platelets and circulating P-selectin Suggests that this may reduce the risk of venous thrombosis.
[Example 5]
Investigation of the effect of purified tomato extract and its subfractions on TF-induced cytokine release in
cultured human umbilical vein endothelial cells (HUVEC cells) Example 4 shows that tomato extract
inhibits P-selectin expression on its platelets. Demonstrates that it may help prevent the onset of venous
thrombosis. P-selectin is an integral part of the tissue factor-mediated initiation of the coagulation
cascade, a pathway thought to be involved in the causative process of venous thrombosis. By reducing
P-selectin expression on the surface of activated platelets or endothelial cells, we can prevent tomato
extract from attaching tissue factor-containing microvesicles to endothelial cells, and thus It was
hypothesized that the intimate contact required for thrombin generation and thrombus formation could be
prevented.
In order to demonstrate that tissue factor action on endothelial cells can be inhibited in the presence of
tomato extract, we designed experiments involving human umbilical vein endothelial cells (HUVEC cells).
HUVEC cells express the protease activated receptors PAR1 and PAR2. PAR2 is a substrate for TF /
FVIIa and FXa and stimulates the release of inflammatory cytokines IL-6 and IL-8 when activated.
Therefore, by measuring TF-mediated IL-6 production and IL-8 production, the effect of preincubation
with tomato extract components can be evaluated.
Experimental details: HUVEC cells were grown (passaged up to 5 generations) in BGM-2 medium. Cells
were serum starved for 5 hours and then treated with 25 nM TF / 10 nM FVIIa and 100 nM FX in the
presence or absence of tomato extract components (treatment agents are fully described below). After
the treated cells were incubated for 20 hours, the supernatant was collected and frozen at -80 ° C until
analysis of IL-6 and IL-8 by ELISA.
Treatment agent: Tomato extract active ingredient concentrate (tAF as described in Example 2) was used
as a “basic” treatment agent, consuming 2.5 raw tomatoes, assuming complete absorption of all
components Calculated as the maximum concentration achievable in the circulating blood of normal
individuals (blood volume 5.5 L). The amount was 43 mg / L for tAF. The AF1-AF3 described in Example
2, which are the three sub-fractions of tAF, are also concentrations reflecting their contribution to tAF on a
dry weight basis, ie 13.6 mg / L, 5.5 for each of AF1, AF2 and AF3. Tested at mg / L and 23.4 mg / L. All
four treatments were tested at their basal concentrations and also at 2 and 10 times the basal
concentrations. Saline was used as a control treatment.
Results: Preincubation with tomato extract components reduced the level of IL-6 produced by HUVEC to
nearly 12% and IL-8 levels to nearly 10-50% (see FIG. 5). reference). This means that tomato extracts
found to inhibit P-selectin expression also reduce the ability of TF to induce a signaling cascade in
endothelial cells via PAR. The AF2 and AF3 components were more effective than the AF1 component—
this is reflected in the results for P-selectin inhibition. The highest concentration used is least effective
and probably reflects greater stress during incubation (significant cell death occurred). IL-8 production
was significantly more affected than IL-6 production, which may be due to differences in the ease of
induction of the two cytokines at the TF concentrations used.
Conclusion: The results of this experiment show that aqueous tomato extract reduces the interaction
between TF and endothelial cells. We have suggested that it occurs at least partly through the effect of
aqueous tomato extract on P-selectin expression (see Example 4). These effects on TF-mediated events
in endothelial cells will reduce thrombin generation as a result of TF / VIIa interaction with endothelial
cells or activated platelets and prevent activation of the coagulation cascade and thrombus formation It
means that. This is supported by the results given in Example 3 where we have shown that plasma
clotting via the extrinsic system could be suppressed by tomato extract components. We suggest that
tomato extract will have a beneficial effect on the important mechanisms affecting venous thrombosis and
even on the broader inflammatory system. The effect on IL-6 suggests that hepatic CRP synthesis may
be reduced in vivo. CRP is an independent risk factor for atherosclerosis and CVD. The effect on IL-8
suggests that neutrophil activation can also be suppressed by tomato extract components.
[Example 6]
Formulation
(i) Capsule formulation The capsule formulation is obtained by drying the tomato extract described in
Example 1 and filling the resulting lyophilized powder into a hard gelatin capsule shell to give a capsule
capacity of 800 mg per capsule. Prepared.
(ii) Capsules containing diluted tomato extract A diluent selected from sucrose, lactose and sorbitol is
added to the aqueous tomato extract of Example 1. The resulting mixture is then lyophilized to form a
powder that is hard gelatin capsules to give a capsule volume of 800 mg per capsule (200 mg of tomato
extract and 600 mg of diluent). To fill.
(iii) Fruit Beverage The aqueous extract of Example 1 is treated with an orange juice matrix, such as
freshly squeezed orange juice, to give a 50 mL and 200 mL volume beverage each containing 18 g of
tomato extract syrup. Can be added. 18 g of tomato extract syrup corresponds to the amount of tomato
extract available with 6 raw tomatoes (total-500 g fresh weight).
The foregoing embodiments are presented for the purpose of illustrating the present invention and should
not be construed as imposing any limitation on the scope of the present invention. It will be readily
appreciated that numerous modifications and changes can be made to the specific embodiments of the
invention described above and illustrated in the examples without departing from the principles underlying
the invention. All such modifications are intended to be included in this application.
The present invention is illustrated by the above-described embodiments with reference to the
accompanying drawings, but is not limited thereto.
2 is an HPLC chromatogram showing the subfractions of the tomato extract (tAF) of the present invention,
as discussed in Example 2, into three subfractions AF1-AF3. FIG. 4 shows the effect of AF1 to AF3 on in
vitro clotting time parameters as discussed in Example 3. FIG. A sub-fraction of tAF (AF1-AF3) was
preincubated with plasma prior to initiation of clotting using PT, TCT or aPTT reagents. All inhibitor
solutions were used at a final concentration of 0.08 g / L and n = 3 for all measurements. FIG. 6 shows
the inhibition of ADP-induced expression of the platelet activation marker P-selectin after preincubation
with whole blood and tomato extract active fraction, tAF and its subfractions AF1-AF3, as discussed in
Example 4. FIG. All inhibitors were used at a final concentration of 0.05 g / L, with n = 5 for each
measurement. All inhibitors showed a significant difference from the control (P <0.001). FIG. 6 shows the
dose response relationship observed for tAF inhibition of P-selectin expression in activated platelets
(shown in FIG. 3) as discussed in Example 4. FIG. 5 shows that preincubation with the tomato extract of
the present invention reduces the levels of interleukin 6 and interleukin 8 produced by human umbilical
vein endothelial cells (HUVEC cells). Significant differences from the control (con-) are shown on the
graph and are discussed in Example 5.
Claims (20)
Hide Dependent
1. Use of tomato extract or an active fraction thereof for the manufacture of a medicament for
preventing or inhibiting the onset of venous thrombosis.
2. Use of a tomato extract or an active fraction thereof for the manufacture of a medicament for
preventing or inhibiting the onset of fibrin thrombus formation in a vein.
3. Tomato extract or an active fraction thereof for use in preventing or inhibiting the onset of
venous thrombosis.
4. A tomato extract or an active fraction thereof for use in preventing or inhibiting the initiation of
fibrin thrombus formation in a vein.
5. A composition comprising a tomato extract or an active fraction thereof for use in preventing
or inhibiting the onset of venous thrombosis.
6. A composition comprising a tomato extract or an active fraction thereof for use in preventing
or inhibiting the initiation of fibrin thrombus formation in a vein.
7. A method of preventing or inhibiting the onset of venous thrombosis in a mammal such as a
human, comprising administering to the mammal an effective amount of a tomato extract or an
active fraction thereof.
8. A method for preventing or inhibiting the onset of fibrin thrombus formation in a vein,
comprising administering to a mammal an effective amount of a tomato extract or an active
fraction thereof.
9. The use according to claim 1 or 2, the extract according to claim 3 or 4, the
composition according to claim 5 or 6, or the method according to claim 7 or 8, wherein
the tomato extract is an aqueous extract.
10. Use, extract, composition or method according to claim 9, wherein the tomato extract
is substantially free of lycopene.
11. 11. Use, extract, composition or method according to claim 9 or 10, wherein the
aqueous tomato extract is substantially free of water-insoluble particulate matter.
12. 12. Use, extract, composition or method according to any one of claims 9 to 11,
wherein the aqueous tomato extract is substantially free of particulate matter.
wherein the aqueous tomato extract is able to pass through a 0.2 [mu] m (0.2 [mu] m)
filter without loss of solids.
wherein the tomato extract has been dehydrated to yield a water-soluble dry extract.
wherein the tomato extract is prepared from whole tomato or cold break tomato paste.
wherein the tomato extract is substantially free of native saccharides.
17. The occurrence of venous thrombosis by the use belonging to any one or two (or any
combination) of the following potentially dangerous subpopulations (a) to (l): Use
according to any one of claims 1 to 16, which is prevention or inhibition of the onset of
venous thrombosis (or prevention or inhibition of initiation of fibrin thrombus formation
in a vein) in a patient at a higher risk than usual Extract, composition or method:
(a) patients over 50 years of age, eg, over 60 years, over 70 years old or over 80 years
of age;
(b) be stationary over a long-term period, for example more than 3, 4, 5, 6, 7, 8, 9, 10,
11 or 12 hours, or more than 1, 2, 3, 4 or 5 days patient;
(c) a patient who is medically obese;
(d) Patients who have undergone surgery recently (eg, in the past month or in the past
21, 14 or 7 days);
(d) a patient with a trauma such as a fracture;
(e) Patients taking oral contraceptives;
(f) Patients undergoing hormone replacement therapy;
(g) a pregnant patient;
(h) Patients who have recently given birth (partum);
(i) patients suffering from cancer and patients undergoing treatment for cancer;
(j) patients suffering from antiphospholipid syndrome;
(k) patients with genetic risk factors; and
(l) Patients with plasma risk factors.
18. 18. Use, extract, composition or method according to claim 17, wherein the patient has
two or more genetic risk factors that are any one or any combination of the following
(ki) to (kv):
(ki) a mutation in the gene encoding antithrombin;
(k-ii) a mutation in the gene encoding protein C;
(k-iii) a mutation in the gene encoding protein S;
(k-iv) Factor V Leiden mutation; and
(kv) Factor II G20210 A mutation.
19. 18. Use, extract, composition or according to claim 17, wherein the patient has two or
more plasma risk factors which are any one or any combination of the following (li) to (l-
vi): Method:
(li) hyperhomocysteinemia;
(l-ii) increased factor II concentration;
(l-iii) increased factor VIII concentration;
(l-iv) increased factor IX concentration;
(lv) increased factor XI concentration; and
(l-vi) Increase in fibrinogen levels.
20. Prevent or inhibit the onset of venous thrombosis (or prevent or inhibit fibrin thrombus
formation in veins) in patients who are members of a subpopulation of people with
recurrent venous thrombosis, such as recurrent deep vein thrombosis 20. Use, extract,
composition or method according to any one of claims 1-19.

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SOP SUBARASHI (Triple Peptide)*

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DETAILED DESCRIPTION OF THE INVENTION

The amount used varies depending on symptoms, age, etc.

MM4 was intraperitoneally injected into C3H / HeN mice.

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION This invention is, Enterococcus (En

[0006] Enterococcus faecalis NF

[0008] (2) Bacteriological and physiological properties {Properties determination} ───────────────

(Culture method) A microorganism belonging to the genus Enterococcus is inoculated into 10 ml of

The amount used varies depending on symptoms, age, etc.

Table 1 エ タ ノ ー ル Days of ethanol administration ───────────────── {728} Control group

Each dog was intravenously administered 10 mg / kg of cyclophosphamide (Endoxane, Shionogi

Table 2 ───────────────────────── Days ５ 5 11 14 ─────────────────────────

(2) 300 mg of dead bacterial cell powder obtained in Example 1

[Brief description of the drawings]

FIG. 1 is a graph showing a white blood cell count recovery effect.

──────────────────────────────────────────────────続 き Continuation of the

1. 1. A preventive agent for leukopenia, comprising a heat-killed cell of a microorganism belonging

DETAILED DESCRIPTION OF THE INVENTION

2. Description of the Related Art In 1989, hepatitis C virus (HC)

The amount used varies depending on symptoms, age, etc.

Embodiment 1 FIG. (Culture of Enterococcus) Enterococcus faecali

The unit is IU / dl for GOT, GPT and LDH.

IGF-1 level increasing agent

The present invention relates to an agent for increasing IGF-1 level .

1. Accelerating pulse wave aging index increasing agent characterized by containing a

TECHNICAL FIELD OF THE INVENTION

Problems to be solved by the invention

Means for solving the problem

Next, neutralization treatment is performed if necessary, followed by holding at a temperature of 70 ° C.

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION

[Problems to be solved by the invention]

[Means for Solving the Problems]

And the peptide SY of this invention is manufactured as follows.

Next, neutralization treatment is performed if necessary, followed by holding at a temperature of 70 ° C.

Peptide SY contains a high concentration of a dipeptide (valyl-tyrosine: hereinafter sometimes referred to

The physicochemical properties of peptide SY are as follows:

(L) Major amino acid composition ─────────────────────

The physicochemical properties of peptide SY-MD are as follows.

【The invention's effect】

1. Peptide SY having the following physicochemical properties:

Thrombosis occurs due to dysfunction or inappropriate activation of components of the hemostatic

The present invention also provides the following:

[Preparation and characteristics of tomato extract]

[Combination of pharmaceuticals and dietary supplements]

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Table 1 エタノール Days of ethanol administration ───────────────── {728} Control group

──────────────────────────────────────────────────続き Continuation of the