JPS5980609A - Preparation of active fraction for lowering cholesterol level, and antiarteriosclerotic agent containing said active fraction - Google Patents

Abstract

PURPOSE:To prepare an active fraction for lowering cholesterol level, useful as an orally administrable non-toxic antiarteriosclerotic agent and its raw material, by culturing bacterial strain of Streptococcus genus, extracting the bacterial cell with water, and separating a fraction having a specific molecular weight from the extract. CONSTITUTION:Bacterial strain belonging to Streptococcus genus (e.g. Streptococcus faecium, Streptococcus faecalis, Streptococcus bovis, etc.) is cultured e.g. aerobically in a liquid medium, and the cultured product is centrifuged to separate the bacterial cells. The cells are extracted with water, and dialyzed with a dialyzing membrane having a cut-off molecular weight of 3,500 (Cellotube; product of Nakarai Chemical Co.) to collect the fraction containing the components having molecular weight of >=3,500 and active to lower the cholesterol level. The fraction is an orally administrable non-toxic material which lowers the cholesterol level in blood, effectively. It is effective to arteriosclerosis, hyperlipemia, hyperlipoproteinemia, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel anti-arteriosclerotic agent and a method for producing the same. Today, several drugs, including clofibrate-related preparations, have been proposed as therapeutic and preventive drugs for arteriosclerotic disease, hyperlipidemia, etc., which are typical adult diseases, but the pharmacological effects and side effects are In these respects, it cannot be said that these drugs are necessarily fully satisfactory, and the desire for more effective drugs is increasing.

As a result of intensive research into new anti-arteriosclerotic agents, the present inventors found that
A specific extract fraction of various microorganisms belonging to the genus Streptococcus can extremely effectively lower blood sterol levels, and its origin is in the so-called intestine.
The present invention was achieved based on the finding that the extracted fraction of these bacterial cells, which are bacteria I11, is substantially non-toxic when administered orally and is suitable as a starting material for higher-level purification. .

Hereinafter, the types of microorganisms that can be used in the present invention, methods for producing active fractions, pharmacological effects, etc. will be described in detail.

Various microorganisms belonging to the genus Streptococcus can be used, among others Streptococcus faecium, Streptococcus fuecalis, Streptococcus bovis, Streptococcus φepium, Streptococcus duulans, Streptococcus ezalivarius, Streptococcus miteis, Streptococcus spp. Equinus etc. can be exemplified as suitable examples.

Further, specific examples of the most useful bacterial strains in the present invention are shown below along with their accession numbers.

Table 1 5treptococcus faecium ADV
1009 FEItM P-6624x faeca
lis ADV9(101w n-6625#-av
ium AD2003 # #-6626y 5
alivariusAT)VlooOI # tr-
6627s clarans AI) V300]
# #-6628tt m1tis AI) V6O
13rr p-6629t equinus ADV8
001 rr r・-6630 Mycological properties In terms of mycological properties, the microorganisms used in the present invention have the same properties as those shown in the known literature regarding bacteria of the same classification.

That is, the following documents are referred to regarding the mycological properties, culture conditions, etc. of the microorganism of the present invention.

]) BergeV's B/Annual of
Det, erminate 13+ct, erjo
Lagy I8 tlled,, 490-509 (
1974) 2) Int, J, 5yst, Bact
, -11114 (1966) 3) Microbiol
, Irrmunol, 25 (31, 257-269
(1981) 4) J, Cl1n, Pathol,
3; 53-57 (1980) 5) J, Gen
eral Microbiol,, 128 7J 3
-720 (1982) 6) Applied Mic
robiol,, 24 f611131-1139
(1972) Here, the main mycological properties of the aforementioned various bacterial strains are summarized as follows.

The following margin (l12,3.5-triphenyltetrazolium chloride) These microorganisms are cultured by conventional methods as described above, but for example, Rogosa H-body medium (
(Note) The cells are cultured aerobically and the resulting culture solution is centrifuged to collect the bacterial cells.

(Note) Distilled water 11 and tributicase 109 Fermented seasonal conyx 57 trif”
1. -s
32 to 2) IP04
3ffKH
2r a) 4
32 Triammonium citrate 22 Tween 80 11 Glucose 20f Cysteine salt rR salt o, 22*Salt solution
5 ml (pH 7, 121℃ 15
(Heat sterilization for 1 minute) *Preparation of active fraction in saline solution distilled water 100% An example of a typical method for producing both active fractions of the present invention is shown below for each step.

1. Bacterial body collection process The above-mentioned strains of each microorganism are placed in the above-mentioned Logoli liquid medium 51.
The culture solution was inoculated at 1 ~, and statically cultured aerobically at 37°C for 5 hours to create a culture solution with a viable cell count of 6 x 10''/-, and the resulting culture solution was subjected to continuous centrifugation at 12.00 Orpm. Collect the applied bacterial cells, wash them 2 to 3 times with physiological saline, and collect the collected bacterial cells).

2. Water extraction step a) The collected bacterial bodies were soaked in physiological saline (0, F!; 5% N
aC1 aqueous solution) A bacterial solution (2X 10”/d) obtained by suspending in 1.5 ynl was heated at 115°C for 10 minutes (
(autoclave) and perform both destruction of the bacterial cells and hot water extraction.

If not obtained, the extracted suspension was centrifuged (2,000G
20 minutes), then the target extract is given as the supernatant.

b) Ultrasonic destruction treatment (15Kc,
60 minutes), and centrifuged the extracted suspension (4).
20,000~25,000G.

30 minutes), the desired extract is obtained as the supernatant.

In addition, as an extraction solvent, not only the above-mentioned physiological saline but also a predetermined p
Various buffer solutions adjusted to H value may also be used as appropriate.

3. Active fraction separation step Each of the above extracts is dialyzed against distilled water for 3 days and nights using a permeable dialysis membrane with a molecular weight of 3,500 or less (trade name "Serochu"; manufactured by Gyudon Chemical Co., Ltd.), The desired active fraction is obtained.

In addition, considering other dialysis data, it is estimated that the active fraction of the present invention is distributed in a concave circle with a molecular weight of 3,500 to 50,000 in the aqueous extract.

Pharmacological Effect 1, Pharmacological Effect As shown in the experimental examples below, the anti-arteriosclerotic agent comprising the active fraction of the present invention can extremely effectively lower blood cholesterol levels.

Therefore, including arteriosclerosis, which is closely related to this indicator, hyperlipidemia, hyperlipoproteinemia, yellow plaque disease,
It can be said to be useful as a therapeutic or preventive drug for diseases such as cholelithiasis, hyperemia, and diabetes.

The agent of the present invention can also be administered by means such as oral administration or intravenous injection, and the dosage thereof is usually several to several 1 Or/Kq body weight, more preferably 1 to several 1 (!) to several 9/Kf body weight when administered orally. The dosage forms include suspensions in physiological saline, powders by freeze-drying, granules, tablets, capsules, etc.
Conventional dosage forms may be used in conjunction with appropriate carriers, thickening agents, diluents, etc., as appropriate.

2. Acute toxicity As shown in the experimental examples below, LDs of the agent of the present invention.

The value is more than 3.7 mg/mouse (intraperitoneal administration), and it is substantially boneless when administered orally.

Experimental Example 1 Using a bacterial solution (suspended in physiological saline) with a viable count of 70 viable bacteria (2 x 10"/-), hot water extraction was carried out from Streptococcus faecium ADV1009 according to the method for producing the active fraction (section 2.a above). ) to obtain the active fraction.

Here, the dry weight of bacterial cells per 10 m of bacterial liquid (2 x I O'' viable bacteria) was 2.02, and the active fraction obtained from this was 435 ml.

Next, the active fraction 2171rq (
Normally, rats (converted to dry type), equivalent amounts of each and 0.5 d of autoclaved dead bacterial cell solution were added to the rat (
Male, 6 weeks old, average weight 219F.

(5 animals in each group) were orally administered daily for 4 weeks.

Arterial blood was then collected from the aorta of these rats and centrifuged to obtain a serum sample, and the cholesterol level in the serum sample was measured using Coleskit (trade name; manufactured by Kanto Kagaku Co., Ltd., Zurkowslci method).

The results are summarized in Table 3.

In addition, in the table, the control is a group of rats to which no sample was administered, and each value is the reduction rate (%) when the control group is taken as 100%.The specific activity is when the number of dead bacteria is subtracted by 1. It shows the relative activity per unit weight of.

In addition, the composition of the diet (heavy charge %) is as shown in Table 4 below, and this was taken as ad libitum intake (hereinafter the same).
.

3rd surface Min. Decrease rate (%) Specific activity hot water extraction residue -1,3- Table 4 Casein 20 Soybean oil 1゜Wheat starch 61 Minerals 4 Vitamin mixture 2 Filter paper powder 3 Streptococcus in the same manner as in [Example] Heat-treated microbial cells and active fractions of various organisms of the genus Kotsucus are obtained, and these are usually injected into rats (
An amount equivalent to 1011 bacteria/day was orally administered to normal and germ-free mice (18 weeks old, average weight 202; 10 mice per group) for 4 weeks. were administered on consecutive days, and the respective reduction rates of serum cholesterol were measured in the same manner as above. The results are shown in Table 5. In the table,
°'Cholesterol load' or 'fructose load' refers to the case where 1% cholesterol was added to the above feed, or when wheat starch was completely replaced with fructose, and the values are for the non-administered group. These are the reduction rate of heat-treated bacterial cells used as a control and the specific activity of the active fraction (values in parentheses).

Table 5 S. S.

S.

(4,3) S.

S.

(4,2) S, Equiyz M) V6O1318,724,4(3
, 6) Fructose-loaded diet Example S with lycholesterol-loaded diet, using the Avirum AD2003 strain, in the same manner as in Experimental Example 1 (however, the destructive extraction step was performed using hot water at 80°C as described in the previous section 2.b). following sonication), the active fraction was obtained.

When this was administered to normal rats in the same manner as in Experimental Example 1, the specific activity was 6.2.

Example ICR mouse (male 6 weeks old, average weight 30.0±0.7
'l), and the active fraction obtained according to the method for producing the active fraction was added per mouse to 9 x J O', 9 x 10'',
9 X I Q' starting bacterial counts in 3 stages (10 bacteria in each group)
A suspension of 0.5 m of physiological saline was intraperitoneally administered to the mice, and the mice were observed for 14 days to see if they were alive or dead.

LD calculated according to Behrens-KMrber method
The so values (■/mouse) are shown in Table 6.

It should be noted that daily oral administration was substantially non-toxic in all cases.

Table 6 S, Faecium ADV10U9 4.7S
, Fuecalis ADV9001 3.8S,
Epiram AD2003 4.2S, Salivarius ADV10001 6. IS, day'yy
xADV3001 5.5S, Mitis A
DV7001 3.7S, Iku (Nuss A
DV8001 5.5 Formulation Example 1゜S, faecium AD obtained according to Experimental Example 1 above
Freeze-dried product of VI O09 active fraction 43 ~ (number of dead bacteria 1
．． 9501 ng of purified starch powder
It was mixed uniformly with the mixture and compressed into tablets for use in the investigation of medicinal purposes. This tablet is for dead bacteria count in adults weighing 50 to 9 [13X]
Corresponds to 0' pieces/Kf body weight.

2. 430 mW of the above freeze-dried product was mixed with 50 mW of purified starch powder.
Similarly, when mixed with 0■ and compressed into tablets, the dose was 3 x 10
Equivalent to 10 pieces/Ky.

In this manner, the agent of the present invention can be prepared into a desired dosage form having a predetermined activity by mixing the active fraction and a pharmaceutically acceptable carrier based on the standard dosage and the like.

Patent Applicant: Advance Development Research Institute Co., Ltd. Written to Sen. Taura J. (Voluntary) July 2, 1982] A
Active 1°1 fraction containing anti-arteriosclerotic agent 3.1! II Correct Person 1'1 Relationship 1. Address of 4 people in r calculation 〒I
O: l lj 5-7 Honbashi Koyocho, 1-1 Nakahi-ku, Kyoto (
]"l':1,03-fi+'i7 1551) Name
Advance Development Institute Co., Ltd. 4, 11 Positive subject specification 9F 1 Detailed description of the invention] Column 5, ? lll Correct Contents Specification No. 4 Correct] Line to line 9, Table 1 is corrected as written in F.

Table 1 - Bacteria - 4 - 1 person It thought - 5t
reptococcus faeciu* ^
DV1009 FEItlI f
il+-290// // faecali
s ADV9001 tt tt -
297〃//' avium AD2003
// tt -298// //
5Alivari++s ADVlooOI
' // // -299pi //
durans ADV3001
// // -300// It m1
nis ADV7001 //
# -3()]” '/cquinus
ADV8001 // It-3
02 Acceptance nL Seat Number Change Notification 1 Call (1158 "i 712-r'r-] 1, + permitted r Copying Officer Kazuhiro Wakasugi 1, Indication of Incident 1982 Patent 3γM No. 189253 2, Invention Name of the production method of the cholesterol-lowering active fraction and the active fraction containing anti-Jll, +1 vein sclerosing agent 3, and its relationship with the person who carried out the procedure Patent Applicant's Address 〒10
3 7'3 (1'EL) 5, Nihon Hyo Komagari-cho, Chuo-ku, Tokyo
03 667 1551) 4.111 Name of deposited Isoseki Ministry of Trade pI Industry-■, Institute of Industrial Science and Technology Hui Institute of Bioindustrial Technology 5,
+t+After consignment -T: 6N 1X'i '+:l' No. 6624 (
F'lzeT
P-6 (32G> Microtechnical Research Institute No. 6627 (FERM 1)-6627) 1sa-■', Research Institute No. 6628 (IFIζIBM ]]-6628) Microtechnology Research Institute No. 6
No. 629 (+, 'ERM I? 6629) Fine 1. Ken IW deposited SG G No. 30 (FEr? M P-6630) 6, tf + deposited machine name Ministry of International Trade and Industry 1 Agency of Industrial Science and Technology Microbial Technology Research Institute 7, new accession number Collected Research Institute No. 296 (FERM B■) -29 (3) Symptom -1゜Kojo Yorihaku No. 297 fFEI? M 13P-297) Hidoken Joyori No. 2! ] No. 8 (FFRM 13P-298) Microtechnical Research Institute No. 299 (FERM BP-299) Feature ■-Fukujo 1ff No. 300 (FEf ii (13I' -300) Zenkoku Ken Joyo No. 301 (Fl ' : RM old) - 301)
IN'-302) 8. Added; I! 1! Chapter 110 (t) Document clarifying M accession number fliF
7 letters (Ukeha ii + 1 million)

Claims (1)

[Scope of Claims] (1) A microorganism belonging to the genus Streptococcus is cultivated, bacterial cells are collected from the culture, the bacterial cells are extracted with water, and the resulting extract is extracted with molecules i: 3,500 or more. A method for producing a cholesterol-lowering active fraction, which comprises separating a fraction containing a component. (21t #! fili'f'Mi'J range number (l
1. K. e. Method 44) An anti-arteriosclerotic agent, characterized in that it contains the active fraction as an active ingredient. (3) The microorganism is Streptococcus faecium, Streptococcus fuecalis, Streptococcus bovis, Streptococcus Oevium, Streptococcus dulans, Streptococcus spp.
The anti-arteriosclerotic agent according to claim (2), further characterized in that the anti-arteriosclerotic agent is one or more microorganisms selected from the group consisting of Streptococcus salivarius, Streptococcus thurimitis, and Streptococcus equiinus.