JPH0534342B2 - - Google Patents

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 arteriosclerosis, hyperlipidemia, etc., which are a type of typical adult disease. 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 novel anti-arterial agents, the present inventors have found that specific extract fractions from various microorganisms belonging to the genus Streptococcus can extremely effectively lower blood cholesterol levels. We have discovered that the extracted fractions of these bacterial cells, which originate from so-called enteric bacteria, are virtually non-toxic when administered orally, and are suitable as starting materials for higher-level purification, leading to the present invention. This is what I did. Hereinafter, the types of microorganisms that can be used in the present invention, methods for producing active fractions, pharmacological effects, etc. will be explained in detail. Microorganisms Various microorganisms belonging to the genus Streptococcus can be used, among others Streptococcus faecium, Streptococcus fuecalis, Streptococcus bovis, Streptococcus spp.
Suitable examples include Streptococcus erectium, Streptococcus duurans, Streptococcus salivarius, Streptococcus miteis, Streptococcus equinus, and the like. Further, specific examples of the most useful bacterial strains in the present invention are shown below along with their accession numbers.

[Table] 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 for the same classification of bacteria. That is, the following documents are referred to regarding the mycological properties, culture conditions, etc. of the microorganism of the present invention. (1) Bergey's Manual of Determinative
Bacteriology, 8th ed., 490−509 (1974) (2) Int.J.Syst.Bact, 16 114 (1966) (3) Microbiol.Immunol. 25 (3), 257−269 (1981) (4) J .Clin. Pathol. 33 53−57 (1980) (5) J.General Microbiol., 128 713−720
(1982) (6) Applied Microbiol., 23 (6) 1131-1139 (1972) Here, the main mycological properties of the various strains mentioned above are summarized as follows.

【table】

[Table] As mentioned above, these microorganisms are cultured using conventional methods. The bacterial cells are then collected. (Note) Composition of Rogosa liquid medium: Trypticase in 1 part distilled water 10 g Yeast extract 5 g Tryptose 3 g K ₂ HPO ₄ 3 g KH ₂ PO ₄ 3 g Triammonium citrate 2 g Tween 80 1 g Glucose 20 g Cysteine hydrochloride 0.2 g *Salt solution 5ml (PH7, 121℃, heat sterilized for 15 minutes) *MgSO ₄・7H ₂ O 11.5g FeSO ₄・7H ₂ O 0.68g MnSO ₄・2H ₂ O 2.4g in 100ml of salt solution distilled water Method for producing active fraction This invention An example of a typical method for producing an active fraction is shown below for each step. 1. Bacterial body collection process The strains of each of the above-mentioned microorganisms were inoculated into the Rogosa liquid medium 5 mentioned above, and cultured for 5 hours aerobically at 37°C to obtain a culture solution with a viable bacterial count of 6 x 10 ⁸ /ml. Making,
The obtained culture solution is subjected to continuous centrifugation at 12,000 rpm to collect bacterial cells, and the cells are washed 2 to 3 times with physiological saline to obtain collected bacterial cells. 2 Water extraction step (a) The collected bacterial bodies were soaked in physiological saline (0.85% NaCl).
Bacterial solution (2×
10 ¹¹ /ml) at 115°C for 10 minutes (autoclave) to destroy the bacterial cells and perform hot water extraction. When the obtained extraction suspension is centrifuged (2000G x 20 minutes), the target extract is obtained as the supernatant. (b) The bacterial solution shown in (a) above is subjected to ultrasonic destruction treatment (15KC, 60 minutes), and the resulting extracted suspension is centrifuged (20000-25000G, 30 minutes), and the supernatant is used for the desired extraction. A liquid is obtained. As the extraction solvent, not only the above-mentioned physiological saline but also various buffer solutions adjusted to a predetermined pH value may be used as appropriate. 3 Active fraction separation step Each of the above extracts is passed through a permeable dialysis membrane with a molecular weight of 3500 or less (trade name "Cerotube"; manufactured by Hanui Chemical Co., Ltd.)
The target active fraction is obtained by dialysis against distilled water for 3 days and nights. Furthermore, considering other dialysis data, the active fraction of the present invention has a molecular weight of 3500 to 3500 in the water extract.
It is estimated that they are distributed within a radius of 50,000. Pharmacological effect 1 Pharmacological effect As shown in the experimental examples below, the anti-arteriosclerotic agent comprising the active fraction of the present invention extremely effectively lowers blood cholesterol levels, and therefore has a close relationship with this index. including arterial effect syndrome, hyperlipidemia, hyperlipoproteinemia,
It can be said to be useful as a therapeutic or preventive drug for diseases such as xanthomatosis, cholelithiasis, hypertension, and diabetes. The agent of the present invention can also be administered orally, intravenously, etc., and the dosage is usually several mg to several 10 g/Kg body weight,
More preferably, the dose is several 10 mg to several g/Kg body weight when administered orally, and the dosage form is usually a suspension in physiological saline, powder by freeze-drying, granules, tablets, capsules, etc. The following dosage forms can be selected and used as appropriate, together with suitable carriers, fillers, diluents, etc. 2. Acute toxicity As shown in the experimental examples below, the LD ₅₀ value of the agent of the present invention is 3.7 mg/mouse (intraperitoneal administration) or more,
It is virtually non-toxic when administered orally. Experimental Example 1 Using a bacterial solution (suspended in physiological saline) with a viable bacterial count of 2 x ¹⁰¹¹ cells/ml, hot water extraction was carried out from Streptococcus faecium ADV1009 according to the method for producing the active fraction (see 2 above). An active fraction was obtained according to section (a)). Here, 10ml of bacterial solution (number of viable bacteria 2 x 10 ^{, 12} )
The dry weight of each bacterial cell was 2.0 g, and the active fraction obtained from this was 435 mg. Next, 217 mg (dry weight equivalent) of the active fraction thus obtained, an equivalent amount of each fraction, and 0.5 ml of the autoclaved dead bacterial cell solution were added to regular rats (male, 6 weeks old, The drug was administered orally every day for 4 weeks to animals (average body weight 219 g, 5 animals in each group). Arterial blood was then collected from the inferior aorta of these rats and centrifuged to obtain a serum sample, and the cholesterol level in the serum sample was measured using Cholescitt (trade name; manufactured by Kanto Kagaku Co., Ltd., Zurkowski 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.
Each value is the reduction rate (%) when the control group is taken as 100%.
Moreover, the specific activity indicates the relative activity per unit weight when the weight of dead bacteria is taken as 1. Also, diet or feed composition (wt%)
As shown in Table 4 below, this was taken as ad libitum intake (the same applies hereinafter).

【table】

[Table] Experimental Example 2 Heat-treated cells and active fractions of various microorganisms of the genus Streptococcus were obtained in the same manner as in Experimental Example 1, and used in regular rats (18 weeks old male, average weight 240 g; 5 animals in each group). , normal and germ-free mice (male 18 weeks old, average weight 20 g; 10 mice in each group) were infected with 10 to ¹¹ bacteria for 4 weeks.
An amount equivalent to 500 mg/day was administered orally on consecutive days, and the respective reduction rates of serum cholesterol were measured in the same manner as described above. The results are shown in Table 5. In addition, in the table, "cholesterol load" or "fructose load" indicates the case where the above feed is further supplemented with 1% cholesterol or when wheat starch is completely replaced with fructose, and the numerical values are These are the reduction rate of heat-treated bacterial cells and the specific activity of the active fraction (values in parentheses) with respect to the non-administered group.

【table】

[Table] Experimental Example 3 Using the S. evium strain AD2003, the same procedure as in Experimental Example 1 was carried out (however, the destructive extraction step was carried out according to the ultrasonic treatment in the previous section 2(b) using hot water at 80°C), and the activity was determined. A 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. Experimental Example 4 ICR mouse (male 6 weeks old, average weight 30.0±0.7
g), and the active fraction obtained according to the above method for producing active fractions was divided into 9 x 10 ⁹ , 9 x 10 ⁸ , 9 per mouse.
A 0.5 ml suspension of the same in physiological saline was administered intraperitoneally in an amount equivalent to 10 × ⁷ starting bacteria in three stages (10 animals in each group).
The survival of the mice was observed for 14 days. Table 6 shows the LD ₅₀ values (mg/mouse) calculated according to the Behrens-Ka¨rber method. It should be noted that daily oral administration was substantially non-toxic in all cases. Table 6 S. faecium ADV1099 4.7 S. fuecalis ADV9001 3.8 S. evium AD2003 4.2 S. salivarius ADV10001 6.1 S. duulans ADV3001 5.5 S. miteis ADV7001 3.7 S. equinus ADV8001 5.5 Formulation Example 1 Obtained according to Experimental Example 1 above Rare S. faecium
43 mg of lyophilized active fraction of ADV1009 (equivalent to 1.5 x ¹⁰ dead cells) and 950 mg of purified starch powder
The mixture was uniformly mixed with the following ingredients and compressed into tablets for oral administration. This tablet corresponds to a dose of 3×10 ⁹ dead bacteria/Kg body weight for an adult with a body weight of 50 kg. 2 Add 430mg of the above freeze-dried product to 500mg of refined starch powder.
Similarly, when mixed with and compressed into tablets, the dose was 3 x ¹⁰
Equivalent to pcs/Kg. 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.

Claims (1)

[Claims] 1 Among the microorganisms belonging to the Streptococcus genus, Streptococcus faecium, Streptococcus fuecalis, Streptococcus evium, Streptococcus salivarius,
Streptococcus duulans, Streptococcus miteis, and Streptococcus equinus are cultured, bacterial cells are collected from the culture, the bacterial cells are extracted with water, and the resulting extract is extracted with components having a molecular weight of 3500 or more. A method for producing a cholesterol-lowering active fraction, which comprises separating a containing fraction.