JPH0343279B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel physiologically active substance (hereinafter referred to as NSH), a method for producing the substance, and a pharmaceutical containing the substance as an active ingredient. The present inventor inoculated with vaccinia virus,
While researching substances extracted from various animal tissues, cultured cells, or cultured tissues that had been infected with pox, we succeeded in obtaining a new physiologically active substance, NSH. NSH is affected by various types of stress, such as biological stress such as viruses, bacteria, protozoa, and Rickettsia, oxygen deprivation, chemical stress such as drugs (ACTH, cortisone, etc.), physical stress such as cold, noise, radiation, fear, It acts on the body's homeostasis maintenance mechanism, which regulates and repairs distortions in the nervous system, endocrine system, immune system, etc. that occur when biological functions are damaged due to mental stress such as anxiety and mania, and the body's natural healing power. It has the effect of increasing body functions and normalizing biological functions. As described below, NSH is not only useful as an anti-stress, anti-ulcer, analgesic, etc. drug, but also widely applicable to various diseases such as autonomic nervous disorder associated with various stresses, and can be used in combination with other drugs. By doing so, side effects of the drug can be reduced and synergistic effects can be expected. The physiologically active substance NSH of the present invention can be produced by the following steps. In the present invention, varicella tissues include virus-infected cultured tissues, cultured cells, virus-infected inflamed tissues of various animals, chorioallantoic membranes of embryonated chicken eggs, etc. obtained by various vaccinia virus inoculation methods or culture methods. (a) Grind the smallpox tissue collected aseptically, and
After adding ~5 times the amount of phenol water and making it emulsified,
Filter or centrifuge. (b) The liquid is adjusted to a pH near its isoelectric point and heated to remove protein, and then filtered using a Seitz filter plate. Furthermore, the filtrate is made slightly alkaline, boiled, and then filtered. (c) After making it acidic with mineral acids such as hydrochloric acid, sulfuric acid, or hydrobromic acid, it is adsorbed on activated carbon. At this time, preferably 5 to 20% activated carbon is used. (d) Add an alkaline aqueous solution to the adsorbent, adjust the pH to 10 to 12, elute under heating, and evaporate or freeze-dry the eluate under reduced pressure to obtain the desired product. Furthermore, if desired, the substance of the present invention obtained by the above method can be purified by fractionation based on molecular weight using means such as ultrafiltration, gel filtration, and dialysis. The following is an example of the method for extracting NSH of the present invention. Example 1 After inoculating the skin of a healthy adult rabbit with vaccinia virus to cause pox, the pox-affected skin was exfoliated aseptically, cut into small pieces, phenol water was added, and the skin was polished with a homogenizer. Crush into a milky state. This was then centrifugally filtered, and the resulting filtrate was diluted to pH4.8 with hydrochloric acid.
-5.5, heated to 100℃ with flowing steam and filtered.
After further filtering the filtrate using a Seitz filter plate,
Adjust the pH to 9 with sodium hydroxide, further heat to 100°C, and then filter. The filtrate was adjusted to pH 4.1 with hydrochloric acid, 7.5% activated carbon was added, stirred for 4 hours, and then filtered. Add water to this activated carbon and add sodium hydroxide to pH 10.9.
The mixture was stirred at 60°C for 1.5 hours, and then filtered. The filtrate was adjusted to pH 6.6 with hydrochloric acid and dried under reduced pressure. The yield from 1 kg of pox skin was 4-6 g. Example 2 The extract obtained by removing proteins in the same manner as in Example 1 was adjusted to pH 3.8 with hydrochloric acid, added with 15% activated carbon, stirred for 5 hours, and then filtered. This activated carbon has a pH of
Add 11.5 aqueous sodium hydroxide solution and heat at 45°C.
After stirring for an hour, filter. The filtrate is neutralized and subjected to ultrafiltration to obtain a fraction with a molecular weight of 1000 or less, which is dried under reduced pressure. The yield from 1 kg of pox skin was 4-5.5 g. Physiologically active substance of the present invention obtained by the above method
NSH has the following physicochemical properties. Properties: Pale yellowish brown amorphous hygroscopic powder Solubility: Soluble in water, methanol, acetone, insoluble in benzene, ether PH: 7.0-8.0 Molecular weight: 1000 or less Ultraviolet absorption: λmax 265-275nm Color reaction: Amino acids (Ninhydrin reaction) ...Positive sugar (Anthrone reaction) ...Positive phosphorus (phosphorus-molybdic acid reaction) ...Positive protein (trichloroacetic acid precipitation) ...Negative phenol (ferric chloride color reaction) ...Negative structure Composition: Total nitrogen...1.7-7.0% Amino nitrogen...0.5-2.0% Ultraviolet absorption substance...0.8-3.5% Sugar...0.1-0.6% Next, the pharmacological action of the physiologically active substance of the present invention, NSH, will be described. . Acute Toxicity Test Acute toxicity tests were conducted on the substance of the present invention using various routes of administration using SD-JCL rats of both sexes, ddY mice of both sexes, and Japanese White rabbits of both sexes in groups of 10 each. The results are shown in Table 1.

[Table] Pharmacological test SART stressed animals used in the following animal experiments were prepared using the method of Kita et al. [Japanese Pharmacological Journal, 71 , 195-210
(1970)]. That is, mice (or rats) were kept at 24°C and 4°C (or -3°C) alternately every hour between 10:00 am and 5:00 pm, and at 4°C (or -3°C) between 5:00 pm and 10:00 am the next day. The animals were reared for 4 to 5 days at a temperature of 3°C. SART-stressed animals raised using this method exhibit severe stress, including body weight loss, increased heart rate, prolonged QRS duration, abnormalities in the circulatory system such as decreased blood pressure, decreased electrodermal resistance values, and abnormalities in the excised small intestine. It can be seen as an animal model that exhibits a state similar to autonomic nervous system imbalance in humans, which is caused by changes in the environment, such as decreased Ach reactivity and decreased pain threshold. (1) Anti-stress effect (1.1) Weight loss suppressing effect Groups of 15 mice were raised under SART stress conditions, and changes in body weight were compared between the group administered with 20 mg/Kg/day of NSH intraperitoneally and the group not administered. At the same time, changes in body weight of mice raised under normal environmental conditions were also measured. The results are shown in Table 2.

[Table] (1.2) Effects on increase in respiration rate, heart rate, shortening of PQ time, and prolongation of QRS time A group of 10 mice was subjected to SART stress for 5 days, and respiratory rate, heart rate, PQ time, and QRS time were We investigated the effect of NSH on. 20mg/NSH
Kg/day when administered intraperitoneally, as shown in Table 3,
respiratory rate induced by SART stress,
It significantly suppressed the increase in heart rate, shortening of PQ time, and prolongation of QRS time.

[Table] (1.3) Normalizing effect on acetylcholine (Ach) sensitivity abnormalities in isolated intestinal tract SART stress-loaded mice restrained and water-immersed stress-loaded mice (after fasting for 18 hours, restrained with a wire mesh and immersed up to the xiphoid process in a water bath at 15℃ for 3 hours) The effect of NSH on the change in Ach (10 ^-7 g/ml) sensitivity of the excised intestines (left for an extended period of time) was investigated. Administration of NSH is
For the SART stress group, 100 mg/Kg was intraperitoneally administered once a day during the stress loading period, and for the restrained water immersion stress group, 100 mg/Kg was intraperitoneally administered twice, once before the start of fasting and before the start of stress. As shown in Table 4, NSH normalized both the decreased and increased Ach sensitivity due to the stress load.

[Table] (2) Anti-ulcer effect (2.1) Restraint water immersion stress ulcer A group of 10 Wistar male rats were fasted for 24 hours and then subjected to restraint water immersion stress (25°C, 20 hours).
[Adami, E. et al., Arch.Int.
Pharmacodyn. 147 , 113 (1964)] using a partially modified method, and the ulcer inhibition rate relative to the control (physiological saline) was calculated. NSH once before stress load,
Or administered continuously for 5 days. The results are shown in Table 5.

[Table] (2.2) Histamine ulcer A group of 10 to 20 male albino guinea pigs was treated with 24
Time fasted, 0.1% histamine diphosphate
0.25 ml/kg was intramuscularly administered 8 times at 30 minute intervals, and 30 minutes after the final administration, the duodenum was removed and the area of the ulcer was measured, as well as the amount of gastric juice, pH, acidity and total acidity. NSH was administered continuously for 5 days before histamine diphosphate treatment. The results are shown in Tables 6 and 7.

【table】

【table】

[Table] (3) Analgesic effect The SART stress mice used in the following analgesic test were subjected to stress for 4 days. (3.1) Acetic acid method 0.1ml/10g of 0.7% acetic acid-added saline to a mouse
The drug was administered intraperitoneally, and the number of writhing events occurring for 15 minutes from 15 minutes after administration was measured. The effectiveness is determined by NSH
An effect was defined as the number of writhing mice subcutaneously administered was reduced to 50% or less of the control group, and the results are expressed as a percentage of the mice. The results are shown in Table 8.

[Table] (3.2) Tail pressure method Stimulation was applied to the ridge of the mouse using a Randall-Selitt type pressure stimulation measuring device, and the pressure was increased until the mouse showed an escape response. 30 days after NSH i.p.
The average value of the pressurized weight after 60, 90, and 120 minutes was divided by the value before administration, and this value was expressed as an analgesic coefficient. The results are shown in Table 9.

[Table] As is clear from the results of the above pharmacological tests, the NSH of the present invention has extremely low toxicity and has a wide range of pharmacological effects, particularly regulating and repairing abnormalities in biological functions caused by various stresses, and improving homeostasis of the body. It is a biological function regulating substance useful for maintenance. Therefore, NSH is not only useful as a drug for anti-ulcer and analgesic purposes, but can also be widely applied as an anti-stress agent, an autonomic nerve modulating agent, and a repairing agent in the event of biological damage. Examples of diseases to which NSH applies are as follows. Stress-induced diseases such as gastric/duodenal ulcer, arteriosclerosis, myocardial infarction, hypertension, diabetes, Meniere's disease, pituitary insufficiency, adrenal cortex insufficiency, various mental and physical abnormalities, neurosis, headache, dizziness, fatigue , autonomic nervous system disorders such as insomnia, anorexia, fecal discharge, diarrhea, and indeterminate complaints; painful diseases such as neuralgia, low back pain, and cervicobrachial syndrome; and other allergies such as bronchial asthma, allergic rhinitis, urticaria, and eczema. Sexual diseases, various immunodeficiency diseases, malignant tumors, liver disorders, hematopoietic dysfunction, etc. NSH can be used alone or in combination with other drugs as a prophylactic agent, therapeutic agent, or therapeutic adjuvant for these diseases. moreover,
When NSH is used in combination with other drugs, it can be expected to reduce the side effects, reduce the dose, and increase the effects of other drugs. Drugs containing the substance of the present invention as an active ingredient are:
For example, tablets, capsules, granules, injections, liquids,
It can be in the form of suppositories, ointments, and aerosols. For producing tablets, capsules, granules, excipients such as starch, lactose, mannitol, etc.
Microcrystalline cellulose, methylcellulose, other cellulose derivatives, gum arabic, gelatin, binders such as polyethylene, polyvinyl alcohol, polyvinylpyrrolidone, wetting agents such as glycerol, disintegrants such as carboxymethylcellulose, microcrystalline cellulose, polyethylene glycol, cetyl alcohol , surfactants such as glycerin and fatty acid esters, lubricants such as talc, magnesium stearate, calcium, and solid polyethylene glycol, other coating agents, coloring agents, flavoring agents, and the like can be used. To produce injections, distilled water for injection, physiological saline, 5-20% glucose injection, vegetable oil for injection, solvents such as ethanol, propylene glycol, polyethylene glycols, sodium pyrosulfite, sodium hydrogen sulfide, l - Stabilizers such as ascorbic acid, thioglycolic acid, citrate, acetate, and phosphate; soothing agents such as procaine hydrochloride and glucose; other tonicity agents, solubilizing agents, preservatives, and suspending agents. , an emulsifier, etc. can be used.
The composition for injection can be made into a sterile solution, suspension, emulsion, or in the form of a dosage form that is dissolved in a solvent before use. Ointments contain fatty oils, paraffin, lanolin,
Suppositories can be formulated using petrolatum, glycols, glycerin, higher alcohols, and other suitable bases, and suppositories can be formulated using bases such as cacao butter, macrogol, glycerogelatin, lanolin fat, etc., as appropriate. Surfactants and preservatives may be added. Furthermore, it can be made into an aerosol in the form of a liquid or fine powder together with a suitable propellant, and if desired, auxiliary agents such as wetting agents and dispersants may be added. The daily dose of the substance of the present invention is 1 μg/Kg to 10
Although mg/Kg is common, it can be changed as appropriate depending on the severity of the disease and symptoms. Examples of formulations of drugs containing the substance of the present invention as an active ingredient are shown below, but the invention is not limited thereto. Formulation example 1 (tablets with a total weight of 150 mg) NSH 10 mg Lactose 80〃 Crystalline cellulose 25〃 Carboxymethylcellulose 30〃 Magnesium stearate 5〃 Total 150 mg Formulation example 2 (Capsules with a filling weight of 200 mg) NSH 20 mg Lactose 125〃 Potato starch 40 〃 Magnesium stearate 5〃 Talc 10〃Total 200mg Prescription example 3 (Capacity: 1ml ampoule for injection) NSH 1mg Dose of distilled water for injection 1ml Prescription example 4 (Total amount: 2g suppository) NSH 20mg Cocoa butter 1980〃Total 2g

Claims (1)

[Claims] 1. A novel physiologically active substance NSH extracted from pox tissue and having the following physicochemical properties. Properties: Pale yellowish brown amorphous hygroscopic powder Solubility: Soluble in water, methanol, acetone, insoluble in benzene, ether PH: 7.0-8.0 Molecular weight: 1000 or less Ultraviolet absorption: λmax 265-275nm Color reaction: Amino acids (Ninhydrin reaction) ...Positive sugar (Anthrone reaction) ...Positive phosphorus (phosphorus-molybdic acid reaction) ...Positive protein (trichloroacetic acid precipitation) ...Negative phenol (ferric chloride color reaction) ...Negative structure Composition: Total nitrogen...1.7-7.0% Amino nitrogen...0.5-2.0% Ultraviolet absorbing substance...0.8-3.5% Sugar...0.1-0.6% 2. Grind the pox tissue and add phenol water to it. In addition, it is extracted and heated after adjusting the pH to near the isoelectric point.
Filter to remove protein, and then dry under acidic conditions for 5 to 20 minutes.
% activated carbon, then adsorbed with an alkaline aqueous solution.
A method for producing a novel physiologically active substance NSH having the following physicochemical properties, which comprises elution under heating at a pH of 10 to 12, followed by molecular weight fractionation. Properties: Pale yellowish brown amorphous hygroscopic powder Solubility: Soluble in water, methanol, acetone, insoluble in benzene, ether PH: 7.0-8.0 Molecular weight: 1000 or less Ultraviolet absorption: λmax 265-275nm Color reaction: Amino acids (Ninhydrin reaction) ...Positive sugar (Anthrone reaction) ...Positive phosphorus (phosphorus-molybdic acid reaction) ...Positive protein (trichloroacetic acid precipitation) ...Negative phenol (ferric chloride color reaction) ...Negative structure Composition: Total nitrogen...1.7-7.0% Amino nitrogen...0.5-2.0% Ultraviolet absorption substance...0.8-3.5% Sugar...0.1-0.6% 3 Extracted from pox tissue and having the following physicochemical properties An anti-stress agent containing NSH as an active ingredient. Properties: Pale yellowish brown amorphous hygroscopic powder Solubility: Soluble in water, methanol, acetone, insoluble in benzene, ether PH: 7.0-8.0 Molecular weight: 1000 or less Ultraviolet absorption: λmax 265-275nm Color reaction: Amino acids (Ninhydrin reaction) ...Positive sugar (Anthrone reaction) ...Positive phosphorus (phosphorus-molybdic acid reaction) ...Positive protein (trichloroacetic acid precipitation) ...Negative phenol (ferric chloride color reaction) ...Negative structure Composition: Total nitrogen...1.7-7.0% Amino nitrogen...0.5-2.0% Ultraviolet absorption substance...0.8-3.5% Sugar...0.1-0.6% 4 Extracted from pox tissue and having the following physicochemical properties An anti-ulcer agent containing NSH as an active ingredient. Properties: Pale yellowish brown amorphous hygroscopic powder Solubility: Soluble in water, methanol, acetone, insoluble in benzene, ether PH: 7.0-8.0 Molecular weight: 1000 or less Ultraviolet absorption: λmax 265-275nm Color reaction: Amino acids (Ninhydrin reaction) ...Positive sugar (Anthrone reaction) ...Positive phosphorus (phosphorus-molybdic acid reaction) ...Positive protein (trichloroacetic acid precipitation) ...Negative phenol (ferric chloride color reaction) ...Negative structure Composition: Total nitrogen...1.7-7.0% Amino nitrogen...0.5-2.0% Ultraviolet absorption substance...0.8-3.5% Sugar...0.1-0.6% 5 Extracted from pox tissue and having the following physicochemical properties Analgesic containing NSH as an active ingredient. Properties: Pale yellowish brown amorphous hygroscopic powder Solubility: Soluble in water, methanol, acetone, insoluble in benzene, ether PH: 7.0-8.0 Molecular weight: 1000 or less Ultraviolet absorption: λmax 265-275nm Color reaction: Amino acids (Ninhydrin reaction) ...Positive sugar (Anthrone reaction) ...Positive phosphorus (phosphorus-molybdic acid reaction) ...Positive protein (trichloroacetic acid precipitation) ...Negative phenol (ferric chloride color reaction) ...Negative structure Composition: Total nitrogen...1.7-7.0% Amino nitrogen...0.5-2.0% Ultraviolet absorption substance...0.8-3.5% Sugar...0.1-0.6%