JPH0469611B2 - - Google Patents

Description

[Detailed description of the invention]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biological defense enhancer containing a garlic extract fraction as an active ingredient. Garlic is a perennial plant cultivated in China, Korea, Japan, and other countries, and is generally known as a tonic.
It is also used as an expectorant, intestinal regulator, bactericidal and anthelmintic. By the way, as humans grow older, their resistance to various diseases generally decreases, making them more susceptible to various diseases. This is thought to be due to a decline in the body's internal organs and various metabolic functions, as well as a decline in the function of phagocytes, which have the ability to dispose of foreign substances and wastes, resulting in a decline in the body's defense ability. As a result of repeated research on garlic components, the present inventors discovered that garlic extract fractions have an effect of enhancing biological defense ability, and thus completed the present invention. SUMMARY OF THE INVENTION The present invention aims to provide a novel biological defense ability-enhancing agent, and is based on the unexpected discovery for those skilled in the art that garlic extract fraction has this effect. Therefore, the biological defense ability enhancer according to the present invention is characterized in that the fraction obtained by the following steps is used as an active ingredient. (b) Extracting garlic with water or a hydrous lower alcohol having 1 to 3 carbon atoms. (b) The obtained extract is subjected to at least one method selected from dialysis, gel filtration, and ultrafiltration to separate and remove low-molecular compounds. (c) Obtaining an adsorbed fraction by subjecting the product treated by the above means to anion exchange chromatography. Effects It was unexpected that this particular extracted fraction of garlic had such a specific physiological activity, and it goes without saying that the present invention provides a biodefense enhancer that is meaningful in countering various diseases. This will make a significant contribution. Detailed Description of the Invention Garlic Garlic as used in the present invention means Liliaceae (Liliaceae).
-ae), Allium sativum L. belonging to the genus Allium
For example, Allium sativum
This is the case for L.forma pekinese Makino). The part that becomes the material for obtaining the desired fraction is particularly preferably the bulb part (which usually contains 5 to 20 blastomere-shaped small bulbs that have been split internally), which can be dried or It can be used for extraction as is. Furthermore, it is also possible to subject these plants to tissue culture by a conventional method and use the resulting culture. Obtaining the desired fraction Garlic extraction and obtaining the desired fraction can basically be performed by any means commonly used for extracting herbal medicines. First, the target for extraction may be any part of the garlic plant, but the bulb part is most preferred. The acquisition method according to the present invention consists of the following unit steps. (b) Extraction Extraction is performed with water or a water-containing lower alcohol. The lower alcohol to be used as the extraction agent is one having 1 to 3 carbon atoms (usually a monohydric alcohol), and ethanol is particularly preferred. Extraction can be carried out either under heating or at room temperature, but extraction time at room temperature is usually long, requiring several hours to several days. Preferably, the extraction is carried out at 40°C or below for 10 to 20 hours. Furthermore, in order to increase the extraction efficiency, it goes without saying that the target plant is preferably crushed. (b) Separation and removal of low-molecular substances After the extraction alcohol is distilled off, the extract is subjected to any polymer purification means such as dialysis, gel filtration, ultrafiltration, or reverse osmosis. It is possible to separate and remove impurities such as low-molecular substances and ions mixed in the water. Preferably, it is subjected to at least one method selected from dialysis, gel filtration and ultrafiltration. Note that the low molecular weight compound as used in the present invention refers to organic and inorganic substances having a molecular weight of 10,000 or less. (1) Dialysis is a method of separating macromolecular components that utilizes the molecular sieving effect of semipermeable membranes, and animal biomembranes, cellophane membranes, collodion membranes, gelatin membranes, etc. are commonly used as semipermeable membranes. ing. Dialysis in the present invention can also be performed using any known means. (1) Gel filtration Gel filtration is a method of separating water-soluble polymer substances with different molecular weights using a polymer gelling agent having a three-dimensional network structure with approximately uniform pore size. Dextran gels (e.g. sephadex) are suitable as gelling agents.
(Pharmacia Fine Chemicals)), polyacrylamide gels (e.g. Bio-Gel (BiO
-Rad Laboratories) or agarose gel can be used. (3) Ultrafiltration Ultrafiltration is an operation in which the polymer component is separated from the dispersion medium by applying a pressure difference by pressurization or suction to both sides of a membrane with pores of a certain size. As the ultrafiltration membrane, a collodion membrane, gelatin membrane, cellophane membrane, etc. can be used as in the case of dialysis. (c) Purification by anion exchange chromatography The above-mentioned treated solution can be concentrated under reduced pressure at low temperature or freeze-dried, and then subjected to anion exchange chromatography to obtain the target fraction as an adsorbed fraction. can. Anion exchange chromatography as used in the present invention refers to resins that have the property of taking in anions in the liquid phase and releasing their own anions.
A method of separating cationic and anionic fractions in a solution using any anion exchanger such as polystyrene divinylbenzene copolymer, polyamine, hydrated zirconium oxide, hydrated titanium oxide or cellulose. It can be carried out using ordinary known means. In addition, the adsorbed fraction obtained by the above chromatography is treated with an inorganic strong acid-strong base salt such as NaCl, KCl, Na ₂ SO ₄ , K ₂ SO ₄ etc. to change the ionic strength. This allows for easy elution and recovery. The above-mentioned eluate can be concentrated under reduced pressure at a low temperature or subjected to lyophilization to obtain the target extracted fraction. Note that pharmacological experiments similar to those in the experimental examples described later were also performed on the above-mentioned pass-through fraction, but no activity was observed. Enhancement of biological defense ability When a foreign substance (bacteria, virus, etc.) invades the body, biological defense reactions such as phagocytosis of macrophages and antibody production by differentiation and proliferation of lymphocytes appear to eliminate the foreign substance. A clever mechanism emerges. However, when immunity or protective factors decrease, aggressive factors (foreign substances) become dominant, making it easier to develop various diseases such as opportunistic infections. In the present invention, the biological defense function enhancing effect refers to the function of activating the biological defense reactions shown above to eliminate foreign substances from the living body, and includes secondary antiviral, antitumor, antiparasitic, and antibacterial effects. effects and the elimination of other foreign antigens. Biodefense enhancer The biodefense enhancer of the present invention is composed of the garlic extract fraction itself or mixed with appropriate excipients, binders, and diluents for formulation, and is available in the form of powder, granules, tablets, and capsules. It can be administered orally or parenterally in the form of tablets, syrups, injections, etc. Further, other drugs may be mixed as necessary. Although the dosage may be adjusted depending on age, body weight, and symptoms, the dose for oral administration is usually about 100 mg to 10 g per day for adults, and more preferably about 500 mg to 5 g per day. A preferred embodiment according to the invention consists of a garlic extract fraction and a pharmaceutical auxiliary ingredient. Another preferred embodiment of the present invention is a unit dosage form in which the above-mentioned daily dose is divided into one or several doses. In addition, the toxicity of the garlic extract fraction in the present invention is, for example, the LD ₅₀ value of the dilute ethanol extract of garlic (extract content 14.5%, alcohol number 1.18).
30ml/Kg or more for all oral, intraperitoneal and subcutaneous routes of administration (The Journal
of Toxicological Sciences., 9 , 57 (1984)) and that garlic is regularly used as a food;
Generally, it has low toxicity. Experimental example: Acquisition of garlic extract fraction: The bulbs of giant garlic crushed to an appropriate size
Add 400ml of 20% ethanol to 200g and heat at 4℃.
Extraction was carried out for 15 hours. This garlic extract was concentrated under reduced pressure at 40° C. or lower to about 80 ml, then transferred to a MWcut off 1000 dialysis tube (manufactured by SPECTRUM), and dialyzed against 10 liters of water at 4° C. for 15 hours. The dialyzed solution was further dialyzed 8 times against 10 liters of water and then freeze-dried (yield: 5.47 g). 4g of the obtained lyophilized product was equilibrated with 0.05M Tris-HCl buffer (PH8) to prepare DEAE-
TOYOPEARL 650M (manufactured by Toyo Soda Co., Ltd.) (2.5φ/20
cm) and the flow-through fraction was separated and removed using the buffer. The adsorbed fraction was eluted using 500 ml of 0.05 M Tris-HCl buffer supplemented with 2 M NaCl, and this was lyophilized to obtain the desired fraction (hereinafter referred to as fraction A). 270 mg). The fraction was a white powder, odorless and tasteless, easily soluble in water, and almost insoluble in organic solvents. Biodefense enhancement effect (1) In vitro macrophage activity effect (1) Experimental method 3 ml of thioglycolate medium was administered into the peritoneal cavity of BALB/c male mice, and after 5 days, the mice were bled to death, and Eagle MEM was intraperitoneally administered.
Inject 5 ml of medium and collect peritoneal exudate cells (hereinafter referred to as
PEC) was collected. Thereafter, the cells were washed with phosphate buffered saline (hereinafter referred to as PBS ^- ) and adjusted to 10 ⁶ cell δ/ml in Dulbecco's modified Eagle's MEM medium supplemented with 10% fetal bovine serum. After dispensing the cell suspension into a 96-well flat-bottom plate at a rate of 0.2 ml/well,
The cells were cultured at 37° C./5% CO ₂ and floating cells were removed. Further wash with ^PBS- and double Betsuko modified Eagle supplemented with 10% fetal bovine serum.
10, 25, 75 and 100μg/in MEM medium
0.2 ml of fraction A test solution adjusted to a concentration of 0.2 ml
It was dispensed in ml/well. After culturing for 48 hours, 20μ of the culture medium was sampled and the amount of glucose consumed was measured using a glycose C-test kit (Wako). The results were statistically processed using Student's t-test. (2) Experimental Results The relationship between the amount of fraction A added and the amount of glucose consumed by PEC was as shown in FIG. Addition of fraction A promoted glucose consumption, and in the addition group of 25 μg/ml or more, a significant promoting effect on glucose consumption was observed (meaning that macrophage activity was activated). Note that * in Figure 1 indicates a significant difference determined by T-test, * indicates significance at a 5% risk rate (hereinafter indicated as *P<0.05),
** indicates significance at a 1% risk rate (hereinafter **P<0.01). (2) Effect of inducing antitumor macrophages (1) Experimental method 3 ml of proteose peptone medium was administered into the peritoneal cavity of DBA/2 male mice. After 5 days, the mice were bled to death, and the PECs were removed and 10%
10 ⁶ cells/ml in RPMI medium supplemented with fetal bovine serum
It was prepared as follows. This cell suspension was dispensed at 0.2 ml/well into a 96-well flat bottom plate, and after culturing for 1 hour, the floating cells were removed and washed with PBS ^- . Next, 0.2 ml/well of 50, 100, 200 and 400 μg/ml of fraction A test solution was dispensed, and after culturing for 6 and 12 hours, the supernatant was removed and PBS ^-
Washed at. Next, add 10% fetal bovine serum.
Target cells (mastcytoma p815 cells) adjusted to 10 ⁵ cells/ml using RPMI1640 medium
0.2 ml/well was dispensed and cultured for 40 hours. 3 hours before the end of culture, add 2μCi/ ^3H -thymidine.
After the culture was completed, the cells were collected using a glass filter and incorporated into the cells.
The amount of ³ H-thymidine was measured using a liquid scintillation counter, and the cytotoxicity index was determined using the following formula. Cytotoxicity index=
Control (cmp) - sample (cmp) / control (cmp) Control (cmp): 3 H-thymidine uptake when cultured with unstimulated macrophages Sample (cmp): Uptake of ³ H-thymidine when cultured with sample-activated macrophages Amount of ³ H-Thymidine Incorporation (2) Experimental Results Changes in macrophage cytotoxicity index due to the addition of Fraction A were as shown in the table below. Macrophages obtained 6 hours after addition of fraction A were found to exhibit strong cytotoxicity towards mastocytoma p815 cells in a dose-dependent manner. In addition, although the activity decreased in macrophages obtained after 12 hours,
A high cytotoxicity index was still obtained in the 400 μg/ml addition group.

[Table] (3) Effect of enhancing infection resistance (1) Experimental animals After raising ICR male mice in an animal house for a certain period of time, 8 healthy 5-week-old mice were placed in one group.
They were used as animals in this experiment. (2) Experimental method The test solution at the concentration shown below was intraperitoneally administered to the above mice, and 24 hours later, E. coli OU-009 strain (5x
10 ⁸ cells/ml) was subcutaneously inoculated into the back of a mouse, and the viability was determined every day for 4 days. (Dose of test solution) (a) Physiological saline (control) 10ml/Kg (b) Fraction A 25mg/ml/Kg (c) 〃 50mg/ml/Kg (d) 〃 100mg/ml/Kg ( e) 250 mg/ml/Kg (3) Experimental results The fluctuations in the number of surviving mice infected with E. coli were as shown in Figure 2. Note that ( ) in the graph indicates the number of survivors out of 8 mice. Administration of Fraction A had a dose-dependent survival effect, and showed an overall high survival rate. (4) Carbon clearance
Activity Effect (1) Experimental Animals ICR male mice were raised for a certain period of time, and then 5-week-old mice that appeared to be healthy were used in this experiment as a group of 10 mice. (2) Experimental method Carbon from Halpern
Clearance method (Halpern, BH, etal., J.
Reticulo endothel. Soc. 1 , 77 (1964)), the above mice were intraperitoneally administered with the test solution at the concentration shown below, and 24 hours later, a carbon suspension (Pelican Inc. (manufactured by Pelican)) was administered to the mice. (diluted 4 times with saline) was administered into the tail vein at a rate of 5 ml/Kg. After 2 and 10 minutes, remove the mouse from the eye socket using a micropipette.
20μ of blood was collected. 0.1 to the blood obtained
After hemolysis by adding 2 ml of % _{Na2CO3 solution} ,
675nm using a spectrophotometer (Shimadzu)
The absorbance (OD ₆₇₅ ) of the carbon suspension was measured to determine the rate of disappearance of the carbon suspension in the blood. (Dosage of test solution) (a) Physiological saline (control) (b) Fraction A 5mg/Kg (c) 〃 10mg/Kg (d) 〃 15mg/Kg The statistical processing of the results is (Student's) t-test. (3) Experimental Results The relationship between the dose of Fraction A and the rate of disappearance of the carbon suspension was as shown in Figure 3. Even in a small dose group of Fraction A, such as 5 mg/Kg, a significant carbon clearance ability (in vivo ability to remove foreign substances) enhancement effect was observed. Note that * in FIG. 3 indicates a significant difference determined by the same T-test as in FIG. 1. (5) Effect of enhancing antibody production ability (1) Experimental animals BALB/c strain male mice were raised for a certain period of time, and 5-week-old mice that appeared to be healthy were used in this experiment in groups of 8 mice. (2) Experimental method The following amount of test solution was added to the above mouse and 5×
10 ⁸ cells/ml of sheep red blood cells (SRBC) 0.1ml
was administered intraperitoneally. After 4 days, the mice were bled to death, the spleen was removed, and the PFC was transferred from Jerne.
The number of antibody-producing cells was measured by the (Plaque Forming Cell) assay method (Science. 140 , 405 (1963)). (Dosage of test solution) (a) Physiological saline (control) 0.1ml/mouse (b) Fraction A 200μg/ml → 0.1ml/mouse (c) 〃 1000μg/ml → 0.1ml/mouse Statistical processing was performed using Student's t-test. (3) Experimental results The enhancement effect of fraction A on antibody-producing cells is as follows:
It was as shown in Figure 4. A significant increase in antibody-producing cells (indicating that immune ability was activated) was observed in the group treated with 4 mg/Kg of fraction A. Note that * in FIG. 4 indicates a significant difference determined by the same T-test as in FIGS. 1 and 3. Formulation Example 1 1050 g of fraction A, 1470 g of crystalline cellulose, 450 g of starch and 30 g of magnesium stearate were uniformly mixed and compression molded using a roller compactor. This is sized according to the usual method, 2g per package.
It was made into a sachet. Example 2 1050 g of fraction A, 1470 g of crystalline cellulose, 450 g of starch and 15 g of magnesium stearate were uniformly mixed and compression molded using a roller compactor. After sizing this, 15 g of magnesium stearate was added and mixed. 1 of this mixture
The tablets were compressed at 500 mg per tablet. Example 3 Fraction 1050g, lactose 1200g, crystalline cellulose 570g
g and 15 g of magnesium stearate were uniformly mixed and made into slugs using a slug tablet machine. This is sized, mixed with 150g of starch and 15g of magnesium stearate, and per tablet.
It was compressed into tablets of 500 mg. Example 4 Approximately 500 ml of purified water was placed in a 2000 ml beaker, and 238 g of fraction A was dissolved therein. Add 1000ml of single syrup to this
After adding and mixing, 17 ml of an ethanol solution in which 0.24 g of butylparaben was dissolved was added and further mixed. Purified water was added to this to make the total volume 1,700 ml, and the mixture was filtered through a membrane filter. This liquid was filled in 30 ml per bottle according to a conventional method, and then heat sterilized to prepare a syrup. Example 5 Approximately 1000 ml of purified water was placed in a 2000 ml beaker, 105 g of fraction A was dissolved and mixed, and then 200 ml of single syrup was added. After dissolving 0.21 g of butyl paraben in this, purified water was added to make the total volume 1500 ml. After passing this through a membrane filter, it was filled with 30ml per bottle according to the usual method and sterilized by heating.
It was made into a so-called drink.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the effect of Fraction A on enhancing glucose consumption by macrophages. Second
The figure is a graph showing the effect of fraction A on enhancing resistance to E. coli infection. FIG. 3 is a graph showing the carbon clearance enhancement effect of fraction A. FIG. 4 is a graph showing the effect of Fraction A on enhancing antibody production ability.

Claims (1)

[Scope of Claims] 1. A biological defense ability-enhancing agent characterized by containing a fraction obtained by the following steps as an active ingredient. (b) Extracting garlic with water or a hydrous lower alcohol having 1 to 3 carbon atoms. (b) The obtained extract is subjected to at least one method selected from dialysis, gel filtration, and ultrafiltration to separate and remove low-molecular compounds. (c) Obtaining an adsorbed fraction by subjecting the product treated by the above means to anion exchange chromatography.