JPH08143462A - Immunostimulating agent and antitumor agent - Google Patents

Abstract

PURPOSE: To produce an immunostimulating agent and antitumor agent having low toxicity, high efficacy and easily forming a pharmaceutical preparation by using a specific propolis extract as an active component. CONSTITUTION: Propolis collected from the hive of honeybee is heated in an aqueous solvent at 35-60 deg.C at a neutral region of pH 7 or thereabout to extract the active component in the water phase. The system is cooled and the precipitate is separated from the cooled liquid to obtain a liquid containing the active component. The precipitate is further extracted at least once with water at 35-50 deg.C to recover the active component. The liquid recovered from the precipitate and containing the active component is mixed with the above liquid containing the active component and the obtained solution containing the active component of propolis is used as it is or after purification. Since the active component of propolis is free from residual particular solvent, it can easily be prepared in the form of a pharmaceutical preparation. The water-soluble propolis is repetitively administered at a daily rate of about 400-800mg/60kg as an immunostimulating agent or antitumor agent.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunostimulant and an antitumor agent containing a specific propolis extract as an active ingredient.

[0002]

2. Description of the Related Art Propolis is a brown-colored lump collected from a honeycomb, and since it contains a medicinal component, it has been used for folk remedies for a long time, and in recent years, effective utilization of the component has attracted attention. Propolis is a viscous resinous substance that contains a large amount of wax components, making it difficult to formulate it.
The one extracted with a high-concentration hydrophilic organic solvent is used. Generally, undenatured ethanol or edible alcohol extract is used, but the use is limited because the undenatured ethanol or edible alcohol is contained at a high concentration, and the wax component is also dissolved. There was a problem such as low. Further, depending on the purpose of use, a step of removing ethanol from the extract is required, and the production is complicated.

Therefore, the present inventors have previously proposed a method of extracting a propolis active ingredient at a high active ingredient concentration in a simple process, without using a specific solvent, which can be easily formulated into a wide variety of applications. Filed a patent application (Japanese Patent Application No. 6-1
24996).

On the other hand, with respect to propolis, various medicinal components thereof have been studied, but conventionally, the propolis extract obtained by a known method is difficult to formulate due to the above-mentioned reasons. At present, no substance suitable for practical use having an effective bioactive effect such as an antitumor effect has been found, and the toxicity is low, the efficacy is high, and the immunostimulant that is easy to formulate A tumor drug was desired.

[0005]

Therefore, an object of the present invention is to use a specific water-soluble propolis extract as an active ingredient,
It is intended to provide an immunostimulant and an antitumor agent with low toxicity, high effect, and easy formulation.

[0006]

Means for Solving the Problems As a result of earnest studies, the present inventors have found that propolis is heated in a water solvent to extract an active ingredient into an aqueous phase, and after cooling, a floating wax component and a precipitate are precipitated. The active ingredient is extracted into the aqueous phase by removing the substance and further by heating the propolis in an aqueous solvent, and after cooling, the precipitate is separated from the cooling liquid to obtain a liquid containing the active ingredient. Got Further, the propolis extract obtained by recovering the active ingredient from the precipitate by extracting the precipitate at least once with water at 35 to 50 ° C. and combining the two has high physiological activity, The present invention has been completed by finding that it is useful as an immunostimulant and an antitumor agent. In the present invention, the water solvent means that water is used as a solvent (extraction medium) for extracting the active ingredient of propolis.

The immunostimulant and antitumor agent of the present invention will be described in detail below with reference to specific examples.

Propolis as a raw material of the propolis extract used for the immunostimulant and antitumor agent of the present invention is known to be produced in Brazil, Australia, China, Japan, etc., and collected by bees. Homogeneity of trees, that is,
Since the active ingredient is highly uniform, it is preferable to use a raw mass produced in Brazil.

The water-soluble propolis extract, which is an active ingredient of the immunostimulant and antitumor agent of the present invention, is characterized by being obtained by heat extraction with water. The extraction method will be described below.

As the raw material propolis, in order to improve the extraction efficiency in a water solvent, it is preferable to use finely ground raw mass of propolis. Therefore, as a pretreatment for dissolution, it is preferable to finely pulverize it to a size of about 100 to 200 mesh. All the equipment to use,
It is preferable to perform high-pressure sterilization at 121 ° C. and 100 to 150 atm. Further, since the extract of the active ingredient from propolis is an aqueous system, it is necessary to sterilize it through a sterilizing filter or No. 131 filter paper manufactured by Advantic Toyo Co., Ltd. to prevent the mixture of mold, microorganisms and the like during the extraction process. .
Therefore, the method for extracting the active ingredient of propolis used in the present invention is preferably carried out in a clean bench, and even in the case of large-scale production, all the steps are preferably carried out under aseptic conditions.

The propolis extraction method used as an active ingredient of the immunostimulant and antitumor agent of the present invention will be described according to the process. After crushing propolis with a sterilized instrument, finely crushed propolis and water are mixed with propolis 1
The mixture is heated at a ratio of 5 parts by weight of water as a solvent to parts by weight. Heating is preferably performed while stirring,
For example, it can be carried out in a three-necked flask reactor equipped with a stirring blade or a rotary evaporator.

The heating temperature is preferably about 40 to 60 ° C., and the active ingredient is extracted from the propolis by maintaining the temperature for 2 hours or more while continuing stirring. The extraction may be performed for about 5 hours. The heating temperature is preferably at least a temperature at which beeswax contained as an impurity is dissolved, and the upper limit of the temperature is preferably about 60 ° C. in order to efficiently dissolve the active ingredient without decomposing it. It is preferably 45 to 50 ° C.

It is also possible to add finely pulverized propolis to warm water at the above temperature and perform extraction with stirring.

As the water used as the solvent for the extraction, any water containing no impurities such as ion-exchanged water and distilled water can be used, but by the magnetic operation under high pressure,
It is preferable to use stable magnetic resonance water having a geotechnical crystal structure (microcluster). this is,
This is because magnetic resonance water has high permeability, is easy to mix, and improves the solubility of the active ingredient. The details of magnetic resonance water are described in "Introduction to the Wave Age" by Masaru Emoto (Sun Road Service Publishing Co., Ltd.), and specific examples thereof include R water (trade name) manufactured by Sun Road Service Co., Ltd. Is mentioned.

Next, the liquid containing the active ingredient and the precipitate extracted from the propolis is left to stand and cooled to room temperature. As a result, wax components, impurities, etc. are separated and float in the upper layer. For cooling, it is preferable to perform cooling at ambient temperature after heating is completed. Therefore, the ambient temperature is 15
It is preferably about 30 ° C.

Impurities in the upper layer containing floating waxes are preferably removed by filtration with gauze. By removing the floating wax component and the precipitate, an aqueous phase containing the active ingredient extracted from propolis can be obtained. The precipitate and the like can be separated by a known method such as centrifugation or filtration.

Further, in order to improve the extraction efficiency, the precipitate can be extracted again with water to recover the active ingredient contained in the precipitate. High-speed centrifugation (for example, 1 × 10 ⁴ rpm, 10 minutes) can also be performed here in order to efficiently separate the liquid and the precipitate.

The precipitate is subjected to extraction treatment with water at least once, and it is preferable that the amount of water is at least 10 parts by weight per 1 part by weight of the precipitate. After adding water to the precipitate, the solution is preferably adjusted to near neutrality in order to enhance the enzyme activity. For example, the pH of the mixture of the residue and water is adjusted to 7. with 1N sodium hydroxide solution. 0-7.
It is more preferable to adjust to about 5, especially about 7.0. Here, the water used is preferably water containing no impurities, that is, pure water, as in the case of the water solvent used for the heat extraction, and particularly magnetic resonance water is preferable in terms of extraction efficiency. . The extraction temperature is 30 to 50 ° C, preferably 45 to 50 ° C. Since the extraction efficiency of the active ingredient is low once in the water extraction step, it is preferable to perform continuous extraction at least twice.

Here, the solution containing the active ingredient recovered from the precipitate and the solution containing the active ingredient can be combined to obtain an active ingredient-containing solution of propolis.

Although the liquid containing the active ingredient of propolis can be used as it is, it is preferable to purify it by performing dialysis, gel filtration, high performance liquid chromatography and the like in order to further raise the purity and remove impurities. Specific examples of the equipment used include a 10-liter pressure filtration (membrane filter) kit for a 90 mm filter. The wax component not removed in the first active ingredient extraction step is also removed in this purification step together with other impurities. The solution containing the water-soluble active ingredient extracted from the obtained propolis can be adjusted in concentration at this point so as to suit the purpose of use. In this way, a liquid propolis active ingredient-containing product is obtained.

Further, it may be provided as a solid product in order to improve long-term storage stability of the active ingredient, or the extract liquid product may be freeze-dried into a powder for solidification.

The active ingredient of the propolis thus obtained does not have any special solvent remaining, and can be easily formulated.

Hereinafter, the present invention will be described in detail with reference to specific examples, but the present invention is not limited thereto.

[0024]

【Example】

[Example 1] (1) Production of water-soluble propolis extract 100 g of an original mass of Brazilian propolis was treated at 100 ° C for 12 hours.
It was autoclaved at 1 atm. 100 g of sterilized propolis was put into a container equipped with a stirrer, and distilled water was added to 50 g.
Pour 0 ml and p with 1N sodium hydroxide solution.
After adjusting H to 7.0, the temperature was raised to 40 to 45 ° C. to maintain the temperature, and the mixture was treated for 2 hours while continuing stirring to extract the active ingredient. After 30 minutes, a yellowish brown solution was confirmed.

Then, the whole amount of the liquid was subjected to high-speed centrifugation (1 × 10 ⁴ rpm) for 10 minutes to separate it into a liquid containing the active ingredient extracted from propolis and a precipitate.

100 g of the precipitate is taken and 500 ml of distilled water
The mixture was put in an extractor together with and the pH was adjusted to 7.0 with 1N sodium hydroxide solution. Extraction was performed at a temperature of 45 to 50 ° C. for 2 hours, and then the total amount of the solution was subjected to high-speed centrifugation (1 × 10 ⁴ rpm) for 10 minutes to separate a solution containing an active ingredient extracted from propolis and a precipitate, A dark brown liquid was obtained.

The solution thus obtained and the solution containing the active ingredient separated in the first extraction are combined and sterilized with a filter (or manufactured by Advantic Toyo Co., Ltd., No. 4).
131 filter paper) was used to remove impurities such as bacteria.
The obtained liquid was freeze-dried to obtain 10.6 g of a yellowish brown water-soluble propolis extract powder.

[Example 2] (2) Purification of propolis extract 1.64 g of the water-soluble propolis extract obtained was taken and
Gel permeation chromatography was performed on Sephadex G-50 (manufactured by Pharmacia) using a / 15M phosphate buffer (pH 6.98). The column size was 58 x 3.1 cm. The absorption curve at 750 nm is shown in FIG. As shown in the graph of Fig. 1, 4
It was confirmed that there were two peaks. Fraction GI,
The yields of fraction G-II, fraction G-III and fraction G-IV are shown in FIG.
As shown inside.

The water-soluble propolis extract thus obtained was confirmed for immunostimulating action, acute toxicity, antitumor action and the like by the following methods.

[Example 3] (3) Measurement of lymphocyte-to-polymorphonuclear leukocyte ratio increasing action (L / P activity) Measurement using a method modified from Metcalf et al. By Hand et al. I went. First, 6-1 after birth
Swiss-Webster within 2 hours (Swiss-Webster)
The littermate newborns of the strain mice were divided into two groups, one of which was intraperitoneally injected with a physiological saline solution of the propolis extract as a sample group, and the other was similarly injected with physiological saline as a control group. Blood was collected from the tail vein before injection and 6 days, 10 days, and 14 days after injection to prepare a thin-layer blood smear, and Wright (Wrig
ht) Staining method, count 100 lymphocytes and polymorphonuclear leukocytes in total using a microscope with mechanical stage (10x40x), and ratio of lymphocytes to polymorphonuclear leukocytes (L / P ratio) I asked.

To determine the efficacy, by the method of Mizutani et al., The increase in L / P ratio was calculated 6 days, 10 days, and 14 days after the blood collection for each of the sample group and the control group, and the risk factor of 5 was calculated by t-test. The case where a significant difference was observed at less than or equal to% was regarded as effective. The results are shown in Table 1.

[0032]

[Table 1]

As shown in Table 1, the propolis extract of the present invention showed significant L / P activity at doses of 200 μg / mouse to 25 μg / mouse. Further, in the subsequent observation, when administered at a dose of 200 μg / mouse, the activity was observed even after 30 days.

Further, the water-soluble propolis of the present invention may be used in an amount of 25 μm.
A graph plotting the L / P ratio 6 days after injection for the log dose of g / mouse to 200 μg / mouse is shown in FIG. 1. The regression of the L / P ratio is shown by the regression line Y = 0.59X +
I got 0.60. That is, good linearity was exhibited within the dose range in this example. Therefore, it is understood that the immunostimulant of the present invention has useful properties suitable for formulation as a medicine.

[Example 4] (4) Acute toxicity test The propolis extract obtained as described above was dissolved in sterile purified water before use so that the dose was 0.2 ml per 10 g of body weight. Was used as the dosing sample. The dose is 3000 mg / kg, 1500 mg / kg, 750
An aqueous solution whose concentration was changed depending on the administration dose was mg / kg, 375 mg / kg, or 187 mg / kg, and was forcibly orally administered once by force using a metal gastric tube. It has been confirmed that the sample preparation is placed in a brown bottle and is stable for several days in an open state at room temperature.

Administration 0 day, 1 day, 3 day, 5 day, 7 day, 10
The body weight was measured on the 14th and 14th. After observing the general condition for 14 days, exsanguination was performed, and the animals were dissected and the tissues of the heart, liver, lungs, kidneys, spleen and digestive tract were observed macroscopically.

No deaths were seen during the study. Observation of changes in average body weight showed that all groups showed weight gain.

In general observation, after 10 days, 187
In the mg / kg group, the behaviors of biting each other were seen, and hypertrophy was observed in 3 of the spleens, which was considered to be stress-related. No abnormalities were found in the other groups. The observed hypertrophy of the spleen is attributed to the bite or trauma, and not the effect of sample administration.

From the above results, the propolis extract of the present invention has an LD ₅₀ value of 3000 mg / kg or more, and no lethal action or acute toxicity symptoms are observed at any dose, and it is considered to be a low toxicity substance. .

[Example 5] (5) Antitumor activity (5-1) Effect on solid cancer Twelve 12-week-old male ICR mice were divided into two groups, and 6 mice were used as a sample group, and the propolis of the present invention was used. 200 extracts once daily
administered by subcutaneous injection at a dose of μg / mouse,
On the other hand, 6 mice were used as a control group, and physiological saline was 0.2 ml / mouse.
Was administered. Ehrlich ascites tumor was used as a tumor, and 4 to 8 × 10 ⁶ cells were subcutaneously transplanted into the right groin of a mouse. Tumor growth was examined by measuring the product of the maximum diameter (major axis) and the minor axis (mm ² ) every week on the skin using a caliper, and the tumor was excised on the 35th day after transplantation, and the tumor size was increased. The length (major axis × minor axis × thickness mm ³ ) and tumor weight were measured. The tumor suppression rate (hereinafter referred to as IR) with respect to the control saline administration group was determined. Further, the fractions GI to G-IV of the water-soluble propolis extract of the present invention obtained in Example 2 by gel chromatography
The same test was conducted for Table 2 shows the results.

[0041]

[Table 2]

As shown in Table 2, as a result of continuous subcutaneous injection of the propolis extract of the present invention for 10 days, a growth inhibitory effect on solid cancer was observed from the third week after transplantation as compared with the control group. Tumor disappeared in 2 out of 6 animals in the week, suppression rate 9
The significance was 3.1% (the risk rate was less than 1%).

Further, the fraction GI which had been fractionated by gel chromatography was continuously administered for 34 days, and a growth inhibitory effect was observed, and tumor disappeared in 4 out of 6 animals at 5 weeks. The inhibition rate was 93.2%, which was significant (risk rate of less than 5%), and the fraction G-II had tumor disappearance in 1 of 6 animals, and the inhibition rate was 83.0%, which was significant (risk rate of less than 5%). )Met. Fraction G-III and fraction G-IV had no antitumor effect. Tumor size was measured over 5 weeks for the water-soluble propolis extract, fraction GI and fraction G-II for which growth inhibitory effects were observed, and the measurement results are shown in FIGS.
It was shown to. Changes in tumor size over time are seen in Figures 3-5.

[Example 6] (5-2) Action against ascites cancer (life-prolonging effect) As tumor, Ehrlich ascites cancer was used, and two 5-week-old male ICR mice in two groups were used in the same manner as in Example 5. The test sample group and the control group were divided into
˜8 × 10 ⁶ cells were transplanted. The propolis extract of the present invention was administered to the test group by subcutaneous injection once a day at a dose of 400 μg / mouse.
The dose was 2 ml / mouse.

The average survival time (hereinafter referred to as MST) of the physiological saline administration group of the sample and the control was calculated by the following formula, and the ratio (T / C) of the MST of the sample group and the control group was calculated.
The effect was judged as T / C> 1.25 being valid and T / C <1.25 being invalid.

MST = Σ (t, f) / n In the formula, f represents the number of animals dead on the t-th day, and n represents the number of observed animals.

The results are shown in the graph of FIG. In ascites cancer, the control group had an average survival time of MST = 12.3, whereas the sample group had MST = 15.4, and T / C = 1.2.
52, the propolis extract of the present invention was also effective for the life prolonging effect in Ehrlich ascites cancer.

From the above examples, it was confirmed that the immunostimulant and antitumor agent according to the present invention have low toxicity and excellent effects. The dose of water-soluble propolis administered as an immunostimulant and an antitumor agent is not particularly limited, but from the results of animal experiments, it is 400-800 mg / day for humans.
It is preferable to continuously administer about 60 kg.

[0049]

INDUSTRIAL APPLICABILITY The propolis extract obtained by the water extraction of the present invention has a low toxicity and shows an excellent effect as an immunostimulant and an antitumor agent.

[Brief description of drawings]

1 is a graph showing an absorption curve at 750 nm of gel chromatography of the water-soluble propolis extract obtained in Example 1. FIG.

FIG. 2 is a graph plotting the L / P ratio 6 days after injection against the logarithmic dose of the water-soluble propolis extract of Example 3.

FIG. 3 is a graph showing changes over time in tumor size in the water-soluble propolis extract-administered group and the control group of Example 5.

FIG. 4 Fraction G of water soluble propolis extract of Example 5
It is a graph which shows the time-dependent change of the tumor size of a -I administration group and a control group.

FIG. 5 Fraction G of the water-soluble propolis extract of Example 5
FIG. 6 is a graph showing changes in tumor size over time in the II administration group and the control group.

FIG. 6 is a graph showing the life-prolonging effect of the water-soluble propolis extract-administered group and the control group of Example 6.

Claims (4)

[Claims] 1. An immunostimulant comprising a water-soluble propolis extract, which is extracted by heating propolis in an aqueous solvent at 35 to 60 ° C., as an active ingredient. 2. The active ingredient is extracted into an aqueous phase by heating propolis in a water solvent at 35 to 50 ° C., and after cooling, the precipitate is separated from the cooling liquid to obtain a liquid containing the active ingredient. In addition, the precipitate is further mixed at least once with 3 times.
An immunostimulant comprising a propolis extract as an active ingredient, which is obtained by recovering an active ingredient from a precipitate by extracting with water at 5 to 50 ° C. 3. An antitumor agent comprising a water-soluble propolis extract extracted by heating propolis in an aqueous solvent at 35 to 60 ° C. as an active ingredient. 4. An active ingredient is extracted into an aqueous phase by heating propolis in a water solvent at 35 to 50 ° C., and after cooling, a precipitate is separated from the cooling liquid to obtain a liquid containing the active ingredient. In addition, the precipitate is further mixed at least once with 3 times.
An antitumor agent comprising a water-soluble propolis extract obtained by combining the above-mentioned active ingredients by recovering the active ingredient from the precipitate by water extraction at 5 to 50 ° C.