KR100814214B1 - Immuno-stimulative composite comprising extract from bamboo sap - Google Patents

Abstract

A composition comprising an extract of Phyllostachys sap is provided to increase the activity of macrophages and natural killing cells directly facing tumor cells and promote the production of antibody against an external including tumor by increasing mitosis materials and physiologically active materials. An immuno-stimulating composition comprises an extract of Phyllostachys sap as an effective ingredient, wherein the extract is lyophilisate of the Phyllostachys sap. The composition is in the form of a food, a food additive or a pharmaceutical composition.

Description

Immunity-enhancing composition containing extract of bamboo sap as active ingredient {IMMUNO-STIMULATIVE COMPOSITE COMPRISING EXTRACT FROM BAMBOO SAP}

1 is a graph showing the lymphocyte proliferation activity by the extract of bamboo sap,

Figure 2 is a graph showing the induction capacity of cytokine IL-2 of bamboo sap extract,

Figure 3 is a graph showing the induction capacity of cytokine IL-4 of bamboo sap extract,

Figure 4 is a graph showing the induction capacity of the cytokine INF-r of bamboo sap extract,

5 is a graph showing the induction capacity (iNOS) of inducible nitric oxide synthase of macrophages of bamboo sap extract,

6 is a graph showing the induction of the proliferation of macrophages of bamboo sap extract.

The present invention relates to an immune enhancing composition containing an extract of bamboo sap as an active ingredient, and more particularly, an immune enhancing composition containing the freeze-dried product of the bamboo sap as an active ingredient, and a functional health food containing the same. Or it relates to food additives and pharmaceutical compositions.

Bamboo (Phyllostachys) is a plant native to Gramineae, and it is known that there are more than 1,250 species in the world. In Korea, bamboo has been cultivated for a long time. There are 13 kinds of bamboo grown in Korea. It is distributed in the area of, and is partly distributed in coastal areas of Gangwon-do and Chungcheongnam-do and Jeju-do.

Since ancient times, bamboo has been used as a folk medicine for the treatment of hypertension, sweating, stroke, etc. It is also known as an antiseptic effect, and stem, epidermis, bamboo shoots and bamboo thread have been used as a medicine for treating diseases, but recently, Like sap, sap is collected and drinking.

While many studies have been conducted on the antimicrobial activity of bamboo leaves, research on bamboo sap has only been conducted on the sampling amount, the sampling method, and the availability of the distilled water.

A sap is a liquid that flows through conduits or dead ends and is a relatively dilute solution in which inorganic salts, nitrogen compounds, carbohydrates, enzymes, and plant hormones are dissolved. The sap is composed of 99.3% water and 0.7% solids, but usually 'Water differentiation' is very different from water.

The custom of drinking plant sap as a healthy drink has a long history in the Soviet Union, China, and Japan. It is a folk medicine that is effective in diuresis, constipation, gastrointestinal diseases, high blood pressure, blood sugar control, neuralgia, etc. In industrialized countries, sap maple syrup is processed in Canada and the United States to produce sugar or syrup, and birch sap is collected and sold as healthy drinks in Hokkaido, Japan.

Bamboo sap can collect more sap than cypress and birch, and it is known to be effective in dislocation, diuresis, nerve stabilization and suppression of heart disease, and elimination of waste products due to its high content of sugar and inorganic components. In addition, compared to the sap collected from other species, the content of sugar and inorganic components is high, the value of drinking water is high, and antimicrobial activity is shown to be great, so it can be used as a natural antibacterial substance.

In Korea, some studies have been conducted on the development of bamboo processing technology, that is, bamboo arts and crafts technology. Until now, however, there has been little development of technology using pharmacological efficacy or functionality of bamboo resources. The research on the activity of antimicrobial activity against is ongoing.

In addition, some research on bamboo resources in foreign countries has been conducted in China and Japan. Especially, in China, bamboo resources have been used in various ways, but mainly focus on industrialization of building materials using bamboo, and bamboo is a functional food. The technology used as a material is still in its infancy, and at present it is a state of developing and distributing bamboo beverages called 'jukgun' and 'juk flavor' coated with rice by preparing bamboo leaf extract has also been developed.

As such, gorgo sap, maple sap, birch sap, etc. have been studied variously on the pharmacological effect, but the research on the bamboo sap has not been much, especially the pharmacological effect has not been reported yet.

An object of the present invention is to provide a composition for immuno-enhancement comprising a freeze-dried product of bamboo sap as an active ingredient, and a functional health food or food additive and pharmaceutical composition containing the same.

In order to achieve the above object, the present invention is the experimental group containing the lyophilized bamboo sap and the control group does not contain the extract activity of the mitotic substance, the inducibility of the bioactive substance, in vivo acute toxicity test, inducible monoxide Immune synthetase gene expression, NO production ability, and natural immune cell activation, and proved the immune enhancing effect of the lyophilized composition of bamboo sap as an active ingredient, functional health food or food additives and pharmaceutical compositions containing the same Will be provided.

Hereinafter, with reference to the accompanying drawings will be described a preferred embodiment of the present invention;

1 is a graph showing the lymphocyte proliferation activity by the extract of the bamboo sap, Figure 2 is a graph showing the induction ability of the cytokine IL-2 of bamboo sap extract, Figure 3 is the induction capacity of the cytokine IL-4 of bamboo sap extract 4 is a graph showing the induction capacity of the cytokine INF-r of the bamboo sap extract, Figure 5 is a graph showing the induction capacity (iNOS) of the induced nitric oxide synthase of macrophages of the bamboo sap extract , Figure 6 is a graph showing inducing the proliferation of macrophages of bamboo sap extract.

Example 1; Lyophilized Powder Preparation of Bamboo Sap

The harvesting of bamboo sap, the material of the present invention, was collected in May-June, and the collected bamboo sap was powdered by freeze-drying and stored at -20 ° C until use, and 500 mg / ml before treatment in animal cells. It was dissolved at a concentration of and filtered through a membrane filter (pore size) of 0.22㎛ (membrane filter) and stored at -20 ℃ was used for the experiment.

Comparative Example 1; Effect of Bamboo Sap on Mitogen in Immune-Related Cells

Harvesting the spleen from the Balb / c mice of 5 weeks of age, and the number of spleen cells (splenocytes) adjusted to a density of 2 × 10 ⁵ / 100㎕ to put into each well (well) to each bamboo sap 200㎍ / ㎖, 100㎍ / Ml, 50 μg / ml and 10 μg / ml were added.

As a positive control, the final concentrations of concavanallin-A (hereinafter referred to as Con-A) and lipopolysaccharide (hereinafter referred to as LPS), which are mitotic substances of T-cell and B-cell, respectively Each well was adjusted to 0.5 μg / ml and 5 μg / ml, and the experimental and control groups were incubated for 3 days at 37 ° C. and 5% CO _2, and the proliferation assay of the lymphocytes by the sample was observed. Was investigated.

Lymphocyte proliferation activity was performed by MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide] method, and the MTT method was 5 mg / ml. 50 μl of the concentration-adjusted MTT reagent was added to each well and incubated for 6 hours to react with the mitochondria dehydrogense produced by the MTT reagent and living cells, resulting in non-aqueous dark blue formazan. The proliferative activity of lymphocytes was measured by measuring the absorbance at 570 nm.

As shown in FIG. 1, the result of Comparative Example 1 showed that the sap freeze-dried product showed higher proliferation activity of splenocytes compared to the sample-free control group.

As a result of this comparative example, it was not confirmed whether the present invention activates any kind of immune cells among B-cells or T-cells, but it was confirmed that there is an activating activity that directly stimulates splenocytes nonspecifically.

In a similar experiment, 1 ml of 5 weeks old Balb / c 3% thioglycollate was intraperitoneally administered and 3 days later, sterile cells of mice were sterilely collected (24). Each well of the well plate (48 well plate) is plated at a concentration of 1 × 10 ⁶ / ㎖, incubated for about 2 hours and washed each well with PBS (macrophage attached to the plate) Was recovered.

The amount of iNOS and NO was quantified by treating bamboo sap with INF-r at 200 µg / ml, 100 µg / ml, 50 µg / ml and 10 µg / ml respectively.

As a result of the experiment, NO and iNOS production change could not be observed in the bamboo sap treatment group, but the increase of NO and iNOS value was observed in the group treated with INF-r depending on the concentration of the sample. .

As a result of this comparative example, the present invention has the effect of enhancing the cellular reactivity of the mature immune cells to induce an effective immune response, when exposed to an external antigen, the living body to which the present invention is administered is not only a nonspecific immune response, Since the number of effector cells for the antigen-specific immune response is increased, it was confirmed that it can induce an effective protective effect against the antigen from the outside.

Comparative Example 2

1. Induction of Cytokine from Bamboo Sap

Effect of Bamboo Sap on Mitogen in Immune-Related Cells

Harvesting the spleen from the Balb / c mice of 5 weeks of age, and by adjusting the number of spleen cells (splenocytes) at a density of 2 × 10 ⁶ / 500㎕ put into each well (well) to each bamboo sap 200㎍ / ㎖, 100 Co-culture was performed for 24 hours by the addition of μg / ml, 50 μg / ml and 10 μg / ml.

In addition, the final concentration of Con A was adjusted to be 0.5 μg / ml as a positive control, treated in wells and incubated for 24 hours. After the incubation, only the supernatant was recovered and the secreted induction into the supernatant was IL-2 and IL-. Various cytokines such as 4, IL-10, INF-r, etc. were measured using each cytokine kit.

As shown in FIGS. 2 to 4, bamboo sap directly activates mouse pancreatic cells to induce immune-related cytokines such as IL-2, IL-4, IL-10, and INF-r. It was confirmed that the immune stimulating effect.

In the present invention, the degree of inducing cytokine was different depending on the type of cytokine and the concentration of the sap of bamboo, but it was confirmed that there is activity as a cytokine derivative (cytokine inducer) depending on the concentration.

2. In vivo acute toxicity test of bamboo sap

25 g of male Balb / c mice were orally administered with 2,000 sag / kg, 500 μg / kg and 100 μg / kg per mouse via oral administration, and the survival and body weight of the mice were examined for 15 days. .

Survival, body weight and liver values of the mice administered bamboo sap according to the comparative example are shown in Tables 1 to 3, respectively.

Table 1. Survival rate of mice administered bamboo sap

Dosing method Dosage Dosing date / survival rate (%) Survival rate (%) 1 day 3 days 5 days 7 days 9th 12 days 15th Oral administration 2mg / kg 100 100 100 100 100 100 100 100 0.5mg / kg 100 100 100 100 100 100 100 100 100 µg / kg 100 100 100 100 100 100 100 100

Table 2. Changes in body weight of mice administered bamboo sap

Dosing method Dosage Dosing date / survival rate (%) Weight change (%) 1 day 3 days 5 days 7 days 9th 12 days 15th Oral administration control 20.3 ± 0.2 20.7 ± 0.4 21.3 ± 0.5 21.5 ± 0.7 22.1 ± 0.6 21.9 ± 0.5 22.3 ± 0.5 109.8 2mg / kg 20.1 ± 0.3 20.4 ± 0.5 20.9 ± 0.4 21.1 ± 0.6 21.7 ± 0.8 22.1 ± 0.6 21.9 ± 0.4 108.9 0.5mg / kg 20.1 ± 0.3 21.0 ± 0.2 21.5 ± 0.6 21.3 ± 0.9 22.3 ± 0.4 22.8 ± 0.8 22.9 ± 0.6 113.9 100 µg / kg 19.9 ± 0.4 20.3 ± 0.6 21.7 ± 0.2 21.4 ± 0.7 21.2 ± 0.8 21.7 ± 0.4 22.4 ± 0.6 112.5

Table 3. Liver Numbers of Mice Administered with Bamboo Sap

Dose No. fo mouse ALT (unit / ml) AST (unit / ml) Mortality control 10 8.8 ± 0.2 19.5 ± 0.4 0 2mg / kg 10 8.7 ± 0.5 18.9 ± 0.6 0 0.5mg / kg 10 8.9 ± 0.1 19.3 ± 0.2 0 100 µg / kg 10 8.6 ± 0.4 18.8 ± 0.3 0

As shown in Table 1, the bamboo sap did not appear to affect the survival rate of the mice regardless of the dose, this result proved that the bamboo sap does not affect the survival of the living body.

In addition, as shown in Table 2, the experimental group treated with the bamboo sap showed a similar weight gain as the sample untreated control group, indicating that the bamboo sap did not induce tissue damage of the living body.

And, as shown in Table 3 above, as a result of examining the liver digits of the bamboo sap treatment group did not show a significant difference with the normal group, it could be observed that did not cause any liver damage.

3. Determination of the effect on the activation of natural killer cells (abbreviated as NK-cells)

22 g of male Balb / c mice were orally administered 200 μg / kg, 50 μg / kg and 10 μg / kg of bamboo sap respectively for 7 days, and after 7 days the spleen cells and NK- The cells were incubated for 6 hours with YAC-1, a sensitive cell line.

Cultured splenocytes were used as effector cells (E) and cultured YAC-1 was used as target cells (T).

The killing effect by NK-cells was centrifuged at 1,400 rpm for 1 minute using chromium-free method (51 Cr-release method), which is a cytotoxicity assay of natural killer cells, and 4 hours at 37 ° C. Incubated.

Supernatant was recovered using a Skatron instrument, counted with a g-counter, and cytotoxic programs measured for experimental release, spontaneous release, and maximum release. In this way, the activity of NK-cells was calculated.

NK-cell activity (%) = [experimental release-spontaneous release / maximum release-spontaneous release] × 100

Table 4. Effect of Bamboo Sap on Natural Killing Cells

Dosing method Dosage E / T ratio 100 50 25 Oral administration control 13.2 ± 4.8 9.3 ± 7.4 7.3 ± 7.9 200 µg / kg 48.5 ± 8.3 34.3 ± 4.8 23.3 ± 4.6 50 µg / kg 29.7 ± 2.5 24.3 ± 5.2 21.3 ± 4.8 10 µg / kg 18.9 ± 7.4 17.3 ± 7.3 16.3 ± 9.3

As shown in Table 4, it was confirmed that the NK-cell activity of bamboo sap is proportional to the E / T ratio and bamboo sap.

Compared with the control group administered with bamboo sap and the control group, the splenocytes of mice treated with 200 μg of bamboo sap showed about four-fold NK-cell activity compared to the control mice. Compared to the effective activity was shown.

As described above, it was proved to be beneficial for lymphocyte proliferation activity due to the sap of bamboo, the ability to induce physiologically active substances, in vivo toxicity test, the ability to induce iNOS and NO, activation of natural killer cells.

The bamboo sap may be used as a composition of food or medicament, and the extract may be used as a composition of food or medicament in the form of lyophilization or concentrate.

Hereinafter, the preparation examples of the composition will be described in detail.

Formulation examples; Preparation of Health Functional Drink

Example; Bamboo sap 1-15% by weight

           1 ~ 2% by weight of bamboo sap

           1 to 10 wt% of liquid fructose

           Citric acid 0.01 ~ 1% by weight

           Bamboo extract 0.05 ~ 0.5% by weight

           Betaine 0.01 ~ 1.0 wt%

           Dietary Fiber 0.1 ~ 1.0% by weight

           Juice concentrate 0.1 ~ 2.0 wt%

           Add 800 ml of purified water

For example, the health functional beverage using the extract of the bamboo sap of the present invention is mixed with the above components according to the conventional health beverage manufacturing method, sterilized for about 30 minutes at 80 ℃ and sealed in a container to secondary pasteurization After packaging in a small packaging container such as glass bottles to prepare a health functional beverage.

As described above, in the detailed description of the present invention, specific embodiments have been described, but various modifications may be made without departing from the scope of the present invention.

Therefore, the substantial scope of the present invention should not be limited by the above-described embodiment, but should be defined by the same structure as the claims as well as the claims described below.

The present invention is configured as described above by extracting the composition for immuno-enhancing bamboo sap as an active ingredient, the administration of the immuno-enhancing composition increases the activity of macrophages and natural killer cells directly against tumor cells, and similar Increasing cleavage and bioactive substances has the effect of enhancing the production of antibodies to foreign antigens, including tumors.

In addition, the present invention has the effect of preventing diseases associated with immunity to daily diet by using the bamboo sap as a composition in food or food additives and pharmaceuticals.

Claims (5)

Immunity-enhancing composition comprising the extract extracted from the sap of bamboo as an active ingredient. The method according to claim 1, The extract is an immune enhancing composition, characterized in that the lyophilized bamboo sap. The method according to claim 1, The composition is an immune enhancing composition, characterized in that provided as a food. The method according to claim 1, The composition is an immune enhancing composition, characterized in that provided as a food additive. The method according to claim 1, The composition is an immune enhancing composition, characterized in that provided as a pharmaceutical composition.

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