KR102188057B1 - Immuno-enhancing compounds and their manufacturing methods using a mixture of tumeric and sargassum coreanum - Google Patents

Abstract

The present invention relates to a composition for enhancing immunity or treating containing a mixture of a Curcuma longa extract and a Sargassum coreanum extract, and to a manufacturing method thereof. A mixed extract in which the Curcuma longa extract and the Sargassum coreanum extract are mixed in a weight ratio of 50 : 50 is a natural substance, thereby being harmless to the human body, and having almost no toxicity and side effects. Therefore, the mixed extract can be safely applied and used for a long period of time as a medical immunity enhancing composition.

Description

Composition for enhancing immunity using turmeric and large leaf capillary extract, and its manufacturing method {IMMUNO-ENHANCING COMPOUNDS AND THEIR MANUFACTURING METHODS USING A MIXTURE OF TUMERIC AND SARGASSUM COREANUM}

The present invention relates to a composition for enhancing immunity and a method for preparing the same using turmeric and leaf capillary extract, and more specifically, a mixture of 50% by weight of turmeric extract and 50% by weight of capillary leaf capillary extract to perform a major function in the immune response. The present invention relates to an extract mixture that increases factor production and prevents cell tissue damage, and a method of manufacturing the same.

In recent years, frequent outbreaks of new pandemic diseases such as SARS, bird flu (AI), swine flu, and novel coronavirus have become a serious economic loss and a risk to public health worldwide.

In the case of these infectious diseases, vaccination and the use of antiviral drugs are known as the best prevention and treatment, but some neurological side effects and the emergence of resistant viruses due to abuse are problematic, and the new virus resistant to antiviral Prevention and treatment are limiting by discovery. Therefore, the consumption and interest in health food and immunity that can protect the body from external bacteria or viruses are increasing.

Immunity is the function of protecting the human body from infectious diseases. It recognizes and neutralizes foreign substances such as bacteria, fungi, parasites, and viruses to maintain the homeostasis of the living body.

Innate immunity, also called natural immunity, is the immune system against infections that exist in our body from birth regardless of age or experience of infection. The main role of innate immunity is to distinguish between self and non-self. do. The human body primarily defends against infection by foreign substances with physical barriers such as the acidic environment of the skin and digestive organs, or bile and mucous membranes. When these physical barriers are destroyed, the innate immune system consisting of complement, natural killer cells, phagocytes, dendritic cells, and mast cells acts and invades from the outside. By recognizing several types of pathogens, they induce inflammatory reactions and interact with adaptive immunity.

The living body acts as a defense against various external factors (pathogenic microorganisms, etc.), which is called the immune system. The body's immune system is a body defense mechanism against infection, and is composed of two mechanisms: innate immunity that has been born since birth and acquired immunity that is obtained through adaptation to life. Innate immunity, also referred to as natural immunity, refers to an immune response that reacts non-specifically to an antigen, and acquired immunity exhibits an immune response by acting specifically against an external antigen, and is activated through immune memory.

Innate immune response plays a role in controlling chronic infection, and as one of the defense mechanisms of innate immunity following pathogen infection, immune factors are secreted, and inflammatory reactions are induced by these factors, so that the defense against pathogens is achieved. Induction of inflammatory response at a level can also be a method of prevention and treatment for various infectious disease pathogens, and research and development of immune enhancing agents that can induce them are required. In recent years, interest in research to improve these immune functions with natural substances is emerging.

Decreased immunity increases the likelihood of developing all diseases such as frequent infections, chronic fatigue, inflammation, etc., promotes regression processes such as cancer and aging, and delays the healing process, while the health of immune cells and the ability to remove infiltrating bacteria It can be greatly enhanced with proper nutrition, rest, exercise, and nutritional supplementation.

Turmeric is a perennial plant belonging to the ginger family and has been used by humans for a long time in tropical South Asia and Southeast Asia as medicinal herbs, spices and edibles.Eulgeum, Geogeum, Jade Gold, and Turmeric (深黃), has a herbal name for the Emperor family (黃帝足).

In addition to curcumin, which is the main ingredient, turmeric has efficacy as demethoxycurcumin, bisdmethoxycurcumin, cyclocurcumin, and calebin. Vegetable sterols and essential oil ingredients β-sitosterol, zingiberene, campesterol, stigmasterol, mono- and di-enoic acids, tumerone , zingiberone, borneol, eugenol, camphor, curdion, α-phellandrene, cineol, etc. are contained within 4.2~4.5% of the total. Studies on the physiological activity of turmeric have reported the antioxidant activity of curcuminoids and the antioxidant, anticancer, antimutagenic, anti-inflammatory and antibacterial properties of curcumin.

Turmeric (Curcuma longa L. or Turmeric) is a perennial plant of the Zingiberaceae family that grows wild in southern Asia, Africa, and Central and South America in hot and humid conditions. Turmeric is also widely known as a natural spice that gives food a yellow color, and is mainly used as a health food.

The roots and stems of turmeric are peeled, cooked, dried, and powdered. This is the world famous curry's yellow beef spice “tameric”. It has detoxification and is effective for hangovers, and is also used in various dishes and drinks. In Indian curry, turmeric is always used as a spice, and in Japan, these pigments are used to color pickled radish. In addition, India, Southeast Asia, and China have been used for dyeing silk and cotton since ancient times.

Recently, in Korea, the mass cultivation of turmeric in Jindo has been successful, and it is cultivated all over the country. The difference in quality of turmeric depends primarily on the content of curcumin. It is reported that the content of curcumin has a correlation between macronutrients (N, P, K), secondary nutrients (Ca, Mg, S), and micronutrients (Zn, Fe, Cu, M) depending on the soil where turmeric is grown. It is known to be affected mainly by the cultivated region, variety and climate.

However, rhizomes and tubers are traditionally used for medicinal purposes, but leaves growing over 150cm after collecting medicinal parts and by-products of the basement are discarded without being used. The development of technology to discover and utilize new materials that can be used for functional foods by utilizing the by-products of turmeric leaves and stems is meaningful in terms of creating a new high-value income source for cultivators and new growth engines for related industries.

Sargassum is a generic term for a bird of the genus Sargassum. It is also used collectively for a specific species Sargassum fulvellum or large brown algae. The body has a distinct distinction between roots, stems, and leaves, and the roots are gaban-shaped and grow 1-3m or more with one central branch. The stem is triangular or triangular and twisted. Leaves grow from the stem toward the base and bend, have a spatula shape or oval shape, and have a middle rib up to the center of the leaf. The upper leaves are lanceolate, with serrated protrusions on the edge, and air bubbles form from the stem on the whole body. It has a dark tan and can be seen on all coasts of Korea.

It is a representative type of half-hats and hats used for food, and is often sold in the market. The genus Mosquito is an incombustible plant that is perennial, and is a representative species that forms a marine forest on the coast of Korea. About 20 species are gathered, including only the worms, half-leaved hats, half-legged hats, half-leaved hats, half-leaved hats, half-leaved hats, and twin-legged hats.

Sargassum coreanum is distributed in the east and south coasts of Korea, and in Jeju. It is an edible brown algae belonging to the family Mosquito family. Young stems and leaves are eaten or whole plants are dried for feed. The big leaf hatch has a 5~8 cm conical root and a 5~7 mm thick cylindrical central stem from the root. It is split into several branches to give out branches and leaves.

The results of the study on the leaf capillary are reported to have anti-cancer, anti-inflammatory, anticoagulant, antihyperlipidemic, antihypertensive and anti-arteriosclerosis effects, and the study on the enzymatic extract of the capillary leaflet has an antioxidant effect. .

However, there are many reports on the component analysis and antioxidant power of seaweeds that have been used only for food until now, but reports on the immunity enhancement of seaweeds are very poor. Recently, the search for natural products for the immune enhancing effect has been actively studied not only in terrestrial organisms, but also in marine organisms. In particular, seaweed, a marine plant used for food, is generally not rich in nutritional value, but has been reported to have various physiological activities. In general, seaweed is rich in carotinoids, contains a lot of mycosporine-glycine, which exhibits high antioxidant effects, and contains anti-carcinogens and antioxidants.

It has already been known that the immune function protects the living body from various pathogens, and vaccination and antitoxin use are typically used as a method of preventing and treating diseases using these immune functions. , As the mechanism of action of the immune function is known, attempts are being made to directly use an immunomodulatory substance capable of regulating the immune function.These immunomodulatory substances non-specifically or specifically stimulate immune cells to inhibit the immune function of the living body or By promoting it, it was possible to enhance the defense of the living body from disease factors.

However, most of the above immunomodulatory substances are chemically synthesized substances, and some side effects or toxicity are found, so there are some limitations to apply them to a living body. In an effort to solve this problem, studies on immunomodulatory substances have recently been conducted through validation of efficacy of non-toxic food materials or active ingredients extracted from natural products, and existing herbal medicines. As such, researches are actively underway to find bioactive substances that promote bioregulation and biodefensive systems from natural products.

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Unexamined Patent Publication No. 10-2015-0036937 (2015. 04. 08.) Registered Patent Publication No. 10-2010654 (2019. 08. 13.)

An object of the present invention is to provide a composition for enhancing immunity comprising a turmeric extract and a large leaf capillary extract and a method for producing the same, thereby enhancing the immunity enhancing effect by promoting the biological regulation and the biological defense system.

The composition for enhancing immunity using the extract of turmeric and large leaf capillary according to the present invention reduces the side effects and toxicity of immunosuppressants having non-selective toxicity and reduces changes in bioimmune function caused by chemical drugs. As a physiologically active substance that can be made, 50% by weight of turmeric extract containing curcumin and 50% by weight of large leaf capillary extract containing fucose are mixed, while doubling the immune-related cell proliferation rate. Viruses by activating the reaction to recognize and kill infected cells of natural killer cells (NL cells) by reducing cytotoxicity caused by cyclophosphamide (cy), including natural products that are harmless to And it is characterized in that it plays an effective role in removing cells infected by bacteria.

In addition, in the method for preparing a composition for enhancing immunity using the extract of turmeric and large leaf capillary according to the present invention, in the method for preparing a composition for enhancing immunity, after obtaining a turmeric extract from turmeric using an alcohol extraction method, the turmeric extract is filtered and Freeze-drying after concentration to obtain a powdery turmeric extract; After obtaining a large leaf capsilla extract by using an alcohol extraction method, the capillary capillary extract is filtered, concentrated, and freeze-dried to obtain a powdery capillary capillary extract; Mixing 50% by weight of the turmeric extract and 50% by weight of the big leaf capillary extract; Including, the step of obtaining the turmeric extract includes: a raw material preparation step of quantitatively weighing 210g of turmeric; An extraction step of adding 10 times 50% alcohol to the prepared turmeric and extracting it at 70° C. for 3 hours to obtain a turmeric extract; A filtering step of filtering the extracted turmeric extract using a two-ply extraction cloth; A concentration step of concentrating the filtered turmeric extract at 40 to 45° C. using a low-temperature decompression concentrator; A drying step of freeze-drying the concentrated turmeric extract at -70°C to -80°C to obtain a powdery turmeric extract; A storage step of sealing the powdery turmeric extract and then storing it at -20°C to -30°C, wherein the step of obtaining the large leaf capillary extract includes: a raw material preparation step of quantitatively weighing 210g of the large leaf capillary; An extraction step of adding 10 times 50% alcohol to the prepared large leaf caps and extracting at 70° C. for 3 hours to obtain a large leaf caps extract; A filtering step of filtering the extracted large leaf capillary extract using a two-ply extraction cloth; A concentration step of concentrating the filtered large leaf capillary extract at 40 to 45°C using a low-temperature decompression concentrator, and freeze-drying the concentrated capillary capillary extract at -70°C to -80°C to obtain a powdery large leaf capillary extract ; And a storage step of sealing the powdery leaf capillary extract and then storing it at -20°C to -30°C.

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It is an object of the present invention to provide a composition for enhancing immunity using a mixture of extracts of turmeric and capillary leaves. More specifically, it is intended to provide a composition for enhancing immunity using natural products harmless to the human body by blending 50% by weight of turmeric and 50% by weight of large leaf caps at a ratio of 50:50, which is the most excellent ratio of immunity enhancing effects in vivo.

1 is a schematic diagram showing a method of preparing a turmeric extract among the manufacturing methods of the present invention.
Figure 2 is a schematic diagram showing a method of manufacturing a large leaf capillary extract in the manufacturing method of the present invention.
Figure 3 is a photograph showing the production of turmeric extract in Example 1 according to the manufacturing method of the present invention.
Figure 4 is a photograph showing the preparation of the large leaf capillary extract in Example 1 according to the manufacturing method of the present invention.
Figure 5 is a photograph showing that the composition for enhancing immunity is good by mixing the turmeric extract of the present invention and the big leaf capillary extract in a 50:50 ratio.
6 is a view showing the molecular structures of curcumin and fucose, which are the main components of the turmeric extract and the big leaf capillary extract of the present invention.
7 is a diagram showing a component analysis graph according to Experimental Example 1 of the present invention.
8 is a photograph showing a part of the component analysis report according to Experimental Example 1 of the present invention.
9 is a diagram showing a graph of the viability of splenocytes according to Experimental Example 2 of the present invention.
10 is a diagram showing a graph of the viability of splenocytes treated with cyclophosphamide according to Experimental Example 3 of the present invention.
11 is a diagram showing a graph of the survival rate of AR42J cells according to Experimental Example 4 of the present invention.
12 is a diagram showing a graph of NK cell activation using AR42J cells according to Experimental Example 4 of the present invention.
13 is a diagram showing a graph of the amount of TNF-alpha produced in splenocytes treated with cyclophosphamide according to Experimental Example 5 of the present invention.
14 is a diagram showing a graph of the amount of TNF-gamma produced in splenocytes treated with cyclophosphamide according to Experimental Example 5 of the present invention.
15 is a diagram showing a graph showing the amount of IL-12 produced in splenocytes treated with cyclophosphamide according to Experimental Example 5 of the present invention.
16 is a view showing a graph showing a weekly weight change of an immunity-compromising animal model in which a mixture of turmeric and large leaf capillary extract according to Experimental Example 6 of the present invention is shown.
17 is a diagram showing a liver tissue weight graph of an immune-compromising animal model in which a mixture of turmeric and large leaf capillary extract according to Experimental Example 7 of the present invention is shown.
18 is a view showing a graph of the weight of the spleen tissue of the animal model of reducing immunity of a mixture of turmeric and large leaf capillary extract according to Experimental Example 7 of the present invention.
FIG. 19 is a diagram showing a graph of the content of leukocytes in the blood of an immunity-compromising animal model in which a mixture of turmeric and capillary extract according to Experimental Example 8 of the present invention is shown.
20 is a view showing a blood lymphocyte content graph of an immunity-compromising animal model of a mixture of turmeric and capillary extract according to Experimental Example 8 of the present invention.

The present invention comprises 50% by weight of a turmeric extract containing curcumin as a physiologically active substance capable of alleviating side effects and toxicity of immunosuppressants having non-selective toxicity and reducing changes in bioimmune function caused by chemical drugs, The present invention relates to a composition for enhancing immunity prepared by using a component harmless to the human body while doubling the rate of immune-related cell proliferation by mixing 50% by weight of an extract containing fucose.

In order to prepare a composition for enhancing immunity, after obtaining a turmeric extract by using an alcohol extraction method from turmeric as shown in FIG. 1, filtering and concentrating the turmeric extract, followed by lyophilization to obtain a powdery turmeric extract, as shown in FIG. After obtaining a capillary capillary extract by using the alcohol extraction method, the capillary capillary extract is filtered and concentrated, and then freeze-dried to obtain a powder capillary capillary extract in powder form, 50% by weight of the turmeric extract and 50% by weight of the large leaf capillary extract. It characterized in that it comprises; mixing step.

In addition, the step of obtaining the turmeric extract includes: a raw material preparation step of quantitatively weighing 210 g of turmeric, an extraction step of adding 10 times 50% alcohol to the prepared turmeric and extracting it at 70° C. for 3 hours to obtain a turmeric extract, the extracted Filtration step of filtering turmeric extract using a two-ply extract cloth, concentration step of concentrating the filtered turmeric extract at 40 to 45°C using a low-temperature decompression concentrator, and -70°C to- A drying step of obtaining a powdery turmeric extract by lyophilization at 80°C, and a storage step of storing the powdery turmeric extract at -20°C to -30°C after sealing.

In addition, the step of obtaining the large leaf capillary extract may include a step of preparing raw materials for quantitatively weighing 210 g of large capillary capillary, and an extraction step of obtaining a capillary extract of large leaf capillary by adding 10 times 50% alcohol to the prepared capillary capillary and extracting at 70°C for 3 hours. ;, Filtration step of filtering the extracted large leaf capillary extract using a two-ply extraction cloth, concentration step of concentrating the filtered large capillary capillary extract at 40 to 45°C using a low-temperature decompression concentrator, and the concentrated large capillary cape It characterized in that it comprises a drying step of freeze-drying the extract at -70 ℃ ~ -80 ℃ to obtain a powdery large leaf capillary extract, and storing the powdery large leaf capillary extract at -20 ℃ ~ -30 ℃ after sealing. do.

The step of obtaining the turmeric extract is a raw material preparation step of quantitatively weighing 210 g of turmeric powder, an alcohol extraction step of extracting 50% alcohol at 70° C. for 3 hours, as shown in FIG. Filtration step of filtering, concentration step of concentrating at 40~45℃ using a low-temperature decompression condenser, drying step of freeze-drying at -70℃~ -80℃, and storage at -20℃~-30℃ after sealing the dried composition Including the extraction and storage step, and the step of obtaining the large leaf capillary extract is a raw material preparation step of quantitatively weighing 210 g of large capillary capillary spot, as shown in FIG. 2, by adding 10 times 50% alcohol, and at 70° C. for 3 hours. Extracting alcohol extraction step, filtration step of filtering using two layers of extract cloth, concentration step of concentrating at 40~45℃ using a low-temperature decompression concentrator, drying step of freeze drying at -70℃~ -80℃, drying The composition is sealed and then stored at -20°C to -30°C.

In the step of obtaining the turmeric extract and the large leaf capillary extract, the alcohol extraction step is extracted at 70° C. for 3 hours. This is because the boiling point of the alcohol used for alcohol extraction, that is, ethanol, is 78.3°C at the standard pressure, so the hydrolyzed alcohol begins to boil around 70°C and the temperature does not rise further. In addition, hot water extraction, such as alcohol extraction, is generally heated for 3 hours, which is an appropriate time for extracting effective ingredients. If heated longer than the above time, the extracted component may be excessively heated and damaged, or impurities may be extracted together and the sample may be contaminated. Therefore, it is heated for 3 hours to extract alcohol.

In addition, when the temperature during low-temperature decompression concentration is 45°C or higher, the recovery rate is too low to reduce production efficiency, so the temperature during low-temperature decompression concentration is maintained at 40 to 45°C.

Example 1.

The turmeric powder used in the present invention was purchased and used at a nearby mart.

210 g of the turmeric powder is weighed to prepare, and 50% alcohol is extracted. At this time, the water is added 10 times and extracted for 3 hours at 70°C. After extracting the alcohol, filter it using two layers of extract cloth. After the filtered composition is concentrated at 45°C using a low-temperature vacuum condenser, the concentrated composition is freeze-dried at -75°C. After sealing the dried composition, it is stored at -25°C.

The turmeric extract prepared through the above process is shown in FIG. 3, and is a turmeric powder, a turmeric composition extracted from alcohol, and a filtered turmeric extract in order from the left.

In addition, the large leaf hat plate used in the present invention was purchased and used at a nearby mart.

210g of the large leaf cap is prepared by weighing, and 50% alcohol is extracted. At this time, the water is added 10 times and extracted for 3 hours at 70°C. After extracting the alcohol, filter it using two layers of extract cloth. After the filtered composition is concentrated at 45°C using a low-temperature vacuum condenser, the concentrated composition is freeze-dried at -75°C. After sealing the dried composition, it is stored at -25°C.

The large leaf capillary extract prepared through the above process is shown in Fig. 4, and is a large capillary capillary composition from the left in sequence, a large capillary capillary composition subjected to an alcohol extraction process, and a filtered large capillary capillary extract.

A composition for enhancing immunity was prepared as shown in FIG. 5 by mixing the powdery turmeric extract and the powdery leaf capillary extract prepared through the above process in a 50:50 ratio.

In the preparation of the composition for enhancing the immunity, the turmeric extract and the big leaf capillary extract, through experimental examples, cell proliferation rate using immune cells, cytokine analysis, NK cell activity analysis, in vivo immune enhancement change study using an immunocompromised animal model The mixture ratio of turmeric extract and large leaf capillary extract was determined, and the immunity enhancing effect was verified.In animal experiments, weekly body weight, dietary intake, tissue weight, blood cell count related to immunity in the blood in an immunocompromising animal model using cyclophosphamide Analysis of cytokine levels and spleen biopsy were performed.

In conclusion, the composition for enhancing immunity in which the turmeric extract and the large leaf capillary extract are mixed has an immune enhancing effect in vivo, and the effect is greatest when the mixing ratio of the turmeric extract and the large leaf capillary extract is 50:50. The mixture of the turmeric extract and the big leaf capillary extract reduces cytotoxicity by Cy and activates the reaction to kill the infected cells of NK cells, which are natural killer cells, thereby playing an effective role in removing cells infected by viruses and bacteria. Perform.

Experimental Example 1.

In order to analyze the specifications and components of the functional ingredients, each functional ingredient was analyzed under the conditions shown in Table 1 below. Each functional ingredient may be Curcumin in the case of turmeric and Fucose in the case of a large leaf hatch, and the molecular structure thereof is as shown in FIG. 6.

Curcumin Fucose Instruments SHIMADZU HPLC
C18 Column 250 x 4.6 mm
5μm SHIMADZU HPLC
NH2P-50 Column Detector / Wavelength PDA Detector / 420*±*nm RI Detector Column oven temperature 30 ℃ 30 ℃ Flow rate 1.0 mL/min 0.8 mL/min Injection volume 20μL 20μL Mobile phase Isocratic, 0.1% H3PO4:Acetonitrile
= 450:505 Isocratic, 250 mM H3PO4:Acetonitrile
= 20:80 Sample preparation Take 1 g of sample, add 25 ml of methanol, and elute
→ Ultrasonic extraction for 40 minutes
→ Filtered with 0.22μm syringe filter
→ HPLC analysis Aliquot 4 g and place it in a round bottom flask
→ 5 ml of 2 M TFA treatment
→ 4 hours hydrolysis treatment
→ Adjust to pH 7~8
→ Filtration with 0.45μm syringe filter
→ HPLC analysis

The index components of the turmeric alcohol extract and the large leaf capillary alcohol extract according to Table 1 were requested to be analyzed by the Korea Testing Institute.

The analysis results are shown in a graph in FIG. 7, and a part of the report is shown in FIG. As can be seen in Figures 7 and 8, the analysis results showed that the curcumin of the turmeric alcohol extract was 81774.4 mg/kg, and the fucose of the big leaf capillary alcohol extract was 1218.9 mg/kg.

In addition, cell viability, cytokine production, and natural killer cell activity were analyzed to determine the immunity-enhancing efficacy of the mixture of turmeric and capillaris extract through experiments.

As for the spleen cells for use in the experiment, the spleen of Wistar rats was excised, and a single cell suspension was prepared using tweezers and a mesh. After washing the single cell suspension by centrifugal precipitation (x1,000g, 5 min, 4℃) 3 times with RPMI-1640 culture solution, red blood cell lysing buffer (Sigma, CA, USA) was treated for 3 minutes to remove red blood cells. It was used in the experiment.

Experimental Example 2.

This experimental example is for analyzing the viability of cells, and the splenocytes and AR42J cells (pancreatic gland cells) cultured through the above process were treated with an extract by concentration at 5x10 ⁵ cells/90 μl/well at 37°C, 5% CO ₂ Incubated for 24 hours each. Thereafter, 10 μl of WST-1 (ITSBio, Seoul, Korea) solution was added to 100 μl of the cell culture solution, and incubated for 1 hour, the absorbance value was measured using a Multi Detection Reader (Infinite 200, TECAN Group Ltd, Switzerland). The control group was set as the test group in which only the solvent in which the sample was dissolved without treatment with the sample was treated at the same concentration as the high concentration test group, and the cell proliferation rate was calculated according to the following equation, thereby analyzing the cell viability.

Cell proliferation assay (%)=(absorbance of sample treatment group/absorbance of control group)*100

Cell viability analysis results are shown in Figure 9, A is turmeric 50% alcohol extract, B is a large leaf cap 50% alcohol extract, C is an extract mixture obtained by mixing turmeric and large leaf caps at 25:75, D is for turmeric and large leaf caps The extract mixture was mixed at 50:50, E is the extract mixture mixed at 25:75 of turmeric and large leaf cap, F is the alcohol extract of 50% red ginseng.

In other words, in order to check the toxic concentration of splenocytes according to the treatment of the sample, turmeric 50% alcohol extract and 50% alcohol extract of large leaf capillary, and turmeric and large leaf capillary extract mixture by mixing ratio based on the control without sample treatment. The cell viability of the test group treated with was analyzed.

Looking at the cell viability of each experimental group and control group shown in FIG. 9, in the case of A 50% alcohol extract of turmeric, 100 to 112% at a concentration of 3 to 500 μg/ml did not show a significant difference from the control group, but at 1000 μg/ml At 80.1±4.2% and 3000μg/ml, it was 60.3±1.2%, showing a significant decrease.

In addition, in FIG. 9B, there was no significant difference from the control group at 3 to 100 μg/ml in the 50% alcohol extract of the big leaf capillary, but at 94.4±2.7% at 300 μg/ml, 57.9±9.1% at 500 μg/ml, and at 1000 μg/ml At 8.9±1.0% and 3000μg/ml, it was 3.9±1.3%, showing a significant difference.

In addition, comparing the cell viability of the extract mixture of turmeric and large leaf hatch in C, D and E of FIG. 9, when processing a sample in which a mixture of turmeric: big leaf hatch is 75:25, 500 μg/ml, 50:50 mixed sample When treated with 300 μg/ml and 25:75 mixed samples, 300 μg/ml was found to be the highest concentration showing no significant difference from the control group, respectively.

Experimental Example 3.

In this experimental example, in order to observe the immune enhancing effect through analysis of splenocyte proliferation rate by treatment with Cy (clophosphamide), the isolated splenocytes were dispensed into a 96 well plate at ⁵ ×10 ⁵ cells/80 μl/well and cultured for 24 hours. Thereafter, 1.6 mg/ml of Cy and the extract by concentration were treated, cultured at 37° C. and 5% CO ₂ for 48 hours, and then splenocyte proliferation rate was analyzed using WST-1 reagent.

The analysis result graph is shown in FIG. 10. In Figure 10, A is a turmeric 50% alcohol extract, B is a 50% alcohol extract of a large leaf cap, C is an extract mixture obtained by mixing turmeric and large leaf caps at 25:75, and D is an extract obtained by mixing turmeric and large leaf caps at 50:50. Mixture, E is an extract mixture of 25:75 turmeric and large leaf hatch, F is an alcohol extract of 50% red ginseng. As a result of the analysis, compared to the control group not treated with Cy and the sample, the cell viability of the test group treated with Cy alone was 55-59%, indicating that the survival rate of splenocytes was significantly reduced due to the treatment of Cy.

The survival rate of the test group treated with 50% alcohol extract of Cy and turmeric was shown in FIG. 10A, and at a concentration of 3-50 μg/ml, there was no remarkable difference compared to the Cy alone treatment group, but at 100 μg/ml It was 62.1±1.3% and 62.6±1.2% at 300 μg/ml, indicating that the survival rate was increased in the high concentration treatment group. In the case of the test group treated with Cy and 50% alcohol extract of leaf capillary, as shown in FIG. 10B, it was 60.2±1.5% at 100 μg/ml, and the survival rate was significantly increased only at the highest concentration compared to the Cy alone treatment group. Even in the case of treatment with the mixture of turmeric and large leaf capillary extract by mixing ratio along with Cy, as can be seen in C, D and E of FIG. 10, a significant difference was mainly observed in the high concentration treatment group from the Cy alone treatment group. 75:25 The mixing ratio was significantly increased from 300μg/ml to 62.0±2.4%, from 100μg/ml to 60.7±1.2% at 50:50, and from 300μg/ml to 63.8±0.5% compared to the Cy alone treatment group. It showed 58.3±0.2% at 300 μg/ml, but there was no significant difference from Cy alone treatment group. In the case of the red ginseng extract, which is the positive control of F in FIG. 10, it was found to be significantly increased from the Cy alone treatment group to 64.4±1.2% at 100 μg/ml and 69.0±2.8% at 300 μg/ml.

In the results of this experiment, comparing the effect of the mixture of turmeric and large leaf capillary extract by mixing ratio on the cell viability by treatment with Cy, the highest cell viability was obtained at the ratio of 50:50 mixture of turmeric extract and large leaf capillary extract. Based on these results, the analysis of natural killer cell activity and cytokine production of splenocytes was performed by setting an extract mixture of 50:50 turmeric 50% alcohol extract and 50% alcohol extract of large leaf hatch as a blank sample.

Experimental Example 4.

In this experimental example, for the analysis of the activity of natural killer cells (NK cells), splenocytes were treated with samples by concentration at 5x10 ⁶ cells/ml in a 96 well plate, and AR42J cells were used. Thus, the effector cells and target cells were added at the ratio (1:20) and incubated for 24 hours in a 37°C, 5% CO ₂ incubator. LDH measurement after cultivation was measured using a CytoTox detection kit (Takara), and the absorbance of formazan formed by oxidation of NAD in the reaction solution was measured at 490 nm and compared with the control group.

The analysis result graph is shown in FIGS. 11 and 12. Figure 11 shows the effect of the extract mixture of turmeric and large leaf capillary on the survival rate of AR42J cells, Fig. 11A is a mixture in which the ratio of turmeric and capillary extract is 50:50, and B is red ginseng 50% alcohol extract to be. Figure 12 shows the effect of the extract mixture of turmeric and large leaf capillary on NK cell activity using AR42J cells, Fig. 12A is a mixture in which the ratio of turmeric and large leaf capillary extract is 50:50, and B in Fig. 12 is red ginseng 50 % Alcohol extract.

As can be seen from Figure 11, the analysis result of the analysis results of the mixture of turmeric and large leaf capillary extract (50:50) showed no significant difference from the control group at a concentration of 500 μg/ml or less, and only red ginseng 50% alcohol extract at a concentration of 1000 μg/ml or less There was no significant difference from the control group. However, considering that the cell viability of splenocyte, which is an effector cell, is lower than that of AR42J cell, which is a target cell, when the sample of the same concentration is treated, the natural killer cell activity test was conducted at a concentration that did not show toxicity of the sample itself in the target cell and effector cell. Phosphorus was set to 300 μg/ml.

In addition, the result of analyzing the effect of the mixture of turmeric and leaf capillary extract on the activity of natural killer cells was shown in FIG. 12, compared with the control group, 108.2±6.4% at 5g/ml and 109.5±7.7 at 10μg/ml. %, 109.5±7.7% at 30 μg/ml, 121.2±7.3% at 50 μg/ml, 137.8±4.5% at 100 μg/ml, 129.6±2.5% at 300 μg/ml, and red ginseng extract 105.6 at 5 μg/ml ±5.0%, 111.5±2.9% at 10 μg/ml, 123.4±5.9% at 30 μg/ml, 158.3±8.2% at 50 μg/ml, 160.6±7.6% at 100 μg/ml, and 173.0±3.0% at 300 μg/ml Became. That is, in the case of the extract mixture of turmeric and large leaf capillary in this experiment, the natural killer cell activity was significantly increased compared to the control at a concentration of 30 μg/ml or more, and the activity of red ginseng extract was significantly increased from 10 μg/ml or more. Became.

Experimental Example 5.

This example is for analyzing the content of cytokines in immune cells by Cy treatment, and the amount of secretion of cytokines was used by an ELISA kit purchased from R&D sstem (Minneapolis, MN, USA). Cy and sample were treated and reacted for 24 hours, and then TNF-α (tumor necrosis factor-α), IFN-γ (Interferon-γ), IL-2 (interleukin-2), and IL-12 (interleukin-12) were analyzed. .

13 is a graph showing the effect of the extract mixture of turmeric and large leaf capillary on the amount of TNF-alpha produced in splenocytes treated with cyclophosphamide, and FIG. 13A is an extract obtained by mixing turmeric and large leaf capillary at a ratio of 50:50. 13B shows the case of red ginseng 50% alcohol extract.

As a result of analysis of the amount of TNF-alpha produced, the test group without Cy treatment (Control) was 86.7 ± 10.2 pg/ml, whereas the test group treated with Cy alone was 77.7 ± 9.7 pg/ml. As shown in FIG. 13A, the test group treated with turmeric and large leaf capillary extract mixture and Cy was 48.1±15.0pg/ml at 5 μg/ml, 45.1±7.4pg/ml at 10 μg/ml, and 86.9±14.6 at 30 μg/ml. pg/ml, 50 μg/ml at 168.3±14.2 pg/ml, 100 μg/ml at 332.0±51.4 pg/ml, 300 μg/ml at 524.4±34.6 pg/ml in all test groups treated with 30 μg/ml or more Appeared to be increased. As shown in FIG. 13B, the test group treated with the positive control red ginseng extract and Cy was 30.3±2.7 pg/ml at 10 μg/ml, 33.4±7.6 pg/ml at 30 μg/ml, 19.1±2.4 pg/ at 50 μg/ml. 12.0±9.1pg/ml at ml and 100μg/ml, 8.9±9.0pg/ml at 300μg/ml, and 2.0±3.4pg/ml at 500μg/ml, showing that TNF-alpha production decreased with higher concentration. .

In addition, FIG. 14 is a graph showing the effect of the extract mixture of turmeric and large leaf caps on the IFN-gamma production amount of splenocytes treated with cyclophosphamide, and Fig. 14A is a mixture of turmeric and large leaf caps at a ratio of 50:50. Of the extract, Fig. 14B shows the case of 50% alcohol extract of red ginseng.

As a result of analyzing the production of IFN-gamma, the control without Cy was 149.8±28.0pg/ml, which was significantly different from 31.6±5.5pg/ml of the test group treated with Cy alone. On the other hand, the test group treated with Cy and the sample showed a difference depending on the concentration, but in the case of the extract mixture of turmeric and large leaf hatchling, 103.1±5.6 pg/ml at 5 μg/ml and 87.9±14.2 at 10 μg/ml as shown in FIG. 14A. pg/ml, 121.8±18.3pg/ml at 30μg/ml, 102.3±21.5pg/ml at 50μg/ml, 125.8±16.5pg/ml at 100μg/ml, and 141.0±42.2pg/ml at 300μg/ml. In the case of red ginseng extract, 81.1±12.9pg/ml at 10μg/ml, 69.5±3.6pg/ml at 30μg/ml, 82.2±0.9pg/ml at 50μg/ml, 78.7± at 100μg/ml, as shown in FIG. 14B. 92.9±11.1pg/ml at 14.9pg/ml, 300μg/ml, and 103.1±5.6pg/ml at 500μg/ml, all of which were significantly increased compared to Cy alone treatment group.

In addition, FIG. 15 is a graph showing the effect of the extract mixture of turmeric and large leaf capillary on the amount of IL-12 produced in splenocytes treated with cyclophosphamide, and Fig. 15A is a mixture of turmeric and large leaf capillary at a ratio of 50:50. Of the extract, Fig. 15B shows the case of 50% alcohol extract of red ginseng.

As a result of IL-12 analysis, the control was 18.2±0.3pg/ml, which was significantly different from 11.1±1.5pg/ml of the test group treated with only Cy. The test group treated with turmeric and leaf capillary extract mixture and Cy was 10.0±1.0 pg/ml at 5 μg/ml, 11.1±0.8 pg/ml at 10 μg/ml, 8.1±3.8 pg at 30 μg/ml as shown in FIG. 15A. /ml, 11.6±2.1pg/ml at 50μg/ml, 14.7±3.1pg/ml at 100μg/ml, 14.4±2.4pg/ml at 300μg/ml, and IL-12 at a concentration of 100-300μg/ml There was a tendency that the production amount of was increased. On the other hand, the test group treated with red ginseng extract and Cy showed a significant difference from the Cy alone treatment group at the time of low concentration treatment (10-50 μg/ml) as shown in B of FIG. 15, but about 10 at the time of high concentration treatment (100 to 500 μg/ml). All showed similar levels at ~18 pg/ml, so no significant difference was confirmed by concentration.

In animal experiments, analysis of weekly body weight, dietary intake, tissue weight, immune-related blood cell count and cytokine level in the blood, and spleen biopsy in an immunocompromised animal model using cyclophosphamide.

Prior to the experiment, cyclophosphamide (Cy), known as an immune-lowering substance, was purchased from Sigma-Aldrich (St. Louis, USA) to form an immune-lowering induction model. As a result of the previous study, this experiment was carried out by setting the dose concentration of 5mg/kg, which was identified as the most suitable Cy dose concentration for the test. In this test, Cy, which is an immune-lowering substance, was administered simultaneously with the test substance, and the administration method was to provide a prepared feed mixed for each concentration in the case of the test substance so that the experimental animals could freely ingest it. Cy (5mg/kg) dissolved in distilled water was forcibly orally administered to supply the same amount.

During the breeding period, regular solid feed (Samtako, Gyunggi, Korea) is fed, and the experimental animals after the acclimatization period are separated so that the average value between groups is uniform using the egg mass method based on body weight, and individual identification is indicated using an ear punch. Was done. The experimental group consisted of a normal group, a control group inducing immunocompromising, a group administered with 30mg/kg of turmeric and large leaf capillary extract, a group administered with 100mg/kg of turmeric and large leaf capillary extract, and a group administered with 300mg/kg of turmeric and large leaf capillary extract. Each group was used for the experiment. Cy, an immune-lowering substance, was treated in the same manner in the control and experimental groups, except for the normal group.

Experimental Example 6.

Weekly weight change was measured once a week by individual body weights of all experimental groups, and diet and drinking water intake were measured at intervals of 1-2 times a week by measuring the remaining amount the next day after feeding. After anesthesia with inhalation anesthesia, blood was collected from the abdominal vena cava and used for blood analysis. The liver, thymus, and spleen tissues were removed and weighed, and only the spleen tissue was fixed to the formalin.

To determine the effect of the extract mixture of turmeric and leaf capillary on the immune-compromising animal model using cyclophosphamide (Cy), the weekly body weight change between each test group was first checked. As a result, as shown in the graph of FIG. 16, the control group administered only cyclophosphamide (Cy) showed a decrease in the gain rate as the concentration of Cy increased compared to the normal group administered only with distilled water. At week 4, the end of the experiment, the weight of each experimental group was 265.8±1.9g in the normal group, 264.4±1.2g in the control group, and 247.8 in the experimental group (CS 30 group) administered 30 mg/kg of a mixture of Cy, turmeric and large leaf capillary extract. The experimental group (CS 100 group) administered ±3.3g, Cy and 100 mg/kg of a mixture of turmeric and large leaf capillary extract was 255.1±1.7g, and an experimental group administered 300 mg/kg of Cy, turmeric and large leaf capillary extract mixture (CS 300) group) was 254.6±3.4g, and the experimental group (RG 300 group) to which 300 mg/kg of red ginseng extract as a positive control was administered was 250.4±3.1g. At the end of the study, the weight of the control group and the test group significantly decreased compared to the normal group, but compared to the control group, the test group and the positive control group administered with a high concentration of turmeric and leaf capillary extract mixture were significantly increased. Was investigated.

Experimental Example 7.

The absolute weight and relative weight of each tissue were analyzed to determine the effect of the extract mixture of turmeric and large leaf hatch on the tissue weight of the immunocompromising animal model.

First, in the case of liver tissue, as shown in Fig. 17A, the absolute weight of the normal group is 8.19±0.06g, the control group is 7.38±0.09g, the CS 30 experiment group is 7.45±0.1g, the CS 100 experiment group is 7.58±0.09g, and the CS 300 The experimental group was 7.79±0.13g and the RG 300 experimental group was 8.14±0.09g. Compared to the liver tissue weight of the control group, there was no significant difference in the low-dose group, but the medium-dose group tended to slightly increase, but the high-dose group showed a significant increase. Appeared. These results were similar in the relative weight, as shown in Fig. 17B, in the normal group 3.13±0.04g, the control group 2.99±0.04g, the CS 30 experimental group 2.97±0.07g, the CS 100 experimental group 2.99±0.04g, and CS 300 The experimental group was 3.13±0.04g, and the RG 300 experimental group was 3.20±0.05, which was found to have increased liver weight in the high concentration administration group compared to the control group.

The spleen tissue showed similar patterns in both absolute and relative weights as shown in FIG. 18, but the weight of the spleen based on absolute weight was 0.72±0.02g in the normal group and 0.43±0.01g in the control group, as shown in FIG. 18A. 30 Experimental group 0.44±0.01g, CS 100 experiment group 0.47±0.01g, CS 300 experiment group 0.47±0.01g, RG 300 experiment group 0.51±0.01g. Compared with the control group, turmeric and large leaf capillary extract mixture were used at medium and high concentrations. It was found to be significantly increased in the administered test group and the test group administered red ginseng extract. In the case of relative weight, as shown in B of FIG. 18, 0.27±0.01g in the normal group, 0.18±0.01g in the control group, 0.17±0.01g in the CS 30 experimental group, 0.19±0.01g in the CS 100 experimental group, and 0.18±0.01 in the CS 300 experimental group. g, RG 300 experimental group showed 0.20±0.01 g, showing that the medium-dose group and the high-dose group showed a tendency to have higher spleen tissue weight than the control group.

Experimental Example 8.

Hematological analysis for each group was performed after the end of the four-week test period to determine the effect of the mixture of turmeric and leaf capillary extract on the number of immune cells in the blood of the Cy-induced immunocompromised animal model.

The results are as shown in FIG. 19, and the number of white blood cells in each test group was 5.30±0.13x10 ⁹ /L in the normal group, which was significantly different from 2.27±0.14x10 ⁹ /L in the control group. On the other hand, in the test group administered with the extract mixture of turmeric and large leaf capillary, the CS 30 test group was 2.68±0.12x10 ⁹ /L, the CS 100 test group was 2.70±0.10x10 ⁹ /L, and the CS 300 test group 2.67±0.08x10 ⁹ /L L, and in the RG 300 test group, the blood leukocyte content was significantly increased in the test group administered with a mixture of turmeric and large leaf capillary extract and the test group administered red ginseng extract at 2.83±0.15x10 ⁹ /L compared to the control group. Was investigated.

As a result of lymphocyte analysis, as shown in FIG. 20, 82.57±0.22% of the normal group and 70.15±0.77% of the control group showed a significant difference. The CS 30 experimental group was 74.50±0.80%, the CS 100 experimental group 74.13±0.85%, and the CS 100 experimental group showed a significant difference. The 300 experimental group was 76.28±0.80% and the RG 300 experimental group was 77.77±0.45%, indicating that all test groups were significantly increased compared to the control group.

In conclusion, when the splenocytes were treated with cyclophosphamide, an immune-lowering substance, the cell-protective effect of each sample was analyzed, and the cell viability was found to be the highest in the sample with a ratio of 50:50 between turmeric and large leaf caps. Based on these results, the mixing ratio of turmeric and large leaf capillary extract was set to 50:50. In the analysis of the activity of natural killer cells (NK cells), it was found that the test group treated with the extract mixture of turmeric and large leaf hatch was significantly higher than that of the control group. In the cytokine-producing ability assay, the mixture of turmeric and large leaf capillary extract increased the production of TNF-alpha, IFN-gamma, and IL-12 compared to the test group treated with cyclophosphamide alone. In the test of measuring weekly body weight change, the control group treated with cyclophosphamide showed a significant decrease compared to the normal group, and the test group treated with the turmeric and leaf capillary extract mixture showed a tendency to increase or significantly increased compared to the control group. It was investigated to be increased. As a result of tissue weight measurement, liver tissue weight was found to be significantly increased in the high concentration administration group compared to the control group, and the spleen tissue was significantly higher in the medium concentration administration group and the high concentration administration group. In general hematological analysis, both white blood cells (WBC) and lymphocytes (LYM) were significantly increased in the sample administration group compared to the control group. Therefore, as a result of this study, the mixture of turmeric and capillaris extract was effective in enhancing immunity, and a composition for enhancing immunity can be formed using this.

Claims (4)

In the composition for enhancing immunity,
As a physiologically active substance capable of reducing the side effects and toxicity of immunosuppressants with non-selective toxicity and reducing changes in bioimmune function caused by chemical drugs, 50% by weight of turmeric extract containing curcumin, and Including natural products that are harmless to the human body while doubling the rate of immune-related cell proliferation by mixing 50% by weight of the big leaf capillary extract containing fucose,
It reduces cytotoxicity caused by cyclophosphamide (cy) and activates a reaction that recognizes and kills infected cells of natural killer cells (NL cells), thereby removing cells infected by viruses and bacteria. A composition for enhancing immunity using turmeric and large leaf capillary extract, characterized in that it plays an effective role.
In the method for producing a composition for enhancing immunity,
After obtaining a turmeric extract from turmeric using an alcohol extraction method, filtering and concentrating the turmeric extract, followed by lyophilization to obtain a powdery turmeric extract;
After obtaining a large leaf capsilla extract by using an alcohol extraction method, the capillary capillary extract is filtered, concentrated, and freeze-dried to obtain a powdery capillary capillary extract;
Mixing 50% by weight of the turmeric extract and 50% by weight of the large leaf capillary extract; including,
The step of obtaining the turmeric extract,
Raw material preparation step of quantitatively weighing 210 g of turmeric;
An extraction step of adding 10 times 50% alcohol to the prepared turmeric and extracting it at 70° C. for 3 hours to obtain a turmeric extract;
A filtering step of filtering the extracted turmeric extract using a two-ply extraction cloth;
A concentration step of concentrating the filtered turmeric extract at 40 to 45° C. using a low-temperature decompression concentrator;
A drying step of freeze-drying the concentrated turmeric extract at -70°C to -80°C to obtain a powdery turmeric extract;
A storage step of sealing the powdery turmeric extract and storing it at -20°C to -30°C; including,
The step of obtaining the large leaf capillary extract,
Raw material preparation step of quantitatively weighing 210g of large leaf caps;
An extraction step of adding 10 times 50% alcohol to the prepared large leaf caps and extracting at 70° C. for 3 hours to obtain a large leaf caps extract;
A filtering step of filtering the extracted large leaf capillary extract using a two-ply extraction cloth;
A concentration step of concentrating the filtered large leaf capillary extract at 40 to 45°C using a low-temperature decompression concentrator,
A drying step of freeze-drying the concentrated large leaf capillary extract at -70°C to -80°C to obtain a powdery capillary capillary extract;
A method for preparing a composition for enhancing immunity using turmeric and large leaf capillary extract, comprising: a storage step of sealing the powdery capillary extract and storing at -20°C to -30°C.

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