KR100549662B1 - Composition comprising the extract or fraction isolated from Russian Mumie for activation of Immunity - Google Patents

Abstract

The present invention relates to a composition for enhancing immune activity containing a fraction of Russian Mumie (Russian Mumie), in particular for enhancing immune activity containing a FA1, FA2 or FA3 fraction derived from the tasteless extract of fulvic acid or Provided are compositions for the prevention and treatment of infectious diseases.

The fulvic acid fraction derived from the tasteless may be used as a drug to increase the immune activity by enhancing the production of reactive oxygen species (ROS) and nitric acid (NO) by macrophages, or by showing a splenocyte proliferation effect. In addition, since there is no toxicity to cells, it can be used as a therapeutic agent for infectious diseases.

Tasteless, humic substances, fulvic acid, reactive oxygen species, nitric acid, macrophages, immune activation, infectious diseases, pharmaceuticals

Description

Composition comprising the extract or fraction isolated from Russian Mumie for activation of Immunity}             

1 is a diagram briefly illustrating the process of fractionation from Russian radish,

2A is a diagram showing electron spectra of FA _total , HA, LMW1 and LMW2,

Figure 2b is a diagram showing the electron spectrum of the tasteless ethanol extract,

3 is the IR spectrum of the tasteless fraction,

4A-4C are emission fluorescence spectra of raw tasteless, tasteless FA fractions and HA high molecular weight fractions,

5 is HPSEC analytical spectra for the tasteless and fractions thereof,

Figure 6a is a diagram observing the ROS generation rate by treating the FA fraction after PMA induction to macrophages,

Figure 6b is a diagram observing the natural ROS production by macrophages after FA fractions treatment,

7 is a view showing the effect on the production of ROS of tasteless fractions,

8 is a diagram showing the effect on the NO production of tasteless fractions,

9 is a diagram showing the effect of tasteless fractions on the splenocyte growth.

The present invention relates to a composition for enhancing immune activity containing extracts or fractions of Russian mumie.

In bacterial, viral infections or inflammatory reactions, regulation of macrophage and lymphocyte activity plays a pivotal role in the determination of the therapeutic effect of a medicinal product. The production of reactive oxygen species such as superoxide anion (O ₂ ⁻ ), hydrogen peroxide (H ₂ O ₂ ) and nitric oxide (NO) by activated macrophages is nonspecific It is an important cytotoxic and cytostatic mechanism in immunity. Many studies have been conducted on which natural compounds affect the production of ROS and NO by macrophages. Macrophages are antigen-presenting, tumor-tumoring, and microbicidal cells that play a central role in cell-mediated or humoral immunity. As control cells, NO produced in activated macrophages exhibits a macrophage, a nonspecific host defense mechanism, and a proliferation inhibitory activity of bacteria and cancer cells.

In addition, during chemotherapy, such as radiotherapy or chemotherapy, hematopoietic stem cells are damaged during the hematopoietic process, thereby reducing successively related hematopoietic cells and immune cells, which often causes impairment in the formation of hematopoietic and immune functions. As a result, patients often experience anemia and lymphocytosis, thrombocytopenia or granulocytopenia, which can lead to serious and fatal infections and increase mortality in patients. Clinically, bone marrow growth factors such as G-CSF, GM-CSF, IL-1-12, M-CSF and EPO are used after chemotherapy or bone marrow transplantation. Promotes cell differentiation and production from BFU, increases cell migration, phagocytosis, superoxide production, and cytotoxicity of leukocytes following antibody stimulation.

On the other hand, geopolymers and natural biominerals have recently received great attention as raw materials for the development of food additives and pharmaceuticals. Currently, based on natural humic products such as peat, sapropel, and mumie, drugs that can be applied to various clinical applications are being developed.

Mumie (silajit, shilajit) used in the present invention is a semi-solid black resin (resin), long-lasting Euphobia and algae (Clover, Trifolium) plants, and lichens inhabiting the mountain It is formed due to the humicization of. Mumie humus consists of 60-80% organic matter, 20-40% mineral matter, and trace elements. Tajikistan people routinely use tasteless foods. Food additives containing tastelessness with several biological activities have been patented and manufactured. Mumi is used in Indian society as a rejuvenator (rasayana) in relation to health. This substance contains an active chemical that promotes the acquisition of knowledge and memory. Dull urinary disease, diabetes (Bhattacharya, SK, Activity of shilajit on alloxan-induced hyperglycemia in rat, Fitoterapia , volume LXVI, No. 4 , pp328-, 1995), digestive disorders, nervous system disease, tuberculosis, chronic bronchitis, asthma, It is prescribed for anemia, bone fractures and other diseases. Although tasteless rice has been used as a traditional medicine in many countries for nearly 3000 years, it has not been fully studied on the structure, physicochemical properties of tasteless ingredients, and the mechanism of its therapeutic efficacy.

For clinical applications, tasteless as well as food additives with immunostimulatory and anabolic activity are used in the form of liquid extracts (processed tasteless). In the tasteless water-soluble fraction, the main component of organic matter is fulvic acids. These humic substances are abundant in peat, sapropel and other humus matters, which are of medical importance. Nevertheless, the biological effects of humic materials vary depending on their chemical structure and physicochemical properties. The chemical composition, structure and functional groups of humic materials are very different and this is due to the humiliation process such as origin, age and humidity, aeration, temperature and mineral micro-environment. Depends on condition A tasteless area is a particular ecosystem that differs significantly from other parts of the world in terms of the humiliation process. Important research activities are currently focused on studying the physicochemical properties of humic materials collected and separated from soil, peat, bunnies, seas, rivers and other reservoirs.

The tasteless water extract activates phagocytosis and cytokine secretion in macrophages of the peritoneum of mice. Application of the drug free results in the proliferation of lymphocytes in the thymus layer of the cortex and causes their lymph nodes and splints to migrate to the thymus-dependent site. Fulvic acid isolated from soil and reservoir stimulates the degree of functional activity of neutrophils and T-lymphocytes.

Humic acid is used to treat eye infections, hemorrhagic eye infections, and livestock infections (Guofan, Tang, Jiangxi; Humic acids , 3 , 1984 in application of Fulvic acid and its derivatives in the field of agriculture and medicine, First edition, June 1993) Fulvic acid is used for the treatment of bronchial cancer and skin ulcers (Yuan, Shenyuan; Fulvic acid , 4 , 1988 in application of Fulvic acid and its derivatives in the field of agriculture and medicine, First edition, June 1993).

However, the effects of tasteless FA fractions on ROS and NO production and lymphocyte proliferation by macrophages have not been identified.

Thus, the present invention is characterized by tasteless fractionation, IR, UV / VIS, fluorescence microscopy, high-pressure exclusion chromatography, ROS and NO production by the macrophage of FA fraction containing fulvic acid and lymphocytes The present invention has been completed by investigating the effect on proliferation and revealing that it enhances immunity.

An object of the present invention is to provide a pharmaceutical composition for immuno-enhancing containing fulvic acid (FA) fractions of the fractions extracted and separated from Russian radish.

The present invention provides a composition for the prevention and treatment of diseases caused by immunocompromised or immune system damage containing Russian extracts or fractions.

The present invention also provides a pharmaceutical composition for the prevention and treatment of infectious diseases containing extracts or fractions of Russian radish, which enhances immunity.

In order to achieve the above object, the present invention provides a composition for immuno-enhancement containing an extract or fraction of Russian Mumie (Russian Mumie).

The present invention provides a composition for the prevention and treatment of diseases caused by immunosuppression or damage to the immune system, which contains Russian radish extract or fraction as an active ingredient and a pharmaceutically acceptable carrier or excipient.

In another aspect, the present invention to provide a pharmaceutical composition for the prevention and treatment of various diseases caused by infection or inflammation containing extracts or fractions of Russian radish having immune enhancing effect.

The composition for the prevention and treatment of diseases caused by immuno-enhancing, immunocompromised or immune system damage, or the prevention and treatment of various diseases caused by infection or inflammation, the Russian radish extract and fractions based on the total weight of the composition 0.5 to 50 It is included by weight.

Russian tasteless extracts and fractions of the present invention can be prepared by the following manufacturing process.

Dried Russian radish can be prepared according to a conventional extraction method, for example, the tasteless material is pulverized or crushed by various grinding machines, such as 0.1 to 2 moles of sodium hydroxide, about 1 to 5 times that of the extract material. A first step of stirring at room temperature for 12 hours to 48 hours using a basic solution, and obtaining a crude extract as a supernatant through centrifugation;

Strong acids such as hydrochloric acid, sulfuric acid and nitric acid were added to the crude extract to acidify to pH 1.0 to 2.0, preferably pH 1.0, followed by centrifugation to obtain supernatant fulvic acid (FA) and humic acid (HA). Next, a strong base such as sodium hydroxide and potassium hydroxide was added to the supernatant of the fulvic acid solution to adjust the pH to 6.5 to 8.0, preferably pH 7.0, and then filtered through a 0.2 to 0.5 μm filter to obtain a molecular weight of 10,000 to 15,000. Obtaining a _total solution having FA;

Ethanol was added to the final _total FA solution obtained in the second step to prepare a final concentration of 60 to 70%, preferably 66%, followed by centrifugation to obtain supernatant and precipitate 1, and ethanol again in the supernatant. To a final concentration of 80-90%, preferably 85%, followed by centrifugation to obtain supernatant and precipitate 2, followed by dialysis of each precipitate 1 and 2, preferably a 10 kDa cut-off dialysis tube. A third step of performing the used dialysis to obtain the FA1 and FA2 fractions;

The final _total FA solution obtained in the second step is subjected to dialysis, preferably using a 10 kDa cut-off dialysis tube, and then passed through a DEAE-cellulose column to obtain a _total FA solution of 0.3 to 2.0 moles, preferably Is a fourth step of eluting stepwise using 0.5 moles, 2.0 moles or 0.3 moles of sodium chloride solutions, respectively, to obtain FA3, FA4 and FA5 fractions;

Then, the fifth step of concentrating the fraction available in the 85% ethanol of the third step to obtain the LMW1 and LMW2 fraction by reprecipitation after adding the same amount of ethanol to a solution saturated with low molecular weight material Provided is a method for preparing a composition for enhancing immune activity from Russian radish.

The Russian tasteless crude extract obtained from the above process was further subjected to various Sephadex column chromatography methods, for example, a porous resin column such as Sephacryl S-300, Sephadex G-50, Sephadex-G-25, and the like. Purified fractions can be obtained.

In addition, the Russian radish extract of the present invention may be further fractionated by a conventional fractionation method (Harborne JB Phytochemical methods: A guide to modern techniques of plant analysis. 3rd Ed. Pp 6-7, 1998).

The present invention also provides extracts or fractions of Russian tasteless radish having the following characteristics isolated from the preparation method.

The FA _total has a molecular weight of 10,000 to 15,000, shows a maximum absorption wavelength at 402nm and 665nm in UV / VIS, absorption bands at 3400, 1450 and 1090cm ^-1 in IR.

The FA1 fraction has a molecular weight of 10,000 to 15,000, a carbohydrate content of 2.3% by weight, the highest absorption wavelength at 402 and 665 nm in UV / VIS, and an absorption band at 3400, 2900, 1720, 1600, 1450 and 1350 cm ^-1 at IR. The fluorescence emission wavelengths are characterized by appearing at 370, 395, 435 and 480 nm.

The FA2 fraction has a molecular weight of 10,000 to 15,000, a carbohydrate content of 1.8% by weight and shows the highest absorption wavelengths at 402 and 665 nm in UV / VIS, absorption bands at 3400, 2900, 1720, 1600 and 1350 cm ^-1 in IR, The fluorescence emission wavelength has a characteristic appearing at 415 nm.

The FA3 fraction has a molecular weight ranging from 10,000 to 15,000, a carbohydrate content of 0.6% by weight, an IR showing absorption bands at 3400 cm ⁻¹ , and a fluorescence emission wavelength at 395 and 480 nm.

The Russian radish extract (FA _total ) and fractions (FA1, FA2 and FA3) were positive in the production of reactive oxygen species (ROS), the production of nitric acid (NO) in the macrophages and the growth of splenocytes The results show the effect of boosting immune activity.

FA _total contains fulvic acid, and falvic acid yellows neutral leukocytes and T-lymphocytes, so the composition containing the same can be expected to enhance immune activity.

The present invention provides a composition for immunoactivation containing one or more fractions selected from Russian radish extract (FA _total ) having the above characteristics or fractions separated by additional purification processes (FA1, FA2 and FA3).

In addition, the present invention of the present invention contains one or more fractions selected from Russian radish extract (FA _total ) having the above characteristics or fractions separated by additional purification process as an active ingredient and pharmaceuticals It can be used for the prevention and treatment of diseases caused by immunodeficiency, immunodeficiency or damage to the immune system containing a carrier or excipient, which is acceptable.

The pharmaceutical composition of the present invention is used for the prevention and treatment of diseases caused by a decrease in immune function by chemotherapy and radiation therapy, or a disease caused by immunosuppression after bone marrow transplantation, AIDS due to immune system damage and a decrease in immune function. Can be used.

In another aspect, the present invention to provide a pharmaceutical composition for the prevention and treatment of various diseases caused by infection or inflammation containing extracts or fractions of Russian radish having immune enhancing effect.

The composition can be used as a prophylactic and therapeutic agent for infectious diseases caused by bacteria or viruses, and because there is no toxicity to cells, eye infections, hemorrhagic eye infections, esophagitis, esophagal tumors, bronchitis, atopy It can be used as a treatment for dermatitis diseases such as sexual dermatitis, pressure sores and skin ulcers.

Compositions comprising Russian tasteless extracts and fractions of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the Russian tasteless extracts and fractions of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds, as well as in a suitable collection.

Compositions comprising Russian tasteless extracts and fractions according to the present invention are oral formulations, external preparations, suppositories, and sterile injections of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., according to conventional methods, respectively. Carriers, excipients and diluents which may be formulated in the form of solutions and may be included in compositions comprising Russian tasteless extracts and fractions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, Maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate Rate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like. Such solid preparations include at least one excipient such as starch, calcium carbonate, and the like in the Russian radish extract and fractions. , Sucrose or lactose, gelatin and the like are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid preparations for oral use may include various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water and liquid paraffin, which are commonly used to include suspensions, solutions, emulsions, and syrups. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The amount of the Russian radish extract and fractions of the present invention may vary depending on the age, sex, and weight of the patient, but generally in an amount of 0.01 to 500 mg / kg, preferably 0.1 to 100 mg / kg, once a day Can be administered in several divided doses. In addition, the dosage of the Russian radish extract and fractions may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The pharmaceutical composition of the present invention can be administered to various mammals such as rats, mice, livestock, humans, and the like. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The composition comprising the Russian tasteless extract of the present invention can be used in various forms, such as drugs, foods and beverages for promoting bone growth and osteoporosis treatment in the above formulation. Examples of the food to which the Russian radish extract can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

Russian tasteless extract itself of the present invention is a drug that can be used with confidence even for long-term administration for the purpose of prevention because there is little toxicity and side effects.

The present invention also manufactures a health functional food comprising at least one fraction selected from the Russian tasteless fractions FA1, FA2 and FA3 obtained from the above production process and a food acceptable additive.

The Russian tasteless fraction of the present invention may be added to food or beverage for the purpose of increasing immune activity and preventing infectious diseases. At this time, the amount of the Russian tasteless fraction in the food or beverage is generally added to the health food composition of the present invention 0.1 to 15% by weight, preferably 1 to 10% by weight of the total food weight, food health beverage composition It can be added in the ratio of 1-30 g, Preferably it is 3-10 g based on 100 ml of silver.

The health beverage composition of the present invention has no particular limitation on the liquid component except for containing the above-mentioned Russian tasteless fraction as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.), and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of said natural carbohydrate is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof. , Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The present invention will be described in more detail based on the following Examples and Experimental Examples, but the present invention is not limited thereto.

Example 1 Preparation of Flavored Crude Extracts

100 g of tasteless rice collected in the mountainous region of Kazakhstan (Ust'-Kamenogorsk) was added to 500 ml of 0.5 M sodium hydroxide solution and stirred at room temperature for 16 hours. 35.4 g of the residue (humin and other insoluble compounds) were centrifuged (3,900 x g, 15 minutes). The supernatant thereof was separated to prepare a crude extract (see FIG. 1).

Example 2. Separation of tasteless fractions and humic substances

The process of separating humic substances from the tastelessness of the present invention was performed by a conventional fractionation method, and fractionation was performed according to the difference in solubility of humic substances in water of various pH (see FIG. 1).

2-1. Manufacture of Humic Acid, Himatomellanic Acid and Mucus

Hydrochloric acid was added to the supernatant of Example 1 to acidify the pH to 1.0 to obtain 6.1 g of a precipitate, humic acid (HA). After the centrifugation, the fulvic acid (FA) solution was adjusted to pH 7.0, filtered using a 0.45 μm membrane filter, and fractionated by ethanol precipitation or ion exchange chromatography in Example 2-2 below.

Hymatomelanic acid (HymA) and dark green mucus were obtained by extraction with 100% ethanol from HA and humin respectively.

2-2. Preparation of LMW Fractions and Fulvic Acid Fractions (FA1, FA2)

Fractions of the fulvic acid (FA) solution by ethanol precipitation were obtained by minor modifications to Yudina's method (Yudina et al . ; Chem. Plant. Mater. , 4 , pp 33-38, 1998). Ethanol was added continuously and stepwise to the fulvic acid solution by the fractionation method, and the resulting precipitate was centrifuged. We used ethanol with varying concentrations to separate the FA _total solution into high and low molecular weight fractions.

In summary, the ethanol precipitation process was carried out as follows.

The fulvic acid (FA _total ) liquid phase solution was mixed with ethanol (final concentration 66% (v / v)) to precipitate high molecular weight insoluble FA (FA1). The precipitate was separated by centrifugation, and then ethanol (final concentration 85% (v / v)) was added to the obtained supernatant to precipitate again insoluble FA (FA2). The 24 g FA _total solution present in the first solution was precipitated with ethanol at 66% and 85% concentrations to yield -14.8g and -9.2g, respectively. Two FA fractions were dialyzed using 10kDa cut-off dialysis tubes (Sigma Co., USA), and each fraction was named FA1 and FA2 (see FIG. 1).

In the above, the ethanol soluble fraction, the supernatant obtained after 85% ethanol precipitation and centrifugation, was concentrated to 10% of the initial volume using a rotary evaporator. 7.5 g of the precipitated gray residue was further purified by centrifugation to add the same amount of ethanol and then reprecipitated from the saturated liquid solution. As a result, a white residue (fractional LMW1) and a solution which were ethanol insoluble were obtained. This solution was evaporated to yield 24.3 g of pellet of bitter dark brown viscous material (fractional LMW2) (see FIG. 1).

2-3. Preparation of Fulvic Acid Fractions (FA3, FA4, FA5)

To obtain the fulvic acid (FA) fraction by ion exchange chromatography, the total FA (FA _total ) solution was dialyzed using a 10 kDa cut-off dialysis tube. A _{total of} 10 g of dialyzed FA was passed through a diethylaminoethyl (DEAE) -cellulose column (Fluka Co., Switzerland). The material adsorbed on DEAE-cellulose was continuously eluted with 0.5M sodium chloride solution, 2M sodium chloride solution and 0.3M sodium chloride solution. The fractions obtained were named FA3, FA4, FA5 and obtained in amounts of -7.3 g, -0.4 g and -1.9 g, respectively. For the following experiments, these fractions were dialyzed against distilled water using a 10 kDa cut-off dialysis tube.

Example 3. Spectroscopic Analysis Using UV / VIS

The UV / VIS spectra of the tasteless fractions were examined using an Ultropec 4000 spectrophotometer (Pharmacia Biotech, Uppsala, Sweden) and 200 to 1-cm quartz cuvette (1-cm quartz cuvette). Scanning at 800 nm.

The electron spectra of FA _total , HA and LMW2 showed a gentle downward curve in absorbance with increasing wavelength (see FIG. 2A). The FA1 to FA5 fractions showed a spectrum of the same shape as FA _total , which is the same as the characteristic of humic materials extracted from other natural materials.

The electron spectra of the LMW1 fraction showed maximum absorption in the range of 275-280 nm specific for benzoic acid and its derivatives.

The electron spectrum of the raw tasteless ethanol extract is similar to the spectrum of Humus samples containing geoporphyrin and chlorins. The highest absorbance for the porphyrin-like pigment was 402 nm and the highest for chlorine rich precipitates at 665 nm (see FIG. 2B).

The chlorine and porphyrins in the precipitate are diagenetic products of chlorophyll molecules, which are thought to be biological markers of chemical fossils or humus materials. Chlorine is the direct product of chlorophyll, whereas porphyrin is the result of long term chlorophyll attribution. Porphyrin-like pigments are characterized by peaks of 410 nm, whereas chlorine-like pigments have the highest absorbance at 665 nm, suggesting that chlorine-rich precipitates have an E ₄₁₀ / E ₆₆₅ (porphyrin / chlorine) ratio between 1 and 5. Porphyrin-rich precipitates have higher values between 5 and 10.

The inventors found that the ratio of E ₄₁₀ / E ₆₆₅ to the crude tasteless ethanol extract was about 3.4. This indicates that the tastelessness of the present invention can be classified as a chlorine-rich humus material.

Example 4 Spectroscopic Analysis Using Infrared (IR)

Infrared spectra of tasteless fractions (650 to 4000 cm ⁻¹ ) were measured on a potassium bromide (KBr) disk using a Speccord 71 IR spectrophotometer (Carl Zeizz, Germany) and a dry sample of 2 mg After mixing with dried 800 mg of potassium bromide (KBr), the mixture was pressed into a disc.

The IR spectrum of the tasteless fraction is shown in FIG. 3. Unlike pure compounds with specific absorption peaks, the humic material isolated from the tasteless group showed a relatively broad band. These wide IR bands are due to the overlap of absorption in all kinds of similar functional groups.

4-1. FA _total,  IR spectra of HA, HymA, raw tasteless ethanol extract and Humin ethanol extract

Absorbent bands specific to these humic materials were also observed in the IR spectra of the FA _total and HA fractions. The strong absorption band at 3400 cm ⁻¹ confirms that the OH groups in the hydroxyl, phenol and carboxyl groups are abundant, and that the tasteless fraction also contains many amino groups. ^{2900cm -1, 1720cm -1, 1600-1660cm -1} , 1420cm -1 and a strong absorption in the visible, near 1240cm ^-1 was reported in other literature. In all spectra, wide bands were observed at 3300-3350 cm ⁻¹ , which is related to the OH groups in the carboxyl groups. The presence of carboxyl functional groups was confirmed by strong absorption at 1650-1720 cm ⁻¹ of COOH group. This indicates that these bands are present at shorter wavelengths (1720 cm ⁻¹ ) in the samples obtained by 100% ethanol extraction, which may correspond to the content of non-ionized COOH groups in the samples.

Spectra of humin and raw tasteless ethanol extract showed 730 and -1570 cm ^-1 additional bands. They are due to the vibration of the polymethylene chain and / or NH bonds. In the spectrum of ethanol extract and HA, the absorption band of 2950 cm ⁻¹ portion is also characteristic and corresponds to the stretching vibration of CH bonds in hydrocarbons. The intensity of absorption increased in the order of HA <HymA <raw tasteless ethanol extract <Humin ethanol extract. This shows that the band at 1090 cm ⁻¹ corresponding to the CO stretching of the polysaccharide in the spectrum of FA _total was better than in the other fractions. The relative decrease in intensity of the peaks at the relatively lipidic portions of 2930 cm ^-1 and 2850 cm ^-1 was evident in the IR spectrum of the FA _total fraction.

Therefore, the crude taste and its ethanol extract, and its HA fraction, in its humins, can be seen that the hydrocarbon chain contains molecules with very long fragments. Relatively low molecular weight compounds previously identified in tastelessness will also be present in the extract. Humic materials consist of the backbone of alkyl / aromatic units, which are mainly cross-linked by oxygen and nitrogen groups, with carboxylic acid, phenol and alcoholic hydroxyl groups as the main functional groups. hydroxyls), ketone groups, and quinone groups. According to the results of the present invention, in the FA _total spectrum, there was no peak characteristic of molecules with long hydrocarbon fragments (see FIG. 3).

In the IR spectrum of the people - Whew ethanol extract, absorption band at 2850cm ^-1 were more strongly than the band of the OH stretching vibration in the vicinity of 3400cm ^-1. This proves to be a smaller fraction of the groups (OH, COOH) with polar oxygen in this sample. And obviously this is associated with low solubility in water and basic medium at about pH 7. Examining other IR spectra from this point of view, it can be seen that the samples of HymA contain very few hydroxyl and carboxyl groups, and the hydrocarbon fragments are increased. This observation shows that HymA is rarely soluble in water and much more soluble in organic solvents such as ethanol. Peaks with low intensity at 2950 cm ⁻¹ are also observed in the IR spectrum of HA, indicating that there is some amount of hydrocarbon chain in the sample.

Quantitative difference analysis of the groups containing oxygen in the composition of HA and FA _total showed that OH, COOH, C═O and methoxy groups were present in FA _total in much larger amounts than in HA.

In general, the relative content of polar and nonpolar hydrocarbon fragments in the tasteless fractions is determined by the IR spectrum, but is highly related to their solubility in solution medium. The decrease in the band strength near 2900 cm ^-1 was due to CH-bonded stretching vibration, and the solubility in solution medium increased in the order of Humin>HymA>HA> FA.

Example 5. Spectroscopic Analysis and HIX using Fluorescence Microscope

Fluorescence measurements were performed using an F-2000 spectrofluorimeter (F-2000 spectrofluorimeter, Hitachi, Tokyo, Japan).

Raw tasteless, FA fractions (FA _total and FA1 to FA5) and LMW fractions (LMW1 and LMW2) were used after adjusting the pH to neutral before analysis. HymA and HA were dissolved in 0.1 M sodium hydroxide solution. For emission fluorescence, samples were excited at a wavelength of 254 nm and their fluorescence intensity was measured in the range of 300 to 500 nm. The slit area for the emission and excitation wavelengths was 10 nm. Co-spectrum was measured by using an excitation monochromator with a wavelength difference (Δλ) of 20 nm and scanning from 250 nm to 600 nm.

Fluorescence spectroscopy can be used to determine the degree of humicization by quantitatively measuring the shift in emission spectrum towards longer wavelengths as the degree of humicization increases. Previous studies have suggested that the relationship between primary and secondary fluorescence inner-filtration effects is important for the accurate presentation of fluorescence data and for the calculation of the humification index (HIX).

HIX values were determined using Equation 1 below.

In Equation 1, I (λ) is a function representing the fluorescence spectrum curve drawn while exciting when the excitation wavelength (λ _ex ) is 254 nm.

In the calculation of the HIX figure modified for the in-filtration effect, the inventors extrapolated linearly for a plot of the transmittance at 254 nm vs. the humic index of the solution with different concentrations of fractions. extrapolation was performed. Modified HIX values correspond to infinite dilution, for example 100% permeability.

All emission fluorescence spectra of FA fractions from tasteless and high molecular weight fractions of HA showed approximately the same morphology (see FIG. 4A). It was also similar to the spectrum of humic materials obtained from other natural materials that exhibited the highest intensity at 420-460 nm.

The characteristic of the emission spectrum was to show a typical wide band with a maximum at the shorter 430 nm wavelength in raw tasteless, LMW1 and LMW2 than in the HA and FA fractions (FA _total , FA1 to FA5) (460 nm). The maximum value at raw λ _em = 430 nm of raw taste can be explained by the high content of low molecular weight fractions in this sample (-31.8%). We used co-fluorescence analysis. As shown in Figures 4b and 4c, the simultaneous spectra of the FA1 to FA5 fractions show distinct spectral form. Significant differences were observed between the spectra of FA2 and FA1, FA3 to 5. The FA2 fraction showed a spectrum with a single peak at 415 nm. Spectra with major peaks were seen at FA1 and FA3 to FA5 450 nm and 495 nm. In addition, 370nm (FA1), 380nm (FA5), 395nm (FA1, FA3, FA5), 415nm, 425nm, 435nm (FA1, FA4, FA5), 480nm (FA1, FA3-5), 520nm (FA1, FA3-5) Showed many different peaks. In general, the FA2 spectra showed shorter peak wavelengths than the FA1 and FA3-5 spectra, and FA1 and FA3-5 were found to contain large amounts of conjugated aromatic groups. The LMW2 fraction showed two major peaks at 275 and 345 nm, whereas LMW1 had only one peak at 270 nm in the co-fluorescence spectrum. Three peaks were observed in the raw taste. One was the peak seen at 270 nm and the other was the broad maximum peak seen at 355-385 and 305-415 nm. Small peaks within 425-520 nm were located at the wavelength as maximum in the spectra of FA fractions. The peak at 270 nm of raw, tasteless, LMWl and LMW2 samples can be referred to as benzoic acid with similar spectral characteristics (see Figure 4b). It is believed that this is due to the benzoic acid derivatives being distributed in some proportion between the LMWl and LMW2 fractions due to the different solubility in the water-ethanol mixture used in the fractionation.

For the calculation of modified HIX values, we used the linear relationship of the transmittances at HIX and 254 nm. Intercepts in the relationship provide modified HIX values for internal filtering effects. The range of HIX values for each fraction dissolved in water (pH = 7.0) ranged from FA3 (11.1)> FA4 (4.26)> FA2 (3.33)> FA5 (3.10)> raw taste (3.13)> FA1 (2.23 )> LMW2 (0.95)> LMW1 (-0.08). The range of deviations in HIX values slightly exceeds the deviations of values (1.23 to 7.65) for samples obtained from soils with different degrees of humicization. Minimal HIX levels were seen in fractions of LMWl and LMW2.

The analysis results of the tasteless fractions of Examples 3 to 5 are summarized in Table 1 below.

Carbohydrate content UV / VIS data (λ _max , nm) FT IR data (cm ^-1 ) Fluorescence data (λ _em , nm) Raw taste 270, 370, 410 FA _total 3400, 1450, 1090 FA1 2.3 402, 665 3400, 2900, 1720 1600, 1450, 1350 370, 395, 435, 480 FA2 1.8 402, 665 3400, 2900, 1720 1600, 1350 415 FA3 0.6 3400 395, 480 FA4 0.6 3400 435, 480 FA5 0.5 3400 380, 395, 435, 480 LMW1 280 3400 270 LMW2 0.3 270 3400 275, 345

Example 6 HPSEC Analysis

Samples were analyzed using a 1100 high performance liquid chromatography system (Helettett-Packard, Palo Alto, Calif.) Equipped with four pumps and UV-detectors of various wavelengths. Zobox PSM 300 columns (Zorbax PSM 300 column, Hewelett-Packard, Calif.) And detection wavelengths of 254, 278, and 340 nm were used for HPSEC analysis. The column was 25 cm in length and 6.2 cm in inner diameter. As a mobile phase a solution having a composition of 0.1M NaCl, 0.02MK ₂ HPO ₄ and 0.02M KH ₂ PO ₄ (pH 6.8) was used. The solvent and all samples were filtered using a 0.2 μm membrane filter. The flow rate was 1.0 ml / min and the sample injection volume used was 20 μl.

In HPSEC analysis, FA _total and FA1 to FA5 fractions were used after 10 kDa cut-off dialysis. Other samples (raw tasteless, LMW1, LMW2, Aldrich HA) were used without dialysis. For screening of fractional eluate, a UV detector was used at 254 nm. In addition, 278 nm wavelength was used to detect benzoic acid and / or derivatives thereof, and absorbance was measured at 340 nm to characterize the FA (see FIG. 2A).

The crude tasteless chromatogram showed three major peaks at 254 nm UV detector with retention times (R _t ) of 5.01, 5.26 and 5.60 minutes. When detected at 340 nm wavelength, the crude tasteless chromatogram showed only one peak at R _t = 4.96 min (see FIG. 5), which is clearly the result by FA. All FA fractions detected at 254 nm eluted from the HPSEC column showed a broad, single-form distribution with fine shoulders in the chromatogram. Similar chromatogram structures also appeared in FA isolated from other natural sources, due to the polydispersion of these materials. The R _t indices for the FA1 to FA5 fractions isolated from tasteless were very similar to each other (4.86, 5.00, 4.83, 4.78 and 4.90 min respectively) and the FA _{total was} also similar to R _t = 4.78. The HPSEC chromatogram (254 nm) of Altrichy's HA showed only one peak, R _t = 4.91, similar to the R _t value of the tasteless FA fractions.

According to literature data, the molecular weights of most humic materials isolated from rivers or soils were measured between 2000 and 15000 Daltons. The average molecular weight of HA of Aldrich is from 132000 Daltons to 15000 Daltons. The average molecular weight of the humic materials isolated from the tastelessness of the present invention has not yet been determined. FAs present in the tasteless will have higher molecular weights than those isolated from soil or natural reservoirs. In the study of the present invention, the retention time of the FA fractions (R _t = 4.78-5.00 minutes) was very similar to that of Aldrich's HA (R _t = 4.91 minutes). From this it can be estimated that the average molecular weight of FA fractions from tastelessness, obtained with 10 kDa cut-off dialysis, will be in the range of 10000 to 15000 Daltons.

At 254 nm, the chromatogram of LMW1 had two large peaks (R _t = 5.30 and 5.61 min), showing a wide shoulder in the region of smaller molecular weight material (see FIG. 5). When searched at 278 nm, the chromatogram of LMW1 had one peak (R _t = 5.28 min), and the R _t at the peak of benzoic acid also coincided with 5.28 min. The results of HPSEC analysis of LMW1 are consistent with UV and fluorescence spectroscopy data (FIGS. 2A, 4B) and confirm that benzoic acid and / or its derivatives are a major component of this fraction.

The first and second peak R _t values of LMW2 were 5.32 and 5.58 minutes (see FIG. 5). These peaks are each due to the extraction of different heterocyclic compounds, long chain lipohydrocarbons and their derivatives.

The molecular weight and retention time in HPSEC of the tasteless fractions of Example 6 are summarized in Table 2.

Molecular weight distribution Retention time (R _t , min) Raw tasteless 254nm 5.01, 5.26, 5.60 278 nm - 340 nm 4.96 FAtotal 10000-15000 254nm 4.78 278 nm - 340 nm - FA1 10000-15000 254nm 4.86 278 nm - 340 nm - FA2 10000-15000 254nm 5.00 278 nm - 340 nm - FA3 10000-15000 254nm 4.83 278 nm - 340 nm - FA4 254nm 4.78 278 nm - 340 nm - FA5 254nm 4.90 278 nm - 340 nm - LMW1 254nm 5.30, 5.61 278 nm 2.58 340 nm - LMW2 254nm 5.32, 5.58 278 nm - 340 nm - HA (Aldrich) 254nm 4.91 278 nm - 340 nm -

Example 7. Total Carbohydrate Content

In order to analyze the total carbohydrate content in the raw and tasteless and tasteless fractions, it was measured using the phenol reaction method (Korean Society for Biochemistry, Rev. 3, Experimental Biochemistry, Cheongdang, 1997, pp249-259).

The total carbohydrate content was mixed by adding 0.5 ml of 50 g / l phenol solution to 0.5 ml of sample (dissolved 4 mg of no-fraction fraction in 1 ml of distilled water), followed by addition of 2.5 ml of concentrated sulfuric acid, and quickly mixed again. The solution is reacted at 27 ° C. for 20 minutes. Its absorbance was then measured at 488 nm and compared with the absorbance of a glucose standard curve previously prepared using a glucose standard solution. Carbohydrate content analysis was performed three times, the results are expressed in mg carbohydrate / g.

78-80% ethanol-water (v / v) mixture was used to separate monosaccharides and oligosaccharides from polysaccharides. The concentration of ethanol can extract low molecular weight carbohydrates dropping polysaccharides from the residue. The carbohydrates of the FA1 to FA5 fractions were therefore polysaccharides or part of the carbohydrates in the FA composition, while the LMW1 and LMW2 fractions were mainly monosaccharides or oligosaccharides. The highest carbohydrate peaks were observed for FA1 and FA2 and were 2.3 mg / g and 1.8 mg / g dry weight, respectively. The carbohydrate content was much lower in FA3, FA4 and FA5 separated by ion exchange chromatography, and was 0.8, 0.6 and 0.5 mg / g dry weight, respectively. It is believed that much of the polysaccharide from the FA _total did not adsorb to DEAE-cellulose or did not elute continuously from the column. In low molecular weight fractions, carbohydrate content analysis showed that there was little monosaccharide and oligosaccharide in LMW2 (0.29 mg / g dry weight) and not even in LMW1. The low carbohydrate content in raw tasteless (0.4 mg / g dry weight) is due to the large fraction of the LMW1 and LMW2 fractions.

Example 8. Isolation of Peritoneal Macrophages

Peritoneal macrophages were isolated 4 days after 8 weeks old male BALB / C mice were injected into the abdominal cavity with saline containing 100 μg Concanavalin A.

Cells exuded in the abdominal cavity were obtained by lavage with 10 ml of cold RPMI 1640 medium added 10 units of heparin / ml. Cells exuded intraperitoneally were then placed in RPMI 1640 medium containing antibiotics (penicillin and streptomycin) and 10% fetal calf serum (FCS), and about 10 ⁶ cells were placed in each well of a 24-well culture plate. After 2 hours, cells not attached to the plate were washed out with RPMI medium without phenol red, and the remaining experiments were carried out by treating the remaining peritoneal macrophages with tasteless fractions at various concentrations.

Example 9 Statistical Analysis

The results were all expressed as mean ± standard deviation, and statistical comparisons were performed by Student's t-test on each pair of values.

Experimental Example 1. ROS formation analysis

ROS formation was analyzed by fluorescence search using 2 ', 7'-dichlorofluorescin diacetate (DCFH-DA). When it enters the cell, it is deesterified to become ionized free acid, or DCFH, which reacts with ROS to form a fluorescent 2'7'-dichlorofluorescein (DCF).

To generate DCFH, 5 mM DCFH-DA solution was mixed with 2.5 mM NaOH and reacted at room temperature without light for 1 hour for deacetylation. Hydrogen peroxide (H ₂ O ₂ ) was produced by the enzymatic reaction of glucose oxidative conversion glucose oxidase in the presence of enzymes in solution. β-D-glucose is oxidized by glucose oxidase to produce D-glucoic acid and hydrogen peroxide.

Assays were performed in 24-well plates and the reaction was started by adding 20 μl glucose oxidase stock. At the end of the reaction, a mixture of 30 μM DCFH (pH 6.5) dissolved in 5.0 mM glucose, 0.2 U / mL glucose oxidase, 0.015 U / mL Hoolradish peroxidase and 10 mM sodium phosphate buffer was added. DCFH oxidation was measured for fluorescence absorbance using a 1420 Wallac (Perkin Elmer, UK) Victor 2 multilabel plate reader temperature control (30 ° C.).

The corresponding scheme is shown below.

In summary, the cells were cultured at 5% CO ₂ , 37 ° C. for 90 minutes or 48 hours after the addition of tasteless fractions, and PMA (phorbol-12-myristate-13-acetate, 1 or 5 μM in the presence of DCFH-DA (3 μM). Activated or not). ROS production was measured as an increase in fluorescence per well (λ _ex = 485 nm, λ _em = 535 nm). Fluorescence was measured at intervals of 3 to 4 minutes, until 90 minutes, with a 1420 Wallac Victor 2 multilabel plate reader (Perkin Elmer, UK) with a 37 ° C. temperature control. .

Activity of peritoneal macrophages of FA in the pre-study of the present invention, a long period of time (24-48 hours) were observed during the culturing FA and _total cells with a short period of time (1-2 hours) of culturing the cells for a _total FA while Only the initial effect on the response of PMA (phorbol-12-myristate-13-acetate) was observed. As the time the cells were exposed to FA _total (0-5 hours), the rate of ROS production by macrophages responding to PMA increased (data not shown). Incubation of FA _total with cells for 90 minutes and then removal from the medium had no significant effect on natural and PMA stimulated ROS production by macrophages. The maximum rate of DCFH oxidation by the cells was observed in response to PMA in the presence of FA _total (see FIG. 6A). These results demonstrate that the presence of FA in cell culture media enhances PMA-induced ROS production.

To analyze the effect of molecular weight and ionization of FA on ROS, cells were pretreated with FA1 to FA5 fractions for 90 minutes and PMA-stimulated ROS production was then measured for 90 minutes. These results showed that the FA1 and FA2 fractions isolated by ethanol precipitation caused significant elevation of ROS in macrophages (see FIG. 6B). Among the FA3 to FA5 fractions separated by ion exchange chromatography, only FA3 had biological activity against macrophages, FA4 had increased ionization, high adsorption capacity for DEAE-cellulose, but high FA4 concentration (70 µg / ml). Rather it shows inhibitory activity.

To investigate the effect of tasteless fractions on natural ROS production by macrophages, cells were cultured by treating FA _total , LMW1 and LMW2 fractions at various concentrations for 48 hours. Cell culture medium was replaced with fresh one containing DCFH-DA, and fluorescence intensity was then measured for 90 minutes (see FIG. 7). Significant activation of ROS production was observed only when cells were treated with FA _total at a concentration of 20-200 μg / ml (p <0.05). Cells treated with LMW1 at a concentration of 20-200 μg / ml showed no significant change in ROS production compared to the control. In contrast, the addition of LMW2 showed a decrease in ROS production. The inhibitory effect of LMW2 is related to the cytotoxic activity of low molecular weight substances in the composition of these fractions.

Experimental Example 2. Measurement of nitric acid (NO) production

After separating the peritoneal macrophages as in the method of Example 5, the cells were administered one of the tasteless fractions or lipopolysaccharide (lipopolysaccharide, LPS, 0.5 μg / ml) at 37 ° C. and 5% CO ₂ conditions. Incubated for 48 hours. After 48 hours of incubation, the supernatant was separated to analyze the amount of NO. The concentration of nitrite ions (NO ₂ ⁻ ) was used as a marker to indicate that NO was produced. The amount of nitrite ion in the culture medium was determined by measuring the degree of color development using NaNO ₂ as a reference.

In summary, the same amount of grease reagent (Griess reagent, 0.1% (w / v) N- (1-naphthyl) ethylenediamine dihydrochloride + 1% (w / v) sulfanilamide in 5% (v / v) phosphoric acid) was added and mixed, and then allowed to stand at room temperature for 20 minutes. Then, the absorbance was measured at 540 nm using Ultrospec 4000 spectrophotometer (Pharmacia Biotech, Uppsala, Sweden).

Macrophages were incubated with tasteless fractions for 48 hours, and the NO concentrations in culture supernatants were measured by grease response, and FA1 and FA2 dose-dependently increased NO synthesis of peritoneal macrophages. The FA1 and FA2 fractions had significant effects on NO production, respectively, when at least 2 μg / ml and at least 20 μg / ml. And, the activation effect at the concentration of 200 μg / ml of the FA1 and FA2 fractions was similar to that of 0.5 μg / ml LPS. LMW1 and LMW2 fractions did not activate NO production (see FIG. 8).

Experimental Example 3 Splenocytes Proliferation

Splenocytes of BALB / c mice were isolated and suspended at 5 × 10 ⁶ / ml in RPMI 1640 medium added with 10% FCS, antibiotics and 0.05 mM β-mercaptoethanol. Samples (dissolved in phosphate buffer (pH 7.2) containing 0.15 mM NaCl) were added to a 96-well plate at a final concentration of 0 (control), 2, 20 or 100 μg / ml. 0.1 ml (5 × 10 ⁵ cells) of cell suspension and 0.1 ml of medium were then added to the wells. A sample containing 5 μg / ml concanavalin A was added as a positive control without adding a fraction. Plates were incubated for 72 hours at 37 ° C., 5% CO _2, 100% humidity.

Again [ ³ H] thymidine (1 μCi) was added to each well, and the mixture was further incubated for an additional 18 hours. After incubation, the cells were collected on a glass fiber mat, then uninserted thymidine was washed off and then irradiated with liquid scintillation spectroscopy. This proliferation experiment was repeated three times and presented representative result data.

By measuring the insertion of [ ³ H] thymidine into the DNA of lymphocytes treated with tasteless fractions, we found a dose dependent increase in [ ³ H] thymidine in FA1 treated cells. This showed statistically significant at concentrations higher than 20 μg / ml compared to the control (p <0.05) (see FIG. 9). Incorporation of [ ³ H] thymidine at a FA1 concentration of 500 μg / ml was about 40% of that caused by Concanavalin A stimulation. FA2, LMW1 and LMW2 fractions had no effect on splenocyte growth.

Russian tasteless extracts or fractions of the present invention enhance the production of reactive oxygen species and NO in macrophages, and promote the proliferation of splenocytes, and thus, pharmaceuticals and health functional foods for immune suppression and regulation of immunity in patients with compromised immune systems. It can be used as a medicine for the prevention and treatment of various diseases caused by infection or inflammation.

Claims (16)

A first step of grinding or crushing the taste-free material with various grinding machines, stirring the basic solution at room temperature for 12 to 48 hours, and obtaining a crude extract as a supernatant through centrifugation; After adding strong acid to the crude extract, centrifugation was performed to obtain supernatant fulvic acid (FA) and humic acid (HA), and then the strong base was added to the supernatant which is a fulvic acid solution to adjust the pH to 6.5 to 8.0. A second step of filtration with a 0.2 to 0.5 μm filter to obtain a FA _total solution; Ethanol was added to the final _total FA solution obtained in the second step to prepare a final concentration of 60 to 70%, followed by centrifugation to obtain supernatant and precipitate 1, and ethanol was added to the supernatant again to final concentration 80. To 90% and then centrifuged to obtain supernatant and precipitate 2, followed by dialysis of the precipitates 1 and 2 to obtain FA1 and FA2 fractions; The FA solution obtained by dialysis of the final _total FA solution obtained in the second step was passed through a DEAE-cellulose column, and eluted stepwise using 0.3 to 2.0 moles of sodium chloride solution to obtain the FA3, FA4 and FA5 fractions. A fourth step of obtaining; Then, the fifth step of obtaining the LMW1 and LMW2 fraction obtained by concentrating the fraction available in the 85% ethanol of the third step to add the same amount of ethanol to the solution saturated with low molecular weight material and then reprecipitate Method of producing a composition for enhancing immune activity from Russian tasteless taste. Obtained by the method of claim 1, the molecular weight is 10,000 to 15,000, UV / VIS shows a maximum absorption wavelength at 402nm and 665nm, IR in the absorption band at 3400, 1450 and 1090cm ^-1 Flavor Extract FA _total. A molecular weight ranging from 10,000 to 15,000, a carbohydrate content of 2.3% by weight, the highest absorption wavelength at 402 and 665 nm in UV / VIS, 3400, 2900, 1720, 1600, 1450 and Russian tasteless fraction FA1 which shows absorption bands at 1350 cm ⁻¹ and fluorescence emission wavelengths at 370, 395, 435 and 480 nm. Obtained by the process of claim 1, the molecular weight is 10,000 to 15,000, the carbohydrate content is 1.8% by weight and the highest absorption wavelength at 402 and 665 nm in UV / VIS, 3400, 2900, 1720, 1600 and 1350 cm ^{-1 in} IR In the tasteless emission wavelength at 415 nm Russian tasteless fraction FA2. Obtained by the process of claim 1, the molecular weight is 10,000 to 15,000, the carbohydrate content is 0.6% by weight, the IR shows absorption bands at 3400cm ^-1 , the fluorescence emission wavelength is characterized by 395 and 480nm Tasteless fraction FA3. The composition for immuno-enhancement containing the FA _total Russian extract of claim 2 as an active ingredient. The composition for immuno-enhancing containing at least one fraction selected from the fractions FA1, FA2 and FA3 of the Russian tasteless rice according to claim 3 as an active ingredient. delete delete delete Infectious diseases of the eyes, hemorrhagic eye infections, esophagitis, esophageal cancer, bronchitis, atopic dermatitis, pressure sores, skin ulcers, containing FA _total Russian extract of claim 2 as an active ingredient and a pharmaceutically acceptable carrier or excipient Composition for the prevention and treatment of dermatitis diseases. Infectious disease of the eye, hemorrhagic eye infection containing at least one fraction selected from FA1, FA2 and FA3, the fraction of the Russian tasteless rice according to claims 3 to 5, as an active ingredient and containing a pharmaceutically acceptable carrier or excipient Esophagitis, esophageal cancer, bronchitis, atopic dermatitis, pressure sores, skin ulcers or dermatitis for the prevention and treatment of diseases. delete The formulation of claim 6, 7, 11, or 12, wherein the formulation is a powder, granule, tablet, capsule, suspension, emulsion, syrup, oral dosage form of aerosol, external preparation, suppository, or sterile injectable solution. A composition, characterized in that it is formulated in the form of. delete delete

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