KR101344244B1 - Method for producing cold water-soluble Gastrodia elata extract and pharmaceutical composition for anti-cancer comprising the extract - Google Patents

Abstract

The present invention is a technique that can dissolve the useful components in cold water by treating the cheonma by high temperature and high pressure conditions (120 ~ 180 ℃, 20 ~ 100 bar) by an extrusion molding method that induces cell tissue collapse and melting.
The current industrial cheonma extraction method has disadvantages such as high energy consumption, long extraction time, excessive labor, and low palatability of cheonma processed products. Therefore, when manufacturing the cheonma processed products by the technology of the present invention can reduce the production cost, simplify the process and produce high-quality cheonma products.

Description

Method for producing cold water-soluble cheonma extract and pharmaceutical composition for anti-cancer containing the extract {Method for producing cold water-soluble Gastrodia elata extract and pharmaceutical composition for anti-cancer comprising the extract}

The present invention relates to increasing the yield of biopolymers through the extrusion molding process technology, improve the flavor of the cheonma itself during the processing of high temperature, high pressure, and to improve the existing energy, labor-intensive extraction method.

Gastrodia elata Blume ) refers to a tuber of perennial herbaceous plants belonging to the Orchidaceae native to Korea, China, Japan, etc. It is known as a medicinal herb. Clinical efficacy of Chunma is found in various primary literature such as primary herbal medicine, Dongbokgam, and it is reported to be effective for hypertension, paralysis, headache, paralysis, neurological diseases, diabetes, epilepsy and anemia. In the private sector in Korea, chimpanzees have been used to treat headaches, anemia, limb paralysis, and stroke.

In order to utilize this effect, the existing hot water extraction system requires a lot of energy in the process and requires several processes, resulting in a low efficiency. Therefore, as a physical process to replace it, The development of biopolymer growth techniques and processes are required.

Extraction of acidic polysaccharide and biopolymer can be greatly enhanced by inducing cell wall hydrophilization by high temperature and high pressure extrusion molding process. In addition, starch molecules can be made low molecular weight and inhibit precipitation during concentration / cooling / storage of extract It is also possible to expect the production of new pharmacologically active substances by controlling the residence time at high temperature and high pressure conditions and also to increase the solubility of the polysaccharide due to cell wall depolarization and to produce a new physiological functionalized extract .

 The hot water extraction process, which currently takes up a large portion of the processing method using cheonma, is immersed in an extraction tank for 24 hours, extracted at a high temperature of more than 95 ℃ for 24 to 48 hours, and then subjected to precipitation, etc. Separation of residues and extracts after extraction is not easy, and starch is eluted together during the extraction process. Aging occurs during cooling / concentration, resulting in precipitates, impairing product quality, and excessive energy and manpower have been put into the process. .

Therefore, the inventors have improved the manufacturing process of simple heating extraction of cheonma, a natural product that has been used as a folk remedy for a long time. By applying an extrusion process with the advantages that can be performed to increase the extraction yield of the acidic polysaccharides, it was developed an extraction method to reduce the elution of starch that inhibits the product quality of the solid content extracted through cold water extraction.

Korean Patent Laid-Open Publication No. 2009-0032753 discloses a method for improving the preference of cheonma extract, which comprises a step of roasting cheonma, extracting the roasted cheonma with hot water, and concentrating the extract to obtain a concentrate of cheonma extract. The manufacturing method is disclosed, and Korean Registered Patent No. 0710707 discloses a method for increasing the effective ingredient extraction of cheonma which can extract a large amount of active ingredient possessed by cheonma under optimum steaming conditions, drying conditions, and extraction conditions. , Korean Registered Patent No. 0526329 discloses a method for preparing cheonma extract that can reduce the negative taste and aroma of cheonma by preparing hot water extract after roasting cheonma, but cold water after extruding cheonma as in the present invention The manufacturing method of the cheon-ma extract including the extraction process is not disclosed at all.

The present invention has been improved to solve the problems as described above, the comparative analysis of the extract produced by the conventional hot water extraction process and the extract produced by the cold water extraction process of the raw material subjected to the extrusion molding preliminary analysis of the index material and acidic In order to increase the extraction yield of polysaccharides, it is to investigate the production conditions of the cheonma extrudate according to the extrusion molding process variables such as extrusion molding temperature and pressure. In addition, through the extrusion molding process to improve the flavor by removing the unique flavor material and the formation of a new fragrance material by the browning reaction, and to establish the optimal extraction conditions of acidic polysaccharides as bioactive substances to produce useful ingredient extract from Chunma To develop a technology that can be carried out in a low-energy economic process.

In order to solve the above problems, the present invention is the pre-treatment of the raw material using an extrusion molding process of cold water soluble cheonma extract showing a similar extraction yield or more extraction yield of biopolymer materials such as acidic polysaccharides compared to hot water extraction even with short time cold water extraction It provides a manufacturing method.

In addition, the present invention provides a cold water-soluble cheonma extract with increased uronic acid content and reduced extraction residue compared to the hot water extraction prepared by the above method.

The present invention also provides a pharmaceutical composition for preventing or treating cancer containing the cold water-soluble cheonma extract as an active ingredient.

According to the present invention, by establishing a new raw material pretreatment technique, the flavor of cheon-ma is improved in the environment of high temperature and high pressure during the pretreatment process. By increasing the yield of biopolymers, which are active substances, energy, manpower, time-intensive production, and carbon generation, which are disadvantages of the existing hydrothermal extraction process, can be reduced.

1 is a process flow chart showing a step of pretreating and extracting a raw material using an extrusion process.
Fig. 2 is a photograph showing the characteristics of the gel-cast extrudate of the present invention.
FIG. 3 is a graph comparing the specific gravity of the residue after extraction and the specific gravity of the solid in the extract by adding 15-20 times the weight of water to the raw material pretreated by the pretreatment and the pretreatment with the untreated water and stirring at room temperature for 6 hours.
Figure 4 is a graph comparing the specific gravity of the residue and the solids in the extract after the extraction of the sample extracted by adding 15 to 20 times the weight of water to the raw material pre-treated by the pre-treatment and extrusion process and heated and stirred at 90 ℃ for 6 hours to be.
5 is added to the extract of the sample extracted by stirring at room temperature for the same time as the sample extracted by adding 15 to 20 times the weight of water to the raw material pre-treated by the pre-treatment and extrusion process and preheated at 90 ° C. for 6 hours. It is a graph comparing the polysaccharide content contained.
6 is added to the extract of the sample extracted by stirring at room temperature for the same time as the sample extracted by heating and stirring at 90 ℃ for 6 hours each weight of 15 ~ 20 times the weight of the raw material pre-treated by the pre-treatment and extrusion process It is a graph comparing total carbohydrate content.
7 is added to the extract of the sample extracted by stirring at room temperature for the same time as the sample extracted by heating and stirring at 90 ℃ for 6 hours each weight of the raw material pre-treated by pre-treatment and raw material pre-treated by extrusion process It is a graph comparing the content of uronic acid contained.
FIG. 8 is a graph comparing the solid content of the raw material with the extraction time according to the raw material after the pretreatment with the raw material.
9 is a graph comparing the total carbohydrate content with the extraction time of the raw materials after the pretreatment with the raw materials of the chewing gum.
10 is a graph comparing the content of uronic acid with the extraction time of the raw materials after the pretreatment with the raw materials of the chewing gum.
Fig. 11 shows the chromatogram of the aroma component of the extruded chymotrypsin.
Figure 12 shows a process for preparing crude cell wall extracts from a chimpanzee and an extruded chymase.
FIG. 13 is a graph showing the proliferation inducing activity of the chimpanzee extract of the present invention against spleen lymphocytes. The splenocytes in culture were treated with GEAP (1-10) and Concanavalin A (ConA) at 2 concentrations (20, 100 ug / ml or 1, 2.5 ug / ml) respectively, followed by 37 ° C., 5% CO for 48 hours. Incubated under ₂ conditions. Splenocyte proliferation was measured with the EZ-Cytox cell viability assay kit. Data is mean ± standard deviation of experiments performed in three replicates. Compared with the control group, * p <0.05, ** p <0.01, *** p <0.001.
FIG. 14 is a graph showing the ability of the extract of Chimpanzee extract of the present invention to kill cancer cells against a colon cancer cell line. FIG. HT-29 (1 × 10 ⁵ cells / well) cells were treated with three concentrations of GEAP or 5-fluorouracil (5-FU) and incubated for 24 hours (A) or 48 hours (B), followed by MTT assay. (#: P <0.05, &: p <0.01, $: p <0.001 vs control)
FIG. 15 shows the results of observing whether cancer cell apoptosis induction was induced in the colon cancer cell line of the present invention. The panel was developed using GEAP-4 (C), GEAP-5 (D), GEAP-6 (E), GEAP-8 (F), GEAP- 6-diamidino-2-phenylindole (DAPI) staining of HT-29 cells after treatment with 5-FU (H) and 5-FU (B) for 24 hours. Compared with control (A), the red arrows indicate apoptotic bodies with typical condensed chromatin of apoptotic cells.

In order to achieve the object of the present invention,

Extruding the cheon-ma powder at a temperature of 120-180 ° C. and a pressure of 20-100 bar; And

It provides a method of producing a cold water soluble cheonma extract comprising the step of extracting the extruded cheonma by adding water 15 to 20 times the weight of the extruded cheonma by stirring for 3 to 7 hours at room temperature.

The preparation method of the cold water soluble cheonma extract of the present invention may be made of a cheonma powder extrusion molding process (first step) and a hydrolysis extraction process (second step).

The first step is a step of extruding the cheonma powder using an extrusion molding machine, specifically, the process of extrusion molding the cheonma powder at a temperature of 120 ~ 180 ℃ and a pressure of 20 ~ 100 bar.

The extrusion molding machine is able to produce various functions and products. The most important thing is to push the plastic material forward by rotating the screw in the barrel, so that the pushing force generates a high rotational force inside the discharge port, Extrusion molding machine. The unit processes that can be performed in the extrusion molding machine include heat treatment, mixing, separation, compression, arrangement, expansion and molding. Physicochemical changes that can occur in the food material through these processes are as follows. First, Swelling, swelling, degradation, protein denaturation, intermolecular binding and organization, inactivation of enzymes, fourth, microbial death and disinfection, fifth, destruction of toxic substances, sixth, removal of odor, seventh, tissue swelling And density control, eighth, browning reaction, ninth, cell wall destruction, and the like.

The raw materials that can be processed using such an extrusion molding machine can be used for almost all kinds of food materials. For example, the present invention can be applied to cereals, pulses, milking pans, meat, fish and the like to develop new types of products. , Instant grain products, confectionery, bread, protein, vegetable protein raw materials, soup or porridge, and livestock feed.

 In this way, it is possible to obtain a great economic benefit by appropriately controlling and controlling various reactions occurring in a single process, thereby developing products capable of satisfying various needs of consumers in the present food industry and shortening or replacing existing product production processes It is considered to be one of the important processes that are widely used.

In addition, by using the extraction method using the extrusion process, it is possible to improve the extraction yield of biomolecules, which are biodegradable polymers having physiological activity in the human body, as well as to increase the preference of the product and reduce the energy consumed in the existing hot water extraction method, Simplification can reduce manpower and can reduce carbon emissions by eliminating the long-time heat extraction process. The present invention enhances the extraction of acidic polysaccharides and improves the process by the pretreatment of raw materials utilizing this extrusion process.

The second step is a hydrolysis step, in which the pretreated raw material of chondroitin is placed in an extraction tank, and about 15 to 20 times the weight of the raw material is added and stirred for 3 to 7 hours at room temperature. The stirring extraction time may be 3 to 7 hours, preferably 4 to 6 hours, and most preferably 4 hours, but is not limited thereto. The room temperature may be 18 to 25 ° C, preferably 20 ° C, but is not limited thereto.

The present invention also provides a cold water soluble cheonma extract prepared by the above method. In the case of the extract of the present invention, the uronic acid content, which can be inferred from the acidic polysaccharide content, was found to be higher than 5.20% when the hot water was extracted from 6.32% to 7.54%. The hot water extract without hot water treatment showed 19.36%, but the residue was extracted from cold water after the extrusion treatment from 17.15% to 15.32%, which was lower than the hot water extraction.

In addition, the cheonma extract of the present invention can be applied as a variety of food materials using cheonma by developing a unique odor improvement process. The content of trichloromethane, toluene, cyclopentasiloxane and d-limonene tended to decrease significantly as the mechanical energy consumption rate, which can be determined by extrusion molding, increased. In other words, the sensory properties of the fragrance component after extrusion molding showed improved sensory characteristics, such as trichloromethane, toluene, cyclopentasiloxane, cyclohexanol, and acetic acid, which are expected to adversely affect palatability, and furan, furfural And the release of other ingredients.

The present invention also provides a pharmaceutical composition for preventing or treating cancer, containing the cold water-soluble cheonma extract of the present invention as an active ingredient. According to the process of the present invention, the pretreatment of the cheon-ma and the cold water-extracted cheonma extracts were evaluated for cancer cell inhibition efficacy, and the results were compared to evaluate the cancer cell inhibition efficacy of the fat-soluble substance and the water-soluble substance contained in the cheonma, Polysaccharides contained in Chunma showed higher efficacy than fat-soluble substances. In the pharmaceutical composition of the present invention, the cancer may be colorectal cancer, gastric cancer, breast cancer, lung cancer, liver cancer, kidney cancer, thyroid cancer, and the like, preferably colon cancer, but is not limited thereto.

The pharmaceutical composition comprising the cheonma extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

The pharmaceutical dosage forms of the extract 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 suitable aggregates.

The pharmaceutical composition containing the extract according to the present invention can be administered orally in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions And can be used as formulations. Examples of carriers, excipients and diluents that can be included in the composition containing the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters 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.

Preferred dosages of the extracts of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The extract of the present invention can be administered to mammals such as rats, mice, livestock, humans and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The present invention also provides a health food for preventing or improving cancer containing the cheonma extract of the present invention as an active ingredient.

Examples of foods to which the extract can be added include various foods, beverages, gums, tea, vitamin complexes, and health functional foods.

The amount of the extract in the food or beverage may be added at 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g based on 100 ml.

The health functional beverage composition of the present invention has no particular limitation on the other ingredients other than the above-mentioned extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary 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 polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tautatin, stevia extract, for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above . The proportion of such natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the extract 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 its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the extracts of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components may be used independently or in combination, and the proportion of such additives is not so critical but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

Hereinafter, the present invention will be described with reference to examples. However, the experimental examples according to the present invention may be modified in various other forms, and the scope of the present invention should not be construed as being limited to the examples described below. Embodiments according to the present invention may be various modifications that can be substituted for them at the time of the present application.

 As shown in FIG. 1, the present invention essentially consists of a chun-ma powder extrusion molding process (first step) and a hydrolysis extraction process (second step), and additionally, a physiological activity evaluation step (third step). .

Extrusion pressure, extrusion temperature and mechanical energy consumption rate sample Raw material supply amount
(kg / h) Quantity of water
(kg / h) Screw rotation speed (rpm) Extrusion pressure
(bar) Extrusion temperature
(℃) SME
(KJ / kg) Ext-1 15 3.8 270 87 170 816 Ext-2 15 3.2 270 75 163 1008 Ext-3 15 3.5 270 60 151 1183 Ext-4 15 3.5 270 40 141 1325 Ext-5 15 3.5 270 20 135 1455

The manufacturing process will be described in detail as follows.

1. Extrusion molding process of Gunma powder (first step)

In the process of pretreatment of the sham horse, extrusion treatment was performed by opening a circular mold exit having a diameter of 2 mm by using a biaxial extrusion molding apparatus. In order to induce a strong shearing force, three reverse screws were combined, 15 kg / h) and extruded by varying the amount of water.

Into the extrusion molding machine using the cheonma powder was prepared extruded starch powder according to various extrusion conditions, the properties are as shown in FIG.

2. The hydrolysis extraction process (the second process)

The pretreated raw horse was placed in an extraction tank, and 15-20 times the weight of the raw material was added thereto, followed by stirring extraction at room temperature for 3-7 hours.

Example  1: raw material after raw material and extrusion molding pretreatment Heat number  And cold water extraction comparison

After extraction of the sample extracted by stirring at room temperature for the same time as the sample extracted by heating and stirring at 90 ° C. for 6 hours by adding 15 ~ 20 times of water to the raw material pretreated by the pre-treatment and extrusion process. 3 and 4, the polysaccharide content in the extract is shown in FIG. 5, and the total carbohydrate and uronic acid content are shown in FIGS. 6 and 7, respectively.

FIG. 3 shows that the ppt and the solids content of the ex1 to ex5 pretreated raw materials were about 68% and 30%, respectively, after filtration when the cold water was extracted at room temperature, 25%, and the solids content of the extract was about 71 ~ 79%. As the mechanical energy consumption rate of the extruder increased, the content of residues tended to decrease.

FIG. 4 shows that about 19% of the raw material and 80% of the solid content of the extract, which were heat-extracted from the raw material without heat treatment, were ex1 ~ ex5 after pretreatment, about 15% to 17% ~ 85%, the amount of components extracted in the extract is higher. Compared with FIG. 3, although the difference between the solid content of the raw extract and the cold extraction at room temperature shows a large difference in the solids content of the extract, the difference in the solids content of the heated extract was not significant have.

Fig. 5 shows the crude polysaccharide content of the extract. In the case of cold extraction at room temperature, the polysaccharide of the crude raw extract without the pretreatment contained about 9% of the polysaccharide and about 53% to 62% of the ex1 to ex5 after the pretreatment, The extracted polysaccharide was 58% raw and the ex1 ~ ex5 after pretreatment was about 62% ~ 64%. The extruded raw material showed no significant difference between the polysaccharide of the cold water extract and the polysaccharide of the heat extracted extract.

FIG. 6 shows the total carbohydrate content of the extract. The total carbohydrate content of the extract obtained by extracting raw raw material without cold pretreatment at room temperature was about 8%, and the raw materials ex1 to ex5 after extrusion molding contained about 49% to 59% In the case of heat extraction, raw is about 45%, which is big difference from cold water extraction, but ex1 ~ ex5 is about 50 ~ 60%.

7 shows the Uronic acid content of the extract. The content of uronic acid was about 1% in the extract obtained by extracting the unprocessed raw material from the cold water at room temperature, and about 5% to 7% of the extruded ex1 to ex5 %, And in the case of heat extraction, about 5% of raw and ex1 to ex5 show about 6 to 7% of uronic acid content.

The results of the above experiments show that the extracts obtained by the heat extraction process have similar contents of the raw materials that have not undergone the pretreatment and those of the extruded raw materials, Compared with the untreated raw materials, the content of each ingredient is much higher than that of the raw extract.

Example  2: Depending on extraction time of raw materials after pretreatment of raw materials and extrusion molding Extraction rate

In order to estimate the optimum extraction time, each raw material was extracted for 1 hour to 6 hours and the extracted components were compared. In order to compare heat extraction (BT) and room temperature extraction (RT), raw samples without heating were treated for 3 hours to 6 hours, room temperature extraction samples were compared with unexposed samples and ex1, ex3 , ex5 were selected and extracted for 1 hour to 6 hours. The results of extraction solids, total carbohydrate and uronic acid by extraction time are shown in Figs. 8, 9 and 10. Fig.

The solid contents of ex1, ex3, and ex5 pretreated by raw materials without extrusion, and ex1, ex3, and ex5 at room temperature for 1 to 6 hours were similar to those of heat extraction and decreased after 4 hours of extraction (Fig. 8).

The contents of total carbohydrates in the extracts were extracted from each raw material for 1 hour to 6 hours and the results are shown in FIG. The increase from 1 hour after extraction is not much changed after 4 hours.

The change in the content of the acidic polysaccharide by the extraction time is shown in FIG. And the increase of the amount of increase is not great since 4 hours after the extraction. Based on the above results, the extraction amount of the raw material is increased as the extraction time is increased even when the raw material is extracted at room temperature through the pretreatment of the extrusion molding. However, when the extraction time is over 4 hours, The extraction time can be calculated as 4 hours.

Example  3: Chamma by extrusion treatment Displeasure Reduction

SPME (solid phase microextraction) method was used as follows. After equilibrating 10 mL of the sample at 60 ° C. for 20 minutes, the fragrance was collected for 60 minutes using a 100 μm polydimethylsiloxane-coded fiber to obtain a Stabilwax-DA column (30 mx 0.25 mm Id x 0.25 μm film thickness: Restek Corp., Bellefonte, USA ) Was analyzed using a Hewlett-Packard 7890A GC / HP-5973 mass selective detector (MSD) (Hewlett-Packard Co., Palo Alto, Calif., USA). The oven temperature of the used GC was maintained at 80 ° C. for 5 minutes and then increased to 200 ° C. at a rate of 3 ° C./min. The injector temperature was analyzed using 250 ° C. and carrier gas helium. The analyzed fragrance components and chromatograms are shown in Tables 2 to 3 and FIG. Tables 2 to 3 show the flavor components of the raw chungme material and the extruded chymase.

The contents of volatile components such as trichloromethane, toluene, cyclopentasiloxane and d-limonene, which have short residence times, were significantly reduced compared to the raw cheonma. On the other hand, furan and furfural components, which were not found in the raw cheonma, were found in the extruded cheonma, which is expected to be due to caramelization following the browning reaction by heating oxide in the extrusion process. In addition, nonanal and benzene components which were not present in raw materials were detected after extrusion molding. The benzaldehyde component was increased after extrusion than the raw cloth, and acetic acid was not found after extrusion.

cyclohexanol, estragole, naphthalene and 2-Cyclohexen-1-one were significantly reduced after extrusion molding. Propanoic acid, benzyl alcohol, and benzoic acid were also decreased after extrusion compared to the raw cloth. On the other hand, alpha-calacorene, benzeneacetaldehyde and benzothiazole components, which did not appear in the raw cloth, were present after extrusion molding.

Therefore, the improvement of palatability of fragrance components after extrusion molding reduced the components such as trichloromethane, toluene, cyclopentasiloxane, cyclohexanol and acetic acid, which are expected to adversely affect palatability, and furan that can meet palatability. It can be expected to be due to the release of furfural and other components.

Example  4: Evaluation of Biological Activity of Extruded Chunma Extract

The fat-soluble constituents of cheonma and soluble biopolymers were compared for their immune activity. Figure 12 shows the process for preparing crude cell wall extracts from chimpanzees and extruded cholesterol.

1. Measurement of proliferation inducing activity on splenic lymphocytes

1) Experimental method

After spleen was isolated from BALB / C mice sacrificed by cervical distal bone and separated into single cells, cells were obtained by centrifugation at 2,000 rpm for 10 minutes, and then the cells were well suspended by adding 5 mL of RBC lysis buffer. Hemolysis of erythrocytes was induced by standing at room temperature for 5-10 minutes. The cell suspension was carefully layered on Histopaque-1077 (Sigma Chemical Co., USA), which was previously placed in a centrifuge tube, and centrifuged at 2000 rpm for 20 minutes. After the centrifugation, the upper layer was removed, and the cells at the interface were recovered, and the cells were washed well by repeating suspension and centrifugation 2-3 times with RPMI 1640 medium containing 10% FBS. Cells were also dispensed 100 μl into 96-well plates at a density of 2 × 10 ⁵ cells / well using the same medium. Samples to be measured were added to 1, 10, 100 μg / μl or Concanavalin A (Sigma Chemical Co., USA) to 1, 2.5 ug / ml. After incubation for 48 hours in an incubator containing 5% CO ₂ at 37 ° C., 10 μl of EZ-Cytox reagent (Itsbio Co., South Korea) was stored for 4 hours. Thereafter, the amount of water-soluble formazan produced was measured at 480 nm using an ELISA reader.

2) Experimental results

As a result of measuring the proliferation-inducing activity of the sample on splenic lymphocytes in order to observe whether the lymphocytes, the main role of adaptive immunity, were induced, the GEAP-5 and GEAP-6 samples of GEAP (1 ~ 10) were higher than the control group, respectively. Phosphorus showed growth rates of 2.78 and 1.89 times at 100 ug / ml. Especially, GEAP-5 showed a better effect than 2.14 fold at a high concentration of 2.5 ug / ml of the control group Concanavalin A (ConA) (FIG. 13). ).

2. Measurement of cancer cell killability

1) Experimental method

To determine the ability of the sample GEAP (1 to 10) to kill cancer cells, anticancer efficacy was observed in colon cancer cell line HT-29 cells. 500 μl of HT-29 cells were injected into 1 × 10 ⁵ cells / well into 24-well plates, and cultured in an incubator at 37 ° C. and 5% CO _{2 for} 18-24 hours, and then the samples were 4, 20, and 100 ug / ml, respectively. Treatment was also carried out incubated in 24 hours and 48 hours. After 24 hours or 48 hours, each well was added with 0.05% (W / V) MTT solution (Sigma Chemical Co., USA) at 10% (v / v) and stored for 4 hours. Thereafter, the supernatant was removed, and the formed formazan was dissolved in DMSO (500 μl), and the absorbance was measured at 540 nm using an ELISA reader. 5-Fluorouracil (5-FU) was used as a control at concentrations of 4, 20 and 100 uM, respectively.

2) Experimental results

Results of Killing Activity against Colorectal Cancer Cell Lines

The killing ability of the sample GEAP against the colorectal cancer cell line HT-29 cells was measured by MTT assay 24 hours or 48 hours after the sample treatment, and all of them showed a marked killing capacity (as shown in 5-Fluorouracil (5-FU) (24 hours at 100 μM). GEAP-4, 5, 6, 9 and 10 were about 20-40% at 4, 20 and 100ug / ml, respectively. The killing ability was found to be concentration dependent. In particular, at a high concentration of 100 ug / ml in GEAP-5, 6, 9, and 10, the killing ability was always 30 to 35% or more (FIG. 14).

3. Induction of cancer cell apoptosis of sample GEAP

1) Experimental method

The DAPI staining experiment was performed to observe whether the sample GEAP induces apoptosis in HT-29 colon cancer cell line. Dispense HT-29 cells into a 24-well plate covered with 0.5x10 ⁵ cells / well circle glass, and incubate in an incubator containing 37 ° C and 5% CO ₂ for 18-24 hours, then apply GEAP (1-10), respectively. 5-Fluorouracil (5-FU), which is a control group of 100 ug / ml, was treated with 100 uM and incubated for 24 hours. After 24 hours incubation, the supernatant was removed and 4% paraformaldehyde (PFA) was treated at 300 / / well for 15 min at room temperature. After washing three times with PBS, 0.2% triton X-100 / PBS was treated with 300ul / well at room temperature for another 15min. Repeat the washing process three times with PBS, and then mix them in RPMI1640 medium containing 2% FBS at a concentration of 1 ug / ml 4 ', 6-Diamidino-2-phenlindole dihydrochloride (DAPI, Sigma Chemical Co., USA). Shaded at 300ul, and treated for 30min at room temperature. After repeated washing with PBS three times, the circle glass was covered on a slide glass with mounting solution and observed through a Fluorescence Microscope (BH2-RFCA, Olympus, Japan).

2) Experimental results

Apoptosis, also called program cell death, was observed in HT-29 cells of the colorectal cancer epithelial cell line in the sample. As a result, the typical chromatin condensing of apoptosis was observed in GEAP 4, 5, 6, 8, 9 and 10 compared to the control group. It was observed that the cell density was much lower than the control group. The other samples of GEAP are observed to have little difference compared to the control (data not shown), so it is considered that there is no apoptosis effect (FIG. 15).

Claims (5)

Extruding the castor oil powder at a temperature of 120 to 180 DEG C and a pressure of 20 to 100 bar; And
Method for producing a cold water soluble cheonma extract comprising the step of extracting the extruded cheonma by adding water 15 to 20 times the weight of the extruded cheonma by stirring for 3 to 7 hours at room temperature. delete delete delete delete

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