KR20100070246A - The method for preparing the pharmacologically-enhanced fraction of magnolia officinalis and the composition comprising thereof showing enhanced anti-cancer activity - Google Patents

Abstract

PURPOSE: A method for manufacturing Magnolia officinalis fraction and a composition containing the Magnolia officinalis fraction for preventing and treating cancer are provided. CONSTITUTION: A method for producing Magnolia officinalis fraction with enhanced anticancer activity comprises: a step of extracting dried Magnolia officinalis with water, low alcohol of C1-C4 by enfleurage extraction, hot water extraction, ultrasonic wave extraction, reflux extraction, and filtrate heating; a step of collecting Magnolia officinalis crude extract; a step of adding water-saturated sodium chloride, potassium, magnesium, calcium, aluminum or ammonium; a step of filtering and removing supernatant; and a step of drying the extract.

Description

The method for preparing the pharmacologically-enhanced fraction of Magnolia officinalis and the composition comprising including enhanced enhanced anti-cancer activity}

The present invention relates to a method for producing a thick extract extract having excellent anticancer activity and a composition containing the same as an active ingredient.

[Patent 1] Publication 10-2008-0009178, published January 1, 2008

Shu-Er Yang et al., British Journal of Pharmacology, 138 , pp. 193-201, 2003

[3] So Yong Lee et al., European Journal of Pharmacology, 582 , pp. 17-25, 2008

Fei Chen et al., World J Gastroenterol, 10 (23) , pp.3459-3463, 2004

[Reference 5] Xianhe Bai et al., THE JOURNAL OF BIOLOGICAL CHEMISTRY, 278 , No. 37, Issue of September 12, pp. 35505-35507, 2003

[6] Junichiro Saito et al., Mutation Research, 609 , pp. 68-73, 2006

7 Koji Ikeda et al., Phytother. Res. 17 , pp. 933-937, 2003

8 Dong-Yup Lee et al., J. Food Sci. Nutr., 11 , pp.191-197, 2006

[9] Se-Jung Lee et al., Biochemical pharmacology, 75 , pp. 2289-2300, 2008

Document 10 Koji IKEDA et al., Biol. Pharm. Bull., 25 (12) , pp. 1546-1549, 2002

[11] Wen-Bin Zhong et al., Anti-Cancer Drugs, 14 , pp. 211-217, 2003

12. Daisuke S. et al., Int. J. Urology, 10 , pp. 160-166, 2003

PJ Hu et al., Gut, 53 , pp. 195-200, 2004

[14] Shim Jae-young et al., Journal of Korean Association of Cancer Prevention , 9 (4) , pp. 267-273, 2004

15. Xiao-Han Tang et al., Clinical Cancer Researc h, 10 , pp. 301-13, January 1, 2004

[16] Birgit Hotz et al., J Gastrointest Surg, 12 , pp. 900-906, 2008

According to the 2003 Statistical Yearbook of the Ministry of Health and Welfare, cancer was the number one cause of death in Korea in 2002, with 130 deaths from cancer per 100,000 population, followed by 77 cerebrovascular diseases, 37 heart diseases and 25 diabetes. appear. The death rate from cancer has increased every year, and cancer has long been regarded as an incurable disease. Therefore, researches for treating such cancers have been very active in the last few years, and some of the methods have shown some therapeutic effects, but there is still no way of showing a dramatic therapeutic effect.

Existing cancer treatments include surgical surgery, radiation therapy and chemotherapy, and recently, biotherapies such as immunotherapy to treat tumors with immunomodulatory methods are on the rise. However, despite the many treatments, cancer deaths continue to increase every year, although traditional treatments can surgically remove cancer and temporarily inhibit cancer growth by radiotherapy and chemotherapy. After treatment, new malignancies develop in tissues that are unpredictable due to invasion and metastasis from residual cancer cells. Consequently, these solid cancers have increased resistance to anticancer drugs. For life cannot be saved.

On the other hand, currently used bladder cancer treatment in Korea, such as surgical surgery, radiation treatment and anti-cancer treatment, etc., but has the disadvantage of having a significant economic burden along with the side effects of the treatment (Published Patent 10-2008-0009178, 2008.01 .25 public)

In Korea, natural products are attracting attention as alternative medicines that have reduced side effects and improved therapeutic effects. This is because, in the case of therapeutic agents using natural product extracts and components that have been used for a long time and have been proved to be harmless to humans, resistance or safety problems caused by existing therapeutic agents can be overcome. In particular, herbal functional items using natural products that have been used as herbal medicine are popular.

Magnolia officinalis contains lignan-based components such as magnool and honokiol, which have various pharmacological effects such as antibacterial, palliation of smooth muscle of the digestive tract, suppression of inflammation and cancer metastasis. It is also traditionally known to have all the five organs, including warm, delicious, non-toxic, old-fashioned chills, stomach swelling, brain storms, hunger, hunger, warming crises, and stopping the transfer of soil.

The anticancer effect of magnols or honokiols and ovobatol contained in groceries is known (Shu-Er Yang et al., British Journal of Pharmacology, 138 , pp. 193-201, 2003; So Yong Lee et al., European Journal of Pharmacology, 582 , pp. 17-25, 2008; Fei Chen et al., World J Gastroenterol, 10 (23) , pp.3459-3463, 2004; Xianhe Bai et al., THE JOURNAL OF BIOLOGICAL CHEMISTRY, 278 , No. 37, Issue of September 12, pp. 35505-35507, 2003; Junichiro Saito et al., Mutation Research, 609 , pp.68-73, 2006; Koji Ikeda et al., Phytother. Res. 17 , pp . 933-937, 2003; Dong-Yup Lee et al., J. Food Sci. Nutr., 11 , pp. 191-197, 2006; Se-Jung Lee et al., Biochemical pharmacology, 75 , pp. 2289-2300, 2008; Koji IKEDA et al., Biol. Pharm. Bull., 25 (12) , pp. 1546-1549, 2002; Wen-Bin Zhong et al., Anti-Cancer Drugs, 14 , pp. 211-217, 2003), these are the effects exerted as separate chemicals or their own thick extracts.

The separation of the active ingredient from the natural product is carried out through various columns and solvents. Since it takes considerable effort, time, and cost to separate a single ingredient, it is traditionally used as a water extract or a constant organic solvent extract or a solid preparation. It has been used in powders or granules.

The inventors of the present invention have disclosed that the vulgaris extract has prophylactic and therapeutic activity of bladder cancer and colorectal cancer (Korean Patent Publication No. 10-2008-0009178, published on January 25, 2008). However, in the case of the water extract, a large amount of bulky preparations should be ingested in the clinic, and the carbohydrate or protein may contain a large amount such that dissolution rate or disintegration cannot achieve pharmaceutical standardization.

Accordingly, the present inventors have improved the disadvantages of the existing manufacturing method for use in clinical trials, and attempted to simply separate the bulk of the anti-cancer active ingredients in large quantities, to achieve pharmaceutical standardization by first extracting the thick husk with a certain organic solvent. It was confirmed that the content of the lignan-based component, which is a poorly soluble substance in water, such as magnool or honokiol of the thick extract fraction, was greatly increased by a simple process of fractionating secondly, and the thick extract fraction obtained by the manufacturing method thereof was By confirming that the cancer cell toxicity test and animal tests have excellent anti-cancer effect, the present invention was completed.

In order to accomplish the above object, the present invention provides a preparation method for producing a thick extract fraction improved anticancer activity.

Specifically, the present invention recovers the filtrate obtained by cold extraction, hot water extraction, ultrasonic extraction, reflux extraction, heating extraction and filtration with water, C ₁ to C ₄ lower alcohols or a mixed solvent thereof in a dry thick foil. A first step of obtaining an extract; The suspension is warmed by adding cold salt water or inorganic salts such as sodium chloride, potassium, magnesium, calcium, aluminum or ammonium, preferably cold water or saturated sodium chloride, to the thick thin crude extract obtained in the first step. Obtaining a second step; A third step of filtering and removing the supernatant after leaving the suspension at cold temperature; Extracting the residue remaining after filtration in the third step once to three times in the same manner as in the second step, and then recovering the primary dry residue purified product by concentrating and drying the residue obtained by filtration under reduced pressure; After the organic solvent was added to the primary dried residue purified product obtained in the fourth step, suspended, extracted and filtered, the filtrate was concentrated under reduced pressure and dried to obtain an organic solvent-soluble secondary purified product. It provides a manufacturing method (1) for preparing a Park extract purified after the anticancer activity is enhanced by a manufacturing process comprising a.

In addition, the present invention recovers the filtrate obtained by cold extraction, hot water extraction, ultrasonic extraction, reflux extraction, heating extraction and filtration with water, C ₁ to C ₄ lower alcohols or a mixed solvent thereof in a dry after foil Obtaining a first step; The thick thin extract obtained in the first step was adsorbed onto silica gel to seal the top, and C _a H _b O _c (where a is 1 to 6, b and c can each exhibit a chemically stable structure). It provides a manufacturing method (2) for producing a thick extract extract with enhanced anticancer activity by a gel column chromatography method using a solvent that can be represented by a number).

More specifically, the present invention provides water containing 1 to 30 times, preferably 5 to 15 times, more preferably 10 times purified water, C ₁ to C ₄ lower alcohols, or a mixed solvent thereof, preferably of a thick thickness. Preferably, alcohol is added for 1 hour to 10 hours, preferably 2 hours to 5 hours at 60 ° C. to 100 ° C., preferably at 80 ° C. to 100 ° C., cold extraction, hot water extraction, ultrasonic extraction, reflux extraction, heating extraction, Preferably, after the reflux extraction, the first step of filtering the extract to 50 to 100 mesh to obtain a filtrate;

The filtrate obtained by filtering the residue remaining after filtration in the first step 1 to 3 times in the same manner as the first step was mixed with the filtrate obtained in the first step to obtain a mixed solution, 10 ℃ to 80 ℃, Preferably, the crude thin extract (hereinafter referred to as “MOE”) may be prepared through an extraction process including a second step of concentrating and drying under reduced pressure at 30 ° C. to 60 ° C. FIG.

The present invention performs a further purification step of the following compatible or combined in addition to the crude thin extract to obtain a purified product of the present invention containing a large amount of lignan-based component content of water-soluble substances such as magnool or honokiol can do.

Therefore, the present invention is an example of an additional purification step to the conventional crude extract manufacturing process,

The present invention is 1 to 30 times, preferably 5 to 15 times, more preferably about 9 times the weight of the cold thick extract obtained in the dry state obtained above, or saturated sodium chloride, potassium, magnesium, calcium, aluminum Or an inorganic salt such as ammonium, preferably cold water or saturated sodium chloride, is added to the crude extract, and the suspension is heated to 40 ° C to 100 ° C, preferably 50 ° C to 70 ° C, more preferably about 60 ° C. A first step of obtaining; After the suspension is left for about 1 to 48 hours, preferably 10 to 30 hours, more preferably about 24 hours at a cold temperature of -10 ° C to 10 ° C, preferably -4 ° C to 4 ° C Filtering and removing the supernatant; A third step of recovering the first dry residue purified product by concentrating and drying the residue obtained by filtering the residue remaining after filtration in the second step in the same manner as in the first step, after filtration and drying the residue obtained; 1 to 30 times, preferably 5 to 15 times, more preferably about 10 times the purified water, methanol, ethanol, ethyl ether, ethyl acetate, 1 to 30 times the weight of the first dry residue purified product obtained in the third step, Organic solvents such as hexane, chloroform, methylene chloride, glycerin or butylene glycol, preferably ethanol, diethether or ethyl acetate, are added to the above-mentioned first dry residue purified, suspended, extracted and filtered. The manufacturing method (1) which manufactures the thick extract extract which improved anticancer activity by the manufacturing process containing the 4th process of concentrating and drying a filtrate under reduced pressure and obtaining an organic solvent soluble secondary refined | purified product is provided.

In addition, the present invention is an example of a purification step further compatible with the conventional crude extract manufacturing process,

1 to 30 times, preferably 5 to 15 times, and more preferably 10 times 80 to 95% ethanol of the dried thick extract obtained in the above-mentioned state, which is suspended in the suspension after adding to the crude extract. 5 parts by weight, preferably 1 to 3 parts by weight of 60 to 100 mesh silica gel is further added to adsorb the suspension to silica gel to prepare a sample preparation solution for loading by gel column chromatography ;

Once in the first stage loading the sample preparation liquid for the loading at the top of the gel column of the adsorption mechanism of the column, the distribution mechanism of the column or ion exchange mechanism columns such as Dia-ion HP resin column closed at the top to the room, and C _a H _b A solvent represented by O _c (where a is 1 to 6, b and c are numbers in which each of these organic solvents can represent a chemically stable structure), preferably purified water firstly, 5 to 20 w secondly / w% ethanol solution, the second step of obtaining the ethanol solution eluted fraction, the ethanol solution eluted fraction and the alcohol eluted fraction through the step of distilling the alcohol in the third step Takhu extract with enhanced anticancer activity It provides a preparation method (2) for preparing a fraction.

The extract obtained from the hupar extract is prepared by containing 60 to 100 times the amount of the active ingredient magnool or honokiol than the extract of the huhu extract, so that the usage amount is small and the effect is easy and the effect is selective. It is characterized by enhanced.

The thick extract extract as defined herein is characterized in that it is obtained from the root, stem, bark, leaf, flower or fruit, preferably of the cortex, of the pepper bark.

The thick pepper as defined herein is Magnolia officinalis Rehd. Et Wils or Magnolia obovata Thunb, and is characterized in that it is preferably sugar tree.

In addition, the present invention provides a pharmaceutical composition for the prevention and treatment of cancer diseases containing the thick extract extract obtained by the above production method as an active ingredient.

Cancer diseases as defined herein specifically include bladder cancer, stomach cancer, colon cancer, esophageal cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, colon cancer, brain cancer, prostate cancer or cervical cancer, and the like. Diseases to be preferred include bladder cancer, stomach cancer, colon cancer, esophageal cancer or pancreatic cancer, more preferably bladder cancer or colon cancer.

The pharmaceutical composition for preventing and treating cancer diseases of the present invention contains the thick extract fraction in an amount of 0.1 to 50% by weight based on the total weight of the composition.

However, the composition as described above is not necessarily limited thereto, and may vary according to the condition of the patient and the type and extent of the disease.

The extract fraction itself of the present invention is a drug that can be used with confidence even when taken for a long time for the purpose of prevention because there is little toxicity and side effects.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The compositions of the present invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Carriers, excipients and diluents which may be included in the composition comprising the extract fractions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate 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, and the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose ( It is prepared by mixing sucrose or lactose and gelatin. In addition to simple brothers, lubricants such as magnesium styrate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . 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 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 preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract fraction of the present invention is preferably administered at 0.5 g / kg to 5 g / kg, preferably 1 g / kg to 3 g / kg per day. The administration may be carried out once a day or divided into several doses. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

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

The present invention also provides a health functional food for the prevention and improvement of cancer diseases containing the thick extract extract obtained by the above production method as an active ingredient.

The composition comprising the extract fraction of the present invention can be used in a variety of drugs, foods and beverages for the prevention and improvement of cancer diseases. Examples of the food to which the dried product or the extract of the present invention may be added include various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like, and are powders, granules, tablets, capsules, or beverages. Can be used as

Extract fractions of the present invention can be added to food or beverages for the purpose of preventing and ameliorating cancer diseases. At this time, the amount of the extract fraction in the food or beverage is generally added to the health food composition of the present invention 1 to 5% by weight of the total food weight, the health beverage composition is 0.02 to 10 g based on 100 ml, preferably Preferably at a ratio of 0.3 to 1 g.

The health beverage composition of the present invention, in addition to containing the thick extract extract fraction as an essential ingredient in the indicated ratio, there is no particular limitation on the liquid component, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. have. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described 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 proportion of said natural carbohydrates 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-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices 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 in 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 relates to a method for preparing a thick extract extract having improved anticancer activity and a composition for preventing and treating a cancer disease containing the same. Specifically, the extract of the thick extract obtained by the method of the present invention is an active ingredient The content of nool or honokiol is enhanced to be useful for the prevention and treatment of cancer diseases.

Hereinafter, the present invention will be described in detail by comparative examples, examples, reference examples and experimental examples.

However, the following Comparative Examples, Examples, Reference Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Comparative Examples, Examples, Reference Examples and Experimental Examples.

Comparative Example 1. Preparation of thick thin crude extract

100 g of bark of Danghu-Pak, purchased from Gyeongdong Market, was dried and shredded, put in a reflux extractor, and reflux extracted for 3 hours in a water bath, followed by filtration with 100 mesh to obtain a filtrate. Again, the residue was repeatedly extracted in the same manner as described above, and the filtrate was mixed with the filtrate to obtain about 1600 ml of the mixed solution, and concentrated and dried under reduced pressure at a temperature of 60 ° C. or lower, to about 10 g (5 to 15 g) of a thin crude extract. ) Was obtained and used as a sample of the following experimental example (hereinafter referred to as 'MOE').

Example 1 Preparation of Thick Extract Extract

1-1. Preparation of Thick Water Extract Fraction (MOE-WI)

90 ml of cold purified water was added to 10 g of the MOE obtained in the comparative example, and the mixture was heated and suspended at 60 ° C. for 1 hour, left at −4 ° C. to 4 ° C. for 24 hours, and the supernatant was filtered off. After repeated extracting twice in the same manner as above, the residue obtained by filtration was concentrated and dried under reduced pressure to obtain about 0.5-1.5 g (1 g average) of the dried thin water extract fraction, which was used as a sample of the following experimental example (' MOE-WI ').

1-2. Preparation of Thick Sodium Chloride-Ethanol Extract (MOE-NaCl-E)

The same procedure as in Example 1-1 was performed except that 90 ml of saturated sodium chloride in water was added twice to 10 g of the MOE obtained in the comparative example, followed by 1.5 g of a thin sodium chloride extract fraction (hereinafter, 'MOE 15 ml of 95 v / v% ethanol was added to the dried product, suspended and filtered, and the filtrate was concentrated under reduced pressure and dried again to give 0.5 g of 1.5 g of sodium chloride-ethanol extract fraction. (Average 1 g) was obtained and used as a sample for the following experimental example (hereinafter referred to as 'MOE-NaCl-E').

1-3. Preparation of Thickened Sodium Chloride-Diethyl Ether Extract Fraction (MOE-NaCl-DE)

100 g of dieth ether was added to 10 g of MOE-NaCl obtained in Example 1-2, suspended, filtered, and the filtrate was concentrated under reduced pressure and dried again to give 0.5 g of 1.5 g of sodium chloride-dimethyl ether extract fraction (average). 1 g) was obtained and used as a sample for the following experimental example (hereinafter referred to as 'MOE-NaCl-DE').

1-4. Preparation of Thickened Sodium Chloride-Ethyl Acetate Extract (MOE-NaCl-EA)

Except that 100 ml of ethyl acetate was added to 10 g of MOE-NaCl obtained in Example 1-2, 0.5-1.5 g of a dried sodium chloride-ethyl acetate extracted fraction was dried in the same manner as in Example 1-3. An average of 1 g) was obtained and used as a sample of the following experimental example (hereinafter referred to as 'MOE-NaCl-EA').

1-5. Preparation of Thick Diion Fractions

About 500 g of DIION HP-20 (Mitsubishi Chemical, Japan) was added to 500 g of purified water and left at room temperature for 24 hours, after which the entire amount was filled in a column having an inner diameter of 5 cm and a length of 100 cm to prepare a column.

10 g of silica gel (60-100 mesh) was added to the liquid dissolved by adding 100 ml of 95% ethanol to 10 g of the MOE obtained in the comparative example, and then suspended and concentrated under reduced pressure and dried to 10 g of the dried product obtained above. After pouring, the top was closed and filtered and carefully poured 1 L of purified water to obtain a thick thin water fraction, and again, 1 liter of 10% (w / w) ethanol was spilled to obtain a thick 10% (w / w) ethanol fraction. Then, 2L of alcohol was distilled off to obtain a alcohol fraction. Each fraction was concentrated under reduced pressure and dried to obtain 1-4 g of thick Dion water fraction dried product, 1-4 g of Thick thin diion 10% (w / w) ethanol fraction dried product, and 2-8 g of Thick thin diion alcohol fraction dried product. It was used as a sample of the experimental example (hereinafter, the thick thin diion water fraction dried 'MOE-HP-W', the thick thin diion 10% (w / w) ethanol fraction dried 'MOE-HP-E' and thick thin diion alcohol Fraction dry matter is named 'MOE-HP- alcohol'.

Reference Example 1. Preparation of Cancer Cell Line

Bladder cancer cell line (5637, KCLB 30009), gastric cancer cell line (MKN-74, KCLB 80104), colon cancer cell line (HCT 116, KCLB 10247), esophageal cancer cell (HET-1A, ATCC; CRL-2692) and pancreatic cancer cell line (PANC -1, KCLB 21469) was used for the following experiment.

Experimental Example 1. Determination of the active ingredient content of the thick extract fraction

The ethanol sample solution (1 → 100) of the thick extract fraction obtained in Comparative Examples and Examples was filtered through a membrane filter, and the content of the active ingredient was measured according to the following high performance liquid chromatography conditions. 7 and Table 1 are shown.

<High Speed Liquid Chromatography Conditions>

Column: Agilent C18, 4.6 mm id × 150 mm

Mobile phase: acetonitrile: water: acetic acid = 50: 50: 1 (flow rate 1.0 ml / min)

Detector: 289nm Absorbance Detector

As a result, as shown in Table 1, in all experimental groups except MOE-HP-W and MOE-HP-E obtained in Example 1-5, the magolol and honokiol content was more effective than the MOE obtained in the comparative example. In the MOE-HP- alcohol obtained in Example 1-5, it was confirmed that the highest content of magnool and honokiol.

Experimental group Magnol (mg / g) Honokiol (mg / g) Total (%) ^b) Comparative Example Thick ethanol crude extract MOE 4.8 ± 1.2 ^a)   2.3 ± 0.8 3.9 to 8.2 Example 1-1 Thick Water Extract Fraction MOE-WI 312.6 ± 13.4 212.3 ± 10.9 50.2 ~ 63.2 Example 1-2 Thickening Sodium Chloride-Ethanol Extract Fraction MOE-NaCl-E 314.3 ± 14.1 232.3 ± 21.3 51.3 to 59.4 Example 1-3 Thick Sodium Chloride-Diethyl Ether Extract Fraction MOE-NaCl-DE 315.8 ± 11.8 242.3 ± 24.5 52.6 to 60.1 Example 1-4 Thickening Sodium Chloride-Ethyl Acetate Extract Fraction MOE-NaCl-EA 354.8 ± 14.7 232.3 ± 15.1 48.9 to 62.3 Example 1-5 Thick Diion Water Fraction MOE-HP-W 0 0 0.1 or less Example 1-5 Thick Diion 10% (w / w) Ethanol Fraction MOE-HP-E   3.8 ± 0.2 0.5 ± 0.1 3.9 to 4.8 Example 1-5 Thick Diion Alcohol Fraction MOE-HP-Liquor 534.8 ± 31.2 322.3 ± 23.8 79.6 to 90.3 a) The content of honokiol and magnool in each fraction is expressed as mean ± standard deviation value for three samples in each group.
b) the combined content of magnool and honokiol in the dry matter

Experimental Example 2. Cytotoxicity test (MTT assay) of thick extract fraction

The thick extract fractions obtained in Comparative Examples and Examples were subjected to MTT assay according to the method described in Fei Chen et al., World J Gastroenterol, 10 (23) , pp.3459-3463, 2004. Measured.

As a result of the measurement, as shown in FIG. 8 and Table 2, the average inhibitory amount (IC50 (μg / ml)) was calculated and displayed for all cancer cell lines, and the MOE obtained in the comparative example and the MOE obtained in Examples 1-5. In all groups except -HP-W and MOE-HP-E, IC50 was twice as low as that of pure compound Honokiol or Magnool, showing the anticancer toxicity expected by synergistic effects of Honokiol and Magnool in fractions. It was confirmed that the cytotoxic effect was elevated.

IC50 (μg / ml) Bladder cancer cells Stomach cancer cell Colorectal cancer cells Esophageal Cancer Cells Pancreatic cancer cell Comparative Example MOE 100 50.0 50.0 50.0 100 Example 1-1 MOE-WI 2.5  1.0  1.0  1.0 2.5 Example 1-2 MOE-NaCl-E 2.5  1.0  1.0  1.0 2.5 Example 1-3 MOE-NaCl-DE 2.5  1.0  1.0  1.0 2.5 Example 1-4 MOE-NaCl-EA 2.5  1.0  1.0  1.0 2.5 Example 1-5 MOE-HP-W > 100.0 > 100.0 > 100.0 > 100.0 > 100.0 Example 1-5 MOE-HP-E > 100.0 > 100.0 > 100.0 > 100.0 > 100.0 Example 1-5 MOE-HP-Alcohol 1.0 0.5 0.5 0.5 1.0 Honokiol 10.0 25.0 25.0 25.0 10.0 Magnool 10.0 20.0 20.0 20.0 10.0 Honokiol + Magnool
Mixture (2: 1) 5.0 5.0 5.0 5.0 5.0

Experimental Example 3 Inhibition of Bladder Cancer Production of Thick Extract

Method described in the literature to investigate the inhibitory effect of bladder cancer development of the thick extract fractions obtained in Comparative Examples and Examples (Daisuke S. et al., Int. J. Urology, 10 , pp . 160-166, 2003 ) Was performed as follows.

ICR mice (male, 25-30 g) were used in groups of 5 per group and 0.5 ml of 0.05% N-butyl-N- (4-hydroxybutyl) Nitrosamine (BBN) aqueous solution was orally administered once daily for 8 weeks (Daisuke S. et al. , Int. J. Urology, 10 , pp . 160-166, 2003), and the thick extract fractions obtained in each of the comparative examples and examples correspond to 100 mg / kg once as a spirit extract once daily after 12 hours of BBN administration. Sheep were orally administered for 8 weeks each to check their survival. As a control group, only BBN was administered.

As a result, as shown in Table 3, in all groups except MOE-HP-W and MOE-HP-E obtained in Example 1-5, the survival rate was increased compared to the control, in particular, Example 1-5 MOE-HP-alk obtained at was able to confirm that the survival rate similar to the normal group was not administered BBN.

                             Time (week) 0 One 2 3 4 5 6 7 8 Normal 5 5 5 5 5 5 5 5 5 BBN
Administration group Control 5 5 5 4 4 3 2 0 0 Comparative Example MOE 5 5 5 5 4 4 4 4 3 Example 1-1 MOE-WI 5 5 5 5 4 4 4 4 3 Example 1-2 MOE-NaCl-E 5 5 5 5 4 4 4 4 3 Example 1-3 MOE-NaCl-DE 5 5 5 5 4 4 4 4 3 Example 1-4 MOE-NaCl-EA 5 5 5 5 4 4 4 4 3 Example 1-5 MOE-HP-W 5 5 5 4 4 3 2 0 0 Example 1-5 MOE-HP-E 5 5 5 4 4 3 2 0 0 Example 1-5 MOE-HP-Alcohol 5 5 5 5 5 5 5 5 5 Honokiol 5 5 5 5 4 4 4 4 3 Magnool 5 5 5 5 4 4 4 4 3 Honokiol + magnool mixture (2: 1) 5 5 5 5 4 4 4 4 3

Experimental Example 4. Suppression of Gastric Cancer Development of Thick Extract Fraction

In order to determine the effect of inhibiting gastric cancer development of the thick extract fractions obtained in Comparative Examples and Examples, PJ Hu et al., Gut, 53 , pp.195-200, 2004 The same experiment was performed.

Oral Wistar rats (Woong, 250-300g) of 5 rats per group for 8 weeks with 1 ml of 3% sodium chloride solution dissolved in N-methyl-N-9-nitro-N-nitrosoguanidine (MNNG, 100 ug / ml) once daily. After administration, the thick extract fractions obtained in each Comparative Example and Example were administered orally at a dose of 100 mg / kg (weight of the rat) for 8 weeks, once every 12 hours after MNNG administration for 8 weeks to examine their survival rate. It was. As a control group, only MNNG was administered.

As a result, as shown in Table 4, in all groups except MOE-HP-W and MOE-HP-E obtained in Example 1-5, the survival rate was increased compared to the control, in particular, Example 1-5 MOE-HP-alk obtained at was able to confirm that the survival rate similar to the normal group was not administered MNNG.

                             Time (week) 0 One 2 3 4 5 6 7 8 Normal 5 5 5 5 5 5 5 5 5 MNNG
Administration group Control 5 5 5 4 4 3 2 0 0 Comparative Example MOE 5 5 5 5 4 4 4 4 3 Example 1-1 MOE-WI 5 5 5 5 4 4 4 4 3 Example 1-2 MOE-NaCl-E 5 5 5 5 4 4 4 4 3 Example 1-3 MOE-NaCl-DE 5 5 5 5 4 4 4 4 3 Example 1-4 MOE-NaCl-EA 5 5 5 5 4 4 4 4 3 Example 1-5 MOE-HP-W 5 5 5 4 4 3 2 0 0 Example 1-5 MOE-HP-E 5 5 5 4 4 3 2 0 0 Example 1-5 MOE-HP-Alcohol 5 5 5 5 5 5 5 5 5 Honokiol 5 5 5 5 4 4 4 4 3 Magnool 5 5 5 5 4 4 4 4 3 Honokiol + magnool mixture (2: 1) 5 5 5 5 4 4 4 4 3

Experimental Example 5. Experiment to inhibit colon cancer development

The method described in the literature to investigate the inhibitory effect of colorectal cancer development of the thick extract fractions obtained in Comparative Examples and Examples (Jae Jae Young et al., Journal of Korean Association of Cancer Prevention , 9 (4) , pp.267-273 , 2004).

Wistar rats (Woongseong, 250-300g) were used in 5 rats per group, and 20 mg / kg of 1,2-dimethylhydrazine (DMH) aqueous solution was injected subcutaneously once every 8 weeks for 8 weeks, followed by the extraction of the pepper paste obtained in each Comparative Example and Example. Fractions were administered orally at a dose of 100 mg / kg (weight of the rat) for 8 weeks, once daily, 12 hours after DMH administration, and their viability was examined. Experiment was performed by administration of only DMH as a control.

As a result, as shown in Table 5, in all groups except MOE-HP-W and MOE-HP-E obtained in Example 1-5, the survival rate was increased compared to the control, in particular, Example 1-5 MOE-HP-alk obtained at was able to confirm that the survival rate similar to the normal group did not receive DMH.

                             Time (week) 0 One 2 3 4 5 6 7 8 Normal 5 5 5 5 5 5 5 5 5 DMH
Administration group Control 5 5 5 4 4 3 2 0 0 Comparative Example MOE 5 5 5 5 4 4 4 4 3 Example 1-1 MOE-WI 5 5 5 5 4 4 4 4 3 Example 1-2 MOE-NaCl-E 5 5 5 5 4 4 4 4 3 Example 1-3 MOE-NaCl-DE 5 5 5 5 4 4 4 4 3 Example 1-4 MOE-NaCl-EA 5 5 5 5 4 4 4 4 3 Example 1-5 MOE-HP-W 5 5 5 4 4 3 2 0 0 Example 1-5 MOE-HP-E 5 5 5 4 4 3 2 0 0 Example 1-5 MOE-HP-Alcohol 5 5 5 5 5 5 5 5 5 Honokiol 5 5 5 5 4 4 4 4 3 Magnool 5 5 5 5 4 4 4 4 3 Honokiol + magnool mixture (2: 1) 5 5 5 5 4 4 4 4 3

Experimental Example 6. Inhibition test of esophageal cancer in the extract of hubac extract

The method described in the literature to investigate the inhibitory effect of esophageal cancer development of the thick extract fractions obtained in the comparative examples and examples (Xiao-Han Tang et al., Clinical Cancer Researc h, 10 , pp.301-13, January 1, 2004) was performed as follows.

 Dissolve 4-nitroquinoline 1-oxide (NQO) in polyethylene glycol 400 to 10 w / w% with 5 ICR mice (male, 25-30 g) per group, and then dilute with water to make 5 mg / ml. Was administered as a negative substitute for 8 weeks, and the thick extract fractions obtained in each Comparative Example and Example were orally administered at a dose of 100 mg / kg (mouse body weight) for 8 weeks each as an alcoholic extract once daily to improve the survival rate. Investigate. As a control group, only NQO was administered.

As a result, as shown in Table 6, in all groups except MOE-HP-W and MOE-HP-E obtained in Example 1-5, the survival rate was increased compared to the control, in particular, Example 1-5 MOE-HP-alk obtained at was found to have a similar survival rate as the normal group was not administered NQO.

                             Time (week) 0 One 2 3 4 5 6 7 8 Normal 5 5 5 5 5 5 5 5 5 NQO
Administration group Control 5 5 5 4 4 3 2 0 0 Comparative Example MOE 5 5 5 5 4 4 4 4 3 Example 1-1 MOE-WI 5 5 5 5 4 4 4 4 3 Example 1-2 MOE-NaCl-E 5 5 5 5 4 4 4 4 3 Example 1-3 MOE-NaCl-DE 5 5 5 5 4 4 4 4 3 Example 1-4 MOE-NaCl-EA 5 5 5 5 4 4 4 4 3 Example 1-5 MOE-HP-W 5 5 5 4 4 3 2 0 0 Example 1-5 MOE-HP-E 5 5 5 4 4 3 2 0 0 Example 1-5 MOE-HP-Alcohol 5 5 5 5 5 5 5 5 5 Honokiol 5 5 5 5 4 4 4 4 3 Magnool 5 5 5 5 4 4 4 4 3 Honokiol + magnool mixture (2: 1) 5 5 5 5 4 4 4 4 3

Experimental Example 7. Inhibition test of pancreatic cancer development

In order to determine the effect of inhibiting pancreatic cancer development of the thick extract fractions obtained in Comparative Examples and Examples, the method described in the literature (Birgit Hotz et al., J Gastrointest Surg, 12 , pp.900-906, 2008) was used. The experiment was as follows.

Pancreatic cancer cell lines (PANC-1, KCLB 21469) were suspended in phosphate isoform using 5 ICR mice (male, 25-30 g) per group, and 10 ⁷ cells per subject were intraperitoneally injected into the flanks, Comparative Examples and Examples. The vitreous extract fractions obtained at were administered once daily to oral doses of 100 mg / kg (mouse body weight) for 8 weeks, and their viability was examined. As a control, only cancer cells were transplanted and tested.

As a result, as shown in Table 7, in all groups except MOE-HP-W and MOE-HP-E obtained in Example 1-5, the survival rate was increased compared to the control, in particular, Example 1-5 The MOE-HP- alcohol obtained in the experiment showed a similar survival rate as that of the normal group without transplanting pancreatic cancer cells.

                             Time (week) 0 One 2 3 4 5 6 7 8 Normal 5 5 5 5 5 5 5 5 5 Cancer cell
Transplant group Control 5 5 5 4 4 3 2 0 0 Comparative Example MOE 5 5 5 5 4 3 3 3 2 Example 1-1 MOE-WI 5 5 5 5 4 3 3 3 2 Example 1-2 MOE-NaCl-E 5 5 5 5 4 3 3 3 2 Example 1-3 MOE-NaCl-DE 5 5 5 5 4 3 3 3 2 Example 1-4 MOE-NaCl-EA 5 5 5 5 4 3 3 3 2 Example 1-5 MOE-HP-W 5 5 5 4 4 3 2 0 0 Example 1-5 MOE-HP-E 5 5 5 4 3 3 One 0 0 Example 1-5 MOE-HP-Alcohol 5 5 5 5 5 5 5 5 5 Honokiol 5 5 5 5 4 3 3 3 2 Magnool 5 5 5 5 4 3 3 3 2 Honokiol + magnool mixture (2: 1) 5 5 5 5 4 3 3 3 3

Hereinafter, an example of the formulation of a composition including the thick extract fraction of the present invention will be described, but the present invention is not intended to limit the present invention, but is only intended to be described in detail.

Formulation Example 1 Preparation of Powder

MOE-HP-Alcohol 20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2 Preparation of Tablet

MOE-HP-Alcohol 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation Example 3 Preparation of Capsule

MOE-HP-Alcohol 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

MOE-HP-Alcohol 10 mg

Mannitol 180 mg

Sterile sterilized water for injection 2974 mg

Na2HPO4,12H2O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

MOE-HP-Alcohol 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding lemon flavor appropriately, mixing the above components, adding purified water, adjusting the whole to 100 ml by adding purified water, and then filling into a brown bottle. The solution is prepared by sterilization.

Formulation Example 6 Preparation of Healthy Food

MOE-HP-Alcohol 1000 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5mg

Mineral mixture quantity

Ferrous Sulfate 1.75 mg

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation Example 7 Preparation of Healthy Drink

MOE-HP-Alcohol 1000 mg

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to a total of 900 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the solution thus prepared was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, &Lt; / RTI &gt;

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

[National R & D project supporting this invention]

[Task unique number]

10018327

[Name of Buddha]

Ministry of Knowledge Economy

[Name of research project]

Development of treatment for intractable diseases using natural products of regional industrial technology development task

[Name of Research Project]

Development and Commercialization of Solid Cancer Treatment Using Natural Magnolia

[Host]

Hanseo Pharmaceutical

[Research period]

October 01, 2004 to September 30, 2008

1 is a high-performance liquid chromatogram results of the honokiol and magnool,

Figure 2 is a high-performance liquid chromatogram results of the thick thin crude extract (MOE) obtained in the comparative example,

3 is a high-performance liquid chromatogram of the thick extract water extract (MOE-WI) obtained in Example 1-1,

4 is a representative high performance liquid chromatogram results of the extract fractions obtained in Examples 1-1, 1-2, 1-3 and 1-4,

5 is a high performance liquid chromatogram of the thick-walled diion water fraction (MOE-HP-W) obtained in Example 1-5,

6 is a high-performance liquid chromatogram of the thick hydroxyaion 10% (w / w) ethanol fraction (MOE-HP-E) obtained in Example 1-5,

7 is a high-performance liquid chromatogram of the thick-walled diion alcohol fraction (MOE-HP- alcohol) obtained in Example 1-5,

Fig. 8 shows the crude thin extract (MOE) (O), pure Honokiol pure (◇), pure Magnool pure (△), a mixture of Honochiol and Magnool (2: 1, □), thick extract water extract (MOE-WI ) (◆), Thick thin diion water fraction (MOE-HP-W) (●), Thick thin diion 10% (w / w) ethanol fraction (MOE-HP-E) (▲) and Thick thin diion alcohol fraction ( The result of MTT analysis by adding MOE-HP-alcohol (■) to gastric cancer cells (MKN-74, KCLB 80104) solution by concentration (extract obtained in Examples 1-2, 1-3 and 1-4) The analysis results of the fractions are similar to the extract fractions obtained in Example 1-1 and are not separately indicated).

Claims (7)

The _first to obtain a thick thin crude extract by recovering the filtrate obtained by cold extraction, hot water extraction, ultrasonic extraction, reflux extraction, heat extraction and filtration with water, a lower alcohol of C ₁ to C ₄ or a mixed solvent thereof in the dried after foil step; A second step of obtaining a suspension after warming by adding cold water or an inorganic salt such as saturated sodium chloride, potassium, magnesium, calcium, aluminum or ammonium to the thick extract obtained in the first step; A third step of filtering and removing the supernatant after leaving the suspension at cold temperature; Extracting the residue remaining after filtration in the third step once to three times in the same manner as in the second step, and then recovering the primary dry residue purified product by concentrating and drying the residue obtained by filtration under reduced pressure; After the organic solvent was added to the primary dried residue purified product obtained in the fourth step, suspended, extracted and filtered, the filtrate was concentrated under reduced pressure and dried to obtain an organic solvent-soluble secondary purified product. Manufacturing method for producing a thick extract extract purified by the anticancer activity in a manufacturing process comprising a. The _first to obtain a thick thin crude extract by recovering the filtrate obtained by cold extraction, hot water extraction, ultrasonic extraction, reflux extraction, heat extraction and filtration with water, a lower alcohol of C ₁ to C ₄ or a mixed solvent thereof in the dried after foil step; The thick thin extract obtained in the first step was adsorbed onto silica gel to seal the top, and C _a H _b O _c (where a is 1 to 6, b and c can each exhibit a chemically stable structure). A method for producing a thick extract fraction having anticancer activity enhanced by a manufacturing process by performing column chromatography using a solvent that can be represented by a number represented by). The method according to claim 1 or 2, The thick extract extract fraction is obtained from the roots, stems, bark, leaves, flowers or fruits of the rear bark. The method according to claim 1 or 2, The thick gourd is a manufacturing method characterized in that the Magnolia officinalis Rehd. Et Wils or Magnolia obovata Thunb. A pharmaceutical composition for the prevention and treatment of cancer diseases, comprising as an active ingredient the thick extract extract obtained by the method of claim 1 or 2. The method of claim 5, Specifically, the cancer disease may include bladder cancer, stomach cancer, colon cancer, esophageal cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, colon cancer, brain cancer, prostate cancer, or cervical cancer. Pharmaceutic composition. A health functional food for the prevention and improvement of cancer diseases containing the thick extract fraction obtained by the method of claim 1 or 2 as an active ingredient.

Images