KR101219650B1 - Process for detoxification of Rhus Verniciflua, and the use of detoxified bark extract - Google Patents

Abstract

The present invention relates to a method for detoxifying lacquer and the use of mushroom extracts comprising the detoxified lacquer component. The detoxification method of lacquer according to the present invention is characterized by using the lacquer and grains together as a nutrient source of mushrooms. According to the present invention, not only the toxic components of lacquer can be efficiently detoxified using grains and mushroom fungi, but also the mushroom hyphae containing the detoxified lacquer and the nutritional food ingredients of mushrooms and grains together with pharmaceutical or food ingredients. There is an advantage that can be used.

Description

Process for detoxification of Rhus Verniciflua, and the use of detoxified bark extract}

1 is a graph showing the results of growing mushroom fungi of basidiomycetes using rice bran and lacquer as a medium,

2 is a TLC chromatogram of detoxified ursiol using basidiomycete,

FIG. 3 is an HPLC chromatogram of purified ursiol (right) separated from silica gel open column (left) of fraction (F-2) isolated from sap of lacquer tree (F-2);

FIG. 4 shows LC-MS data of isolated 3- (8'Z, 11'E, 13'Z-pentadecatenyl) catechol,

FIG. 5 is an HPLC chromatogram of ursiol from sumac bark detoxified by basidiomycete,

Figure 6 is a graph showing the change over time of the urushiol content by the growth of longevity mushroom ( F. fraxinea ) mycelium in rice bran lacquer medium,

7 is a graph showing the nitrite scavenging activity of the water extract of lacquer bark detoxified with basidiomycete,

8 is a graph showing the effect on the LPL activity of water and ethanol extract of detoxified lacquer bark,

9 is an electrophoresis result showing the induction of tyrosine hydroxylase expression by detoxified lacquer bark extract in the rat brain region,

10 is a graph showing the induction of tyrosine hydroxylase expression by detoxified lacquer bark extract in PC12 cells,

11 is a photograph showing the results of immunohistochemical detection of TH by detoxified sumac extract at 4 hours after substantia nigra of brain.

12 is a graph showing the cytotoxicity of detoxified sumac bark extract against normal cells (NIH3T3),

Figure 13 is a graph showing the effect on the cytotoxicity of the detoxified sumac extract against hematological cancer cells (T24) and bone marrow cancer cells (Saos).

The present invention relates to a method for detoxifying lacquer and the use of mushroom extracts containing detoxified lacquer. More specifically, a method for detoxifying lacquer by decomposing and removing toxic components of lacquer using cereals and mushroom fungi, A detoxified lacquer-containing mushroom extract prepared by this method and its various uses.

In general, lacquer refers to the sap coming from the lacquer tree when the tree of the lacquer family is wounded. The lacquer family includes 60 genera and 400 species distributed in subtropical or tropical regions, and among them, five species of lacquer tree, lacquer tree and red tree are inhabited.

Such lacquer has been used in Korea, China, Japan, etc. for a long time as a coating material for metal and woodworking, and also as an medicinal herb because it has the effect of promoting the blood circulation of the human body, tonic effect, diameter, Jinhae, etc. come. In addition, recently, there have been reports of extracting an anticancer substance (MU2) from lacquer (Korean Patent 10-0257448; 10-2004-0077984).

When the lacquer having various pharmacological effects is ingested directly in the sap state, side effects such as rash and itching are caused by a toxic substance called urushiol.

In order to exploit the pharmacological and physiological activity of lacquer, the purification process is used to neutralize the urushiol toxicity or to roast the lacquer at high temperature or to carbonize it at a high temperature of 200 ℃ or higher in a closed kiln. It causes evaporation of urushiol, which is an inducing ingredient and a main pharmacologically active ingredient, to remove the pharmacological action of lacquer.

In addition, according to the method for cooking chickens and ducks in jubilee, jujube, chestnut, astragalus, etc. with lacquer, one out of three people who consumed lacquer chickens or ducks showed various side effects including dermatitis. There is an urgent need for a way to completely detoxify.

The present inventors have developed a method of remarkably detoxifying lacquer by growing mushroom strains on a medium containing lacquer in the patent application No. 2003-0071302 (2003.10.14) as a result of earnest research to remove the poison of lacquer tree. In addition, as a result of further studies, mushrooms were grown by incorporating lacquer and grains into the medium, and when grains such as rice and by-products were added than when the lacquer alone was used as a culture substrate. As a result, the growth of mushrooms is promoted over a short period of time, so that the toxic components of lacquer are removed more efficiently, and the components extracted therefrom have various functionalities as described below by supplementing the nutrition of mushrooms and supplementing growth promoting substances. The present invention has been completed.

Accordingly, it is an object of the present invention to provide an improved method for detoxifying lacquer using mushrooms, grains and by-products thereof.

Another object of the present invention is a pharmaceutical composition, a food composition or a beverage composition comprising the components obtained by extracting the mycelium component of detoxified lactose and mushrooms by hot water or an organic solvent (for example, alcohol) by treating as described above. To provide.

In one aspect, the present invention,

a) inoculating a mushroom strain on a medium containing grains selected from the group consisting of rice, brown rice, brown rice glutinous rice, rice bran, black rice, cereals, barley, beans and mixtures thereof and by-products and lacquer components, and

b) culturing the grain-containing lacquer medium for 7 days to 30 days at a temperature of 15 ~ 37 ℃

It provides a method for removing the toxicity of lacquer comprising a.

The present invention has been made as a result of a thorough study on the formulation of health food that can be easily used by consumers, and added the lacquer medium component to cereals or crops such as brown rice, brown rice glutinous rice, black rice, and cereals, which are usually used for diet, and used as a medium. By cultivating the mushrooms to ingest the crops containing the beneficial substances of the mushrooms, which show various effects such as preventing various adult diseases, and the nutritious ingredients of lacquer and the cereals, which show various functionalities as described above, through daily meals. It is characterized by providing a formulation that can prevent various diseases in advance.

According to the present invention, a mixture of lacquer bark and grains is added to the cultivation process of the mushroom to absorb the mushroom, thereby removing the poison of the lacquer in the process of absorbing the nutrients by the metabolic process of the mushroom, thereby making the beneficial component of the lacquer and the useful ingredient of the mushroom It is possible to produce useful substances such as functional agricultural products or high functional foods that have been detoxified in a safe and efficient manner by incorporating them into the grain.

Specifically, the present invention uses grains and lacquer powder selected from the group consisting of rice, brown rice, brown rice glutinous rice, rice bran, black rice, cereals, barley, and soybeans as a medium for growth of mushrooms (small fungi). Provides a method of supplying raw materials of safe forms of food or pharmaceutical ingredients, including detoxified lacquer ingredients absorbed by the medicinal ingredients of lacquer and nutrients of cereals in the cultivation process of mushrooms, while being naturally removed from metabolism. do. Therefore, when grains such as rice and rice by-products are added to the culture substrate, it is good for promoting the growth of mushrooms and detoxifying urushiol in a short period of time since the nutritional enhancement and growth promoting substances of mushrooms are supplemented. There is a synergistic effect. Of course, it is obvious that the addition of these components to the culture substrates commonly used for the cultivation of mushrooms, such as chaff or sawdust medium or other nutrient medium components, can achieve even more desired effects.

A number of commercially available functional crops commercialized by adding natural substances or functional substances have been proposed. For example, products containing chitosan, mushrooms, and green tea components are introduced.

Mushroom has been used for edible and medicinal for about 3,000 years, and it is reported that the mushroom-derived materials are useful for the treatment or prevention of various cancers and for the prevention of cancer caused by immune enhancement. Mushrooms are divided into Mycelium and Fruiting Body. Mycelium means that spores of mushrooms germinate to form a silky shape. Abundant physiologically active substances and the mycelium germinate form fruiting bodies. It is called a mushroom.

As the strain used for the detoxification, it is possible to use basidiomycetes for artificial cultivation of edible and medicinal mushrooms, and such examples are not particularly limited as long as the white rot fungi having laccase secretion ability. Examples of mushrooms include, but are not limited to, longevity mushrooms ( Fomitella fraxinea ), Ganoderma lucidum ( Ganoderma lucidum ), Roe deer fungus ( Hericium erinaceus ), Lyophyllum cinerascens , Pleurotus eryngii , Pleurotus ostreatus , Shiitake mushrooms ( Lentinus edodes ), Rabbit Fur Songp Mushroom ( Trametes trogii ), Janna Butterfly ( Ganoderma applanatum ), Fingerling mushroom ( Coriolus versicolor ), Phellinus linteus Tyromyces palustris or mixtures thereof.

The strains may be used in combination as needed, and combined use may result in more desirable results for maximum efficiency of the medicinal component. Their selection can be carried out by conventional methods.

When the specific mushroom strains or mixed strains are incubated in a medium containing lacquer and grains for an appropriate period, the mushroom strains grow in a mycelial state, and the laccase enzyme released from the mushrooms during the fermentation process. The urushiol component, which is a toxic component of lacquer in lacquer medium, is converted to quinone form, and the unstable quinone compound is polymerized and detoxified.

As used herein, the term “grains and by-products thereof” refers to all grains and their by-products having commercial properties that are grown in an agricultural or biological manner. Therefore, in a specific embodiment of the present invention will be described for a specific grain, but the present invention is not limited to these, for example, rice, brown rice, rice bran, brown rice glutinous rice, black rice, cereals, barley, soybeans, etc. It is obvious.

This method of detoxification of lacquer has the advantage that it is possible to use not only the detoxification of toxic components of lacquer, but also to use the mushroom hyphae component and the nutritional component of the grain which contribute to the detoxification treatment. Alternatively, only the lacquer medium itself, which has undergone mushroom strain growth, can be boiled in water to use the extracted solution as a pharmacological component.

Accordingly, in another aspect, the present invention provides a pharmaceutical composition or a functional food composition for treating a related disease comprising the detoxified lacquer extract of the present invention.

The composition according to the present invention

a) inoculating a mushroom strain on a medium containing grains selected from the group consisting of rice, brown rice, brown rice glutinous rice, rice bran, black rice, cereals, barley, beans and mixtures thereof and by-products and lacquer ingredients,

b) culturing the grain-containing lacquer medium at a temperature of 15-37 ° C. for 7 days to 30 days, and

c) The mixed extract of detoxified lacquer, cereals and mushroom mycelium is obtained as a main component, which is obtained by distilling the culture with water or an organic solvent to filter the solids and concentrate the extract.

Since the extract of the present invention exhibits various functionalities such as lowering blood lipid content and anticancer action as can be seen in the following examples, it is an active ingredient of a food or beverage composition in addition to its use as a pharmaceutical composition for improving or treating related diseases. It can be usefully used as. Examples of the disease to be targeted include, but are not limited to, various cancers including dementia, hyperlipidemia, bone marrow cancer, and blood cancer.

The pharmaceutical composition of the present invention can be formulated in combination with a known medical carrier. Generally, the active ingredient is blended with a pharmaceutically acceptable liquid or solid carrier, and a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, a lubricant and the like are added as necessary and purified. And solid solutions such as granules, powders, powders, and capsules, liquids such as ordinary liquids, suspensions, and emulsions. Moreover, it can also be made into the dry goods which can be made into a liquid state by addition of a suitable carrier before use.

The pharmaceutical composition of the present invention can be administered by any of parenterals such as oral preparations, injections, and drops.

A medical carrier can be selected according to the said dosage form and formulation, and, in the case of an oral preparation, starch, lactose, white sugar, mannite, carboxymethylcellulose, corn starch, an inorganic salt, etc. are used, for example. Moreover, in preparation of an oral preparation, a binder, a disintegrating agent, surfactant, a lubricating agent, a fluidity promoter, a copper, a coloring agent, a fragrance | flavor, etc. can also be mix | blended.

On the other hand, in the case of parenteral preparations, the composition comprising the lacquer mushroom extract, the active ingredient of the present invention, is a diluent for injection as a diluent, physiological saline, aqueous glucose solution, injectable vegetable oil, sesame oil, peanut oil, soybean oil, corn. It is melt | dissolved or suspended in oil, propylene glycol, polyethyleneglycol, etc., and it is prepared by adding a disinfectant, a stabilizer, an isotonicity agent, a non-fatting agent, etc. as needed.

The pharmaceutical composition of the present invention is administered by a suitable route of administration according to the formulation form. The administration method need not be particularly limited and can be administered by internal content, external application, and injection. Injections can be administered, for example, intravenously, intramuscularly, subcutaneously, intradermally or the like.

The dosage of the pharmaceutical composition of the present invention is appropriately set according to the form of the preparation, the method of administration, the purpose of use, and the age, weight, and symptoms of the patient to be applied thereto. Amounts are, for example, 10 μg-200 μg / kg per adult. Of course, since the dose varies depending on various conditions, an amount smaller than the dose may be sufficient, or it may be necessary beyond the range.

In still another aspect, the present invention provides a functional food composition comprising the detoxified lacquer component, cereals, and mushroom culture, and a beverage composition.

Extracts of detoxified lacquer ingredients, cereals and mushrooms can be provided as functional foods such as easy-to-take beverages containing such pharmacologically active or physiologically active substances since they exhibit a lipid content lowering, anticancer activity and the like.

Auxiliary components of the food, their blending ratio and the like can employ those conventionally used in the art without particular technical difficulties.

The food composition according to the present invention may add one or more ingredients selected from the group consisting of conventional auxiliaries, such as aspartame, saccharin, pectin, guar gum, skim milk powder and oligosaccharides, depending on the form of the desired composition.

As the sweetener in the food composition of the present invention, aspartame, saccharin, malitol, sorbitol, and xylitol commonly used may be used. They are suitably used at about 0.1-20% by weight based on the total weight of the food composition of the present invention.

The food composition according to the present invention may be provided, for example, in the form of a beverage such as tablets, capsules, confectionary or drink.

For example, drink the detoxified lactose-containing mushroom extract at 0.1-30% by weight, add citric acid at 0.01-2%, and add vitamins such as vitamin C, vitamin B1, and biotin, and the sweetness is 0.01-2%. To prepare functional drinks.

The lacquer-containing medium is a bark of lacquer trees, that is, used as is or powdered lacquer bark or sap or lacquer bark extracted from the lacquer tree in water (for example, 10 times) or an organic solvent and distilled to extract sap and It is preferable to add a conventional agar medium.

The composition of the lacquer medium is preferably set in consideration of the optimum incubation temperature, moisture content, optimal pH, incubation period, such conditions can be easily set by those skilled in the art. Specifically, as the culture substrate added to the lacquer medium may be used a synthetic medium, semi-synthetic medium, natural medium containing grains and by-products thereof. Synthetic medium is preferably formulated to contain a carbon source, nitrogen source, minerals, vitamins, semi-synthetic medium is added to the appropriate natural product components to improve the mycelia production based on the synthetic medium. The natural medium may include components for mycelial growth, such as lacquer, potato, molasses, malt, skim soybean meal, and other plant extracts. The pH of the lacquer medium was good at 5.5-7.5 according to the experimental results and the best result was obtained at pH 7.

Next, mushroom strains are inoculated in lacquer medium and cultured. The culture period does not need to be particularly limited, but the culture period of the mushroom strain is sufficient to uniformly decompose the toxic components of the lacquer, and the mushroom strain is grown to a certain length, for example, about 3 to 15 cm. Is preferable, and about 7 cm-about 15 cm are more preferable. Therefore, although depending on specific culture conditions in time, it is preferable to culture about 7-30 days normally. It may be most desirable to incubate for 15 to 20 days. It may be more preferable to arrange the mushroom strains in a lacquer medium at equal intervals. If the culture period of the mushroom strain is too long, it is difficult to use the pharmacological components of the lacquer bark because all of the components of lacquer to be detoxified are used as nutrients of the mushroom. In addition, if the incubation period is too short, the toxic components contained in the bark of the bark are not sufficiently removed by the mushroom mycelia. The mushroom strain is appropriate to incubate around 15 ~ 37 ℃, it is most preferable to incubate at 25 ℃. The culture may be any type as long as mushroom culture can grow well in stationary culture, shake culture, fermenter culture, bottle culture, pot culture, and the like.

Preferably, the lacquer medium containing the cultured lacquer medium and mushroom mycelia is placed in a solvent such as water and boiled for a predetermined time (1 hour at 85 ° C., 2 hours at 95 ° C., 3 to 5 hours at 100 ° C.). In addition to water, an organic solvent may be used as an extraction solvent, and an organic solvent may be water, methanol, acetone, ethanol, propanol, butanol, alcohol, or a mixed solvent thereof, but water, ethanol, or alcohol is more preferable. . Usually, it is extracted with an organic solvent for about 3 days. Water extract can be used by adding distilled water about 10 times of the sample and attaching a reflux condenser and filtering it for 3 hours at 100 ° C (Filter paper, No. 2). After adding 5 times the amount of ethanol (95%), it is better to use the resultant after filtering for 10 days at room temperature (Filter paper, No. 2), the acetone extract after adding 10 times acetone at room temperature overnight It is good to use it by filtering (Filter paper, No. 2). If necessary, the solids are filtered to obtain an extract solution containing lacquer components and used as an active ingredient in the form of a solution or a dry powder.

<Examples>

Hereinafter, the present invention is described in more detail by the following representative examples, but the present invention is not limited in any way by these examples.

Example 1: Cultivation of Mushroom Bacteria Using Lacquer Medium

Enteric mushrooms ( Fomitella fraxinea ), which are isolated and stored in the mycology laboratory of Iksan University, Ganoderma lucidum ( Ganoderma lucidum ), Roe deer fungus ( Hericium erinaceus ), Lyophyllum cinerascens , Pleurotus eryngii , Pleurotus ostreatus , Shiitake mushrooms ( Lentinus edodes ), Rabbit Fur Songp Mushroom ( Trametes trogii ), Janna Butterfly ( Ganoderma applanatum ), Fingerling mushroom ( Coriolus versicolor ), Phellinus linteus Basidiomycetes, such as chaga ( Inonotus obliquus ) and dog-rice mushrooms ( Tyromyces palustris ), were used as test strains.

After powdering the lacquer lacquer in Wonju, Gangwon-do, it was put into a pp bottle for mushroom culture, and then aseptically inoculated with the bacteriophage which is separated and stored in the bacteriology laboratory of Iksan University, and incubated at 25 ° C for about 30 days before detoxification of lacquer Investigation was also conducted (cm / day). The results are shown in Table 1 below.

Effect of Lacquer Powder Medium on Growth of Basidiomycetes Mushroom Growth length (cm / days) 4 8 12 16 20 24 28 Longevity Mushroom 0.90 1.83 2.76 4.07 5.31 6.23 7.58 Ganoderma lucidum mushroom 1.06 2.51 4.32 6.87 8.83 11.00 13.03 Roe deer mushroom 1.11 1.64 2.52 4.18 5.26 6.90 8.52 Mandala Mushroom 0.44 0.81 1.10 1.94 2.38 2.85 4.24 King Mushroom 0.73 1.57 2.35 3.63 4.53 5.63 6.68 Oyster mushroom 0.79 1.59 2.33 3.39 4.23 5.28 6.27 Shiitake mushrooms 1.16 2.18 3.27 4.95 6.40 8.07 9.67 Rabbit Fur Songpyeon Mushroom 0.84 1.83 2.95 4.68 5.93 7.43 9.06 Jan Butterfly Stool Mushroom 0.61 1.81 3.23 5.66 7.42 9.47 11.31 Fingering mushroom 1.46 2.88 4.98 7.85 10.04 12.32 14.48 Situation Mushroom 0.67 1.55 2.40 3.39 4.25 5.48 6.77 Dog Rice Cake 1.26 2.98 5.08 8.00 9.88 11.91 13.76

As a result of comparing the growth rate of 12 strains in lacquer medium, there were some differences according to the kinds of mushrooms. However, most mushrooms did not interfere with growth even when lacquer was used as a culture substrate. It was found to grow with detoxification, and especially the growth of woody fungi such as ganoderma lucidum and fingering was good.

Example 2: Mushroom growth rate test in rice mixed lacquer medium

20% (w / w) of rice bran was added to finely crushed lacquer, sterilized at 121 ° C. for 60 minutes, cooled to room temperature, and filled into a test tube (φ 21 cm x 20) at a height of 15 cm. Then, the bacteria were inoculated and then incubated at 25 ° C. for 30 days. In addition, the culture bottle was shaken every 2 days to prevent the medium from clumping. The growth length of the mycelium was measured over time and the results are shown in FIG. 1.

1 is a graph showing the results of growing mushroom fungi of basidiomycetes using rice bran and lacquer as a medium, and all three strains of Ganoderma lucidum mushroom, Janna hibiscus mushroom, and longevity mushroom are by-products such as rice or rice bran In addition, the growth rate was more than 10% over the whole period. Especially, in the Ganoderma lucidum and Jangnabi stool mushroom, rice bran added showed a slightly higher growth rate from the beginning of growth than other rice and rice by-products added. In addition, the growth rate of each strain was relatively low in longevity mushroom, but showed no statistical significance. Therefore, the addition of rice and rice by-products is more effective in promoting mushroom growth and detoxification of urushiol over a short period of time, since the addition of rice and rice by-products is supplemented with nutrient-enhancing and growth promoting substances. Could check.

Example 3: Preparation of extract containing active ingredient from lacquer medium

In order to extract the active ingredient for drugs or food from the culture, the extract was prepared by the following method using water, ethanol, acetone as the extraction solvent.

The water extract was added by distilled water 10 times of the sample and attached to a reflux condenser and filtered for 3 hours at 100 ° C. (Filter paper, No. 2), and the ethanol extract was 5 times ethanol (95%) of the sample. ) Was added and then extracted by standing at room temperature for 10 days and filtered (Filter paper, No. 2), and the acetone extract was added to 10 times acetone and left overnight at room temperature (Filter paper, No 2) and then used.

Example 4 Analysis of Urushiol in Mushroom Culture Lacquer Using TLC

After harvesting lacquer and lacquer cultures according to Example 2, lyophilization was isolated and crude urushiol was isolated by Nacheonsu et al. (1998, Journal of the Korean Forest Society, Vol. 87: 260-269) and the change of urushiol was confirmed by TLC. It was. 2 shows a TLC chromatogram of detoxified urushiol using soak fungus. 1: Tyromyces palustris , 2: Cloudy mushroom ( Coriolus versicolor ), 3: Pleurotus eryngii , 4: Ganoderma applanatum , 5: Shiitake ( Lentinus edodes ), 6: Herringium mushroom ( Hericium erinaceus ), 7: Ganoderma lucidum , 8: Chaga Mushrooms ( Inonotus obliquus ) , 9: Fomitella fraxinea , 10: Phellinus linteus , 11: Trametes trogii , 12: Rhus verniciflua Bark.

As a result of examining the change of crude urushiol according to the growth in lacquer cultured mushroom fungus on TLC plate, urushiol removal effect was high in longevity mushroom, ganoderma lucidum mushroom, and Janna mori mushroom.

Example 5 Determination of Lacasease Activity Involved in Ursiol Removal

The laccase activity from lacquer cultures was measured with a syringaldazine substrate according to the method of Petroski et al. (1980, Applied and Environmental Microbiology, 40: 1003) to determine the laccase activity and the results are shown in Table 2 below. Indicated.

Results of the measurement of laccase activity from culture medium of basidiomycetes Strain name Lacquer addition amount
(%, v / w) Lacasease Activity (Unit / days) 5 8 12 Longevity Mushroom 0 0.681 0.912 0.963 2 0.795 1.112 1.063 Gongji mushroom 2 0.408 0.593 0.865 Roe deer mushroom 2 0.055 0.065 0.081 Mandala Mushroom 2 0.002 0.001 0.001 King Mushroom 2 0.389 0.502 0.708 Oyster mushroom 2 0.211 0.153 0.351 Shiitake mushrooms 2 0.366 0.176 0.068 Earthenware Songpyeon Mushroom 2 0.019 0.001 0.012 Jan Butterfly Stool Mushroom 2 0.475 0.503 0.141 Fingering mushroom 2 0.001 0.002 0.001 Situation Mushroom 2 0.001 0.001 0.001 Dog Rice Cake 2 0.001 0.002 0.000

As can be seen from the above results, it was confirmed that when lacquer (2%) was added to the culture substrate, the production of laccase increased by more than 14%, and longevity mushroom ( F. fraxinea ) , belonging to the perforated mushroom , jannabi stool mushroom (G. applanatum), Ganoderma lucidum (G. lucidum) from me highly active show results similar to urushiol analysis by TLC analysis poison Blood wooreusi come to remove it was found that the closely related and lacquer kinase production .

Example 6: Preparation of Reference Material for Analysis of Ursiol in Lacquer

In order to quantify urushiol, an allergen in lacquer, silica gel absorption column chromatography, 3- (8'Z, 11'E, 13'Z-pentadecatrienyl) catechol as a main component from lacquer sap (raw chile), Purification was carried out using preparative LC and HPLC, and the molecular weight was confirmed by LC-MS analysis. 3 and 4 are shown. FIG. 3 shows an HPLC chromatogram of purified ursiol (right) separated from silica gel open column (left) of fraction (F-2) isolated from sap of lacquer tree (F-2) and FIG. 4 shows LC-MS data of isolated 3- (8'Z, 11'E, 13'Z-pentadecatenyl) catechol, respectively.

Urushiol was isolated from sequencing containing high concentrations of urushiol by column chromatography and preparative LC to isolate the main peak. As a result, it was possible to prepare a standard product of about 0.25 g showing a purity of 99% or more on HPLC. LC-MS identified the component as a 3- (8'Z, 11'E, 13'Z-pentadecatrienyl) catechol having a molecular weight of 314. Subsequent analysis was performed using this component as a reference substance. The analysis could be done.

Example 7: Change of urushiol content in lacquer skin according to mushroom growth

Based on the standard material, the urushiol content in the lacquer in which mushrooms were grown was analyzed by HPLC, and the results are shown in FIGS. 5 and 3. Figure 5 shows an HPLC chromatogram of ursiol from sumac bark detoxified by basidiomycetes. A: otpi (Rhus vernicifulua), B: detoxified otpi with longevity mushroom (Rhus verniciflua).

Urushiol Contents of Detoxified Lacquer Tree Using Basidiomycetes Strain Urushiol Content (mg / 100g dry weight) C _{15: 3} C _{15: 2} C _{15: 1} C _{15: 0} Total D.T. (%) Lacquer 95.70 9.09 42.57 6.79 154.15 - Longevity Mushroom 5.10 0.53 4.35 0.75 10.73 93.0 Gongji mushroom 7.39 0.31 5.39 0.57 13.66 91.1 Roe deer mushroom 21.11 0.95 15.20 3.19 40.45 73.8 Mandala Mushroom 46.65 4.10 26.75 4.58 82.08 46.8 King Mushroom 19.57 0.85 11.61 2.58 34.61 77.6 Oyster mushroom 16.53 1.14 12.79 1.78 32.24 78.9 Shiitake mushrooms 8.30 0.74 6.73 1.46 17.23 89.8 Rabbit Fur Songpyeon Mushroom 9.50 0.54 6.88 1.30 18.22 88.2 Jan Butterfly Stool Mushroom 10.49 0.56 5.89 0.88 17.82 88.4 Fingering mushroom 99.78 3.38 42.65 6.08 151.89 1.46 Situation Mushroom 16.71 0.79 11.01 2.29 30.8 80.0 Dog Rice Cake 81.42 6.18 39.95 7.56 135.11 12.4

DT (%) shows the degree of detoxification, C _{15: 3} : 3- (8'Z, 11'E, 13'Z-pentadecathenyl) catechol, C _{15: 2} : 3- (8 'Z, 11'Z-pentadedecenyl) catechol, C _{15: 1} : 3- (8'Z-pentadecenyl) catechol, C _{15: 0} : 3-pentadedecyl catechol.

As a result, it was very similar to the TLC pattern, and it was confirmed that the mushroom can effectively remove allergy-inducing urushiol. In particular, longevity mushroom, ganoderma lucidum mushroom, and Jang butterfly mushrooms showed high removal rate of 93%, 91%, 89%, respectively, and longevity mushroom was suitable as detoxification strain of lacquer.

Example 8: Changes in Ursiol Content in Lacquer with Growth of Mushroom Bacteria

Time- dependent changes in urushiol content by the growth of F. fraxinea mycelium in rice bran lacquer medium and the results are shown in the graph of FIG. All of the rice bran (20%, w / w) and non-additives were removed about 30% of urushiol by the sterilization process, ie, simple heat treatment before inoculation, but the urushiol was decreased at a high rate as the bacteria grew. In particular, rice bran added showed a higher removal rate at the beginning of growth than non-added rice, but the difference was relatively small after 5 days of growing more than 90% of toxic ursiol. When proliferated for 15 days, 93% of non-added rice and 98% of rice bran added showed very high ursiol removal ability. In addition, toxic ursiol, which is poorly soluble in water in detoxified lacquer biologically modified by mushroom bacteria, is not detected at all in hot water extracts even in the presence of thorough detoxification by mushroom bacteria and the presence of various emulsifying components. I could confirm it.

Example 9: Physiological Activity of Lacquer Extract Extracted with Mushrooms

In order to confirm the functionality of the detoxified lacquer prepared by rice bran and mushroom fungus treatment in Example 3, polyphenol content analysis and antioxidant activity, ACE (Angiotensin Converting Enzyme) inhibitory activity, Tyrosinase inhibitory activity, nitrite scavenging activity as functional ingredients Physiological activities such as hyperlipidemia inhibitory effect, gene expression associated with geriatric neurodegenerative disease and cytotoxicity against cancer cell line were investigated.

9-1: Total Polyphenol Content Analysis

The total polyphenol content of physiologically active functions such as antioxidant effect in lactobacillus detoxified by mushroom fungi was determined by Folin-Ciocalteu's coloration method according to the method of Rhee et al. (1981, J. Food science, 46: 75-77). Was measured and the results are shown in Table 4.

Polyphenol Contents of Lacquer Tree Detoxified Using Basidiomycetes Strain name Total phenols
(mg%) Monomer
(mg%) Monomer content ratio in total phenols Lacquer 171.50 16.69 9.73 Longevity Mushroom 41.42 2.83 6.84 Gongji mushroom 43.90 2.82 6.42 Roe deer mushroom 51.20 8.16 15.94 Mandala Mushroom 60.69 13.06 21.51 King Mushroom 53.92 7.94 14.72 Oyster mushroom 50.98 5.47 10.72 Shiitake mushrooms 54.29 4.09 7.53 Rabbit Fur Songpyeon Mushroom 53.77 5.00 9.30 Jan Butterfly Stool Mushroom 46.69 2.30 4.94 Fingering mushroom 154.56 19.86 12.85 Situation Mushroom 46.99 5.78 12.31 Dog Rice Cake 159.07 18.85 11.85

As a result, 7 ~ 75% (catechol weight / w) was reduced by mushroom growth, and the ratio of monomeric phenol in the total polyphenol content was relatively similar or rather high.

9-2: antioxidant activity

Antioxidant activity of the detoxified lacquer extract was searched for DPPH as a substrate according to Blois (1958, Nature, 181: 1990-1200) method and the results are shown in Table 5 below.

Antioxidant Activity of Lacquer Bark Detoxified with Basidiomycetes Strain name IC ₅₀ (μg) Water extract Ethanol Extract Acetone Extract Lacquer 21.27 25.80 22.14 Longevity Mushroom 83.57 51.66 55.77 Gongji mushroom 65.58 44.69 50.82 Roe deer mushroom 59.67 48.99 65.62 Mandala Mushroom 52.12 26.49 45.62 King Mushroom 65.42 78.50 43.69 Oyster mushroom 59.11 40.20 63.74 Shiitake mushrooms 105.96 54.84 68.90 Rabbit Fur Songpyeon Mushroom 52.56 35.18 59.54 Jan Butterfly Stool Mushroom 74.08 44.41 43.26 Fingering mushroom 33.26 15.30 26.11 Situation Mushroom 41.44 62.81 44.11 Dog Rice Cake 32.13 19.08 25.39 BHA 14.09 BHT 83.62

As a result, the antioxidant activity of the detoxified lacquer extracted from each of the three solvent systems had similar activities regardless of the solvent, and showed a similar pattern to the urushiol content. In addition, each of the solvent-based extracts were mostly slightly lower than the antioxidant activity of BHA but had 1.5 to 4 times higher or similar activity than the antioxidant activity of BHT.

9-3: nitrite scavenging activity

Nitrite scavenging activity of detoxified lacquer was measured in accordance with the methods of Kato et al. (1987, Agricultural and Biological Chemistry, 51: 1333-1338) and Kim et al. 7 is shown. 7 is a graph showing the nitrite scavenging activity of the water extract of lacquer bark detoxified with basidiomycete.

The extracts showed pH dependence regardless of solvent system and showed the best scavenging activity at pH 1.2. In particular, when the pH of the water and ethanol extracts were adjusted to 1.2, most of the treatments showed a complete inhibitory effect, and all treatments had more than 90% nitrite scavenging activity. In particular, the ethanol extract of the Zinabi hibiscus mushroom showed a complete inhibitory effect over the entire pH range.

9-4: ACE (Angiotensin Converting Enzyme) Inhibitory Activity

The ACE inhibitory activity of the detoxified lacquer was measured according to the method of Cushman and Cheung (1971, Biochemical Pharmacology, 20: 1637-1648), and the results are shown in Table 6 below.

ACE Inhibitory Activity of Lacquer Bark Extracted as a Molecular Fungus Strain name % Inhibition Water extract Ethanol Extract Acetone Extract Lacquer 25.6 27.3 29.7 Longevity Mushroom 14.2 21.4 32.0 Gongji mushroom 19.4 27.9 26.5 Roe deer mushroom 24.6 20.6 25.2 Mandala Mushroom 20.6 27.7 33.1 King Mushroom 28.1 25.6 38.0 Oyster mushroom 7.3 27.3 25.4 Shiitake mushrooms 20.7 12.6 35.2 Rabbit Fur Songpyeon Mushroom 22.6 25.6 32.1 Jan Butterfly Stool Mushroom 26.1 12.0 30.1 Fingering mushroom 25.9 24.8 38.0 Situation Mushroom 29.6 22.7 28.0 Dog Rice Cake 29.0 30.0 37.0

As can be seen from the results of the above table, the ACE inhibitory activity of the detoxified lacquer showed a little higher activity in the acetone extract than in the water extract and the ethanol extract, but did not show any significant inhibitory activity.

9-5: Tyrosinase Inhibitory Activity

Tyrosinase inhibitory activity of the detoxified lacquer was measured according to the method of Jung et al. (1995, Korean Food Science Society, 27: 891-896) and the results are shown in Table 7 below.

Tyrosinase Inhibitory Activity of Water Extracts of Lacquer Bark Extract Using as a Basidiomycetes Mushroom % Inhibition Lacquer 89.02 Longevity Mushroom 94.82 Ganoderma lucidum mushroom 99.39 Roe deer mushroom 74.24 Mandala Mushroom 98.78 King Mushroom 90.40 Oyster mushroom 68.90 Shiitake mushrooms 81.40 Rabbit Fur Songpyeon Mushroom 64.94 Jan Butterfly Stool Mushroom 100.00 Fingering mushroom 85.98 Situation Mushroom 98.02 Dog Rice Cake 100.00

As can be seen from the above results, the result showed that the tyrosinase inhibitory activity of the lacquer water extract, which was not treated, was about 89%, while the water extracts of the worms, chaga, and fungi were almost completely inhibited. The longevity treatment with the highest urushiol removal activity also showed high inhibitory activity of about 95%.

7-6: Antihyperlipidemic Effect

In order to confirm the therapeutic effect of hypertriglyceridemia among the bioactive functions of the lacquer extract, 7F2 cells were grown on a 75cm culture flask using DMEM as a standard medium for the water extract and ethanol extract of Example 3. (adipogenesis) was promoted, Lipoprotein Lipase (LPL) activity was measured according to the method of Nilsson-Ehle and Schotz (1976, J. Lipid Research, 17: 536-541) on the 6th day of culture and the results are shown in FIG. It was. Figure 8 is a graph showing the effect on the LPL activity of water and ethanol extract of detoxified sumac bark. A; Sumac bark, B; Ganoderma lucidum, C; Longevity mushroom, D; Roe deer fungus E; Fingering mushroom, F; Dog rice cake, G; Situation mushroom.

As a result, when the same amount of water and EtOH extracts were treated, lipoprotein lipase activity was increased even in the lacquer extract treatment, which was not treated, but it was confirmed that the activity of lipoprotein lipase was significantly increased in the fungus treatment compared to the lacquer treatment, which was the control. Japanese lacquer was considered to have a high antihyperlipidemic effect.

9-7: Expression of Gene (TH; Tyrosine hydroxylase) Related to Geriatric Degenerative Diseases

(a) gene expression

After treatment with detoxified lacquer extract in PC 12 cells and mouse, cell harvest or organs were extracted at predetermined time intervals to separate total RNA and perform RT-PCR to obtain cDNA. After PCR, TH gene expression was confirmed by electrophoresis on 1.2 ~ 1.5% agarose gel.

(b) In-vitro (PC12)

The mRNA of TH, a rate-limiting enzyme of Dopamine biosynthesis in PC12 cells, was dependent on the throughput and treatment time, and showed twice as strong expression when treated at 10 ㎍ for 8 hours.

(c) In-vivo (rat)

Similar to pc12, rat brain was also dependent on treatment concentration and time, and was expressed at 10mg / kg concentration rather than 20mg / kg concentration. In particular, after the treatment at 10mg / kg concentration was increased by 1.6 times the amount of expression up to 4 hours, then decreased and then strongly expressed again after 12 hours. In addition, we observed changes in TH-IR (immunoreactive) neurons through immunohistochemical staining. As a result, VTA (ventral tegmental area), SNR (substantia nigra, reticular), SNC ( Substantia nigra, compacta) showed higher densities of TH-IR neurons. In particular, dopamine was strongly expressed in the SNC region where dopamine was synthesized and secreted by dopaminergic neurons, and increased cell body size due to increased expression of TH-IR neurons, which exist independently. It seems to be.

Figure 9 is an electrophoresis result showing the induction of tyrosine hydroxylase expression by detoxified lacquer bark extract in the rat brain region. N: untreated, PCR product was observed as a band on 1.2% agarose gel.

  10 is a graph showing induction of tyrosine hydroxylase expression by detoxified lacquer bark extract in PC12 cells. The insert shows over time the induction of TH mRNA at 10 μg. N: untreated, PCR product was visualized as a band on 1.2% agarose gel.

11 is a photograph showing the results of immunohistochemical detection of TH by detoxified sumac extract in substantia nigra after 4 hours (# 200). A, B: untreated, C, D: detoxified lacquer extract, VTA: Ventral Tegmental Area, SNC: Substantia nigra, compact, SNR: Substantia nigra, reticular.

9-8: Cytotoxicity Against Cancer Cell Lines

To examine the cytotoxicity of hot water and ethanol extracts of detoxified lacquer, first, cytotoxicity was measured against normal cell line NIH3T3 (Rat fibropblast cell line) cells. Did.

Subsequently, blood cancer (T24) and bone marrow cancer (Saos) cells were grown on 75 cm ² culture flasks using RPMI 1640 and MEM containing 10% FBS as standard medium and harvested to obtain 1 x 10 ³ cell / well. After adjusting to the detoxified lacquer extract, cytotoxicity was measured by MTT assay. The results are shown in FIGS. 12 and 13. 12 is a graph showing the cytotoxicity of detoxified sumac bark extract against NIH3T3. Figure 13 is a graph showing the effect on the cytotoxicity of the detoxified sumac extract against hematological cancer (T24) and bone marrow cancer (Saos) cells.

As a result, hematocrit (T24) and bone marrow (Saos) cell lines were dependent on incubation time and does, and cytotoxicity increased with increasing time and throughput, and showed similar tendency regardless of the extraction solvent. In addition, blood cancer cells showed higher cytotoxicity than bone marrow cancer cells, and when treated at a concentration of 150 µg / 200 µl or more, cytotoxicity against cancer cells was about 80% after 72 hours.

Example 10: Pharmaceutical Formulations

Form of formulation (tablets)

50 mg of active ingredient (lyophilized product of extract of Example 3)

Lactose 50 mg

Starch 15 mg

2 mg magnesium stearate

10 mg of crystalline cellulose

Tablets were prepared by conventional methods and contain the above ingredients as raw material in one tablet. Tablets may be added as usual, enteric coat (eg hydroxypropylmethylcellulose phthalate), sugar coating or film (ethyl cellulose).

Example 11: Preparation of Beverages

10 g of the detoxified lacquer mushroom extract obtained in Example 3 (pH 6.5) was added to 1 L of water, and 3 mg of citric acid, 1.7 g of aspartame, and 30 mg of vitamin C were added thereto to prepare a beverage and pasteurization. As a control of drinking water, Cordyceps sinensis (Ilyang Pharmaceutical) was used. As a result, the beverage of the present invention was not different from conventional beverage products in terms of color, flavor, mouth feel, sweetness, and overall evaluation.

As described so far, according to the present invention, it is possible to efficiently detoxify the toxic components of lacquer using grains and mushroom fungi, as well as the nutritional food of mushroom hyphae and mushrooms and cereals containing detoxified lacquer. Not only can the ingredients be used together as a medicinal or food ingredient, but also grains have the advantage of significantly shortening the culture rate of mushrooms and the duration of detoxification accordingly.

Claims (6)

a) inoculating a mushroom strain on a medium containing grains selected from the group consisting of rice, brown rice, brown rice glutinous rice, rice bran, black rice, grains, barley, soybean, and mixtures thereof and by-products and lacquer ingredients, and b) culturing the grain-containing lacquer medium for 7 days to 30 days at a temperature of 15 ~ 37 ℃ Method for removing the toxicity of lacquer, characterized in that it comprises a. According to claim 1, The lacquer medium is prepared by using the bark of the lacquer tree as it is or powdered, or by adding a conventional agar medium to the sap extracted from the sap or lacquer bark extracted from the lacquer tree in water or an organic solvent and distilled Using a medium. The method of claim 1, wherein the mushroom strain is Fomitella fraxinea , Ganoderma lucidum ( Ganoderma lucidum ), Roe deer fungus ( Hericium erinaceus ), Lyophyllum cinerascens , Pleurotus eryngii , Pleurotus ostreatus , Shiitake mushrooms ( Lentinus edodes ), Rabbit Fur Songp Mushroom ( Trametes trogii ), Janna Butterfly ( Ganoderma applanatum ), Fingerling mushroom ( Coriolus versicolor ), Phellinus linteus Tympyceps mushroom ( Tyromyces palustris ) or a mixture thereof. A composition for the treatment of dementia, hyperlipidemia, bone marrow cancer and hematological cancer, which contains a mixed extract of detoxified lacquer extract, cereals and mushroom mycelium according to claim 1 as a main component. delete delete

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