KR100314608B1 - Methods and foods using the extract of Scutellaria baicalensis as an inductive material of Quinone reductase activity - Google Patents

Abstract

The present invention relates to a method of using the gold extract as an activity inducer of quinone reductase (Finone reductase) and to a food containing the same, more specifically, the gold extract induces the activity of quinone reductase to prevent cancer By combining it with foods such as chewing gum, candy, biscuits, ice cream, beverages, sausages and chocolates, it can be easily consumed anytime, anywhere to a wide range of teenagers, adults and the elderly, and expect continuous drinking effects. It relates to a food compound containing a golden extract having a cancer prevention effect.

Description

Methods and foods using the extract of Scutellaria baicalensis as an inductive material of Quinone reductase activity}

The present invention relates to a method of using the gold extract as an activity inducer of quinone reductase (Finone reductase) and to a food containing the same, more specifically, the gold extract induces the activity of quinone reductase to prevent cancer By combining it with foods such as chewing gum, candy, biscuits, ice cream, beverages, sausages and chocolates, it can be easily consumed anytime, anywhere to a wide range of teenagers, adults and the elderly, and expect continuous drinking effects. It relates to a food compound containing a golden extract having a cancer prevention effect.

Cancer is one of the causes of death in Korea, and 51.1% of all cases die within a year after knowing the onset of disease, and the treatment results have been steadily improving, but medical researchers report that the cure rate will not exceed 50% by the end of the 20th century. Each year, there are 8 million cases worldwide, of which 5 million are the worst of all.

Carcinogens that cause such cancers include food, smoking, ultraviolet rays, chemicals, and other environmental factors, and the reality is that humans have to be exposed to these carcinogens by will or by other means.

Therefore, there is an urgent need for the development of cancer prevention substances, and the development of such cancer prevention substances can be very useful for inhibiting or treating all diseases as well as cancer. For this reason, as the development of cancer prevention substances is recognized as a new direction of cancer research and attention is focused, the development of cancer prevention substance materials is active not only in the real medicine field, but also in the food chemical field, so that research on natural product materials is active. In fact, it is also being introduced into the food sector.

As a result, many analytical systems using microorganisms and animal cells have been established and utilized for the search for new cancer prevention substances, and active biological studies of pure separation of active ingredients and molecular mechanisms of action are also in progress.

Early onset of various tumors is caused by damaging DNA by carcinogens, mutagenic substances, and reactive oxygen species, and mammalian cells have complex and sophisticated mechanisms of action that protect against such damage. In other words, when carcinogens with electron affinity are inactivated by detoxifying enzymes and released into the body, they do not progress to cancer, but they form a complex with DNA and undergo a mutation or transformation process. It is going to proceed.

In particular, among the above detoxification enzymes, Phase II detoxification enzyme exhibits specific cytotoxicity against various cancer cells and thus damages DNA caused by carcinogens, mutagenic substances, and reactive oxygen species such as having anticancer function. It is known to have a protective action against. Such Phase II detoxification enzymes include glutathione sulfur transferase (GST), NAD (P) H: quinone reductase (QR), and epoxide hydrolase. ), Glucuronosyltransferase, aldehyde reductase and the like.

Of the Phase II detoxification enzymes, quinone reductase (EC 1.6.99.2) is an enzyme that blocks interaction with DNA by mutations or carcinogens, and uses NAD (P) H. It is a flavoprotein that catalyzes the reduction of quinones. In addition, the molecular weight is 54,000 daltons, the same structure of two (homodimer) each composed of 273 amino acids, the active site flavin containing flavin adenine dinucleotide (FAD) Boproprotein (flavoprotein), also known as menadione reductase (menadione reductase), vitamin K reductase (vitamin K reductase), DT-diaphorase (DT-diaphorase).

These quinone reductases have a number of characteristics, firstly, using NAD (P) H as an electron donor, and secondly, not shifting two electrons to form a semiquinone, and thirdly, dicumanol It is strongly inhibited by anticoagulants such as dicoumarol. Fourth, it is induced by various foreign substances in many cells and tissues. In particular, these quinone reductases have a protective effect on quinones themselves, and are commonly induced with other cancer prevention enzymes and are induced by many compounds with anticancer activity. Ⅱ) Among the detoxification enzymes, it can be selected as a representative enzyme for the search for cancer prevention substance [Lind, C., Vady, H. and Ernster, L. 1978, Arch. Biochem Biophys. 190: 97-108; Wefers, H., Komai, T., Talalay, P. and Sies, H. 1984. FEBS Lett. 169: 63-66.

Meanwhile, various kinds of animal cells can be used to more quickly, simply and accurately measure the quinone reductase, and in the present invention, a mouse liver cell line (Hepa 1c1c7 murine hepatoma cell line) has been introduced. Substances exhibiting quinone reductase-inducing activity by these cell lines include butyl hydroxy anisole (BHA), butyl hydroxy toluene (BHT) and tertiary-butyl hydroxy quinone (t-BHQ). Phenolic antioxidants; Lipophilic azo dyes of 1,1'-azonaphthalene and sudan I, III; Polycyclic aromatic carbons; Coumarin and lactones; Flavonoids; Benzyl isothiocyanate, sulfur compounds including dithiolthiones and dithiocarbamate. Among them, cruciferous plants (broccoli, cabbage, kale, cauliflower, etc.), liliaceae plants (onions, garlic) and Vaccinium plants (lowbush, blue-berry) ), Bilberry (cranberry), cranberry (lingonberry)] is reported to have a strong quinone reductase (kinin reductase) induction activity. Among the above, in particular, the most active research to date and the strongest inducing agent is sulfoaphane (CH ₃ -S (O)-(CH ₂ ) ₄ -N, an active substance of Brassica oleraceae ). = C = S) is a kind of isothiocyanate and has been studied a lot of induction mechanisms [Gerhauser, C., You, M., Liu, J., Moriarty, RM, Hawthorne, M. , Mehts, RG, Moon, RC and Pezzuto, JM 1997. Cancer Research 57: 272-278; Posner, GH, Cho, CG, Green, GV, Zhangg, Y. and Talalay, P. 1994, J. Med. Chem 37: 170-176.

The present inventors have made efforts to keep pace with these trends, and as a result, the gold extract extracts a higher or equivalent amount of quinone reductase activity compared to the active inducers of the existing quinone reductase. It was confirmed that it has a preventive effect.

Scutellaria baicalensis George is a perennial herb belonging to the labiatae , and its main origin is Northeast Asia including Korea and China, and is grown or grown in Northeast Asia including China and Siberia in the north of Yangtze River. In particular, in China, a variety of Scutellaria ( Scutellaria ) plants are distributed in the mid-North to the north-east. Gold is also known as deciduous grass, and other names are general managers (new agricultural plants), inner gardens, factories, gold plants, yellow gates (namebooks), and pharynxes (obovones). It is 20 ~ 60 ㎝ high and is a single plant that blooms purple flowers between July and September. It can be cultivated throughout Korea. It is mainly used for medicinal purposes, but also for ornamental or edible use.

The root of the golden root used in the present invention is fusiform or semi-tubular, and has a length of 8 to 20 cm and a diameter of 5 to 20 mm. The outer surface is coarse in shape and has a horizontal wrinkle, and the marks are attached to the entourage in various places. It is mainly used as a medicine and is used for the treatment of anti-inflammatory antipyretic, analgesic, branch, and atherosclerosis hypertension. In addition, it is recorded in the dictionary of medicinal herbs, 'the death of the true story, the elimination of the moist heat, hemostasis, has the effect of anxiety'.

The representative component of the gold is known as flavonoids (flavonoids), and about 4,000 kinds of flavonoid compounds (flavonoid) compounds are known in the plant kingdom to date. Since the 1970s, studies on the composition of gold began in earnest using scientific analyzers. Since then, the ginseng, etc. (Bussam, Gillonil Bum, Jung Bon Gil-lung, Mokjin Jig. 1984. Research on plant composition of Scutellaria (3rd) (Investigations have been carried out by Golden Flavorinoid Ingredients. Pharmacy Magazine 104: 524-528). So far, baicalin, baicalein, and ogonin-7-O -Glucuronide (Wogonin-7-O-glucuronide), Ogonin (Wogonin), Oroxylin AO-Glucuronide (Oroxylin A-7-O-glucuronide), Oroxylin A (Oroxylin A), Schoolcap About 60 flavonoid compounds such as flavone I (Skullcapflavone I), Dihydrooroxylin A (Baicalein-7-O-glucoside) and the like have been identified. These ingredients were not so noticeable because they were so much in the plant. In recent years, various pharmacological activities have been reported, and more studies have been reported on the production of higher amounts of flavonoids through plant culture of gold [Seo, WT, Park, YH and Choe, TB 1993. and production of flavonoids in a cell suspension culture of Schutellaria baicalensis G. Plant Cell Reports.12: 414-417.

In addition, a golden alcohol disorder prevention and treatment composition is known in [Korean Patent Publication No. 96-40370], and a cataract treatment agent of a golden flavonoid component is known in [Korean Patent Publication No. 96-3725], A periodontal disease prevention and treatment composition comprising the golden extract as an active ingredient is proposed in [Korean Patent Publication No. 94-11006]. However, the above described technology is not yet widely used.

In particular, despite many studies of natural substances that exhibit cancer prevention effects today, there has been no research showing that the golden extract has a cancer prevention effect as shown in the present invention, and it is also used as a beverage, chewing gum, candy, biscuits. And no application to foods such as ice cream.

The inventors have confirmed that the golden extract has the effect of preventing cancer by inducing the activity of quinone reductase (Quinone reductase), and completed the present invention by applying it to food.

Therefore, the present invention by adding and blending the golden extract to food to avoid the conventional method of using the golden raw material and its extract as a conventional herbal medicine, the various pharmacological activity of the golden itself, namely anti-inflammatory antipyretic, analgesic, It provides foods containing golden extracts that can prevent cancers by inducing quinone reductase activity, as well as treating the effects of branch offices, atherosclerosis hypertension and cataracts and preventing alcohol disorders. Its purpose is.

Figure 1 shows the effect of inducing quinone reductase (Quinone reductase) at a concentration of 25 ~ 100 ㎍ / ㎖ of gold extract using mouse hepatocytes (mouse, hepatoma cells; Hepa 1c1c7 cells),

Figure 2 shows the effect of inducing quinone reductase after administration of Sprague-Dawley rats at a concentration of 200 ~ 1,000 mg / kg golden extract.

The present invention is characterized by a method of using the gold extract as an activity inducer for quinone reductase.

In addition, the present invention includes a food extract containing 0.1 to 1.0% by weight of the total extract as an activity inducing substance for quinone reductase (quinone reductase).

The present invention will be described in more detail as follows.

The present invention relates to a new use of the golden extract to have a cancer prevention effect by using the golden extract as an activity inducer of quinone reductase.

In general, a method of obtaining a golden extract may include a solvent extraction using ethanol or the like, or a single hot water extraction. In the present invention, ethanol is used as a solvent and extracted using ultrasonic waves. .

First, ethanol was added to gold, a sample, and extracted repeatedly by ultrasonic wave for 1 to 4 times for 20 to 40 minutes. Then, the filtrate was obtained using Toyo filter No. 2 at 45 to 55 ° C. with a rotary vacuum evaporator. Concentrated under reduced pressure and the concentrate was lyophilized to obtain a yellow golden dry powder, which was stored at a temperature of -70 ℃. At this time, if the temperature under reduced pressure concentration is more than 55 ℃ damage to the active material and the like.

Here, as described above, in obtaining the golden extract from the golden raw material, since ethanol is used, it is possible to obtain the advantage that it is possible to extract a substance having a low polarity as compared to simply using water, and by using ultrasonic waves, it is more effective than the extraction or shaking extraction There is an advantage that can be extracted effectively.

As a result of measuring the quinone reductase induction effect on the golden extract prepared by the above manufacturing method, it was confirmed that the cancer prevention effect is quite excellent, and chewing gum, candy, biscuits, ice cream, drinks, sausage and It can be applied to chocolate bar, etc. By combining it with food, it can be easily consumed anytime, anywhere up to a wide range of adolescents, adults and the elderly can be expected continuous drinking effect.

As in the present invention, when the golden extract is applied to food, it is preferable to use the golden extract in an amount of 0.1 to 1.0 wt% based on the total composition. If the content is less than 0.1% by weight, the present invention may not achieve the desired effect, and if it is added in excess of the above range, it may be uneconomical and affect the taste and color of the finished product. It is not desirable because it can be crazy.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited by Examples.

Comparative Experimental Example: Screening of Quinone Reductase Inducer from Natural Products

To select the most inducible inducers of quinone reductase, 72 medicinal plants, 10 cereals, 66 vegetables and fruits, spices and 27 other ethanol known as quinone reductase active inducers A total of 218 species, including 43 supernatants of the microorganisms extracted from the extract and Chunggukjang, were investigated for their effects on quinone reductase activity in the Hepa 1clc7 cell culture system. The results are shown in Tables 1a to 1j below.

As shown in Tables 1a to 1j, cheonnyeon, dill, juniper, yew, golden, pine, safflower, oak, chestnut, prunus oak, walrus, cacao bean husk, cucurbita, broccoli, A total of 23 species, including celery, garlic stem, watermelon, pineapple, deraware, coriander, acaricus, c18-4 and c19-2, showed more than 50% induction activity compared to the control group, and quinone reductase induction activity. This strongest golden extract was finally selected.

Reference Example: Animal cell line culture and experimental animal breeding

Mouse-derived hepatocytes (Hepa 1c1c7 cell line) used in the present invention were purchased from the American Type Culture Collection (ATCC CRL 2026), 2 mM glutamine (glutamine), 100 U Minimum essential medium alpha modification (α-MEM) medium containing / ml penicillin G, 100 μg / ml streptomycin, 10% petal bovine serum, and the like Was used in the experiment while subcultured in a CO ₂ incubator at 37 ℃ 5% CO ₂ /95% air environment.

On the other hand, the experimental animals were used to receive a three-week-old Sprague-Dawley rat females of about 85g body weight from the animal nursery of the National Institutes of Health. The animal breeding environment was maintained under the conditions of artificial lighting (AM: 08-PM: 08), illumination intensity 300-500 lux, temperature 23 ± 1 ° C, exhaust 10-18 times / h at 12 hour intervals, and solid feed (Samyang feed). ) And drinking water freely.

Example: Isolation of Golden Extract

The disclosure material used in the present invention was purchased by domestic gold in the Gyeongdong market to remove foreign matter and finely used, the extraction solvent was used as a first-class reagent, the remaining reagents are US Sigma (Sigma) products and special reagents It was.

5 g of ethanol was added to 500 g of gold, a sample, and extracted three times for 30 minutes using ultrasonic waves (Nihonseiki kaisha, Model No. NS-300), and the filtrate was obtained using a Toyo filter No. 2 to obtain a vacuum vacuum evaporator. The mixture was concentrated under reduced pressure at 50 ° C. and the concentrate was lyophilized to obtain a yellow golden dry powder. The sample was then used while stored at -70 ° C.

Experimental Example 1 Measurement of Inhibitory Activity of Quinone Reductase Using Mouse Hepatocytes

Quinone reductase (E.C. 1.6.99.2) deactivation assay was measured by Prochaska H. J. and Santamaria A. B. 1988. Anal. Biochem. 169: 328-336, 1988] and some modifications of the method presented.

First, the same number (1.0 × 10 ⁴ cells / ml) of mouse hepatocytes (Hepa 1c1c7 cells) were dispensed into each well of a 24 well plate, and cultured 24 hours before. After adding the golden extract prepared through, incubated again for 24 hours, and then the medium was removed. Next, 250 μl of Lysis buffer (lysis buffer (10 mM Tris-Cl, pH 8.0, 140 mM NaCl, 15 mM MgCl ₂ , 0.5% NP-40)) was added to each well, followed by Cells were lysed by shaking slowly for 10 minutes under temperature conditions. To this, 1 ml of enzyme solution was added and allowed to react for 5 minutes, followed by 250 µl of reaction stopping solution [0.3 mM dicoumarol, 0.5% pyridine, 5 mM potassium phosphate, pH 7.4)], and then the absorbance at 610 nm was measured. In addition, the amount of protein was quantified by crystal violet staining on the same set of well plates. At this time, the enzyme solution is the final concentration of the reaction solution 10 mM Tris buffer (Tris-Cl, pH 7.4), 0.5 mg / ml bovine serum albumin (BSA), 0.008% Tween-20, 40 μM flavin adenine dinucleotide (FAD), 0.8 mM glucose 6-phosphate, 2 U / ml glucose 6-phosphate dehydrogenase, 25 μM nicotinamide adenine dinucleotide (NADP), 40 μg / ml 3- (4,5-dimethyl-thiazo-2-yl) -2,5-diphenyltetrazolium bromide [3- (4,5-dimethyl-thiazo-2-yl)- 2,5-diphenyltetrazoliumbromide (MTT)] and 1 mM menadione were used together.

Then, the quinone reductase activity was calculated using Equation 1, and the results are shown in the accompanying drawings. In this case, a solvent in which the golden extract was dissolved was used as a control.

According to the accompanying drawings, the yellow gold extract was treated with hepatic cells (Hepa 1c1c7 cells) of mice at concentrations of 25, 50, 75, and 100 ㎍ / ml, and then examined for quinone reductase induction activity. As the concentration increased, they increased 1.36, 1.52, 1.67, and 1.81 fold, respectively. Through the above results, it was found that the golden extract has quinone reductase inducing activity.

Experimental Example 2 Measurement of Inhibitory Activity of Quinone Reductase Using Splog Dooli Rats

Sprague-Dawley rats were tested with gold extract divided into 5 groups, including the normal group, and the dosage was 0, 200, 500, 1000 mg / kg for 2 weeks. Oral administration over Then, the liver of the mouse was homogenized with a homogenizing buffer (homogenizing buffer (0.25 M sucrose, pH 7.4)), centrifuged at 9,000 xg for 20 minutes (4 ° C), and the supernatant was taken again for 1 hour at 100,000 xg. The supernatant obtained by ultracentrifugation (4 ° C) was used for the measurement of quinone reductase induction effect. And quinone reductase activity was calculated using the same method as Experimental Example 1, and the results are shown in the accompanying drawings.

According to the accompanying drawings, when the gold extract 200, 500, 1000 mg / kg treatment showed 1.2, 1.46, 1.59-fold induction activity, respectively. This indicates that quinone reductase activity is induced by gold extract not only in mouse hepatic cell system (Hepa 1c1c7 cell culture system), but also in vivo system.

In addition, as a result of measuring the body weight after oral administration for 2 weeks, no significant difference was observed between the normal group and the golden extract administration group. It did not cause change.

In addition, blood glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) activities, which are indicators of hepatotoxicity, were prepared using conventional methods. The measured results did not show any significant difference compared to the normal group.

Preparation Example 1 Preparation of Chewing Gum (A)

Gum base 20%

Sugar 76.36-76.76%

Golden extract (Example) 0.24-0.64%

1% fruit flavor

Water 2%

Chewing gum was prepared using the conventional method according to the composition and content.

Preparation Example 2 Preparation of Chewing Gum (B)

Gum base 20-30%

Sorbitol 66.86-77.26%

Golden extract (Example) 0.24-0.64%

Peppermint Flavor 2.5%

Chewing gum was prepared using the conventional method according to the composition and content.

Preparation Example 3 Preparation of Candy (A)

50 to 60% sugar

Starch syrup 39.26-49.66%

Golden extract (Example) 0.24-0.64%

Orange flavor 0.1%

Candy was prepared using the conventional method according to the composition and content.

Preparation Example 4 Preparation of Candy (B)

Maltitol 50-60%

Reduced syrup 39.22-49.62%

Golden extract (Example) 0.24-0.64%

Natural Herbal Flavor 0.14%

Candy was prepared using the conventional method according to the composition and content.

Preparation Example 5 Preparation of Biscuits

Force Class 1 88 kg

Gravity First Class 76.4 ㎏

16.5 kg per white

2.5 kg of salt

2.7 kg of glucose

Palm shortening 40.5 kg

5.3 kg of ammo

Medium kg 0.6 kg

0.55 kg sodium bisulfite

Rice flour 5.0 kg

Vitamin B1 0.003 kg

0.003 kg of vitamin B2

Milk Flavor 0.16 ㎏

71.1 kg of water

Whole milk powder 4 kg

Substitute powder 1 kg

0.1 kg of calcium phosphate

Spray salt 1 kg

25 kg of spray oil

Golden extract (example) 0.1-0.5 kg

Biscuits were prepared using conventional methods according to the composition and content.

Preparation Example 6 Preparation of Ice Cream

Milkfat 10.0%

Non-fat solids 10.8%

Sugar 12.0%

Starch syrup 3.0%

Emulsifying stabilizer (span) 0.5%

Spices (Strawberries) 0.15%

Water 63.31-62.91%

Golden extract (Example) 0.24-0.64%

Ice cream was prepared by the conventional method according to the composition and content.

Preparation Example 7 Preparation of Beverage

522 mg of honey

Chioctosanamide 5 mg

Nicotinamide 10 mg

Riboflavin Sodium Hydrochloride 3 mg

Pyridoxine hydrochloride 2 mg

Inositol 30 mg

Orthoic acid 50 mg

Golden extract (Example) 0.48-1.28 mg

200 ml of water

The beverage was prepared by the conventional method according to the composition and content.

Preparation Example 8 Preparation of Sausage

Pork 63.6%

Chicken 27.5%

Starch 3.5%

Soy Protein 1.7%

Saline 1.62%

Glucose 0.5%

Other additives (glycerine) 0.94-1.34%

Golden extract (Example) 0.24-0.64%

Sausage was prepared using the conventional method according to the composition and content.

Preparation Example 9 Preparation of Chocolate

Sugar 34.36-34.76%

Cocoa Butter 34%

Cocoa Mass 15%

Cocoa Powder 15%

Lecithin 0.5%

0.5% vanilla

Golden extract (Example) 0.24-0.64%

Chocolate was prepared using the conventional method according to the composition and content.

As described above, the present invention can provide a functional food having cancer prevention effect by preparing various foods (eg, chewing gum, candy, biscuits, ice cream, beverages, sausages, chocolates, etc.) containing golden extracts.

As described above, the present invention was confirmed that the golden extract has the anti-cancer effect by inducing the activity of quinone reductase (Quinone reductase), it is chewing gum, candy, biscuits, ice cream, beverages, sausages and foods such as chocolate By blending together, a wide range of adolescents, adults and seniors can be easily consumed anytime, anywhere, and can be expected to have a continuous drinking effect.

Claims (3)

Activity inducer of quinone reductase consisting of golden extract. Food extract containing golden extract, characterized in that the golden extract is contained 0.1 to 1.0% by weight based on the total composition as an active inducer for quinone reductase. 3. The food containing gold extract according to claim 2, wherein the food is chewing gum, candy, biscuits, ice cream, beverages, sausages and chocolates.