KR100859984B1 - Submerged-Liquid Culture Extract of Fomitopsis pinicola Mycelium and Composition Comprising the Same for Lowering Blood Sugar - Google Patents

Abstract

The present invention relates to a pine myrtle mushroom culture mycelium extract cultured in a medium containing citrus peel or green tea extract and a blood glucose lowering composition containing the same. By using the pine myrtle mushroom culture mycelium extract of the present invention, it can exhibit a significantly greater effect than the hypoglycemic effect of the culture mycelium extract cultured using a conventional potato medium, can be widely used in functional foods for diabetic patients will be.

Pine myrtle mushroom culture mycelia extract, green tea medium, tangerine medium, hypoglycemic composition

Description

Submerged-Liquid Culture Extract of Fomitopsis pinicola Mycelium and Composition Comprising the Same for Lowering Blood Sugar}

The present invention relates to a pine myrtle mushroom culture mycelium extract cultured in a medium containing citrus peel or green tea extract and a blood glucose lowering composition containing the same. More specifically, the extract of pine myrtle mushroom culture mycelium and the hypoglycemic composition containing the same, which have better hypoglycemic effect and exhibit serum lipid improving effect and antioxidant effect than the culture mycelium extract cultured with a general synthetic medium such as potato medium. It is about.

Due to economic growth and changes in dietary culture and overnutrition, the incidence of degenerative adult diseases such as cancer, stroke, high blood pressure and diabetes is increasing. Among them, diabetes is increasing every year with a prevalence rate of more than 3% of the total population of Korea, especially for adults aged 65 years or more, and more than 6.5%. Excess sugar in the blood causes cardiac circulatory disorders, neurological and renal disorders and causes various immune dysfunctions and complications.

In addition, diabetes mellitus of insulin receptors such as overproduction of TNF-αmRNA and protein in adipocytes and increased activity of intraperitoneal macrophages due to insulin-dependent and obesity, which increase various immune-related cytokines due to damage of β-cells in the Langerhans islet of the pancreas. It is classified as insulin-independent type caused by dysfunction of, and more than 90% of Korean diabetic patients are known as insulin-independent diabetes. Representative metabolic features of diabetics are abnormal blood glucose levels and abnormal lipid metabolism. In the insulin-independent type, the development of coronary artery disease due to an increase in triglycerides and a decrease in HDL-cholesterol.

As described above, the treatment of diabetes is a combination of drug therapy and diet, which affects the immune function of the body when taking drugs such as sulfonyl urea, biguanide and troglitazone preparation for a long time. There is a known risk of side effects, so the role of diet is very important. In order to control blood sugar and normalize serum lipid levels, the diet recommends a method of suppressing post-prandial blood sugar and blood insulin elevations by ingesting low-fat diets and low starch index carbohydrates. Acarbose) and dietary fiber, and the recent research results that the biologically active substances contained in red ginseng, goji berry, buckwheat, mushrooms, mushrooms, etc. are effective in lowering blood sugar. Mushrooms belong to fungi, and generally have a low lipid content, but contain abundant nutrients such as proteins, sugars, minerals, nucleic acids, and vitamins, and unique flavors and aromas.

Known physiological activities in mushrooms have been reported to be anti-cancer, immune-enhancing, lowering cholesterol and blood sugar, preventing and curing stroke and heart disease, and defense against infection, and the effects are due to the polysaccharides contained in them. Representative polysaccharides include shiitake mushrooms ( Lentinus) β-1,3-glucan extracted from edodes ), PS-K extracted from culture mycelium of Coriolus versicolor , and the like have been reported.

Until now, the main researches on mushrooms have been reported on nutrition analysis, artificial culture and polysaccharide extraction. Among them, solid culture and liquid culture are used for artificial culture of mushrooms. High efficiency is low, while the latter is high, but the problem that the underlying technology is required. Lee et al suggested temperature 25 ℃, pH 4.0, agitation speed 300rpm, inoculum 10%, oxygen aeration 1.0v / v / m as optimal conditions for shiitake mycelium culture (Refer to Lee, BW, et al., 'Cultural characteristics and pilot scale fermentation for the submerged mycelial culture of Lentinus edodes ', Kor. J. Appl. Microbial Biotechnol., 21, 609-614, (1993)), Fraser reported that yeast extract and casein were very effective nutrient sources for mycelial growth in mushroom mycelium cultures (Fraser, IM 'The growth promptive effect of several amino acids). on the common cultivated mushroom ', Mushroom Sci., 3, 190-200, (1956)). In addition, the growth of mycelium during the cultivation has been reported that environmental conditions such as vitamins and metal ions in addition to the carbon source or nitrogen source have a significant effect on the growth.

Since mycelium liquid culture is slower than other microorganisms and grows more likely to be contaminated during culture, it is possible to shorten the incubation period, mass production of uniform quality mycelium, economical efficiency, extractability of useful substances and bioactive substances, as well as mycelia and culture medium. It is very important to determine the media components that can be considered in terms of food development, and there are studies using natural materials such as ginseng and brewer's yeast extract to increase the yield of mycelia, but cultivation using synthetic media is still the mainstream. However, research on natural medium is insufficient.

In addition, as a method for extracting mushrooms of polysaccharides, a method of obtaining polysaccharides by dividing the fruiting body, adding distilled water, extracting hot water at 85 to 110 ° C. for 2 to 5 hours, and dialysising the precipitate obtained by adding ethanol to obtain polysaccharides It is used. Other methods of extracting polysaccharides include a method of extracting extracellular crude polysaccharides from Ganoderma lucidum mushrooms, which are concentrated for 24 hours in an ultrafiltration device with a molecular exclusion amount of 100,000, added with 2 times of acetone and left at 4 ° C for 24 hours. Bacillus obtained by diluting the culture solution with distilled water, adding NaOH to 1% (w / v) to free the cells and neutralizing the supernatant obtained by centrifugation and ethanol precipitation. It is known to extract polysaccharides from polymyxa KS-1. Lee et al. Used polysaccharides of cloud mushrooms in hot water (97 ℃), cold water (4 ℃), methanol (70 ℃), ethanol (70 ℃), acetone (50 ℃), hexane (50 ℃), 0.1N NaOH (60 ℃). ) And 0.1N HCl (60 ° C.), and extracted by solvent, and found that the yield was high in the range of 10.4 to 18.4% in hot water, cold water, 0.1N HCl, and 0.1N NaOH.

Meanwhile, Fomitopsis pinicola ( Fomitopsis pinicola ) is a native mushroom in Korea, which contains antibacterial steroids and unknown protein polysaccharides that are functional and is known to have an antidiabetic effect. Korean Patent Publication No. 2005-60726 A hypoglycemic agent and a method for preparing the same, which include the extract of pine beetle mushrooms cultured using potato medium, have been disclosed. Recently, the present inventors have been shown to prevent the diabetic complications of the pine beetle mushroom and to improve diabetes-induced hypercholesterolemia ( Korea Patent Application No. 2006-1430), pancreatic cell damage inhibition effect (Korea Patent Application No. 2006-1431), active oxygen production inhibitory effect due to diabetes (Korean Patent Application No. 2005-133990), The protein polysaccharide exhibiting activity is the one to which β-1,3-glucan and β-1,6-heterogalactomannan are bound. It has been revealed that the protein is bound to β-1,3-glucano-β-1,6-heterogalactomannan in a weight of 300,000 to 500,000.

However, there was a limit in cultivating pine beetle mushrooms by the conventional culture method, and in order to solve the problems such as hygiene problems caused by the use of synthetic medium and lowered yield of mycelia, there are many possibilities regarding the availability of natural medium. Research is ongoing.

The present inventors noted green tea and tangerine as a composition for natural medium, green tea is a food containing a small amount of free amino acid, caffeine and vitamin C and polyphenols, antioxidant activity, anticancer, anti-inflammatory, blood cholesterol lowering effect and It is known to show physiologically active functions such as prevention of hypertension, and tangerine is rich in vitamin A precursors β-carotene and flavonoids and terpenes, which are effective for antioxidant, anti-aging, prevention of circulatory diseases, enhancement of immunity and anticancer. Known.

Thus, the present inventors cultivated pine myrtle mushroom mycelium in a natural medium to solve the hygiene problems caused by using a synthetic medium and further utilize the culture medium as a beverage, such as pine green berry mushroom in a medium added with green tea and tangerine extract Optimal conditions for mycelial culture have been established (see Korean Patent Application No. 2005-133989), and besides, fruiting body hydrothermal extract, fruiting body alkali extract and cultured mycelium extract for extracting polysaccharides from pine fruit Manufacturing method has been established (see Korean Patent Application No. 2006-1430).

Therefore, among the pine needles fruiting fruit extract, the extract from the mycelium cultured in natural medium and the mycelium extract cultured from the conventional synthetic medium, the study on the method of obtaining an extract showing the best hypoglycemic effect and the best hypoglycemic effect There is a need for research on a blood sugar lowering composition comprising an extract.

Accordingly, the present inventors have investigated the results of blood glucose, serum lipids, etc. by administering to the rats inducing diabetic rats mushrooms hot water extract, mycelium extract cultured in general synthetic medium, mycelia extract cultured in green tea medium or tangerine blood medium, etc. By confirming the optimal extract conditions, antidiabetic effect was enhanced, the present invention was completed.

After all, the main object of the present invention is to provide a pine myrtle mushroom culture mycelium extract enhanced in the anti-diabetic effect, cultured in green tea or tangerine medium.

It is another object of the present invention to provide a composition for lowering blood glucose, which comprises the extract of the pine myrtle cultivated mycelium.

The present invention is cultured in green tea or tangerine medium, provides a pine myrtle mushroom culture mycelium extract enhanced anti-diabetic effect.

In addition, the present invention provides a composition for lowering blood glucose, which comprises the extract of the mycelium fungus mushroom culture mycelium.

The blood sugar lowering composition may include pine berry butterfly mushroom culture mycelium extract alone, but more preferably comprises an extract mixed with pine berry butterfly fruit body hydrothermal extract, and may further include a culture solution.

The method for preparing the green tea or tangerine medium is based on the method disclosed in Korean Patent Application No. 2005-133989 to the present applicant, which is hereby incorporated by reference in its entirety. Distilled water was added to the green tea powder, and a cooling tube was attached and heated for 2 to 3 hours. Then, the filtered solution was used as the green tea extract stock solution to prepare a green tea medium containing 2.0% (v / v). In addition, distilled water was added to the dried pulverized tangerine, and a cooling tube was attached and heated for 2 to 3 hours, and then the filtered solution was used as a tangerine extract stock solution to prepare a tangerine medium containing 2.0% (v / v).

In addition, the hot water extract of pine fruit, nasal mushroom, extracted by distilled water to the pulverized, pulverized fruiting body by using an extraction tank attached with a cooling tube for 24 hours, using a concentrated solution.

The culture of the mycelia of pine beetle mushroom culture using the potato medium, which is a general medium, was inoculated on YM medium and subcultured, and then sterilized and cooled potato medium (potato hot water extract 10-20% (v / v), sugar 1 ~ A solution incubated and inoculated in 3% (w / v), corn powder 0.1-0.5% (w / v) and drinking water 76.5-88.9% (v / v) is used (see Korean Patent Publication No. 2005) -60726).

The hypoglycemic effect and the serum lipid reduction effect of various pine needles butterfly extracts obtained from the above method were investigated as follows. Dietary feed containing the extracts were fed to streptozotocin (STZ) -induced diabetic rats for 4 weeks, and then blood was collected from the tail vein of the mice to analyze blood glucose levels, and from the abdominal aorta of the mice. Serum lipids were analyzed by blood collection, liver and kidney were extracted to compare changes in weight, and xanthine oxidase activity of liver tissue was measured.

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

Example

Example 1 : Preparation of pine myrtle mushroom culture mycelium extract using green tea medium and tangerine medium

Example 1-1 Separation of Pine Pinnacle Mushroom Mycelium

The mycelia of pine myrtle were isolated from the fruiting bodies of annual pine weaver butterfly which was sold from regenerated agricultural products in Pohang, Gyeongbuk. After washing a certain amount of fruiting body cut into 2 × 2 × 2 mm with 70% (v / v) ethanol, potato 2.0% (w / v), sugar 1.0% (w / v) and peptone 0.6% (w / v) was transplanted to 5.0% (w / v) in a medium containing v) and shaken at 150 rpm for 10 days. The pine myrtle mycelium produced by the electroculture was prepared in YM agar medium (yeast extract 0.5% (w / v), peptone 0.5% (w / v), malt extract 0.2% (w / v), glucose 1.0% (v / v) ) And agar was inoculated at 2.0% (w / v), pH 6.5), and cultured at 30 ° C. for 5 to 6 days to identify mushroom mycelia, and then passaged at 15-day intervals to be used as strains.

Example 1-2 Preparation of Green Tea and Tangerine Medium and Preparation of Pine Sprout Mushroom Culture Mycelia Extract

The green tea extract was prepared by the same hot water extraction method as that of the tangerine extract using commercially available Sulloc tea (Pacific). That is, 2.5L of distilled water was added to 100 g of dried green tea leaves (100mesh), and then a cooling tube was attached and heated for 2 hours, followed by preparing a green tea extract using the filtrate filtered with Miracloth (BioChem Co. USA) as a stock solution. It was. In addition, dried pulpy (dermis) purchased from Daegu Yangnyeongsi (100 mesh), 2.5L of distilled water was added to 100g of electrically dried pulverized tangerine, and then attached to a cooling tube and heated for 2 hours, followed by Miracloth (BioChem Co. USA) was prepared by extracting the tangerine extract as a stock solution.

Green tea medium containing 2% (v / v) of the prepared green tea extract (2% green tea extract (v / v), 5% glucose (v / v), yeast extract 0.5% (w / v), peptone 0.5% (w / v) and malt extract 0.3% (w / v), pH 5.0) and 2% (v / v) green tea extract stock solution of 2% (v / v) tangerine extract stock solution The tangerine medium was prepared.

The pine needles mycelia obtained in Example 1-1 were inoculated in green tea medium and tangerine medium, and cultured in a shaker at 30 ° C. and 150 rpm for 12 days. Thereafter, the culture medium containing the pine myrtle mushroom culture mycelium was adjusted to pH 7.0 with sodium bicarbonate (NaHCO ₃ ), homogenized and concentrated under reduced pressure to 1/20 at 40 ° C. to extract the pine myrtle mushroom culture mycelium extract using green tea and tangerine medium. Prepared.

Example 2 Preparation of Cultured Mycelium Extract Using Pine Shrimp Fruit Fruit Hot Water Extract and Potato Medium

* Example 2-1 : Preparation of pine needles mushroom fruiting hot water extract

After cutting 150g of fruiting body of 1 year old pine tree butterfly, which was sold from regenerated agricultural product in Pohang, Gyeongbuk, into 2 × 2 × 2cm, 4L of distilled water was added and extracted for 24 hours using an extraction tank with a cooling tube. The solution was concentrated to give a solution of 3.75 g / 100 mL.

Example 2-2 Preparation of Cultured Mycelia Extract Using Potato Medium

Pine myrtle mushroom mycelium, which was preserved and preserved by regenerated agricultural products in Pohang, Gyeongbuk, was prepared by YM medium (1% (glucose), yeast extract 0.5% (w / v), peptone 0.5% (w / v) and malt extract 0.3% (w / v), pH 6.5), subcultured at 30 ° C. for 10 days, sterilized and cooled at 121 ° C. for 1 hour [potato hot water extract 15% (v / v), dry weight 4% (w / v)), sugar 2% (w / v), corn powder 0.3% (w / v), pH 6.5] inoculated to 5% (v / v) and shaken at 30 rpm at 150 rpm. The incubator was incubated for 12 days. Subsequently, the culture medium containing pine myrtle cultivated mycelium was adjusted to pH 7.0 with sodium bicarbonate, homogenized, and concentrated under reduced pressure to 1/20 at 40 ° C to prepare a culture mycelium extract.

Example 3 Comparison of Antidiabetic Effects of Various Pine Grass Butterfly Extracts

Example 3-1 : Experimental group and experimental method

To obtain a total of 70 Sprague-Dawley male rats with an average weight of 225 ± 5.2g for each experimental group, they were adapted to the environment at room temperature of 23 ± 2 ℃, humidity of 60 ± 5%, and 12hr contrast cycle. After a week of pre-breeding with a general feed (Purina Co., Seoul Korea) was carried out an experimental diet.

The seven experimental diet groups (see Table 1) are as follows.

1) NC: Normal Control

DM: Diabetic Control

3) DM-PD: 2% administration group of mycelial culture concentrate (1/20) cultured in potato medium (PD medium) after diabetes induction

4) DM-CP: 2% administration group of mycelial culture concentrate (1/20) cultured in tangerine medium (CP medium) after diabetes induction

5) DM-GT: 2% administration group of mycelial culture concentrate (1/20) cultured in green tea medium (GT medium) after diabetes induction

6) DM-WE: fruiting body hot water extract (WE) concentrate 2% administration group after diabetes-induced

7) DM-WC: 1% mixture of 1% mixed solution of fruiting body hot water extract concentrate and cultured mycelium concentrate (1/20) cultured in K.

Table 1 : Basic Dietary Composition for Animal Experiments (g / kg)

Diet NC DM DM-PD DM-CP DM-GT DM-WE DM-WC Casein 200 200 195 200 195 200 195 cornstarch 150 150 142.5 150 142.5 150 142.5 Sugar 500 500 500 500 500 500 500 Cellulose 50 50 45 49.5 45 49.5 45 Corn oil 50 50 48 50 48 50 48 AIN-mineral mix ^{1 )} 35 35 34.5 35 34.5 35 34.5 AIN-Vitamin Mixture ^{2 )} 10 10 10 10 10 10 10 DL-Methionine 3 3 3 3 3 3 3 Cholinevitatarate 2 2 2 2 2 2 2 Mycelia PD culture solution (1/20 concentration) - - 20 - - - - Mycelia CP culture solution (1/20 concentration) - - - 20 - - 10 Mycelia GT culture (1/20 concentration) - - - - 20 - - Fruiting body hot water extract (3.75g / 100mL) - - - - - 20 - Fruiting body hot water extract + mycelium CP culture solution (1: 1) - - - - - - 10     total 1,000 1,000 1,000 1,000 1,000 1,000 1,000

¹⁾ AIN-mineral mixture (g / kg): calcium lactate 620.0, sodium chloride 74.0, potassium diphosphate 220.0, potassium sulfate 52.0, magnesium oxide 23.0, manganese carbonate 3.3, iron citrate 6.0, zinc carbonate 1.0, copper carbonate 0.2 , 1,000 g of potassium iodide 0.01, sodium selenite 0.01 and potassium chromium sulfate 0.5 as fine powder

²⁾ AIN-vitamin mixture (mg / kg): Thiamine hydrochloride 600, Riboflavin 600, Pyridoxine hydrochloride 700, Nicotinic acid 3,000, D-calcium pantothenate 1,600, Folic acid 200, D-biotin 20, Vitamin B12 2.5, Vitamin A 400,000 IU, 1,000 g of Vitamin D3 100,000 IU, Vitamin E 7,500 IU and Vitamin K 75 as fine powder

Example 3-2 : Antidiabetic Effect Measurement

Weight gain, dietary intake, Dietary efficiency

Streptozotocin (STZ, Sigma Chem. Co. MO, USA) was dissolved in 0.1M citric acid buffer (pH 4.3) and injected into the femoral muscle at a concentration of 40 mg / kg per 250 g of body weight. Fasting tail vein at 48 hours after STZ administration Diabetes was considered when the blood glucose concentration from the blood was 200 mg / dl or more.

Blood glucose analysis was measured at 10 to 12 am daily using Gluco-Tester (Life Scan Lnc., USA), and blood was collected from the vessels of the fasting tail with lancet.

Body weight, diet and drinking water intake were measured at regular times every day throughout the entire experimental period, and food efficiency ratio (FER) was the weight gain for the same period divided by the intake of the same period. Dietary intake and dietary efficiency are shown in Table 2 below.

In the diabetes-induced group (DM), body weight was significantly reduced (58% decrease) compared to the normal group (NC), dietary intake increased by 14%, water intake increased by 84%, and urinary excretion increased by 108%. Because of this, the phenomenon of triad and weight loss occurred. In the other diet-administered groups, the above-described trivalent phenomenon and weight loss were observed, but the symptoms were alleviated significantly compared to the DM group, showing anti-diabetic effect. Among them, the anti-diabetic effect in the DM-WC and DM-GT groups Was high and showed a significant difference compared to the potato medium which is a conventionally used medium. That is, the weight loss rate was 17%, 16%, 32%, 23% and 15% in DM-GT group, DM-CP group, DM-PD group, DM-WE group and DM-WC group, respectively. The mixed solution of the culture medium, the fruiting body hot water extract and the culture medium cultured in the tangerine medium was about twice as effective as the culture medium grown in the potato medium. Other dietary intakes, dietary efficiency, water intake, and urine excretion were the most effective in the DM-CP and DM-WC groups (Table 2).

Table 2 : Dietary Effect of Four-Week Diets Containing Various Extracts of Pine Needles on the Weight Gain, Dietary Intake, Dietary Efficiency, Water Intake, and Urinary Excretion in STZ-Induced Diabetic Rats

Group ^{1 )} Final weight (g) Weight gain (g / week) Dietary Intake (g / week) Dietary Efficiency ^{2 )} Water intake (mL / week) Urine excretion (mL / day)  NC 371.2 ± 11.2 36.5 ± 5.3 176.5 ± 6.1 0.21 ± 0.04 231.0 ± 16.1 74.1 ± 16.0  DM 213.4 ± 10.3 -2.9 ± 0.9 201.7 ± 8.9 -0.14 ± 0.01 424.3 ± 31.7 154.4 ± 25.6  DM-PD 250.6 ± 6.4 6.3 ± 4.5 187.4 ± 5.1 0.03 ± 0.03 345.6 ± 25.2 124.7 ± 12.4  DM-CP 312.7 ± 7.6 21.8 ± 5.7 180.9 ± 4.7 0.12 ± 0.04 298.3 ± 20.4 82.7 ± 13.8  DM-GT 307.0 ± 7.5 20.5 ± 6.4 182.0 ± 6.7 0.11 ± 0.04 326.4 ± 29.5 99.6 ± 7.5  DM-WE 286.5 ± 8.4 15.4 ± 6.2 185.6 ± 6.9 0.08 ± 0.04 320.0 ± 30.8 110.7 ± 7.5  DM-WC 314.3 ± 5.7 22.1 ± 4.4 179.8 ± 5.4 0.12 ± 0.03 296.2 ± 21.3 81.9 ± 14.1

¹⁾ : NC: normal control group, DM: diabetes control group, DM-PD: after diabetes induction, mycelium culture concentrate (1/20) 2% administration group cultured in potato medium (PD medium), DM-CP: after diabetes induction, 2% administration group of mycelial culture concentrate (1/20) cultured in tangerine medium (CP medium), DM-GT: 2% administration of mycelial culture concentrate (1/20) cultured in green tea medium (GT medium) after diabetes induction, DM-WE: 2% administration of fruiting body hot water extract (WE) concentrate after induction of diabetes mellitus, DM-WC: 1: 1 mixture of cultured mycelium concentrate (1/20) cultured in fruiting body hot water extract concentrate and gulpie medium after diabetes induction 2% administration group

²⁾ : Dietary efficiency: total weight gain / total dietary intake

Long-term weight

Diabetes is known to be a metabolic system of acetyl-CoA due to abnormal insulin metabolism due to lowering insulin and resistance of the body, and lipid components are accumulated in the liver, especially carbohydrate metabolism in streptozotocin-induced diabetic rats Increasing the activity of glucose-6-phosphatase in the liver, which is involved in the liver, doubles the liver. In addition, diabetes mellitus increases the kidneys because the concentration of plasma glucose due to diabetes is metabolized by glycogen and accumulate in the blood vessels (mesangial cells) in the glomeruli.

Of rats bred for 4 weeks after diabetes As a result of comparing the changes in weight of liver and kidney, the weight percent of DM in the DM group increased by 48% compared to the NC group, and the hypertrophy of liver due to diabetes was confirmed. Hypertrophy was alleviated. In the DM-CP group and DM-WC group, the alleviating effect was greatest, and the kidney showed the same tendency as the liver (see Table 3).

Table 3 : Effect of Dietary Supplements Containing Various Pine Sprout Mushroom Extracts on Soy and Kidney Weight (% of Weight) for 4 Weeks after Diabetes Induction

Experimental group ^{1 )} Soy weight Soy weight  NC 2.47 ± 0.04 ^d 0.64 ± 0.07 ^a  DM 3.65 ± 0.16 ^a 0.83 ± 0.12 ^a  DM-PD 3.08 ± 0.09 ^b 0.74 ± 0.06 ^a  DM-CP 2.60 ± 0.12 ^cd 0.65 ± 0.07 ^a  DM-GT 2.83 ± 0.17 ^bC 0.66 ± 0.08 ^a  DM-WE 2.92 ± 0.18 ^b 0.73 ± 0.07 ^a  DM-WC 2.59 ± 0.11 ^cd 0.64 ± 0.06 ^a

* ¹⁾ Same as in Table 2

²⁾ The values of 10 animals in each experimental group are expressed as mean ± standard deviation, and different characters are significant within 5%.

Blood sugar content

Diabetes-induced rats were examined for changes in blood glucose during the four-week diet. As a result, DM group maintained 523.5 ~ 546.5 mg / dL during the four weeks diet. However, in the experimental diet group, there was a difference in the degree of blood glucose, and the blood glucose reduction rate at the 4th week of feeding was 69% in the DM-GT group, 74% in the DM-CP group, 53% in the DM-PD group, and DM. 58% of the WE groups and 75% of the DM-WC groups. These results showed a difference in efficacy according to the culture conditions of the mycelium. As a result, it was found that there was a high effect when the mixture of citrus fruit or green tea medium or fruiting body hot water extract was compared to the conventional potato medium (see Table: 4). In particular, the improvement effect after 4 weeks in the case of the culture mycelium extract cultured using the conventional potato medium, the blood sugar was reduced by about 292 mg / dl before and after administration showed an improvement effect of about 53%, In the case of cultured mycelium extracts, the blood glucose was decreased by about 406 mg / dl before and after administration, which showed about 74% improvement in blood glucose.

Table 4 : Comparison of Blood Glucose Changes in Rats During Dietary Diet Containing Pineapple Butterfly Extract for 4 Weeks After Diabetic Induction

Group ^{1 )} Dietary period (Note)          One 2 3 4  NC 117.6 ± 12.3 ^{bA , 2)} 124.3 ± 11.3 ^cA 118.5 ± 7.3 ^dA 120.6 ± 12.4 ^dA  DM 546.5 ± 24.5 ^aA 550.4 ± 32.3 ^aA 549.6 ± 32.4 ^aA 523.5 ± 32.8 ^aA  DM-PD 538.7 ± 19.6 ^aA 525.3 ± 35.6 ^aA 435.5 ± 36.1 ^bB 254.6 ± 22.5 ^bC  DM-CP 532.4 ± 20.8 ^aA 453.9 ± 30.8 ^bB 344.2 ± 37.6 ^cC 140.2 ± 27.1 ^cdD  DM-GT 528.7 ± 28.7 ^aA 549.8 ± 56.2 ^aA 347.4 ± 51.2 ^{cB 165.4} ± 19.7 ^cC  DM-WE 540.5 ± 21.4 ^aA 500.7 ± 32.3 ^abA 382.7 ± 35.4 ^bcB 224.5 ± 23.5 ^bC  DM-WC 535.3 ± 21.2 ^aA 456.5 ± 31.4 ^bB 345.8 ± 338.3 ^cC 135.8 ± 25.6 ^cdD

¹⁾ Same as in Table 2

²⁾ The values of 10 animals in each experimental group are expressed as mean ± standard deviation, and different characters are significant within 5%.

Example 4 Improvement of Serum Lipids and Inhibitory Effect of Xanthine Oxidase on Liver Tissues from Experimental Diet Containing Pineapple Butterfly Extract

Experimental group and experimental method was carried out in the same manner as in Example 3, the rats were fed for 4 weeks as an experimental diet, except water, fasted for 24 hours, anesthetized with ethyl ether, and opened, after which blood was collected from the abdominal aorta, The liver was perfused with ice-cold saline and the organs were extracted. The collected blood was left at refrigeration temperature for 7 hours, and then serum was separated by centrifugation at 3,000 rpm for 10 minutes, and homogenized by grinding with 4 times of ice-cold 0.25M sucrose solution in a predetermined amount of liver. After centrifugation for 10 minutes at, the supernatant was further centrifuged at 10,000 × g for 30 minutes to separate the supernatant as a cytosol fraction. All samples were stored in -70 ℃ deep freezer, was taken out of the experiment.

Serum lipid  analysis

Serum neutral lipid, total cholesterol, HDL-cholesterol content was measured by kit reagent (AM 157S-K, AM 202-K, AM 203-K, Asanpharm Co., Korea). That is, the content of neutral lipid was added to each of 0.02 ml of serum and 3.0 ml of enzyme solution and reacted at 37 ° C. for 10 minutes, and the absorbance was measured at 550 nm. The serum total cholesterol content was obtained by the reaction formula (mg / dL = (absorbance of standard solution / absorbance of sample solution) × 300) by measuring absorbance at 500 nm for 5 minutes at 37 ° C at the same dose ratio as that of neutral lipid. . In addition, the HDL-cholesterol content was measured by adding 3.0 ml of enzyme solution to 0.1 ml of serum and reacting at 37 ° C. for 5 minutes, and then measuring the absorbance at 500 nm. The content was calculated by LDL-cholesterol content was calculated as total cholesterol- (HDL-cholesterol content)-(neutral lipid / 5).

Serum triglyceride content increased by 73% in the DM group compared to the NC group, which was normal, compared with 16% in the DM-GT group, 8% in the DM-CP group, 47% in the DM-PD group, and 39% in the DM-WE group. In addition, DM-WC increased by 7%, with the DM-WC group and DM-CP group having the same level of increase. The average content of total cholesterol was the same as that of neutral lipid, but there was no significant difference. The HDL-cholesterol content was 19% lower in the DM group than in the NC group, but there was no significant difference in the experimental group in the NC group. The LDL-cholesterol content was 42% higher in the DM group than in the normal group, but the DM-WC, DM-CP, and DM-GT groups showed little or no difference from the normal group. The effect of improving was even higher (see Table 5).

Table 5 : Effect of Dietary Containing Pineapple Butterfly Extract on Serum Lipids During 4 Weeks after Diabetic Induction

Group ^{1 )} Neutral Lipids (mg / dL) Total Cholesterol (mg / dL) HDL-cholesterol (mg / dL) LDL-cholesterol (mg / dL) ²⁾  NC 72.4 ± 13.5 ^c 121.5 ± 12.4 ^{a, 3)} 56.5 ± 5.9 ^a 50.5 ± 5.6 ^bc  DM 125.6 ± 13.4 ^a 142.5 ± 17.5 ^a 45.8 ± 4.7 ^b 71.6 ± 6.4 ^a  DM-PD 106.3 ± 12.0 ^ab 134.6 ± 10.3 ^a 49.0 ± 2.4 ^ab 64.3 ± 5.3 ^a  DM-CP 78.2 ± 11.5 ^c 112.4 ± 13.3 ^a 53.5 ± 4.5 ^ab 43.3 ± 4.7 ^c  DM-GT 84.3 ± 10.8 ^bc 120.7 ± 12.4 ^a 52.3 ± 3.3 ^ab 51.5 ± 5.6 ^bc  DM-WE 100.3 ± 12.5 ^abc 130.5 ± 12.4 ^a 50.3 ± 3.1 ^ab 60.1 ± 6.4 ^ab  DM-WC 77.8 ± 10.3 ^c 112.4 ± 13.3 ^a 53.5 ± 4.5 ^ab 43.3 ± 3.9 ^c

¹⁾ Same as in Table 2.

²⁾ Total Cholesterol-(HDL-Cholesterol Amount + Neutral Lipid / 5).

³⁾ The mean and standard deviation of 10 experimental animals. Different letters on the vertical axis (ac) indicate significance within 5%.

Liver tissue Xanthine oxidase  activation

The activity of xanthine oxidase (XOD) in liver tissue was measured according to the method of Stirpe et al., Which measured absorbance at 292 nm for uric acid produced by reacting xanthine as a substrate for 10 minutes at 30 ° C. In the active unit of the enzyme, 1 mg of liver tissue protein was reacted for 1 minute, and the amount of uric acid generated from the substrate was expressed in nmol concentration.

It has been reported that xanthine oxidase increases its activity when it damages the liver, and its activity increases due to increased oxidation of xanthine upon administration of toxic substances such as ethanol, carbon tetrachloride and streptozotocin. Higher O and T ratios (%) indicate the production of reactive oxygen species.

Table 6 below compares the activity of xanthine oxidase when diabetic rats were fed an experimental diet for 4 weeks. As a result, there was no significant difference in the total xanthine oxidase activity, but O type activity and O / T (%) were significantly higher in DM-CP group, DM-WC and DM-GT group than DM-PD group. Not only was it low, it was found to have an effect of significantly inhibiting the production of reactive oxygen species produced by diabetes in parallel with the normal group.

Table 6 : Comparison of xanthine oxidase activity of rat liver tissue fed experimental diet for 4 weeks after diabetes induction

Group ^{1 )} Total O type O / T (%)  NC 4.61 ± 0.19 ^{a, 2)} 1.10 ± 0.39 ^d 23.86 ± 7.18 ^d  DM 4.30 ± 0.27 ^a 3.15 ± 0.54 ^a 73.26 ± 7.48 ^a  DM-PD 4.48 ± 0.12 ^a 2.40 ± 0.20 ^b 53.57 ± 5.74 ^b  DM-CP 4.60 ± 0.13 ^a 1.16 ± 0.39 ^d 25.22 ± 8.94 ^d  DM-GT 4.56 ± 0.18 ^a 1.35 ± 0.34 ^cd 29.61 ± 8.30 ^cd  DM-WE 4.52 ± 0.14 ^a 1.97 ± 0.32 ^bc 43.58 ± 8.17 ^bc  DM-WC 4.60 ± 0.12 ^a 1.18 ± 0.32 ^d 25.65 ± 8.51 ^d

¹⁾ Same as in Table 2.

²⁾ The mean and standard deviation of 10 experimental animals. Different letters on the vertical axis (ac) indicate significance within 5%.

As described and demonstrated in detail above, the present invention relates to a pine myrtle mushroom culture mycelium extract with enhanced anti-diabetic effect, cultured in a medium containing green tea or tangerine extract. By using the pine myrtle mushroom culture mycelium extract of the present invention, it can exhibit a significantly greater effect than the hypoglycemic effect of the culture mycelium extract cultured using a conventional potato medium, can be widely used in functional foods for diabetic patients will be.

Claims (5)

2.0% (v / v) undiluted juice, 5.0% (v / v) glucose, 0.5% (w / v) yeast extract, 0.5% (w / v) yeast extract In a tangerine medium containing 0.3% (w / v) of malt extract and 91.7% (v / v) of purified water and having a pH of 5.0, Inoculated with pine mycelium mycelium mycelium and incubated for 12 days in a shaker at 30 ℃, 150rpm conditions, the resulting culture was adjusted to pH 7.0 with sodium bicarbonate (NaHCO ₃ ), homogenized and concentrated under reduced pressure to 1/20 at 40 ℃ Pine myrtle mushroom culture mycelium extract using the obtained tangerine medium,  Distilled water was added to the shredded and crushed pine needles fruiting body and extracted for 24 hours using an extraction tank attached with a cooling tube, and then concentrated to 1/4. The pineapple butterfly mushroom culture mycelium extract using the tangerine medium, and the weight ratio of the pine tree butterfly mushroom fruiting body hydrothermal extract is 1: 1 composition for lowering blood sugar. delete delete delete delete