KR102413556B1 - Extract of Pine Bark with Enhanced pharmacological Activity toward Metabolic Diseases and a Method for Preparing the Same - Google Patents

Abstract

The present invention relates to a method for preparing a pine bark extract, a pine bark extract prepared through the method, and a pharmaceutical composition or functional food composition for preventing or treating metabolic diseases comprising the same as an active ingredient. The present invention can be usefully used as an efficient extraction process capable of obtaining a pine bark extract with significantly improved pharmacological activity against metabolic diseases in high yield.

Description

Pine bark extract with enhanced pharmacological activity for metabolic diseases and a method for preparing the same { Extract of Pine Bark with Enhanced pharmacological Activity toward Metabolic Diseases and a Method for Preparing the Same}

The present invention relates to a novel method for preparing a pine bark extract with enhanced pharmacological activity for metabolic diseases, specifically diabetes.

All parts of the pine tree have been used as food and medicine since ancient times, for example, shoots for cancer, blood circulation, stomach, diabetes and neuralgia, pine cones for bronchial asthma, stroke and diabetes, and pine needles for joints, neuralgia, high blood pressure, and It is known that the stem is effective in recovering from fatigue and blood clotting, the bark is effective in blood circulation and preventing blood clotting, and the pollen and pine needles are also used as ingredients for food.

Among them, it is reported that extracts extracted from pine bark with hot water, alcohol, etc. have antioxidant and anti-inflammatory effects, as well as neuroprotective effects, atopic sedative effects, adipocyte production inhibitory effects, and DNA protection effects.

The main components contained in pine bark include catechin [(+)/(-)-catechin], rutin, quercetin, kaempferol, epicatechin [(-)-epicatechin], taxifolin ( taxifolin), taxifolin-3'-O-β-glucoside, and catechin complex procyanidins, such as flavonoids.

Procyanidin or procyanidin oligomer is a low molecular weight component composed of less than a tetramer of catechin and epicatechin molecules, and is known to have effects such as antioxidant, anti-inflammatory, connective tissue protection, and blood vessel protection.

The most widely known extract of pine bark is Pycnogenol®, which mainly contains procyanidins extracted from the bark of French coastal pine ( Pinus pinaster ). Pycnogenol® has been used as a powerful antioxidant for a long time, and in addition to its antioxidant activity, it is known to be effective in the treatment and prevention of chronic inflammation, circulatory disorders, and asthma.

New Zealand pine ( P. radiata ) is the most well-known tree species along with French coastal pine. The 15-30 year old New Zealand pine bark extract (Enzogenol®) contains a significant amount of procyanidin, and has antioxidant, anti-inflammatory, anticancer, and cardioprotective properties. , is known to have various effects such as neuroprotection.

P. massoniana Lamb also known as Chinese red pine, bark, leaves, pollen, and essential oil have been used in traditional Chinese medicine to treat bleeding, rheumatism, arthralgia, inflammation, and cancer, as well as Chinese red pine bark extract through recent studies. It has been found that silver can help in cell growth and reproduction, and the main component exhibiting this physiological activity is procyanidin. Recently, NOW®, prepared from bark extract of Chinese red pine, was launched based on its strong antioxidant effect. The main components of this product also consist of flavonoids and proanthocyanidin oligomers.

In addition to the pine species mentioned above, Pinus densiflora (Korean red pine) is a tree species widespread in Asia, especially in Korea, whose stems are very expensive and are used as materials for hanok construction in Korea, while the bark is very large. It is emitted as a by-product of the wood industry. Studies have been conducted on the extracts of red pine bark and pine needles as well as their physiological activity against various diseases and the constituents of the extract.

Recently, the pine needle extract of domestic red pine has been found to have effects such as improving memory and preventing cataract formation in animals. It has been found to have biological activity. The components of red pine bark extract, which exhibit such physiologically active effects, also contain a large amount of procyanidin, like other tree species, and are known to contain polyphenols such as catechin and taxifolin.

However, the content and composition of these active ingredients contained in the pine extract varies depending on the region, growth conditions, age, etc., and the content, composition, physiological activity, etc. difference appears. In addition, since most of the existing pine bark extracts target a specific component such as procyanidin, there is a difficulty in manufacturing, and there are problems such as importing raw materials from abroad.

In the present invention, using the bark of domestic red pine ( Pinus densiflora Sieb & Zucc.), which is relatively easy to supply and supply, an extract showing excellent physiological activities such as antioxidant, anti-inflammatory, anti-diabetic, and liver function improvement through the search for optimal extraction conditions was obtained.

In addition, the antidiabetic activity in animals has not yet been evaluated for the above-mentioned domestic red pine bark extract.

Diabetes mellitus is a type of metabolic disease such as insufficient insulin secretion or failure to function normally. It is characterized by high blood sugar in which the concentration of glucose in the blood increases. . Such diabetes is divided into type 1 and type 2, and type 1 diabetes, previously called 'juvenile diabetes', is a disease that occurs due to the inability to produce insulin at all. Type 2 diabetes mellitus, which is relatively deficient in insulin, is characterized by insulin resistance (insulin resistance; the inability of cells to effectively burn glucose due to the decreased ability of insulin to lower blood sugar). In type 2 diabetes, environmental factors such as high calorie, high fat, high protein diet, lack of exercise, and stress due to westernization of diet seem to play a major role. It can also be caused by infection or medication.

Chronic high blood sugar caused by diabetes causes damage and dysfunction of each body organ. In particular, microvascular complications appearing in the retina, kidneys, and nerves, and macrovascular complications such as arteriosclerosis, cardiovascular and cerebrovascular diseases, etc. Diabetes mellitus is regarded as one of the major diseases that threaten the health of modern people.

Even though they do not fall under the category of diabetes, people with high blood sugar often present with impaired glucose tolerance (IGT). Glucose absorbed into the body is burned in peripheral tissues, or blood sugar can be controlled by stimulating beta cells to secrete insulin. In general, diabetes has been referred to as a case in which the concentration of glucose in venous blood is 15 mmol/l (270 mg/dl) or higher, but the WHO has more standardized guidelines: ① Blood glucose concentration during fasting is 7.8 mmol/l (140 mg/dl) or higher; , ② Diabetes is prescribed when the blood glucose concentration is 11.1 mmol/l (200mg/dl) or higher after 2 hours of oral glucose tolerance (OGT) administration of 75g. In contrast, IGT refers to when the blood glucose concentration is between 7.8-11.1 mmol (140-200 mg/dl) after 2 hours in the 75g-OGT test.

In the case of impaired glucose tolerance, it means that the blood sugar is outside the range of normal blood sugar but is not high enough to be diagnosed as diabetes. This means that it is not a normal condition and is a high-risk group that can soon develop diabetes. In this pre-diabetic stage, the risk of developing diabetes in the future is 3 to 5 times higher than that of normal people, and the risk of cardiovascular disease by itself is already 2 to 3 times higher.

Most diabetic patients and people suffering from impaired glucose tolerance know the purpose of the diet, but complain of difficulties in its practice.

Numerous papers and patent documents are referenced throughout this specification and their citations are indicated. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention pertains and the content of the present invention.

Patent Document 1. Republic of Korea Application No. 10-1998-0050169

The present inventors made intensive research efforts to develop a method for extracting pine bark in which a stable yield is maintained while an extract showing a high level of pharmacological activity is obtained by containing beneficial ingredients in a high content. As a result, when extracting 15 - 45% alcohol, specifically 20% to 40% ethanol, at 50 - 70°C for 8 - 16 hours using 20% to 40% ethanol as an extraction solvent, the total phenol content is lower than that of the extraction result using the conventional method. By discovering that an excellent extract with significantly increased and significantly improved pharmacological activity for metabolic diseases can be obtained in high yield, the present invention has been completed.

Accordingly, it is an object of the present invention to provide a method for preparing a pine bark extract.

Another object of the present invention is to provide a pine bark extract prepared by the method of the present invention and a pharmaceutical composition or a functional food composition for preventing or treating metabolic diseases comprising the same as an active ingredient.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for producing a pine bark extract comprising the steps of:

(a) adding 15 - 45% alcohol to the dried pine bark and extracting at 50 - 70 °C for 8 - 16 hours; and

(b) pulverizing the extract obtained in step (a).

The present inventors made intensive research efforts to develop a method for extracting pine bark in which a stable yield is maintained while an extract showing a high level of pharmacological activity is obtained by containing beneficial ingredients in a high content. As a result, when extracting 15 - 45% alcohol, specifically 20% to 40% ethanol, at 50 - 70°C for 8 - 16 hours using 20% to 40% ethanol as an extraction solvent, the total phenol content is lower than that of the extraction result using the conventional method. It was found that an excellent extract with significantly improved pharmacological activity and antioxidant activity against metabolic diseases can be obtained in high yield.

As used herein, the term “alcohol” refers to a compound in which a hydroxyl group is bonded to a carbon of an alkyl group. Alcohol in the present specification is, for example, a lower alcohol, for example, C ₁ alcohol (methanol), C ₂ alcohol (ethanol) or C ₃ alcohol (butanol) may be.

As used herein, the term “extract” includes not only the meaning of a crude extract commonly used in the art, but also a fraction obtained by additionally fractionating the extract. That is, the pine bark extract obtained in step (a) includes not only the result obtained using the alcohol extraction solvent described above, but also the result of applying an additional purification process thereto. For example, a fraction obtained by passing the extract through an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (prepared for separation according to size, charge, hydrophobicity or affinity), etc. The fraction obtained through the purification method is also included in the pine bark extract of the present invention.

According to a specific embodiment of the present invention, in the method of the present invention, between step (a) and step (b), a filtration step and a concentration step for the extract obtained in step (a) are additionally performed in any order include

According to the present invention, before finally powdering the extract of the present invention, it is possible to make properties and volume suitable for powdering through spray drying, etc. by going through filtration and concentration processes. Specifically, the filtration step and the concentration step are sequentially performed.

According to a specific embodiment of the present invention, the filtering step is performed using a filter paper having a pore size of 9-13 μm. More specifically, filter paper having a pore size of 10-12 μm is used, and most specifically, filter paper having a pore size of 11 μm is used.

According to a specific embodiment of the present invention, the alcohol in step (a) of the present invention is added so that the weight ratio with the dried pine bark is 1:8 - 1:12. More specifically, it is added so that the pine bark sample and alcohol are 1:9 - 1:11, and most specifically 1:10.

According to a specific embodiment of the present invention, the alcohol in step (a) of the present invention is 35-45% ethanol, more specifically 38-42% ethanol, and most specifically 39-41% ethanol.

According to a specific embodiment of the present invention, step (a) of the present invention is carried out by extraction at 55 - 65 °C for 14 - 16 hours. More specifically, extraction is performed at 57-63° C. for 15 hours.

According to a specific embodiment of the present invention, the pine tree used in the present invention is Pinus densiflora ( Pinus densiflora ). Pinus densiflora is a pine tree species that inhabits East Asia. It is widely distributed in Korea, so it is easy to supply and supply, and it is widely used as a building material in Korea. very good In addition, as domestic supply and demand are easy, it has the advantage of being able to mass-produce on an industrial scale without the restrictions of international treaties such as the Nagoya Protocol.

According to a specific embodiment of the present invention, the dried pine bark of the present invention is obtained by pulverizing the collected pine bark into a sample having a size of 3 cm or less after drying in a dark room.

According to another aspect of the present invention, there is provided a pine bark extract having a total phenol content (TPC) of 250 GAE mg/g extract or more, prepared by the method of the present invention as described above. As used herein, the term “phenol” is meant to encompass phenol-based compounds including compounds in which a hydroxyl group is substituted on a 6-carbon aromatic ring and derivatives thereof. Phenolic compounds are known to have strong pharmacological activity against chronic diseases such as diabetes, Alzheimer's disease, cancer, oxidative stress and certain bacterial infections. become one According to the present invention, the pine bark extract extracted by the method of the present invention has a remarkably high total phenol content and can be used as an excellent and effective pharmacological ingredient.

According to a specific embodiment of the present invention, the extract of the present invention comprises Massonianoside B. More specifically, it contains masonianoside B in an amount of 90 to 190 μg/g of extract, more specifically, includes it in an amount of 110 to 170 μg/g of extract, and most specifically includes it in an amount of 130 to 150 μg/g of extract.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating metabolic diseases comprising the above-described extract of the present invention as an active ingredient.

Since the extract of the present invention has already been described above, description thereof is omitted to avoid excessive duplication.

As used herein, the term “metabolic disease” is a clinical term that conceptualizes a phenomenon in which the risk factors of various cardiovascular diseases and type 2 diabetes cluster with each other as one disease group. It is a concept that encompasses both metabolic abnormalities and clinical features. In 1988, Reaven argued that the common cause of these symptoms was insulin resistance, in which the body's insulin does not work well, and named it insulin resistance syndrome. Because it could not be done, the term metabolic syndrome or metabolic disease was introduced.

As used herein, the term “treatment” refers to (a) inhibiting the development of a disease, disorder or symptom; (b) alleviation of the disease, condition or condition; or (c) eliminating the disease, disease or symptom. The composition comprising the pine bark extract prepared by the method of the present invention increases glucose absorption, protects hepatocytes, improves oral glucose tolerance, reduces glycated hemoglobin, and blood triglyceride concentration in individuals with metabolic diseases. By significantly reducing the diabetes, dyslipidemia and fatty liver, it serves to inhibit the progression of various metabolic diseases, or to eliminate or alleviate its symptoms. Accordingly, the composition of the present invention may be a therapeutic composition for a metabolic disease by itself, or may be administered together with other pharmacological components to be applied as a therapeutic adjuvant for a metabolic disease. Accordingly, as used herein, the term “treatment” or “therapeutic agent” includes the meaning of “therapeutic adjuvant” or “therapeutic adjuvant”.

As used herein, the term “prevention” refers to inhibiting the occurrence of a disease or disease in a subject who has never been diagnosed with a disease or disease, but is likely to have the disease or disease.

As used herein, the term “administration” or “administering” refers to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that the same amount is formed in the subject's body. A “therapeutically effective amount” of a composition means an extract content sufficient to provide a therapeutic or prophylactic effect to a subject to be administered the composition, and includes a “prophylactically effective amount”. As used herein, the term “subject” includes, without limitation, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon or rhesus monkey. Specifically, the subject of the present invention is a human.

According to a specific embodiment of the present invention, the metabolic disease treated with the composition of the present invention is selected from the group consisting of diabetes, fatty liver and dyslipidemia.

As used herein, the term “diabetes” refers to a chronic disease characterized by a relative or absolute lack of insulin resulting in glucose-intolerance. In the present invention, “diabetes” includes all types of diabetes satisfying the above definition, and includes, for example, type 1 diabetes, type 2 diabetes, and hereditary diabetes. Type 1 diabetes is insulin-dependent diabetes mellitus mainly caused by destruction of β-cells, and type 2 diabetes is non-insulin-dependent diabetes mellitus caused by insufficient insulin secretion or insulin resistance.

As used herein, the term “insulin resistance” refers to a state in which the function of insulin to lower blood sugar is lowered, so that cells cannot effectively burn glucose. When insulin resistance is high, the body produces an excess of insulin, which leads to high blood pressure, dyslipidemia, heart disease, diabetes, and the like. In particular, in type 2 diabetes mellitus, an increase in insulin in muscle and adipose tissue is not noticed, and the action of insulin does not occur.

As used herein, the term "fatty liver" refers to a state in which fat is accumulated in hepatocytes in an excessive amount due to a fatty metabolic disorder of the liver, which causes various diseases such as angina pectoris, myocardial infarction, stroke, arteriosclerosis and pancreatitis. Specifically, the fatty liver that can be prevented or treated by the composition of the present invention is non-alcoholic fatty liver. As used herein, the term “non-alcoholic fatty liver (NAFL)” refers to a disease in which an excessive amount of fat is accumulated in liver cells regardless of excessive alcohol absorption.

As used herein, the term “dyslipidemia” is a concept that includes hyperlipidemia, and appears as a problem such as hypercholesterolemia, hypertriglyceridemia, and low HDL-cholesterolemia, which appear due to an increase in the level of fat in the blood, as well as problems such as abnormal metabolism of lipoproteins. It means an abnormal lipid state.

As used herein, the term “hyperlipidemia” encompasses all diseases caused by an increase in blood fat concentration due to poor metabolism of fats such as triglycerides and cholesterol. More specifically, hyperlipidemia includes hypercholesterolemia or hypertriglyceridemia with a high incidence in a state in which lipid components such as triglycerides, LDL cholesterol, phospholipids and free fatty acids in the blood are increased. Specifically, the hyperlipidemia that can be prevented or treated by the composition of the present invention is hypertriglyceridemia.

When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil; it's not going to be The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, specifically, it is administered in a parenteral manner.

A suitable dosage of the pharmaceutical composition of the present invention is variously prescribed depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity of the patient. can be A preferred dosage of the pharmaceutical composition of the present invention is within the range of 0.001-100 mg/kg for adults.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person of ordinary skill in the art to which the present invention pertains. or it may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in oil or an aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.

According to another aspect of the present invention, the present invention provides a functional food composition for improving metabolic diseases comprising the above-described extract of the present invention as an active ingredient.

Since the extract of the present invention has already been described above, description thereof is omitted to avoid excessive duplication.

When the composition of the present invention is prepared as a food composition, it may include not only the pine bark extract as an active ingredient, but also carbohydrates, seasonings and flavoring agents that are commonly added during food production. Examples of carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol, and erythritol, but are not limited thereto. As the flavoring agent, natural flavoring agents (taumatine, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is prepared as a drink, citric acid, high fructose, sugar, glucose, acetic acid, malic acid, fruit juice, licorice root extract, jujube extract, licorice extract, etc. are added in addition to the pine bark extract, which is the active ingredient of the present invention. can be included as

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a method for producing a pine bark extract, a pine bark extract prepared through the same, and a pharmaceutical composition or functional food composition for preventing or treating metabolic diseases comprising the same as an active ingredient.

(b) The present invention can be usefully used as an efficient extraction process capable of obtaining a pine bark extract with significantly improved pharmacological activity against metabolic diseases in high yield.

1 is a diagram showing the area under the oral glucose tolerance response curve (AUC) of experimental animals treated with pine bark extract.
2 is a diagram showing the measurement results of glycated hemoglobin (HbA1c) in experimental animals treated with pine bark extract.
3 is a diagram showing the blood triglyceride measurement results of the experimental animals treated with the pine bark extract.
4 is a diagram showing the muscle weight measurement results of the experimental animals treated with the pine bark extract.
5 is a diagram showing the results of H&E staining of liver tissues of experimental animals treated with pine bark extract.
6 is a diagram showing the results of masson trichrome staining of liver tissues of experimental animals treated with pine bark extract.
7 is a diagram showing the results of masson trichrome staining of liver tissues of experimental animals treated with pine bark extract.
8 is a diagram showing the results of HPLC-MS spectrum of pine bark extract.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Example 1: Establishment of conditions for extraction of pine bark

Materials and Methods

The pine bark used in this study was purchased from the Dongbu Wood Distribution Center (Donghae, Korea). The pine bark was washed in running water and dried in a dark room, and the dried pine bark was homogenized using a blender. Mix 20 g of dried bark and 200 ml of solvent and use a 6-necked heating mantle (EAM-9202-06, 250 ml capacity, 150 WATTS, Medline Scientific Limited, Oxon, UK, OX44 7XZ) at 60 ± 5 ° C. , 9, and 15 hours were extracted. The extract was filtered using Whatman filter paper No. 1, and then concentrated under reduced pressure at 50°C. The concentrated pine bark extract was prepared at a concentration of 15 mg/ml using DMSO (dimethyl sulfoxide, Sigma, D8418) with a purity of 99.9%, and was stored at -20°C before use. Finally, the concentrated pine bark extract was powdered by spray-drying at 170°C.

Total phenol content of pine bark extract according to the change of extraction conditions

The total phenol content of the pine bark extract was measured using the Folin-Ciocalteu method. 0.8 ml of the extract was dissolved in distilled water to a concentration of 50 μg/ml, and then mixed with 0.1 ml Folin-Ciocalteu (Cat. No. 47641, Sigma, USA) reagent diluted at a ratio of 1:4. After standing at room temperature for 3 minutes, 0.1 ml of 10% sodium carbonate was added and left in the dark for 30 minutes. Then, 0.1 ml of the mixed solution was taken and placed in a 96-well plate, and the absorbance was measured at 760 nm. 0-20 μg/ml of gallic acid Cat. No. G7384, SIgma, USA) was used as a standard, and the total phenol content was expressed as gallic acid equilibrium (GAE) mg/g.

Table 1 below shows the total phenol content of the pine bark extract according to the extraction solvent. When extraction was performed under the same conditions except for the extraction solvent, when 20% ethanol was used as the extraction solvent, the total phenol content was the highest, followed by extraction with 40% ethanol showed the highest total phenol content. Confirmed.

Effect of extraction solvent on total phenol content in Pinus densiflora bark (PDB) extract extraction solvent extraction time TPC (GAE mg/g extract) water 9 h 146.35 ± 0.01 20% ethanol (v/v) 9 h 266.05 ± 4.30 ^*** 40% ethanol (v/v) 9 h 255.57 ± 4.44 ^*** 60% ethanol (v/v) 9 h 171.38 ± 1.09 ^*** 80% ethanol (v/v) 9 h 144.60 ± 3.22 ^ns 100% ethanol (v/v) 9 h 92.75 ± 2.04 ^***

Each value was expressed as gallic acid equilibrium (GAE) mg/g (extract), and was expressed as the mean ± standard deviation for three independent experiments. * **p < 0.001, ns - not significant compared to E0 (water extract).

Extraction yield of pine bark extract according to the change of extraction conditions

Table 2 below shows the change in yield of the pine bark extract according to the extraction solvent (0, 20, 40% ethanol), the extraction time (6, 9, 15 hours), and the extraction temperature (60 ℃, 100 ℃). The condition showing the highest extraction yield was extraction for 15 hours using 40% ethanol, followed by 40% ethanol/9 hours extraction showing a high extraction yield.

Effect of extraction solvent and extraction time on the yield of Pinus densiflora bark extract menstruum temperature Ethanol content extraction time transference number(%) sample number DW 60℃ 0 6hr 6.9 ± 0.5 #One 9hr 7.8 ± 0.7 #2 15hr 9.1 ± 0.7 #3 100℃ 0 6hr 9.1 ± 0.6 #4 9hr 10.3 ± 1.8 #5 15hr 10.9 ± 2.2 #6 ethanol 60℃ 20% 6hr 9.5 ± 1.8 #7 9hr 10.0 ± 2.1 #8 15hr 11.2 ± 1.9 #9 60℃ 40% 6hr 11.7 ± 2.1 #10 9hr 12.6 ± 2.1 #11 15hr 16.9 ± 8.2 #12

Antidiabetic activity of pine bark extract according to the change of extraction conditions (in vitro)

The in vitro antidiabetic efficacy was evaluated for the pine bark extracts for each of the 12 conditions shown in Table 2 above. The test substance was dissolved in DMSO (Sigma-Aldrich Co.) to prepare a 100 mg/ml stock solution, and added to the cell culture solution to achieve the treatment concentration set for each test.

Antidiabetic activity was evaluated on L6 cells, which are myoblasts derived from skeletal muscle of rats, and the cells were purchased from the Korea Cell Line Bank and used. L6 cells were cultured in DMEM medium (Welgene) with 10% FBS (fetal bovine serum), 100 units/mL penicillin, and 100 μg/mL streptomycin added to a cell culture medium at 37° C. in a humidified CO ₂ incubator (5% CO ₂ / 95% air). When the cells were about 80% full of the culture dish, the cell monolayer was washed with PBS (phosphate buffer saline, pH 7.4), and then the cells were removed and subcultured by adding trypsin-2.65 mM EDTA, and the medium was exchanged every 2 days. .

L6 cells were aliquoted in a 48-well plate so as to become 5 × 10 ⁴ cells/well, and the cells were cultured for 24 hours. After culturing the cells for 24 hours, each test substance was exchanged with a cell culture medium containing various concentrations, and the cells were cultured for 24 hours. After culturing the cells for 24 hours, the MTT assay (Denizot F and Lang R. J Immunological Method 89: 271-277, 1986) was performed to measure the number of viable cells. The MTT assay method is based on the principle that mitochondrial dehydrogenase reduces MTT (Amresco) to make formazan, a blue substance. In this test, formazan was dissolved in isopropanol and absorbance was measured at a wavelength of 570 nm.

L6 cells were aliquoted in a 96-well plate to 1 × 10 ⁴ cells/well, and the cells were cultured for 24 hours. Thereafter, the cells were cultured for 4 days by exchanging them with a cell culture medium containing 2% FBS to induce cell differentiation. After inducing cell differentiation, each test substance was exchanged with a cell culture medium containing various concentrations, and the cells were further cultured for 2 hours. Then, using the glucose uptake Colorimetric Assay Kit (BioVision), measure the glucose uptake according to the manufacturer's instructions. did

As shown in Table 3 below, in the case of insulin used as a positive control, glucose uptake in L6 muscle cells was 169.4 ± 8.3% compared to the control, and when 2 mM metformin was treated, glucose uptake of 172.4 ± 7.0% occurred. appeared to be It was confirmed that glucose absorption was significantly increased in #8, #9, #11, and #12 when #7 to #12, which had excellent total phenol content and yield among the 12 kinds of pine bark extracts in Table 2, were treated, In particular, in the case of #8 and #12 extracts, when treated at concentrations of 50 ug/ml and 100 ug/ml, glucose absorption of more than 200% was observed, indicating superior efficacy than insulin and metformin used as positive controls.

Effect of Extraction Solvent and Extraction Time of Pinus densiflora Bark Extract on Glucose Uptake in L6 Cells Glucose uptake (% versus control) #7 0 μg/mL 12.5 μg/mL 25 μg/mL 50 μg/mL Insulin (1 μM) metformin
(2 mM) 100.0 ± 6.6 89.9 ± 5.5 132.0 ± 21.5 122.3 ± 4.7 169.4 ± 8.3*** 172.5 ± 7.0*** #8 0 μg/mL 25 μg/mL 50 μg/mL 100 μg/mL Insulin (1 μM) metformin
(2 mM) 100.0 ± 6.6 161.1 ± 12.2** 217.1 ± 13.5*** 216.1 ± 5.0*** 169.4 ± 8.3*** 172.5 ± 7.0*** #9 0 μg/mL 12.5 μg/mL 25 μg/mL 50 μg/mL Insulin (1 μM) metformin
(2 mM) 100.0 ± 6.6 141.0 ± 11.3* 158.1 ± 15.0** 173.1 ± 14.4*** 169.4 ± 8.3*** 172.5 ± 7.0*** #10 0 μg/mL 50 μg/mL 100 μg/mL 200 μg/mL Insulin (1 μM) metformin
(2 mM) 100.0 ± 6.6 185.3 ± 12.8*** 143.5 ± 14.5* 128.1 ± 18.7 169.4 ± 8.3*** 172.5 ± 7.0*** #11 0 μg/mL 50 μg/mL 100 μg/mL 200 μg/mL Insulin (1 μM) metformin
(2 mM) 100.0 ± 6.6 182.4 ± 13.2*** 176.4 ± 3.8*** 176.3 ± 19.5*** 169.4 ± 8.3*** 172.5 ± 7.0*** #12 0 μg/mL 25 μg/mL 50 μg/mL 100 μg/mL Insulin (1 μM) metformin
(2 mM) 100.0 ± 6.6 165.0 ± 7.1*** 203.2 ± 11.4*** 203.0 ± 10.1*** 169.4 ± 8.3*** 172.5 ± 7.0***

Each value is expressed as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001 - Significantly different from 0 μg/mL-treated group

Efficacy of hepatocellular damage protection of pine bark extract according to changes in extraction conditions (in vitro)

Among the pine bark extracts for each of the 12 conditions shown in Table 2, the in vitro hepatocellular damage protection efficacy was evaluated for #7 to #12, which had excellent total phenol content and yield. The test substance was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich Co.) to prepare a 100 mg/ml stock solution, and added to the cell culture solution to achieve the treatment concentration set for each test.

HepG2 cells, which are liver cancer cells derived from human liver, were purchased from ATCC (American Type Culture Collection) and used. HepG2 cells were cultured in DMEM/F12 medium (Welgene) with 10% FBS (fetal bovine serum), 100 units/mL penicillin, and 100 μg/mL streptomycin added to a cell culture medium at 37°C in a humidified CO ₂ incubator (5% CO2). ₂ /95% air). When the cells were about 80% full of the culture dish, the cell monolayer was washed with PBS (phosphate buffer saline, pH 7.4), and then the cells were removed and subcultured by adding trypsin-2.65 mM EDTA, and the medium was exchanged every 2 days. .

HepG2 cells were aliquoted in a 48-well plate so as to become 5 × 10 ⁴ cells/well, and the cells were cultured for 24 hours. After culturing the cells for 24 hours, each test substance was exchanged with a cell culture medium containing various concentrations, and the cells were cultured for 24 hours. After culturing the cells for 24 hours, MTT assay was performed to measure the number of viable cells.

ALT (Alanine aminotransferase) is an enzyme present in hepatocytes and its release is increased due to hepatocellular injury. Therefore, to investigate the hepatoprotective effect of various test substances, ALT released from HepG2 cells induced by ethanol was measured. HepG2 cells were seeded into 48 wells at 5 × 10 ⁴ cells/well, and were stabilized for 24 hours. After 24 hours, the cells were exchanged with a serum free medium (SFM) and serum-free for 2 hours, and then the test substance was added at various concentrations together with ethanol (EtOH, 400 mM) used as a hepatotoxicity inducer in SFM. After treatment and incubation for 4 hours, the supernatant was taken. The ALT level in the cell culture was measured according to the method suggested by the manufacturer using a test kit for GPT measurement (Asan Pharmaceutical).

As shown in Table 4 below, when ethanol was not treated, ALT activity was significantly lower than that of the ethanol-treated group, and when all pine bark extracts were treated, the ALT activity induced by ethanol was significantly reduced. confirmed that. In particular, when #11 and #12 were each treated with a high concentration of the test substance, the ALT activity induced by ethanol was reduced to 50% or less.

Effect of PDB extract on ALT activity of HepG2 cells ALT activity (% versus control) EtOH (-) (+) (+) (+) (+) extract 0 ug/ml 0 ug/ml 50 ug/ml 100 ug/ml 200 ug/ml #7 46.8 ± 4.8 100.0 ± 6.0 ^### 83.3 ± 12.7 77.2 ± 4.0* 79.2 ± 7.0 #8 46.8 ± 4.8 100.0 ± 6.0 ^### 77.8 ± 6.6* 58.8 ± 3.9** 57.4 ± 2.3*** #9 46.8 ± 4.8 100.0 ± 6.0 ^### 79.9 ± 7.6 60.1 ± 6.6** 63.5 ± 1.9** #10 46.8 ± 4.8 100.0 ± 6.0 ^### 59.4 ± 4.2** 60.8 ± 1.1** 57.4 ± 5.7** #11 46.8 ± 4.8 100.0 ± 6.0 ^### 36.3 ± 6.2*** 26.7 ± 6.8*** 19.9 ± 7.3*** #12 46.8 ± 4.8 100.0 ± 6.0 ^### 58.8 ± 5.3** 41.0 ± 10.9*** 33.5 ± 5.6***

Each value is expressed as mean ± standard deviation. ### p < 0.001 - Significant difference compared to (-)+0 μg/mL-treated group. * p < 0.05, ** p < 0.01, *** p < 0.001 - significantly different from (+)+0 μg/mL-treated group.

Example 2: in vivo Antidiabetic effect of pine bark extract in

The in vivo antidiabetic activity of pine bark extract was evaluated using diabetic model mice (db/db) as follows.

animal model

6-week-old, male C57BL/6J mice and db/db mice without specific pathogens were purchased from Dooyeol Bio Co., Ltd. and used. After one week of quarantine and adaptation, healthy animals without weight loss were selected and used for the experiment. The animals had a temperature of 23 ± 3 °C, a relative humidity of 50 ± 10%, a ventilation rate of 10 to 15 times/hour, and a lighting time. It was bred in a breeding environment set at 12 hours (08:00~20:00) and an illuminance of 150~300 Lux. During the entire period of the test, the experimental animals were allowed to freely ingest solid feed for laboratory animals (Cargill Agripurina Co., Ltd.) and drinking water.

Test group and pine bark extract administration

After a one-week adaptation period, healthy animals were selected according to the egg mass method, (G1) normal control group, (G2) hyperglycemic control group, (G3) hyperglycemia + 20 mg/kg body weight (BW) pine bark extract (40% ethanol) #12 extract extracted for 15 hours) administration group, (G4) hyperglycemia + 50 mg/kg BW pine bark extract administration group, (G5) hyperglycemia + 100 mg/kg pine bark extract administration group, (G6) hyperglycemia + 100 mg/kg pycnogenol (Positive control group) It was classified into administration group (Table 5). Ten experimental animals were used in each test group, and food and drinking water were freely ingested to the experimental animals during the entire test period.

The test substance (pine bark extract) and the positive control substance (Pycnogenol) were dissolved in distilled water and orally administered according to the dose at a fixed time every day for 6 weeks. G3, G4, G5, G6) was administered orally.

Administration of Pine Bark Extract G1 G2 G3 G4 G5 G6 laboratory animal C57BL/6J db/db db/db db/db db/db db/db test substance
(mg/kg BW) - - pine tree
Extract (20) pine tree
Extract (50) pine tree
Extract (100) - control
(mg/kg BW) - - - - - Pycnogenol (100) number of experimental animals 10 10 10 10 10 10

Evaluation of the effect of pine bark extract on oral glucose tolerance test (OGTT)

After the test animals were fasted for 12 hours, the test substance was administered, and 2 g/kg BW glucose was orally administered 30 minutes later. After 30, 60, and 120 minutes, blood was collected from the tail of the experimental animal and blood glucose was measured using a blood glucose meter (ACCU-CHEK, Roche Diagnostics Korea).

Table 6 below shows the oral glucose tolerance of the experimental animals treated with the pine tree extract. After oral administration of the test substance and 2 g/kg of glucose, blood glucose was measured using tail vein blood 30, 60, and 120 minutes later. As a result, all groups showed the maximum blood glucose level 30 minutes after oral administration of glucose, and the blood glucose level gradually decreased over time. Blood glucose for 30 minutes was 993.1 ± 32.0 mg/dL, 924.6 ± 26.8 mg/dL, and 881.8 ± 31.4 mg/dL in the pine bark extract administration group (G3, G4, G5), respectively, and 1179.1 ± 44.0 mg/dL in the hyperglycemic control group (G2). They showed significantly lower blood glucose levels compared to dL. Blood glucose at 60 minutes was 865.5 ± 96.1 mg/dL, 917.4 ± 38.1 mg/dL, and 866.0 ± 30.5 in the pine bark extract-administered group (G3, G4, G5), respectively, compared to 1020.4 ± 36.8 mg/dL in the hyperglycemic control group (G2). It showed low blood glucose levels in mg/dL, but there was no significant difference. Blood glucose at 120 minutes was 694.2 ± 45.2 mg/dL and 686.0 ± 34.9 mg/dL in some pine bark extract-administered groups (G3, G5), respectively, which was significantly lower than 887.8 ± 48.1 mg/dL in the hyperglycemic control group (G2). blood sugar levels were indicated. Also, the area under the curves (AUC) was 1570.6 ± 96.2, 1612.7 ± 44.3, and 1504.5 ± 54.7 in the pine bark extract administration group (G3, G4, G5), respectively, and 1888.6 ± 70.4 in the hyperglycemic control group (G2). AUC was significantly decreased compared with .

Oral glucose tolerance of experimental animals (mg/dl) G1 G2 G3 G4 G5 G6 laboratory animal C57BL/6J db/db db/db db/db db/db db/db test substance
(mg/kg) - - pine tree
Extract (20) pine tree
Extract (50) pine tree
Extract (100) Pycnogenol (100) 0 minutes 64.5 ± 2.9 359.5 ± 27.7 311.2 ± 23.8 314.3 ± 29.8 284.2 ± 24.5 304.3 ± 36.6 30 minutes 206.9 ± 12.3 1179.1 ± 44.0 993.1 ± 32.0 924.6 ± 26.8 881.8 ± 31.4 822.8 ± 63.7 60 minutes 141.8 ± 5.9 1020.4 ± 36.8 865.5 ± 96.1 917.4 ± 38.1 866.0 ± 30.5 810.8 ± 76.1 120 minutes 115.8 ± 6.2 887.8 ± 48.1 694.2 ± 45.2 767.6 ± 32.1 686.0 ± 34.9 655.6 ± 63.4

Evaluation of the effect of pine bark extract on glycated hemoglobin (HbA1c)

Anesthesia prepared by diluting tribromoethanol with quaternary tert-amyl alcohol was used to anesthetize the experimental animals, and then blood was collected from the orbit. A portion of whole blood was taken and the glycated hemoglobin in the blood was measured using a glycated hemoglobin meter (HLC-723G7, Tosoh).

As a result of measuring the glycated hemoglobin content, which is an indicator of the long-term blood glucose level, using whole blood collected at the end of the test, the glycated hemoglobin in the normal control group (G1) was 4.33 ± 0.15%, and the glycated hemoglobin level in the hyperglycemic control group (G2) was 8.43 It was significantly increased as compared to the normal control group (G1) at ± 0.19%.

The glycated hemoglobin levels of the 50 mg/kg BW pine bark extract group (G4) and the 100 mg/kg BW pine bark extract group (G5) were 7.41 ± 0.37% and 7.51 ± 0.12%, respectively, which were significant compared to the hyperglycemic control group (G2). was decreased (Fig. 2).

Blood collection and serum lipid analysis of experimental animals administered with pine bark extract

Experimental animals administered with pine bark extract were fasted for 16 hours before sacrifice, and then anesthetized using an anesthetic prepared by diluting tribromoethanol with quaternary tert-amyl alcohol, and then blood was collected from the orbit. Blood was placed in a serum separation tube, left at room temperature for 30 minutes, centrifuged at 3,000 rpm for 20 minutes to separate serum, and stored at -70° C. until analysis. The content of glucose and triglycerides in serum and the activity of ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase) and γ-GT (gamma glutamyl transpeptidase) were analyzed using a blood biochemical analyzer (KoneLab 20 XT, Thermo Fisher Scientific). was used for analysis.

To evaluate the effect of the test substance on serum lipids, the level of triglycerides in the serum collected at the end of the test was measured using a blood biochemical analyzer. The triglyceride level was significantly increased in the hyperglycemic control group (G2) compared to the normal control group (G1), and was significantly increased in the group administered with 50 mg/kg BW pine bark extract (G4) and the group administered with 100 mg/kg BW pine bark extract (G5). was reduced to (Fig. 3).

Analysis of changes in muscle weight of experimental animals administered with pine bark extract

After sacrificing the experimental animals to which the pine bark extract of the present invention was administered, skeletal muscle was extracted, rinsed with cold physiological saline, and excess moisture was removed with filter paper, and then the weight was measured. As a result, the muscle weight of the normal control group (G1) was 14.8 ± 0.39 mg, and the hyperglycemic control group (G2) was 12.4 ± 0.31 mg, which significantly decreased muscle weight compared to the normal control group. The muscle weight of the 100 mg/kg BW pine bark administration group (G5) was 14 ± 0.47 mg, which was significantly increased compared to the hyperglycemic control group (G2) ( FIG. 4 ).

Example 3: in vivo Hepatocellular damage protective effect of pine bark extract in

Animal model used to evaluate the hepatocellular damage protection efficacy of pine bark extract

6-week-old, male C57BL/6J mice and db/db mice without specific pathogens were purchased from Dooyeol Bio Co., Ltd. and used. After one week of quarantine and adaptation, healthy animals without weight loss were selected and used for the experiment. The animals had a temperature of 23 ± 3 °C, a relative humidity of 50 ± 10%, a ventilation rate of 10 to 15 times/hour, and a lighting time. It was bred in a breeding environment set at 12 hours (08:00~20:00) and an illuminance of 150~300 Lux. During the entire period of the test, the experimental animals were allowed to freely ingest solid feed for laboratory animals (Cargill Agripurina Co., Ltd.) and drinking water.

Test group and pine bark extract administration

After a one-week adaptation period, healthy animals were selected according to the egg mass method, (G1) normal control group, (G2) hyperglycemic control group, (G3) hyperglycemia + 20 mg/kg body weight (BW) pine bark extract administered group, (G4) ) Hyperglycemia + 50 mg/kg BW pine bark extract administration group, (G5) hyperglycemia + 100 mg/kg pine bark extract administration group, (G6) hyperglycemia + 100 mg/kg pycnogenol (positive control) administration group (Table 5). Ten experimental animals were used in each test group, and food and drinking water were freely ingested to the experimental animals during the entire test period.

The test substance (pine bark extract) and the positive control substance (Pycnogenol) were dissolved in distilled water and orally administered according to the dose at a fixed time every day for 6 weeks. G3, G4, G5, G6) was administered orally.

Effect of pine bark extract on histomorphologic changes in liver tissue

Liver tissues fixed with 4% PFA were embedded in paraffin, and tissue sections of 5 μm were prepared from the embedded tissues. After paraffin removal, tissues were hydrated by lowering the percentage of alcohol sequentially, starting with 100% alcohol and reaching 0% ethanol (H ₂ O). For histomorphological observation of liver tissue, ① Accustain ^® Hematoxylin and Eosin Stains (Sigma-Aldrich Co.) were used and the tissue was stained according to the method suggested by the manufacturer. To observe the degree of histological fibrosis of liver tissue, ② Sirius red (abcam) staining and ③ Massaon trichrome (Sigma-Aldrich Co.) staining were performed according to the method suggested by the manufacturer. Thereafter, histological changes in liver tissue were observed using an optical microscope (Carl Zeiss).

8 shows the results of observation under a microscope after H&E staining of liver tissue to investigate histomorphological changes in liver tissue. The liver tissue of the normal control group (G1) showed a uniform shape without accumulation of fat, whereas the liver tissue of the hyperglycemic control group (G2) showed fat accumulation and showed a shape significantly different from that of the normal control group (G1). It was observed that the distribution of accumulated fat cells was somewhat decreased in the liver tissue morphology of the test substance, pine bark extract administered group (G3~G5) compared to the hyperglycemic control group (G2). As a result of the masson trichrome staining shown in FIG. 6 , it was observed that the level of collagen production in the pine bark extract administered group (G3 to G5) was somewhat decreased compared to the hyperglycemic control group (G2), and as shown in FIG. 10, Sirius red staining results of pine In the liver tissue of the bark extract-administered group (G3 to G5), it was observed that the level of collagen production was somewhat decreased.

Effect of pine bark extract on liver function indicators

In general, ALP (alkaline phosphatase), one of the important biochemical test items used for the diagnosis of liver disease, is an enzyme involved in the transport of metabolites through the cell membrane and is present on the surface of bile duct epithelial cells. disease or bone disease. ALP (alkaline phosphatase) activity in the serum of experimental animals was analyzed using a blood biochemistry analyzer (KoneLab 20 XT, Thermo Fisher Scientific). Table 7 below shows ALP activity in experimental animals treated with pine bark extract. The increased ALP activity in the hyperglycemic control group (G2) was 20 mg/kg BW pine bark extract administered group (G3) and 50 mg/kg BW pine bark extract It was significantly decreased in the administration group (G4).

ALP activity by concentration of pine bark extract G1 G2 G3 G4 G5 G6 laboratory animal C57BL/6J db/db db/db db/db db/db db/db test substance - - pine bark
extract pine bark
extract pine bark
extract Pycnogenol 20 mg/kg 50 mg/kg 100 mg/kg 100 mg/kg ALP
(U/L) 218.2 ± 11.5 449.5 ± 12 384.4 ± 15.5 377.7 ± 13.1 420.6 ± 18.4 438.2 ± 19.3

Effect of pine bark extract on lipid peroxide formation and antioxidant enzyme activity

Representative antioxidant enzymes responsible for the body's antioxidant system include SOD, CAT, and GPx, and oxygen free radicals are converted into hydrogen peroxide (H ₂ O ₂ ) by SOD, which is again converted to water by CAT and GPx and detoxified. To evaluate the oxidative stress caused by diabetes and its effect on the antioxidant system, the level of lipid peroxide (MDA) in liver tissue and the antioxidant enzymes SOD, CAT, and GPx activity were measured. As shown in Table 8 below, the level of lipid peroxidation in the liver tissue was significantly increased to 22.69 ± 0.34 μM in the hyperglycemic control group (G2) compared to 19.38 ± 1.13 μM in the normal control group (G1), and 50 mg/kg BW pine bark The extract administration group (G4) and the 100 mg/kg BW pine bark extract administration group (G5) showed 20.90 ± 0.39 μM and 20.62 ± 0.57 μM, respectively, which were significantly reduced compared to the hyperglycemic control group (G2). The lipid peroxidation level of the pycnogenol-administered group (G7) was 19.72 ± 0.55 μM, which was significantly reduced compared to that of the hyperglycemic control group (G2). SOD activity in liver tissue was significantly reduced to 1.22 ± 0.07 U/mg protein in the hyperglycemic control group (G2) compared to 1.58 ± 0.07 U/mg protein in the normal control group (G1), and compared to the hyperglycemic control group (G2). It was significantly increased in the group administered with 50 mg/kg BW pine bark extract (G4), group administered with 100 mg/kg BW pine bark extract (G5), and group administered with pycnogenol (G7). CAT activity and GPx activity in liver tissue were significantly decreased in the hyperglycemic control group (G2) compared to the normal control group (G1), and compared to the hyperglycemic control group (G2), 100 mg/kg BW pine bark extract administered group (G5) and Pycnogenol administration group (G7) significantly increased.

Antioxidant activity of pine bark extract G1 G2 G3 G4 G5 G6 laboratory animal C57BL/6J db/db db/db db/db db/db db/db test substance - - pine bark
extract pine bark
extract pine bark
extract Pycno
xenol 20 mg/kg 50 mg/kg 100 mg/kg 100 mg/kg MDA
(μM) 19.38 ± 1.13 22.69 ± 0.34 21.52 ± 0.55 20.90 ± 0.39 20.62 ± 0.57 23.93 ± 0.84 SOD activity
(U/mg protein) 1.58 ± 0.07 1.22 ± 0.07 1.43 ± 0.07 1.47 ± 0.08 1.51 ± 0.08 1.50 ± 0.06 CAT active
(nmol/min/mg protein) 8.94 ± 0.45 4.66 ± 0.30 4.56 ± 0.42 5.86 ± 0.46 6.15 ± 0.39 4.96 ± 0.48 GPx active
(nmol/min/mg protein) 117.9 ± 8.9 78.6 ± 4.7 80.5 ± 4.2 82.3 ± 5.7 99.9 ± 3.9 80.8 ± 7.3

Example 4: Component Analysis of Pine Bark Extract

LC and MS

To analyze the components in the pine bark extract, a HALO.5 C18 (2.1 x 150 mm, 5 μm) column, mobile phase A with 0.1% TFA in 5% acetonitrile and mobile phase B with 0.1% TFA in acetonitrile were used. Liquid chromatography (LC) was performed. Analysis was performed under gradient elution conditions in Table 9 below at a sample injection volume of 10 μL, a flow rate of 0.2 ml/min and a column temperature of 30°C:

Gradient elution conditions for both mobile phases of LC hours (minutes) A (%) B (%) 0 100 0 3 100 0 10 90 10 30 90 10 40 70 30 50 0 100 55 0 100 60 100 0 70 100 0

Mass spectroscopy (MS) is Capillary voltage 3.57 kV, cone voltage 30 V, source temperature 130°C, desolute temperature 400°C, desolute gas flow rate 500 L/hr, cone gas flow rate 50 L/hr and detection molecular weight value [M+ Na] ⁺ 515.

Calibration curve creation and measurement

Precisely weigh 10 mg of masonianoside B, put it in a 10 mL volumetric flask, set it up to the mark with methanol, and dissolve it by sonication to prepare a standard stock solution, which is diluted with methanol and compared to the standard concentration (25 μg/mL, 100%) Five concentrations were prepared so as to be 25% (6.25 μg/mL), 50% (12.5 μg/mL), 100% (25 μg/mL), 200% (50 μg/mL) and 400% (100 μg/mL).

The area of the masonianoside B peak with a molecular weight [M+Na]+515 that appears at a retention time of 24.6 minutes by analysis by HPLC according to the experimental conditions was measured, and the A calibration curve was prepared using the area.

For the pine bark extract, 1.6 g of the sample was precisely weighed, placed in a 10 mL volumetric flask, and 9 mL of methanol was added, ultrasonically treated for 15 minutes, and then placed at room temperature for 30 minutes to lower the solvent temperature to room temperature. In addition, methanol was added, adjusted to the 10 mL mark, thoroughly mixed, and centrifuged to precipitate methanol-insoluble residues in the sample. After that, 1 mL of the supernatant was filtered through a 0.45 μm membrane filter and analyzed by HPLC.

According to the experimental conditions, the area of the masonianoside B peak with a molecular weight of [M+Na]+ 515 that appears at 24.6 minutes of retention time by HPLC analysis according to the experimental conditions is substituted into the above calibration curve and included in the pine bark extract according to the formula below Measure the content of masonianoside B.

Massonianoside B content in PDE sample (ug/g) =

A: Total amount of test solution (mL) (= 10 mL)

B: Masonicanoside B concentration of the test solution calculated from the calibration curve (ug/mL)

C: Sample amount of pine bark (g)

As a result of the measurement, it was confirmed that the pine bark extract of the present invention contained 139 μg/g extract of masonianoside B, which has not been reported to be contained in Pinus densiflora ( FIG. 8 ).

마소니아노사이드 B

Masonicanoside B

As the specific parts of the present invention have been described in detail above, for those of ordinary skill in the art, these specific descriptions are only preferred embodiments, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (13)

A method for preparing a pine bark extract comprising Massonianoside B, comprising the steps of:
(a) adding 38 to 42% alcohol to the dried pine bark and extracting at 55 to 65° C. for 14 to 16 hours; and
(b) pulverizing the extract obtained in step (a).
The method of claim 1,
The method characterized in that it further comprises a filtration step and a concentration step for the extract obtained in step (a) in any order between step (a) and step (b).
3. The method of claim 2,
The filtration step is a method characterized in that it is performed using a filter paper (filter paper) having a pore size of 9 to 13 μm.
The method of claim 1,
The method, characterized in that the alcohol in step (a) is added so that the weight ratio with the dried pine bark is 1:8 to 1:12.
delete delete The method of claim 1,
The pine is Pinus densiflora ( Pinus densiflora ) Method, characterized in that.
The method of claim 1,
A method, characterized in that the dried pine bark is obtained by pulverizing the collected pine bark into a sample having a size of 3 cm or less after drying in a dark room.
A total phenol content (TPC) of at least 250 GAE mg/g of extract, prepared by the method of any one of claims 1 to 4, 7 and 8, and containing Massonianoside B pine bark extract.
delete A pharmaceutical composition for preventing or treating diabetes, fatty liver or hypertriglyceridemia comprising the extract of claim 9 as an active ingredient.
delete A functional food composition for improving diabetes, fatty liver or hypertriglyceridemia comprising the extract of claim 9 as an active ingredient.

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