KR20180058160A - Extract of Cirsium japonicum var. maackii (Maxim) having anti-diabetic effect and the manufacturing method thereof - Google Patents

Abstract

The present invention relates to a Cirsium japonicum var. maackii (Maxim) extract having an anti-diabetic activity and a method for preparing the same and, more specifically, to a Cirsium japonicum var. maackii (Maxim) extract having an anti-diabetic activity and a method for preparing the same, wherein the Cirsium japonicum var. maackii (Maxim) extract is prepared by preparing dry Cirsium japonicum var. maackii (Maxim), adding alcohol to perform extraction, and conducting filtration, concentration, lyophilization, and pulverization steps, and the Cirsium japonicum var. maackii (Maxim) extract includes linarin, pectolinarin, apigenin, hispidulin, cirsimarin, and cirsimaritin, thereby increasing insulin secretion, protecting pancreatic cells from streptozotocin, and significantly enhancing anti-diabetic efficacy. The method of the present invention comprises: a step of preparing dry Cirsium japonicum var. maackii (Maxim); a step of adding alcohol to the dry Cirsium japonicum var. maackii (Maxim) and extracting the same; a step of filtering the extracted dry Cirsium japonicum var. maackii (Maxim) by using a filtration means; a step of concentrating the filtered dry Cirsium japonicum var. maackii (Maxim); a step of lyophilizing the concentrated dry Cirsium japonicum var. maackii (Maxim); and a step of pulverizing the lyophilized Cirsium japonicum var. maackii (Maxim) into powder. The prepared Cirsium japonicum var. maackii (Maxim) extract is characterized by including linarin, pectolinarin, apigenin, hispidulin, cirsimarin, and cirsimaritin.

Description

Extract of Cirsium japonicum var. ≪ RTI ID = 0.0 > I. < / RTI > maackii (Maxim) having anti-diabetic effect and the manufacturing method thereof}

The present invention relates to a thistle extract having an anti-diabetic activity and a method for preparing the same. More specifically, the present invention relates to a thistle extract having an anti-diabetic activity and, more particularly, Wherein the thistle extract comprises components of linarin, feticolin, apigenin, hethipalin, cirsimarin and cirsimaritin, whereby the thistle extract and components increase insulin secretion, The present invention relates to a thistle extract having anti-diabetic activity which can protect the pancreatic cells from the mastocytoma and significantly improve the antidiabetic effect, and a method for producing the same.

Diabetes mellitus is a type of metabolic disease that lacks insulin secretion or does not function normally. It is characterized by hyperglycemia in which the concentration of glucose in the blood rises. Hyperglycemia causes various symptoms and signs and excretes glucose from the urine .

This type of diabetes is divided into type 1 and type 2, type 1 diabetes was formerly called 'child diabetes' and is caused by the failure to produce insulin at all. Type 2 diabetes, which is relatively insufficiently insulin-dependent, is characterized by insulin resistance (insulin resistance that lowers blood sugar and cells do not burn glucose effectively). Type 2 diabetes may be due to environmental factors such as high calorie, high fat, high protein diet, lack of exercise, and stress due to westernization of dietary habits. However, diabetes mellitus may also be caused by defects of specific genes, Infection, or drug.

Chronic hyperglycemia due to diabetes results in damage and dysfunction of the organs of the body, especially microvascular complications in the retina, kidneys, nerves and macrovascular complications such as arteriosclerosis, cardiovascular and cerebrovascular diseases, and the mortality rate Diabetes is considered to be one of the major health threats to modern people.

Thistle (Cirsium japonicum var. Maackii (Maxim)) is a perennial plant belonging to the family Asteraceae, distributed in Korea, Japan, and northeastern China. It is 50 ~ 100 ㎝ in height. It has hairs like cobwebs with white hair as a whole. Root leaves remain till petals and larger than stem leaves. The stem leaf is fibrillated elliptical, and is split like a feather. The lower part encircles the base, and the most split part is split again and there is a thorn with the teeth on the acute angle. Flowers bloom from June to August, purple to red. Arms of guns are arranged in 7 to 8 rows, and the inside is longer. The fruit is achenium and the tubular length is 16 ~ 19 ㎜. Leaves are narrow and green, some thorns are narrow leaf thistles, and thorns are often visible thorns.

This thistle is known as a pharmacological agent and has been known to have the efficacy of hematopoietic stem (hemostasis), hemorrhagic detoxification (hemostatic detoxification), and inflammation (Jang Changsoo et al., 1998; In particular, thistles are known to have excellent hemostatic properties. The major component of hemostatic activity is pectolinarin, which is reported to be dose-dependent in mouse experiments (Ishida et al., 1987 ). In addition, thistle promotes the proliferation of lymphocytes, increases the activity of NK cells, and inhibits the tumor. Thus, the anti-cancer and immune enhancement

(Liu et al., 2006). It is also known to be used in the treatment of symptoms such as colds, pertussis, enteritis, nephritis, hematemesis, hematuria, hemorrhage, postpartum bleeding, (Lee, 2002; Kim, 1997).

In addition, components having such various physiological activities are separated and purified from thistles. The physiologically active components of thistle that have been found so far include aplotaxene, dihydroaplotaxene, tetrahydroafflotaxene, such as tetrahydroaplotaxene, hexahydroaplotaxene, cyperene, caryophyllene, thujopsene, and alpha-himachalene, are separated, and alpha -Amyrin and β-amyrin have been isolated. In addition, polyacetylene compounds have recently been isolated.

However, studies on the antidiabetic activity of the maritin component of the thistle extract, especially the thistle extract, have not been made at present.

Korean Patent No. 10-1352591 (2014.01.10) Korean Patent No. 10-1281710 (2013.06.27)

The present invention has been made in order to solve the problems of the prior art as described above, and it is an object of the present invention to provide a thistle extract by preparing dry thistle, adding alcohol, extracting, filtering, concentrating, lyophilizing and pulverizing, The thistle extracts include components such as linarin, fektrnarin, apigenin, hethipidulin, sullimarin and sulcimarotin, whereby the thistle extract and components increase insulin secretion and protect pancreatic cells from streptozotocin, It is an object of the present invention to provide a thistle extract having anti-diabetic activity which can remarkably enhance the diabetic effect and a method for producing the same.

In order to accomplish the above object, the present invention provides a thistle extract having antidiabetic activity and a method for preparing the same, comprising: preparing a dried thistle; Adding alcohol to the dry thistle and extracting it; Filtering the extracted dried thistles using filtration means; Concentrating said filtered dried thistle; Lyophilizing the concentrated dried thistle; Pulverizing the lyophilized dried thistle; Lt; / RTI >

The prepared thistle extracts are characterized in that they contain linalin, fektorinin, apigenin, histidine, sulcimarine and dessalmitin components.

The thistle extract having anti-diabetic activity according to the present invention and the method for producing the same according to the present invention include thalidomide extract, linalin, fektorinin, apigenin, hethipalin, sullimarin and dessalmitin, And components have the effect of increasing insulin secretion and protecting pancreatic cells from streptozotocin, thereby significantly improving anti-diabetic efficacy.

More specifically, the sulciparital component of the present invention has an effect of excellently insulin secretion effect superior to gliclazide corresponding to an insulin secretagogue (a therapeutic agent for diabetic patients).

Also, there is an effect that the cell death rate is decreased and the cell survival rate is increased when treating the streptozotocin substance which induces apoptosis and stops insulin secretion by destroying the pancreatic cell.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flow chart illustrating a method of preparing a thistle extract having anti-diabetic activity according to an embodiment of the present invention. FIG.
FIG. 2 shows results of measurement of caspase-8, -3, Bid, Bcl-2 and PARP protein expression of thistle extract having anti-diabetic activity according to an embodiment of the present invention.
FIG. 3 is a graph showing the results of the cytotoxicity assay of a thistle extract having anti-diabetic activity according to an embodiment of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail. These examples are for further illustrating the present invention, and the scope of rights of the present invention is not limited to these examples.

The present invention relates to a thistle extract having antidiabetic activity and a method for preparing the same, comprising the steps of: (S10) preparing a dried thistle; Adding alcohol to the dried thistle to extract (S20); Filtering the extracted dried thistles by filtration (S30); Concentrating the filtered dried thistle (S40); Lyophilizing the concentrated dried thistle (S50); Powdering the lyophilized dried thistle (S60); .

At this time, the thistle extract prepared by the above method is characterized in that the extract contains Linarin, Pectolinarin, Apigenin, Hispidulin, sulcimarine and dessalmitin components .

Meanwhile, the step of preparing the dried thistle (S10) is characterized by preparing a dried thistle outpost.

Also, the extracting step (S20) comprises adding 25 to 35% of alcohol to the dried thistle and extracting it at 75 to 85 ° C for 2 to 4 hours, wherein the alcohol includes alcohol and ethanol.

The reason why 25 to 35% of the alcohol is used is that the activity is lowered when the concentration of the alcohol is less than 25% or exceeds 35%.

In addition, 25 to 35% of alcohol is added to the dried thistle, and the ratio of the dried thistle to the alcohol is 1:20.

The filtering step S30 is characterized in that the filtering means includes a filter paper.

Also, the concentration step (S40) is characterized in that it is concentrated at 75 to 85 DEG C for 2 to 4 hours so as to be a final 10 to 13 Bricks.

The concentrated dried thistles were lyophilized and then pulverized to give Linarin, Pectolinarin, Apigenin, Hispidulin, Sulfimarin and Sullivan, Tin components can be identified and separated by HPLC analysis for the analysis of the components, more specifically using a Waters 1525 Binary HPLC instrument as well as the INNO column C18 (4.6 × 250 mm, 5 μm ) Column may be used for analysis. The solvent composition of the mobile phase was determined by slanting of water and acetonitrile, starting with water and acetonitrile (83:17) for 10 minutes, flowing at a rate of 70:30 for 20 minutes, The flow rate is 1 ml / min, and all the experiments can be carried out in 3 repetitions.

In general, thistle (Cirsium japonicum var. Maackii (Maxim)) is a perennial plant belonging to the family Asteraceae. It is known as a medicine and is known to have the efficacy of hematopoietic (hemostatic) have. In particular, thistle is known to have excellent hemostatic function. The major component of hemostatic function is pectolinarin, which is reported to be dose-dependent in mice. In addition, thistle promotes the proliferation of lymphocytes, increases the activity of NK cells and inhibits tumors, and thus has anticancer and immunity enhancing effects. In addition, it is considered that thistle has effects of cold, pertussis, enteritis, nephritis, , Bleeding in the stool or postpartum is not stopped, or in the treatment of symptoms such as major depression and boils.

Among the components of the thistle extract, Linarin is one of the flavonoids and is contained together with Pectolinarin in flower, has antioxidative activity, has anti-inflammatory and antiviral efficacy, and Apigenin also has flavonoid Is one of the antioxidant, anti-inflammatory effect, as well as the effect of stimulating the production of brain cells.

Particularly, the sialic acid component of the present invention is superior in insulin secretion effect to gliclazide, which is an insulin secretagogue (therapeutic drug for diabetic patients), and also has an effect of inducing apoptosis and destroying pancreatic cells, In the case of the crude toxic substance, the cell death rate is reduced during the treatment with the maritimin component to increase the cell survival rate.

Thus, the thistle extract having anti-diabetic activity according to the present invention contains linarin, fektornerin, apigenin, histpidalin, sullimarin and sullimaritin components in the thistle extract, so that the thistle extract and components increase insulin secretion And protects the pancreatic cells from streptozotocin, thereby remarkably improving the anti-diabetic effect.

< Example  1> Anti-diabetic  Preparation of active thistle extracts

Prepare dried thistle.

At this time, 15 g of the powder obtained by crushing the dried thistle with a shrunken dried thistle outpipe is prepared.

30% alcohol is added to the prepared dried thistle, and the mixture is extracted at 80 캜 for 3 hours.

At this time, 30% of alcohol is added to the dried thistle, and the ratio of the dried thistle to the 30% alcohol is 1:20. That is, 300 g of 30% alcohol is added to 15 g of dried thistle.

The extracted dried thistle is filtered with filter paper (HYUNDAI Mircro, No. 20).

The filtered dried thistle is concentrated at 80 &lt; 0 &gt; C for 3 hours to a final 11.8 brix.

The concentrated dried thistle is lyophilized.

The lyophilized dried thistle is pulverized.

Thereby identifying and isolating Linarin, Pectolinarin, Apigenin, Hispidulin, sulcervicin and sullimaritin components in the pulverized dried thistle, which is a finished thistle extract, do.

More specifically, the apparatus used for the analysis was a Waters 1525 Binary HPLC. The column used for the analysis was INNO column C18 (4.6 x 250 mm, 5 &lt; RTI ID = 0.0 &gt; μm). The solvent composition of the mobile phase is carried out using a slanting technique of water and acetonitrile, starting with water and acetonitrile (83:17) for 10 minutes and flowing at a rate of 70:30 for 20 minutes. For the next 5 minutes, flow at a rate of 20:80. The injection amount is 10 μl, the wavelength is 270 nm, the flow rate is 1 ml / min, and all experiments are carried out in three iterations.

< Experimental Example  1> Thistle extract and insulin of ingredient Secretory function  Evaluation (In vitro, in vitro test)

Insulin secretory potency was evaluated as follows using rat insulinoma cell line INS-1 (Biohermes, Shanghai, China).

(1) Toxicity measurement of thistle extract and components against INS-1 cells

INS-1 cells were incubated with 100 ug / ml of 10% fetal bovine serum (Gibco BRL, Life Technologies), penicillin G 100 units / ml (Gibco BRL, Life Technologies), streptomycin (Gibco BRL Life Technologies) , Gibco BRL Life Technologies), 2 mM L-glutamine (Gibco BRL Life Technologies), 1 mM sodium pyruvate (Gibco BRL Life Technologies) and 0.05 mM 2-mercaptoethanol Was cultured in a 95% air / 5% carbon dioxide incubator maintained at 37 ° C using RPMI1640 (Cellgro, Manassas, VA, USA) medium supplemented with 2-mercaptoethanol (Gibco BRL Life Technologies). The cultured INS-1 cells were dispensed into 96-well plates at 1 × 10 ⁴ cells per well and the cells were allowed to stabilize for 24 hours. Then, the thistle extract and components were added to the designated concentrations, and after culturing for 24 hours, 10 μl of CCK-8 (CCK-8, Dojindo Laboratories, Japan) reagent was added to each well and cultured at 37 ° C. After 1 hour, the viability of the cells was measured by measuring the absorbance using a 450 nm detection wavelength in a BIO-TEK (Winooski, VT, USA) microplate reader.

Table 1 below shows the toxicity of thistle extract and components to INS-1 cells, which are insulinoma cells in rats. Thistle extract was not cytotoxic when treated with 1 μg / ml, 2.5 μg / ml, 5 μg / ml and 1 μM, 2.5 μM and 5 μM components.

This indicated that the thistle extract and components were not toxic and safe for INS-1 cells.

division Concentration (μg / ml) Cell survival rate (%) Thistle extract One 88.9 ± 3.2 2.5 87.2 ± 0.0 5 88.4 ± 2.0 division Concentration (μM) Cell survival rate (%) Linarin One 90.4 ± 3.2 2.5 90.9 ± 2.6 5 90.4 ± 1.3 Pectolinarin One 91.1 ± 3.7 2.5 96.4 ± 3.2 5 91.9 ± 2.4 Apigenin One 94.4 ± 4.1 2.5 88.0 ± 1.1 5 95.7 ± 3.3 Hispidulin One 86.8 ± 1.2 2.5 89.9 ± 2.4 5 90.0 ± 2.2 Cirsimarin One 97.5 ± 3.8 2.5 91.3 ± 2.8 5 93.5 ± 1.9 Maritin
(Cirimaritin) One 102.7 ± 3.4 2.5 101.0 ± 0.3 5 101.0 ± 0.7

(Toxicity of Thistle Extract and Ingredients to INS-1 Cells)

(2) Determination of insulin secretion by INS-1 cells from thistle extract and components

The cultured INS-1 cells were dispensed into 12-well plates at 5 × 10 ⁵ per well and the cells were allowed to stabilize for 24 hours. Sigma, St. Lois), 4.4 mM potassium chloride (KCL; Sigma, St. Louis), 1 mM magnesium sulfate (MgSO4; Sigma, St. Louis), 1.28 mM calcium chloride (CaCl2; Sigma, St. Louis, Sigma, St. Lois), 10 mM Hepes (Gibco BRL Life Technologies), 0.1% bovine serum albumin (BSA; Sigma, St. Louis) The hydrogen ion concentration was washed twice with Krebs-Ringer buffer (pH 7.4), and the mixture was incubated for 1 hour. Krebs-Ringer buffer ) For 30 minutes. After incubation for 1 hour with Krebs-Ringer buffer containing 3.3 mM and 16.7 mM glucose, the cells were centrifuged at 12,000 rpm for 10 minutes at 4 ° C, (Gentaur Molecular Products, Belgium). Stimulation index (SI) is calculated from the value measured at high (the amount of insulin secreted by 16.7 mM glucose stimulation) divided by the value measured at low (the amount of insulin secreted by 3.3 mM glucose stimulation).

Table 2 shows the insulin secretion enhancement (SI) according to the concentration of thistle extract and ingredient concentration in order to examine the glucose-dependent insulin secretion enhancement pattern by the thistle extract and components in INS-1 cells. 2.5 μg / ml, 5 μg / ml, and 1 μM, 2.5 μM, and 5 μM treatments of thistle extracts in the presence of low glucose (3.3 mM) and high concentration of glucose (16.7 mM) After one hour of insulin secretion, the stimulation index (SI) increased in a concentration-dependent manner.

Among the above components, SI was similar to SI after treatment with Gluclazide (Gliclazide) 20 μM in the positive control and 1 μM of SSM in treatment of SSM.

Gliclazide, which is used as a positive control, is a therapeutic drug for diabetic patients and is an insulin secretagogue. Therefore, it can be seen that the SI value after treatment with Gliclazide (20 μM) and the SI value and 1 μM of sarsimitin are similar, suggesting that the insulin secretion effect of sarsimitin is remarkably excellent.

division Concentration (μg / ml) Stimulation index (SI) Thistle extract One 1.4 ± 0.0 2.5 2.2 ± 0.0 5 2.7 ± 0.2 division Concentration (μM) Stimulation index (SI) Linarin One 3.1 ± 0.0 2.5 4.8 ± 0.0 5 6.7 ± 0.1 Pectolinarin One 3.0 ± 0.0 2.5 4.8 ± 0.1 5 4.9 ± 0.1 Apigenin One 7.0 ± 0.0 2.5 7.8 ± 0.1 5 8.1 ± 0.2 Hispidulin One 5.2 ± 0.0 2.5 6.0 ± 0.0 5 6.7 ± 0.1 Cirsimarin One 3.1 ± 0.0 2.5 4.8 ± 0.0 5 6.7 ± 0.1 Maritin
(Cirimaritin) One 8.5 ± 0.2 2.5 17.3 ± 1.1 5 17.1 ± 1.0 Gliclazide 2.5 6.1 ± 0.0 5 5.7 ± 0.0 10 6.1 ± 0.1 20 9.3 ± 0.0

(Results of insulin secretion measurement by INS-1 cells and thistle extracts and components)

< Experimental Example  2> with 30% ethanol thistle extract Maritin  ( Cirimaritin )of Streptozotocin (streptozotocin) -induced pancreatic toxicity (In vitro, in vitro)

The protective effect on pancreatic toxicity was evaluated using the mouse insulinoma cell line INS-1 (Biohermes, Shanghai, China) as follows.

(1) Protection effect on pancreatic toxicity

INS-1 cells were incubated with 100 ug / ml of 10% fetal bovine serum (Gibco BRL, Life Technologies), penicillin G 100 units / ml (Gibco BRL, Life Technologies), streptomycin (Gibco BRL Life Technologies) , Gibco BRL Life Technologies), 2 mM L-glutamine (Gibco BRL Life Technologies), 1 mM sodium pyruvate (Gibco BRL Life Technologies) and 0.05 mM 2-mercaptoethanol Was cultured in a 95% air / 5% carbon dioxide incubator maintained at 37 ° C using RPMI1640 (Cellgro, Manassas, VA, USA) medium supplemented with 2-mercaptoethanol (Gibco BRL Life Technologies). The cultured INS-1 cells were dispensed into 96-well plates at 1 × 10 ⁴ per well and allowed to stabilize for 24 hours.

Then, 30% ethanol thistle extract and cirsimaritin were added to the solution to the specified concentration. After 2 hours, 0.1 ml of 50 μM streptozotocin dissolved in the medium was added and cultured for 24 hours. 10 μl of CCK -8 (CCK-8, Dojindo Laboratories, Japan) reagent was added to each well and cultured at 37 ° C. After 1 hour, the viability of the cells was measured by measuring the absorbance using a 450 nm detection wavelength in a BIO-TEK (Winooski, VT, USA) microplate reader.

Table 3 shows the effect of 30% ethanol thistle extract and cirsimaritin on INS-1 cells, which are insulinoma cells treated with streptozotocin. The number of INS-1 cells was reduced to less than 50% of streptozotocin-treated group by treatment with 50 μM streptozotocin.

30% ethanol thistle extract and cirsimaritin inhibited such cell damage. The 30% ethanol thistle extract and cirsimaritin significantly decreased cell viability by 50 μM streptozotocin treatment in a concentration dependent manner. The 30% ethanol thistle extract showed 88.4 % Of the cells.

In particular, cirsimaritin increased the cell viability to 92.6% at a concentration of 5 μM.

Streptozotocin specifically destroys the beta cells of the pancreatic islets of Langerhans to stop insulin secretion. It causes experimental diabetes in animals. When streptozotocin is treated with INS-1 cells, As described, the number of cells is reduced to 50% or less as compared with the untreated group. However, treatment with the 30% ethanol thistle extract and salsimaritin significantly increased the cell survival rate, and the 30% ethanol thistle extract and sarsimaritin

Cirsimaritin has the effect of protecting pancreatic cells from the streptozotocin.

division Concentration (μg / ml) Cell survival rate (%) 30% ethanol thistle extract 0 58.3 ± 2.7 10 80.6 ± 3.2 25 80.6 ± 1.2 50 87.5 ± 2.3 100 88.4 ± 2.9 division Concentration (μM) Cell survival rate (%) Maritin
(Cirimaritin) 0 62.0 ± 1.0 One 72.1 ± 0.8 2.5 85.6 ± 3.0 5 92.6 ± 3.0

(Protective effect test for pancreatic toxicity)

(2) Protein detection test (western blot) [caspase-8, -3, Bid, Bcl-2, measurement of PARP protein expression]

The cultured INS-1 cells were divided into 6-well plates at 4 × 10 ⁵ cells per well. Cirsimaritin was treated at a concentration of 2.5 μM and 5 μM, and after 2 hours, 50 μM streptozotocin ). After incubation for 24 hours, the cells were collected, washed once with phosphate-buffered saline, and then treated with 1 mM phenylmethylsulfonyl fluoride in RIPA buffer (Cell Signaling, MA, USA ) And left on ice for 20 minutes. The cell lysate was isolated by centrifugation at 12,000 rpm for 20 minutes at 4 ° C. The separated cell lysate was quantitated using a protein analysis kit (Thermo Scientific, Rockford, Ill., USA) and 20 μg of cell lysate per well ) Were denatured and separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein was transferred to a polyvinylidene fluoride membrane (Merck Millipore, Darmstadt, Germany), and the membrane was immersed in 5% nonfat milk powder to prevent nonspecific binding of the anti- (skim milk) at room temperature for 2 hours. The cells were then washed three times with TBST buffer (20 nM tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.5) for 10 min and the cleaved caspase-8,3, Bid, Bcl-2, PARP and GAPDH To measure the expression level, the primary antibody (Cell Signaling, Danvers, MN, USA) was diluted 1: 1,000 and reacted at room temperature for 1 hour and washed three times with TBST buffer for 10 minutes. Then, the secondary antibody (goatantirabbit IgG, Calbiochem, La Jolla, CA, USA) was diluted 1: 2,000, reacted at room temperature for 2 hours, and the protein was confirmed with an ECL detection kit (GE healthcare).

As shown in FIG. 2, the protein level of truncated-Bid, a pro-apoptotic Bcl-2 protein, among the Bcl-2 family proteins, which is one of important proteins acting in the apoptosis process, is detected by streptozotocin While the level of anti-apoptotic Bcl-2 protein, Bcl-2 protein, decreased. In addition, the protein level of cleaved caspase-8, -3, which is an active form of caspase that carries out apoptosis, was increased by treatment with 50 μM of streptozotocin, and an inactive form of PARP, one of the target proteins of caspase- The protein level of cleaved PARP also increased remarkably. These results were significantly reduced by treatment with cirsimaritin 2.5 μM and 5 μM.

As a result, the streptozotocin induces apoptosis. On the contrary, treatment with cirsimaritin reduces apoptosis caused by the streptozotocin.

(3) Image-based cell analysis (Tali) [Quantitative analysis of cell death]

The cultured INS-1 cells were divided into 6-well plates at 4 × 10 ⁵ cells per well. Cirsimaritin was treated at a concentration of 2.5 μM and 5 μM, and after 2 hours, 50 μM streptozotocin ). After 24 h incubation, the cells were collected, washed once with phosphate-buffered saline, resuspended in 100 μl of Annexin binding buffer ( ⁵ × 10 ⁵ to ⁵ × 10 ⁶ cells / mL) Mix 5 μl of Alexa Fluor 488 and fix and dye in a dark room at room temperature for 20 minutes. The supernatant was discarded by centrifugation and the resuspended cells (cell pellet) were resuspended in 100 μl of Annexin binding buffer. Propidium (1 μl) was added and mixed well for 1 ~ 5 minutes at room temperature. After staining and staining, stained cells were analyzed by TaliImage-based cytometer and TaliPCApp (version 1.0). The apoptotic cells are divided into surviving cells (annexin V-negative / PI-negative), early apoptosis cells (annexin V-positive / PI-negative), late apoptosis or necrosis cells (annexin V-positive / PI-positive) annexin V-positive / PI-negative or positive cells.

3 and Table 4, the cells were subjected to Annexin-V / PI staining to quantitatively analyze the degree of apoptosis. As a result, apoptosis ratio was increased from 1.0% to 23.0% in untreated group by treatment with streptozotocin 50 μM %, Respectively, and decreased by 9.6% and 7%, respectively, when treated with 2.5 μM and 5 μM of cirsimaritin.

Thus, as shown in the results of the quantitative analysis of cell death as well as the level of the protein level involved in the cell death that has been proven in the protein detection test, the streptozotocin increases the cell death rate, whereas the cirsimaritin increases the cell death rate It is possible to confirm that the cell death rate due to Jocosan can be reduced.

division Concentration (μM) apoptosis (%) Cirrhimaritin 0 23.0 + 1.7 2.5 9.6 ± 1.5 5 7.0 ± 1.0

(Results of quantitative analysis of cell death)

Claims (7)

Preparing a dried thistle (S10);
Adding alcohol to the dried thistle to extract (S20);
Filtering the extracted dried thistles by filtration (S30);
Concentrating the filtered dried thistle (S40);
Lyophilizing the concentrated dried thistle (S50);
Powdering the lyophilized dried thistle (S60); To produce a thistle extract,
Wherein said thistle extract comprises linarin, fektronine, apigenin, histidine, sulcimarine and sullimaritin components.
The method according to claim 1,
The step of preparing the dried thistle (S10)
A method for preparing a thistle extract having antidiabetic activity, which comprises preparing a dried thistle outpost.
The method according to claim 1,
The extracting (S20)
Wherein the dried thistle is extracted with 25 to 35% of alcohol at 75 to 85 ° C for 2 to 4 hours, and the alcohol comprises alcohol and ethanol.
The method according to claim 1,
The extracting (S20)
Wherein the dry thistle is added with 25 to 35% of alcohol and the ratio of the dried thistle to the alcohol is 1:20.
The method according to claim 1,
The filtering step (S30)
Wherein the filtration means comprises filter paper. &Lt; Desc / Clms Page number 20 &gt;
The method according to claim 1,
The concentration step (S40)
Wherein the concentrate is concentrated at 75 to 85 캜 for 2 to 4 hours to be a final 10 to 13 Bricks.
A thistle extract having antidiabetic activity produced according to the method of any one of claims 1 to 6.

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