TWI586360B - Use of taraxacum mongolicum extract in the manufacture of a medicament for activating adenosine monophosphate-activated protein kinase - Google Patents

Description

Use of dandelion extract for the preparation of adenylate-activated protease activator

This invention relates to the use of an adenylate-activated protease (AMPK) activator, and more particularly to the use of a dandelion extract for the preparation of adenylate-activated protease activators.

Taraxacum mongolicum , also known as yellow flower diced, is a common plant in the temperate to subtropical zone. The growth is very strong, and people generally regard it as a weed, and even remove the dandelion for the beauty of the garden or lawn. However, dandelion leaves are rich in vitamin A and vitamin C. The young leaves can be used as a wild vegetable for cold salad, soup or fried food. Europeans also used dandelion flowers to make wine in the Middle Ages. Dandelion has a wide range of medicinal uses, such as diuretic, anti-oxidant, anti-inflammatory, treatment of ovarian insufficiency, infertility and relief of menopausal symptoms, bactericidal effects on Staphylococcus aureus, hemolytic streptococcus and other fungi, also It clears the obstruction of the milk vasculature and promotes the function of lactation. In addition, it is widely used in the treatment of various respiratory diseases and gastroenteritis.

Adenosine monophosphate-activated protein kinase (AMPK) is an enzyme composed of three kinds of proteins. The presence of AMPK can be seen from yeast to human, and its main function is to maintain cell energy balance. Many tissues in the human body exhibit AMPK, including the liver, brain, and skeletal muscle. The net effect of AMPK activation is to stimulate liver fatty acid oxidation and production, inhibit cholesterol synthesis, fat synthesis and triglyceride synthesis, inhibit fat cell lipogenesis and lipogenesis, skeletal muscle fatty acid oxidation and muscle glucose uptake, regulate insulin Secretion of pancreatic β -cells. Therefore, in past studies, AMPK has been implicated in many biological functions including cancer, diabetes, and appropriate immune function.

Therefore, dandelion is easy to colonize. If it can find active ingredients that activate AMPK in dandelion, it will be of great help to those who need to regulate fatty acids, cholesterol, fat, triglycerides, and sugar, and for cancer and immunity. The function is also quite helpful.

In view of the above, the present invention provides the use of a dandelion (taraxacum mongolicum ) extract for the preparation of an adenylate-activated protease (AMPK) activator, wherein the dandelion extract is obtained by extracting dandelion with an alcohol.

In a preferred embodiment of the present invention, the dandelion extract further comprises an extract obtained by extracting a dandelion ethanol extract by a solvent, and the solvent is n-Hexane or Dichloromethane. ), ethyl acetate (Ethyl acetate) or water.

In another preferred embodiment of the present invention, the dandelion extract is obtained by extracting an alcohol and comprises dandelion (Taraxacerin A; structural formula I) and oleracea (All-trans-Violaxanthin; structural formula II) Or a mixture of the two.

The above-mentioned compounds dandelion and violaxanthin can be used to activate adenylate-activated protease (AMPK), which can regulate fatty acid, cholesterol, fat, triglyceride, sugar metabolism, cancer and immune function related diseases. In one embodiment of the invention, the dandelion extract activates a single adenylate-activated protease, preferably an individual.

The embodiments of the present invention are further described in the following description, and the embodiments of the present invention are set forth to illustrate the present invention, and are not intended to limit the scope of the present invention. In the scope of the invention, the scope of protection of the invention is defined by the scope of the appended claims.

The first picture is a preliminary extraction flow chart of dandelion.

The second figure is an extraction flow chart for further separation of 50% ethanol (TM-2).

The third panel is the cell viability after 48 hours of each fraction of 50% ethanol (TM-2) analyzed by MTT. The experiment was repeated 3 times, and the bar represented standard error (SE).

The fourth graph is that TM-4 reduces fatty acid synthesis activity by inhibiting the performance of FASN and the activity of ACC. (A) HepG2 cells were cultured at 12.5 μg/mL TM-4 for a period of time. (B) HepG2 cells were cultured in TM-4 at concentrations of 6.25, 12.5 and 2.5 μg/mL for a period of time. After collection, the cells were lysed and Western blot analysis was performed using antibodies against FASN, phosphorylated ACC (p-ACC, Ser79), and β-actin. (C) HepG2 cells were treated with 12.5 μg/mL TM-4 for a specified period of time, and phosphorylated AMPK (p-AMPK ^Thr172 ) and β-actin content in the extract were analyzed by Western blotting. (D) HepG2 cells were cultured with TM-4 at concentrations of 6.25, 12.5 and 2.5 μg/mL for 24 hours, and the content of phosphorylated AMPK (p-AMPK ^Thr172 ) and β-actin in the extract was analyzed by Western blotting. Western blot data is the average of at least three experimental results. The results of the Western ink point method are quantified based on the performance of the control group.

The fifth picture shows the activation of human liver cancer HepG2 cells by Taraxacerin A and All-trans-Violaxanthin. (A) HepG2 cells were treated with DMSO (control group), dandelion at concentrations of 10, 20, 40, 80 μM for 48 hours. (B) HepG2 cells were treated with DMSO (control group), violax at concentrations of 10, 20, 40, 80 μM for 48 hours. After collecting the cells, the cells were lysed and Western blot analysis was performed using antibodies against phosphorylated AMPK (p-AMPK ^Thr172 ), AMPK, and β-actin. Western blot data is the average of at least three experimental results. The results of the Western ink point method are quantified based on the performance of the control group.

The antibody to β-actin used in the examples of the present invention was purchased from Sigma (St. Louis, MO). The whole plant of dandelion was purchased from the local Chinese medicine market (Pingtung, Taiwan). Fatty acid synthase (FASN), phosphorylated acetyl-CoA carboxylase (ACC, Ser 79), and phosphorylated AMPK (phospho-AMPK, Thr 172) were purchased from Cell Signaling Technology (Beverly, MA). Antibodies to LKB1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rats and rabbits were labeled with horseradish peroxidase (HRP) antibodies from Chemicon (Temecula, CA). Western chemiluminescent HRP substrate was purchased from Millipore Corporation (Billerica, MA).

All experimental values are expressed as mean ± standard deviation. Each value is the average of at least three experiments. The difference between the experimental group and the control group was analyzed by the student's test.

The following is the preparation of dandelion extract, the activity of dandelion extract prepared by Western blotting method to activate AMPK, and the cytotoxicity of dandelion extract by MTT; and further using gas chromatography mass spectrometer (GC- MS) Analyze which active ingredients in the dandelion extract have the experimental process and results of activating AMPK, as detailed in Examples 1 to 4.

Embodiment 1 Preparation of dandelion extract

The whole plant of dandelion ( Taraxacum mongolicum ) was chopped and extracted three times with 1.5 L of water (TM-1), 50% ethanol (TM-2), and 95% (TM-3) of ethanol for three hours, see for three hours. The first picture. Preliminary experiments found that in TM-1, TM-2 and TM-3, the effect of activating AMPK was best with TM-2. The 50% ethanol extract (TM-2) was filtered and the solvent was evaporated and dissolved in water, and 50% ethanol (TM-2) was separated into different liquids by solvent-solvent separation according to the flow chart of the second figure. : n-hexane, dichloromethane, and ethyl acetate tri-solvent are separated into different liquid fractions by solvent-solvent separation from low to high polarity. Store 50% ethanol extract (TM-2), n-hexane (TM-4), dichloromethane (TM-5), ethyl acetate (TM-6) and water (TM-7) Reserve in the refrigerator.

In addition, the dicing action of dandelion before water or ethanol extraction is to obtain smaller pieces of dandelion to facilitate subsequent extraction, but not limited to chopping, other physical treatment methods that can make dandelion into smaller pieces, for example Methods such as mashing or powder grinding can also be applied thereto.

Embodiment 2 Cytotoxicity analysis of dandelion extract

Cells were seeded in 24-well plates at a density of 1 x 10 ⁴ cells/well overnight. The cells were treated with 50% ethanol (TM-2) fractions (TM-4, TM-5, TM-6 and TM-7) for 48 hours. Next, 40 μL of MTT (concentration: 2 mg/mL; purchased from Sigma Chemical Co.) was added to each well having a volume of 500 μL, and cultured at 37 ° C for 2 hours. MTT-formamidine crystals were formed, and then 250 μL of DMSO was added to melt the crystals. Finally, the absorbance at 550 nm was measured by an enzyme-linked immunosorbent assay (ELISA) reader. The experimental results are shown in the third figure. The MTT analysis showed that none of the four fractions of TM-2 caused cytotoxicity to the cells.

Embodiment 3 Effect of dandelion extract on AMPK activation 3-1 HepG2 cell culture

HepG2 cells of human liver cancer were cultured in a humidified environment at 37 ° C, 5% CO ₂ in 10% fetal bovine serum (purchased from Invitrogen Carlsbad, CA) and 1% penicillin-streptomycin (puricillin-streptomycin). Dulbecco's modified Eagle's medium (DMEM, available from Invitrogen, Carlsbad, CA) from Invitrogen, Carlsbad, CA).

3-2 Western blot analysis

The cells were seeded at a density of 1 × 10 ⁶ cells/well on a 100-mm tissue culture plate and cultured in DMEM containing 10% fetal bovine serum. The cells were then cultured in DMEM containing 10% fetal bovine serum and briefly illustrated for treatment with the different agents listed in Figures 4 and 5 for 24 hours. After treatment, the cells were placed on ice and rinsed with ice phosphate buffer solution, which was then lysed with a lysis buffer solution. The Western method of ink dot method was completed according to Way et al., 2009.

FASN and ACC are key enzymes for lipid production. To further investigate the mechanism by which TM-4 lowers blood lipids, ACC phosphorylation was studied. To assess the activity of ACC, phosphorylation of ACC to serine 79 was analyzed by Western blotting. The results of the four fractions of TM-2 (not shown) were the best for the activation of AMPK by TM-4; among them, the phosphorylation of HepG2 cells treated with TM-4 in ACC at serine 79 Increase in a dose- and time-dependent manner, see Figure 4. Among them, (A) HepG2 cells were cultured at 12.5 μg/mL TM-4 for a period of time; (B) HepG2 cells were cultured for TM-4 at concentrations of 6.25, 12.5 and 2.5 μg/mL for a period of time; (C) HepG2 cells were 12.5. Gg/mL TM-4 was treated for a specified period of time; (D) HepG2 cells were incubated with TM-4 at concentrations of 6.25, 12.5 and 2.5 μg/mL for 24 hours. In addition, HepG2 cells treated with TM-4 increased in a dose- and time-dependent manner in FASN proteins (Fig. 4A, B). These results indicate that dandelion inhibits the enzyme activity of fatty acid synthesis and causes a decrease in total lipid content. FASN inhibitors are markedly activated to reduce the body weight pathway; therefore, compounds that modulate the hypohalal pathway and/or the novel neuropeptide system via FASN inhibitors may be candidates for anti-obesity.

AMPK activation is considered to be the key to cellular energy balance response, and the currently accepted indicator of AMPK activation is the phosphorylation of AMPK in hydroxybutyrate 172 (threonine 172), ie p-AMPK ^Thr172 . Therefore, we determined AMPK phosphorylation of HepG2 cells treated with TM-4. Western blot analysis indicated that TM-4 can stimulate AMPK phosphorylation in a time- and dose-dependent manner (fourth C, D map). Among them, (C) HepG2 cells were treated with 12.5 μg/mL TM-4 for a specified period of time; (D) HepG2 cells were cultured with TM-4 at concentrations of 6.25, 12.5 and 2.5 μg/mL for 24 hours. These results show that dandelion inhibits the activity of fatty acid synthase via the activated AMPK pathway. The role of AMPK in lipid metabolism has been an important research in recent years and has played an important role in fatty acid synthesis. This important metabolic modulator functions at least in regulating the phosphorylation and dephosphorylation cycles of ACC and the amount of FASN expression. Rapid activation of AMPK phosphorylation and inhibition of ACC. Slow activation of AMPK reduces the expression of sterol regulatory element binding protein-1c (SREBP-1c), while inhibiting the synthesis of ACC, FASN and other lipid generating enzymes. A "low-energy state" is caused by pharmacy, which causes AMPK to cause ACC phosphorylation and FASN degradation in vivo and in vitro .

Embodiment 4 Analysis of the composition of dandelion extract by gas chromatography mass spectrometry (GC-MS)

Since the previous results show that TM-4 is most effective in activating AMPK, we further studied the activated AMPK active compounds in TM-4.

In the composition analysis, helium gas was used as a carrier gas, and the micropipe column (DB-5, 30 m × 0.250 mm, J&W Scientific, USA) of Shimadzu GCMS-QP2010 GC-MS system was used. The injection amount and flow rate were 1 μL and 0.82 ml/min, respectively. The injection temperature was set to 250 °C. The column temperature was initially maintained at 45 ° C and then ramped up to 270 ° C at a rate of 5 ° C/min. Mass spectra were obtained by electron impact ionization at 70 eV. Peak chemical identification in the GC-MS database.

Finally, the two main compounds, tarane (Taraxacerin A; structural formula I) and violax (All-trans-Violaxanthin; structural formula II) were isolated. The structure I has the appearance of a pale yellow syrup having a molecular weight of 198.07 and a melting point of 283 °C. The structure I has the appearance of an orange crystal having a molecular weight of 600.85 and a melting point of 200 °C.

To test the effect of the two compounds on the activation of AMPK, the degree of AMPK phosphorylation of human HepG2 liver cancer cells treated with dandelion and violaxon diphosphate was determined by Western blotting of Example 3. As a result, it was found that dandelion (Fig. 5A) and violaxanthin (figure B) exhibited a dose-dependent stimulation of AMPK phosphorylation in human HepG2 liver cancer cells. From this, it can be seen that the dandelion and the violaxanthin compound have an effect of activating AMPK.

In summary, the dandelion ethanol extract and the aforementioned compound of formula I, formula II: dandelion and violaxanthin have an activity of effectively activating AMPK. Therefore, dandelion extract, dandelion or violaxanthin can be used as an activator of adenylate-activated protease (AMPK). In addition, the results of the examples of the present invention also highly show that dandelion and violaxanthin can be an effective compound for modulating AMPK activation, and can be used for regulating fatty acids, cholesterol, fat, triglycerides, and sugar by such natural substances. Metabolism, cancer and immune function.

Claims (9)

Use of a dandelion (taraxacum mongolicum ) extract for the preparation of an adenylate-activated protease (AMPK) activator, wherein the dandelion extract comprises a compound of formula I, or formula II, or a mixture thereof: The use of claim 1, wherein the dandelion extract is obtained by extracting dandelion with an alcohol. The use of claim 2, wherein the alcohol is ethanol. The use according to claim 3, wherein the dandelion extract is an extract obtained by further extracting a 50% ethanol extract of dandelion with a solvent, the solvent being n-hexane (n-Hexane), Dichloromethane, Ethyl acetate or water. The use according to claim 1, wherein the compound of the formula I or the formula II is obtained by further extracting the ethanol extract of dandelion with n-hexane. The use according to claim 1, wherein the dandelion extract activates a cell Adenylate-activated protease within. The use of the invention of claim 6, wherein the cell is a human liver cancer HepG2 cell. The use of claim 1, wherein the dandelion extract activates a single adenylate-activated protease. The use of claim 8 wherein the individual is a human.