TW201620540A - Use of Toona sinensis leaf extract for activating peroxisome proliferators activated receptor [alpha] - Google Patents

Abstract

The invention relates to a use of a Toona sinensis leaf extract (TSLE) for activating peroxisome proliferators activated receptor[alpha]. The TSLE comprising 3.5-6.3 % linolenic acid, 1.5-2.7 % linoleic acid, 1.5-2.7 % palmitic acid, 3.9-4.3 % gallic acid, and 3.5-3.9 % rutin has ability of activating PPAR[alpha] gene and protein expression.

Description

Use of Toona sinensis extract as a peroxisome proliferator-activated receptor alpha activator

The invention relates to the use of a toon extract as a peroxisome proliferator-activated receptor alpha activator, in particular to a natural extract of a camphor pure extract having the ability to activate PPARα gene and protein expression; It may further be applied to extract pure toon PPAR α activation of preparing a pharmaceutical composition or a food.

According to the peroxisome proliferators activated receptor (PPAR), the "nuclear hormone receptor superfamily" is activated by the activation of certain ligands, and Binding to a promoter of a partial target gene affects the expression of the gene. It is known that there are three main types of PPAR family, namely PPARα, β, and γ; PPARα is mainly expressed in liver, skeletal muscle and heart; PPARγ is mainly expressed in adipose tissue, but also in tissues of the intestine and chest; PPARβ/δ can be expressed in a large number of skin and skeletal muscles. In recent years, research has pointed out that PPAR plays different roles in different tissues and plays an important role in the regulation of metabolism. Therefore, relevant researchers or researchers are all looking for active substances that can be used to regulate PPAR, especially for modern people. In particular, how to find an active substance derived from natural substances to regulate PPAR has become an important issue.

Toona sinensis Roem is a naturally edible plant. According to the Republic of China Patent Publication No. I394567, the inventors have found that the extract of camphor has the effect of activating PPARγ and lowering blood sugar; and previous studies have also found that feeding high fat Feed-induced hyperglycemia in mice (HFD mice), 95% alcohol extract (TSLE), reduced fasting blood glucose in HFD mice, increased PPARγ gene and protein expression in adipose tissue, and aP2 gene expression, and treated 3T3 with TSLE -L1 fat cells also increase the expression of PPARγ and aP2 genes, and at the same time reduce the accumulation of cellular lipids; however, the effect of camphor extract on PPARα is unknown.

The main object of the present invention is to provide a citron extract as a peroxisome proliferator-activated receptor alpha activator, wherein the citron extract comprises 3.5 to 6.3% linoleic acid, 1.5 to 2.7% linolenic acid, 1.5 to 2.7. % palmitic acid, 3.9~4.3% gallic acid and 3.5~3.9% muscidin; and the citron extract is obtained by the following steps: dissolving a crude citron leaf powder in an alcoholic solution (for example, 95%) The ethanol solution) is used to obtain a scented extract, and is concentrated and lyophilized to obtain a citron extract which can activate the peroxisome proliferator-activated receptor alpha (PPARα) gene and protein.

Another object of the present invention is to provide a use of a toon extract for the preparation of a pharmaceutical composition for activating a peroxisome proliferator-activated receptor alpha, wherein the camphor extract comprises 3.5 to 6.3% linoleic acid, 1.5~. 2.7% linolenic acid, 1.5~2.7% palmitic acid, 3.9~4.3% gallic acid and 3.5~3.9% muscidin; and the citron extract is obtained by the following steps: extracting a citron leaf with an alcoholic solution to obtain A fragrant sputum extract is extracted and the citron extract is filtered to obtain a citron extract which activates the peroxisome proliferator-activated receptor alpha (PPARα) gene and protein.

In one embodiment of the present invention, the camphor extract can increase the lipid breakdown gene The performance of ATGL, lipid-oxidative metabolism-related genes ACO, CPT-1 and UCP-3.

In one embodiment of the present invention, the camphor extract further comprises 0.3-0.9% catechin, 0.3-0.7% methyl gallate and 0.20-0.24% quercetin.

Thereby, the extract of the natural extract of the camphor has the ability to regulate the activity of PPARα, and is very suitable for further preparation of a pharmaceutical composition or food for activating PPAR α , and can also solve the lack of safety concerns of the user.

First panel: Schematic diagram of the effect of TSLE on the expression of PPAR α protein in adipose tissue of HFD mice.

Figure 2: A graph of Bezafibrate's evaluation of the effect of PPARα activation.

Figure 3: Figure of the results of TSLE evaluation of PPARα activation.

Figure 4: Experimental results of the effect of TSLE on the expression of PPARα protein in 3T3-L1 cells in the early stage of differentiation.

Figure 5: Experimental results of the effect of TSLE on the expression of PPARα protein in mature 3T3-L1 cells after differentiation.

Figure 6: Results of the evaluation of the effect of TSLE liquid phase distribution extract on PPARα activation.

Figure 7: Figure of the results of the evaluation of the effect of known components of TSLE on PPARα activation.

The object of the present invention and its structural and functional advantages will be explained in accordance with the structure shown in the following drawings, in conjunction with the specific embodiments, so that the reviewing committee can further deepen the present invention. Specific understanding.

Toona extract present invention provides a Life use as overshoot α activator receptor bodies of oxide, cedar extract comprises 3.5 to 6.3% linolenic acid, 1.5 to 2.7% linolenic acid, 1.5 to 2.7% acid brown beam at the eaves, 3.9 ~ 4.3% gallic acid and 3.5 to 3.9% of ruthenium; and the citron extract is obtained by the following steps: extracting a citron leaf using an alcoholic solution (for example, a 95% ethanol solution) to obtain a scented scented extract And filtering the first extract of the camphor to obtain a citron extract which activates the peroxisome proliferator-activated receptor alpha (PPARα) gene and protein; wherein the camphor extract further increases the lipid breakdown gene ATGL and is associated with lipid oxidative metabolism The performance of genes ACO, CPT-1 and UCP-3.

The invention also provides a use of the citron extract for preparing a pharmaceutical composition for activating a peroxisome proliferator-activated receptor α, wherein the citron extract comprises 3.5 to 6.3% linoleic acid, 1.5 to 2.7% linolenic acid, 1.5. ~2.7% palmitic acid, 3.9~4.3% gallic acid and 3.5~3.9% muscidin; and the citron extract is obtained by the following steps: dissolving a crude citron leaf powder in an alcoholic solution (for example 95% ethanol solution) to obtain a scented scented extract, and concentrated and lyophilized to obtain a citron extract having the ability to activate the peroxisome proliferator-activated receptor alpha (PPARα) gene and protein; Toona sinensis extract further increased the performance of lipid breakdown gene ATGL and lipid oxidative metabolism related genes ACO, CPT-1 and UCP-3.

In one aspect of the invention, the camphor extract further comprises 0.3-0.9% catechin, 0.3-0.7% methyl gallate and 0.20-0.24% quercetin.

In addition, the scope of the invention may be further exemplified by the following specific examples, which are not intended to limit the scope of the invention.

The invention mainly discusses Toona sinensis leaf extract ( The effect of TSLE) on PPARα-related genes and protein expression in high fat diet (HFD) mice. The TST was used to treat the mature 3T3-L1 adipocytes in the early stage of differentiation and differentiation, and the effect of PPARα on the activity of PPARα was detected by Western blotting.

<Preparation of Toona sinensis extract>

The crude extract of Toona sinensis was purchased from Taiwan Shannon Company (Kaohsiung, Taiwan). The preparation method of Toona sinensis extract (TSLE) used in this case is as follows. Each gram of crude extract of camphor leaves is mixed with 10 mL of 95% ethanol solution. Shake for 12 hours at room temperature, then concentrate and freeze-dry to obtain a camphor extract. In the examples of the present invention, the freeze-dried toon extract is used, and the extract of the camphor is dissolved in different solvents according to the experiment, the animal experiment is dissolved in soybean oil, and the cell experiment is dissolved in DMSO to be added to the culture solution.

Embodiment Example: Analysis of Effect of Feeding TSLE HFD PPAR α protein in adipose tissue of mice expressed

Six-week-old male C57BL/6 mice were purchased from the Experimental Animal Center of the National Experimental Research Institute of Taipei. The experimental animals were randomly divided into 3 groups, 4 in each group: after one week of domestication, they were divided into control group (n=4) and induction group (n=8), and began to feed high-fat diet with a caloric source of 54% fat, 23 % is protein and carbohydrates. Six weeks after induction, the mice in the HFD group were randomly divided into two groups, HFD and HFD+TSLE 2g/kg/day, respectively. After 14 weeks of feeding, the tissue was sacrificed for protein performance analysis and control diet. ) Comparison.

Results As shown in the first panel, the HFD group significantly reduced the expression of PPAR α protein in adipose tissue compared with the normal feeding control group. The HFD+TSLE group was significantly higher than the HFD group in the HFD+TSLE group. It increases the performance of PPAR α protein in adipose tissue, and no significant differences were fed with normal control group.

Example 2: Analysis of the effect of TSLE on the activation of PPAR α <PPAR α -LBD enzyme fluorescence (of Luciferase) Analysis>

24 hours after transfection of human hepatoma cell line (HepG2 cells), a new DMEM medium containing 10% FBS and 1% P/S (containing high glucose) was replaced and treated for 24 hours for cold light analysis. After the culture solution was blotted dry, it was washed twice with 1X PBS. Remove PBS, add 250 μl of cell lysis buffer, shake in a plate shaker for 20 minutes, allow the cells to fully dissolve, collect the lysate, take 50 μl of firefly luciferase substrate, add 10 μl The cell lysate was quickly and thoroughly mixed and immediately placed in a Luminometer to detect the luminescence value of firefly luciferase. Then 50 μl of renilla lucifearse substrate was added to detect the luminescence value of renilla luciferase, and the firefly luciferase luminescence value was divided by renilla. luminescence measurement value, and then with the blank control group (not a member of any material) and positive control group 50 μ M Bezafibrate (Sigma-Aldrich , Missouri, USA) , and determines toon extracts ability to activate PPAR α of (n = 6).

Analysis of PPAR α TSLE for enzyme activity using fluorescence assay (luciferase assay), the results as shown in FIG second to PPAR α -LBD Luciferase detection ability of different concentrations of PPAR α activation Bezafibrate, and so select 50 μ M For the positive control group, follow-up experiments were performed. Please refer to FIG third to Bezafibrate 50 μ M is a positive control group, the effect of the test TSLE 50,100,150 μ g / ml of the activation of PPAR α. As the concentration of TSLE increases, the effect of activation increases, indicating that TSLE activates PPAR alpha and is dose-dependent.

Example 3: Analysis of the effect of TSLE on the protein expression of 3T3-L1 cells and mature 3T3-L1 cells after differentiation

The 3T3-L1 mouse embryo fibroblast cell line was purchased from the Bioresource Conservation and Research Center of the Food Industry Development Research Institute and cultured in 10% bovine calf serum (BCS). A 1% P/S DMEM medium (containing high glucose) was cultured at 37 ° C, 5% CO ₂ .

<Differentiation of 3T3-L1 adipocytes>

3T3-L1 adipocytes were cultured at a density of 5× ^{10 4} cells/well in a 12-well plate or 12.5×10 ⁴ in a 6-well plate in DMEM containing 10% BCS and 1% P/S (containing high glucose) at 37 °C. , 5% CO _{2 was} cultured for 48 hours to 100% full plate, and the new culture solution was replaced, and differentiation was started after 48 hours of culture. Upon differentiation, replacing containing 1.7 μ M insulin, 1 μ M dexamethasone, 0.5mM 3 -isobutylmethylxanthine, 10% FBS and 1% P / DMEM culture solution of S (including high glucose) at 37 ℃, 5% CO ₂ for 96 hours, the medium was changed to complete differentiation containing 1.7 μ M insulin, 10% FBS, 1% P / DMEM culture solution of S (with high glucose), once every 48 hours to replace the culture medium.

<Protein extraction>

After the culture solution was removed, it was washed twice with 1X PBS to remove PBS. The dishes were placed on ice, was added 100 μ l lysis buffer (lysis buffer), using a cell spatula and scraped cells were collected in 1.5ml vial, and placed on ice using ultrasonic oscillator Cells Shattering, 10,000 Xg, centrifugation at 4 ° C for 15 minutes, the supernatant was taken to a new 1.5 ml vial and stored at -80 ° C.

<protein quantification>

Protein quantification was detected and quantified using a commercially available combination reagent (BCATM, Pierce Co., Ltd). The extracted protein solution with the concentration of the standard 10 μ l per well was added to 96-well culture plate (each sample were done in triplicate) was added 150 μ l working reagent (Bio- Rad Reagent: Deionized water = 5 :1) To each well, the reaction was protected from light for 15 minutes at room temperature, and the absorbance of OD 595 nm was measured by a protein enzyme immunoassay, and then converted to a protein content by a standard curve calculated according to the standard. The antibody information used in the examples of the present invention is shown in Table 1.

Please refer to the fourth figure. After 3T3-L1 differentiation, different concentrations of TSLE were directly treated for 48 hours, and then cultured in adipocyte growth medium for 48 hours, and the protein was extracted for analysis (n=3). The results showed that treatment of TSLE 50, 100 and 150 μg/ml significantly increased the performance of the PPARα protein. As the concentration of TSLE increases, the effect of activation increases, indicating that TSLE activates PPAR alpha and is dose-dependent.

Please refer to the fifth figure. After 3T3-L1 differentiation, the cells were cultured for 4 days to more than 90% with lipid growth medium, and lipid accumulation was performed. TSLE (100 μg/ml) was treated for 5 days, and proteins were extracted for analysis (n=3). . The results showed that the treatment of TSLE significantly increased the expression of PPARα protein of 3T3-L1 after differentiation and with lipid accumulation.

Example 4: Analysis of the effect of TSLE liquid phase distribution extract on the activation of PPAR α <Liquid phase distribution extraction method>

The liquid-liquid partition method is a treatment method that separates components by using solvent polarity differences. 0.1 g of TSLE was dissolved in 2 ml of 80% methanol, uniformly mixed with an equal amount of n-hexane, centrifuged at 3,000 x g for 5 minutes, and the upper n-Hexane fraction was taken out and transferred to another tube. Add methanol to adjust the methanol concentration to 60%, add equal amount of chloroform, mix well, centrifuge 3,000xg for 5 minutes, remove the upper layer and transfer to the other tube. The remaining chloroform fraction in the tube is concentrated under reduced pressure. (about 40 degrees, 650 mmHg), the methanol in the removed supernatant liquid was removed. Add equal amount of butanol and mix well. Centrifuge at 3,000xg for 5 minutes. The upper layer is a butanol fraction and the lower layer is a water fraction. All the layered solution is concentrated under reduced pressure. Remove, place in a vacuum dish overnight, and evacuate to help remove the solvent.

See FIG. Sixth, the use of liquid phase distribution TSLE extraction separation, the hexane layer was separated, the chloroform layer, the aqueous layer with butanol layer to PPAR α -LBD Luciferase the detected concentration is equal to TSLE 150 μ g / ml of The ability of each extract to activate PPAR alpha (n = 3) showed that the n-hexane layer had the highest ability to activate PPAR alpha .

Example 6: Analysis of the effect of known components of TSLE on the activation of PPAR α

The TSLE component was identified by high performance liquid chromatography (HPLC), and the component having activated PPAR α activity was screened by luciferase assay.

<HPLC analysis>

The HPLC analysis system used a Hitachi pump L7100 pump, a Jasco UV1575 UV visible light detector and a Glison autoinjector 234. The column is made of Ascentis C18, 25 cm x 4.6 mm ID, 5 μm particles. During the analysis, 20 μl of the analyte was injected into the column and subjected to gradient elution. The eluents were A: 20% methanol + 0.05% TFA and B: 100% methanol + 0.05% TFA, and the gradient elution step was 0. -9 minutes, 0-15% B; 9-12 minutes, 15% B; 12-17 minutes, 15-20% B; 17-35 minutes, 20-25% B; 35-55 minutes, 25-53% B; 55-65 minutes, 53% B. The flow rate was 1 ml/min, the detection wavelength was 280 nm, and the temperature was room temperature. Using gallic acid, catechin, methyl gallate, rutin and quercetin as standard samples, dissolved in 50% methanol solution The standard solution with the concentration of 1, 5, 10, 25 and 50 mg/ml was set; the sample of TSLE was also dissolved in 50% methanol solution, the concentration of the sample was 1 mg/ml, and the peak time of each component was gallic acid 4.5. Minutes, catechin 8.9 minutes, methyl gallate 10.8 minutes, rutin 30.4 minutes and quercetin 49.4 minutes, the standard curve concentration and component peak area were used as a standard curve to calculate the proportion of each component contained in TSLE.

<Fatty acid derivatization and analysis>

Since the fatty acid itself has a similar structure and is not easy to be directly separated and analyzed by HPLC, the fatty acid and fatty acid components in TSLE are first derivatized by fatty acid to form a structure suitable for HPLC analysis. Fatty acid standard (0.0125 ~ 1mg / ml) or TSLE sample (10mg / ml) 300μl and 10mg / ml α-bromoacetophenone 200μl and 10mg / ml triethylamine 200μl mixed in a dark tube, heated to 50 ° C and ultrasonic shock for 30 minutes, It was slowly dried at 40 ° C and stored at -20 ° C. It was dissolved in 300 μl of acetone:acetonitrile (1:1; v/v) solution before analysis. The remelted and filtered fatty acid derivative was further injected into HPLC for analysis. The HPLC analysis system was as described above, and the liquid phase system was intensity-eluted with 99% ACN, 1% THF, etc., the flow rate was 1 ml/min, the detection wavelength was 258 nm, and the detection temperature was room temperature.

<Quantification of fatty acid components in TSLE>

It is a standard sample derivatized with Linoleic acid, Linolenic acid and Palmitic Acid, dissolved in acetone:acetonitrile (1:1; v/v) solution, linoleic acid configuration Standard solutions with concentrations of 0.125, 0.25, 0.5, and 1 mg/ml, linolenic acid and palmitic acid are formulated at concentrations of 0.0625, 0.125, 0.25, and 0.5 mg/ml; TSLE-derived products are used as analytical samples and are also soluble in acetone: In the acetonitrile (1:1; v/v) solution, the concentration of the sample was 10 mg/ml, and the peak performance time of each component was 6.7 minutes for linoleic acid, 8.6 minutes for linolenic acid and 12.4 minutes for palmitic acid, respectively. The peak area of the component is used as a standard curve to calculate the proportion of each fatty acid component contained in the TSLE.

The results are shown in Figure 7 and Table 2. It was found that TSLE has gallic acid (4.1%), methyl gallate (0.5%), catechin (0.6%), and rutin (3.7%). Quercetin (0.2%). Is further PPAR α -LBD Luciferase the detected concentration is equal to TSLE 150 μ g / ml of each component PPAR α activation ability (n = 3), the results show, gallic acid and rutin significant activation ability of PPAR α.

Please refer to the seventh figure. The 75% alcohol extract (TSL-E5) is the closest extract to the extract of T. chinensis. The TSL-E5 has a great difference in the ability to activate PPAR α . The extracts of different extracts of different concentrations of alcohol were different; therefore, the difference between the components of TSLE and TSL-E5 was further analyzed by HPLC, and the main components of activated PPAR α in TSLE were determined. The results showed TSLE and TSL-E5. The main difference in composition is the composition of the fatty acids.

Table II

The above experimental results show that the toon extract of the present invention can indeed achieve the effect of activating the PPARα gene or protein expression, and can further increase the performance of the lipid breakdown gene ATGL and the lipid oxidation metabolism related genes ACO, CPT-1 and UCP-3. For a specific example of this part, reference may be made to another application filed by the inventor of the present invention on the same day. The use of a pharmaceutical composition for reducing fat accumulation is not described in detail herein, and all of its contents are incorporated herein by reference; therefore, the citron extract can be used as a PPARα activator or a pharmaceutical combination for the preparation of activated PPARα. Things.

In summary, the use of the toon extract of the present invention as a peroxisome proliferator-activated receptor alpha activator can indeed achieve the intended efficacy by the above disclosed examples, and the present invention has not Before being disclosed to the application, Cheng has fully complied with the requirements and requirements of the Patent Law.爰Issuing an application for a patent for invention in accordance with the law, and asking for a review, and granting a patent, is truly sensible.

The illustrations and descriptions of the present invention are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention; those skilled in the art, which are characterized by the scope of the present invention, Equivalent variations or modifications are considered to be within the scope of the design of the invention.

Claims (8)

The use of a camphor extract as an activator of a peroxisome proliferator-activated receptor alpha, the camphor extract comprises 3.5 to 6.3% linoleic acid, 1.5 to 2.7% linolenic acid, 1.5 to 2.7% palmitic acid, 3.9 to 4.3 % gallic acid and 3.5 to 3.9% of ruthenium; and the citron extract is obtained by dissolving a crude citron leaf powder in an alcohol-containing solution to obtain a scented scent extract and concentrating And lyophilization to obtain a citron extract having the ability to activate the peroxisome proliferator-activated receptor alpha (PPARα) gene and protein. The use of the camphor extract of claim 1 as a peroxisome proliferator-activated receptor alpha activator, wherein the alcohol-containing solution is 95% ethanol. The use of the extract of Toona sinensis as described in claim 1 is a peroxisome proliferator-activated receptor alpha activator, wherein the extract of the camphor tree increases the lipid breakdown gene ATGL and the lipid oxidation metabolism related genes ACO, CPT- 1 and the performance of UCP-3. The use of the citron extract as described in claim 1 of the patent application as an activator of a peroxisome proliferator-activated receptor alpha, wherein the citron extract It also includes 0.3~0.9% catechin, 0.3~0.7% methyl gallate and 0.20~0.24% quercetin. The use of a camphor extract for preparing a pharmaceutical composition for activating a peroxisome proliferator-activated receptor alpha, the camphor extract comprising 3.5 to 6.3% linoleic acid, 1.5 to 2.7% linolenic acid, 1.5 to 2.7% Palmitic acid, 3.9~4.3% gallic acid and 3.5~3.9% muscidin; and the citron extract is obtained by the following steps: extracting a citron leaf with an alcoholic solution to obtain a scented scent extract and filtering The camphor extract is used to obtain a citron extract which activates the peroxisome proliferator-activated receptor alpha (PPARα) gene and protein. A pharmaceutical composition for activating the expression of a peroxisome proliferator-activated receptor alpha according to claim 5, wherein the alcohol-containing solution is a 95% ethanol solution. The pharmaceutical composition for activating the expression of a peroxisome proliferator-activated receptor α according to claim 5, wherein the pharmaceutical composition increases the lipid breakdown gene ATGL and the lipid oxidation metabolism related genes ACO, CPT- 1 and the performance of UCP-3. a pharmaceutical composition for activating the expression of a peroxisome proliferator-activated receptor alpha according to claim 5, wherein the pharmaceutical composition is more It includes 0.3~0.9% catechin, 0.3~0.7% methyl gallate and 0.20~0.24% quercetin.