TWI409075B - Extract of zanthoxylum avicennae (lam.) dc., and the preparation process and uses thereof - Google Patents

Abstract

Disclosed herein is an extract of Zanthoxylum avicennae (Lam.) DC., and the preparation process thereof. Also disclosed is the extract of Zanthoxylum avicennae (Lam.) DC. in the manufacture of pharmaceutical compositions for use in the treatment of cancers.

Description

Eagle non-boo extract, preparation method and use thereof

The invention relates to an extract of eagle berth and a preparation method thereof. The invention also relates to the use of the extract of the eagle to prepare a pharmaceutical composition for the treatment of cancer.

Cancer is one of the most important causes of death in humans worldwide, and liver cancer is the most important cancer in humans. Although the formation of cancer is still not fully understood, it is believed that the occurrence of cancer can be attributed to signal transduction pathways in cells when genetic accumulation of exogenous or endogenous factors leads to genetic abnormalities. An error occurs that causes cell division to lose control, which in turn causes the cells to gradually form cancer cells, which at the same time proliferate and will pass through the lymphatic system or the vascular system (vascular System) Transfer (metastasize) to other body parts.

In general, the intracellular apoptosis signal pathway (including the death receptor-dependent apoptosis signal pathway and the mitochondria-dependent cell lineage) The mitochondrial dependent apoptosis signal pathway is activated by the deactivated and/or activated cell survival signal pathway (proliferation). In addition, the intracellular urokinase plasminogen activator signal pathway, mitogen-activated protein kinase signal pathway (MAPK signal pathway) or Activation of the β-catenin signal pathway leads to metastasis of cancer cells. Therefore, in recent years, by inhibiting the deactivation and/or activation of the above-mentioned signal transduction pathways, inhibiting the proliferation and metastasis of cancer cells and inducing apoptosis of cancer cells has become an important direction for the global research of anticancer drugs. One.

The effects of anticancer drugs used in Western medicine today on cancer treatment are still not satisfactory. The main reasons include: individual differences in patients themselves, side effects of anticancer drugs, and drug resistance of cancer cells. (drug resistance). In view of this, some researchers have tried to find active components that can be used to treat cancer from traditional Chinese medicines (TCM).

Eagle not poem [Latin name: Zanthoxylum avicennae (Lam.) DC.; English common name: avicennia pricklyash; Hanyu Pinyin: ying bu bo; Chinese common name: dog pepper, chicken scorpion, soil pepper, thorn root, bird not stay, The crows are not perennial, scorpion, thrush, thorny, and stalked peppers. They are perennial evergreen shrubs or arbors belonging to the genus Rutaceae . The trunks and branches are reddish brown. Prickle. Eagles are mainly distributed in Taiwan, China, Vietnam, Laos and Cambodia. According to the literature, the various plant parts of the eagle peony (such as roots, stems, leaves and fruits) have health or therapeutic functions (Nanjing University of Traditional Chinese Medicine, Chinese Medicine Dictionary, Shanghai Science and Technology Press, No. 3824-3825). Page, second edition of 2006, the second volume).

In terms of medicinal use, according to the literature, the fruit of the eagle is effective in the treatment of stomachache and abdominal pain; the leaves of the eagle are in the treatment of bone-setters injury, lumbar muscle strain (strain of lumbar muscles), mastitis (mastadenitis), and swollen boil; and eagle roots in the treatment of jaundice hepatitis (icterohepatitis), jaundice edema (hepatitis), hepatitis B (hepatitis) B), hepatocirrhosis, edema due to nephritis, bruises, rheumatoid arthritis, lumbar muscle strain, leucorrhea, common cold, sore throat (sore throat), cough, malaria, colitis, and stomatitis are effective.

In TT Thuy et al . (1999), Phytochemistry , 50: 903-907, TT Thuy et al . used 95% methanol (MeOH) at room temperature to extract the leaves of the eagle-free, and by distillation under vacuum ( Distillate to remove methanol, whereby the resulting aqueous solution was extracted with n -hexane ( n -hexane), followed by extraction with EtOAc and n- BuOH. After removing n- BuOH under vacuum, the residue was partitioned with CHCl ₃ and H ₂ O, and a CHCl ₃ layer was taken. After removing CHCl ₃ under vacuum, a methanol extract of eagle-free leaves was obtained. The methanol extract of the eagle leaves can be further purified by further separation to obtain three novel alkaloids, including: (-)-culantraramine N -oxide [(-)-culantraramine N- oxide], ( -)-culantraraminol N -oxide [(-)-culantraraminol N- oxide] and avicennamine (avicennamine).

In JJ Chen et al . (2008), Journal of Natural Products , 71: 212-217, JJ Chen et al . mentioned the use of ice-cold methanol to extract stem wood in a previous study. the resulting extract was concentrated under reduced pressure, then with EtOAc and H ₂ O to allocate separate, EtOAc- soluble charge separating portion (EtOAc-soluble fraction), and does not give an eagle poise methanol extract stem material. The methanol extract of the eagle stem can inhibit formyl-L-methionine-L-alkamine-L-phenylalanine/cellulolytic B (formyl-L-methionyl-L-leucyl) -L-phenylalanine/cytochalasin B, FMLP/CB)-induced superoxide anion generation and elastase release, thereby achieving anti-inflammatory activity. Therefore, JJ Chen et al. further isolated and purified the methanol extract of the eagle peony stem in this literature to obtain 8 novel compounds, including: 4 new xylans (neolignans), 1 kind of peas Coumarinolignan, two lignan derivatives, and one Chromene, and the anti-inflammatory activity of the eight compounds on FMLP/CB-induced superoxide anion generation and elastase release was investigated.

As far as the Applicant is aware, there has been no literature or patents that have previously revealed that the extract product of Eagle Bingbo can be used to treat cancer.

Summary of invention

Thus, in a first aspect, the present invention provides an extract of Zanthoxylum avicennae (Lam.) DC. which is obtained by a method comprising the steps of: heating an eagle The botanical plant material is subjected to an acid treatment using a first acid, thereby obtaining an acid-treated eagle-non-bottle plant material; extracting the acid-treated eagle-free plant material with an alcohol to obtain an alcohol An extract; and mixing the alcohol extract with a second acid to form the eagle-free extract.

In a second aspect, the present invention provides a method for preparing an eagle-free extract comprising: subjecting an eagle-free plant material to an acid treatment using a first acid under heating, thereby Obtaining an acid-treated eagle-free plant material; extracting the acid-treated eagle-free plant material with an alcohol to obtain an alcohol extract; and mixing the alcohol extract with a second acid to form the Eagle's extract.

In a third aspect, the present invention provides a pharmaceutical composition for inhibiting proliferation of a tumor/cancer cell comprising an extract of eagle-free, as described above.

In a fourth aspect, the present invention provides a pharmaceutical composition for inhibiting metastasis of a tumor/cancer cell comprising an extract of eagle-free, as described above.

In a fifth aspect, the present invention provides a pharmaceutical composition for inducing apoptosis of a tumor/cancer cell comprising an extract of eagle-free, as described above.

The above and other objects, features and advantages of the present invention will become apparent from

Detailed description of the invention

It is to be understood that if any of the previous publications is quoted here, the prior publication does not constitute an acknowledgement that in Taiwan or any other country, the pre-existing publication forms a common general knowledge in the art. Part of it.

For the purposes of this specification, it will be clearly understood that the term "comprising" means "including but not limited to" and the term "comprises" has a corresponding meaning.

All technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains, unless otherwise defined. A person skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used to practice the invention. Of course, the invention is in no way limited by the methods and materials described.

In order to develop a new drug with anti-cancer effect, the applicant tried to extract the eagle-free in various treatments, and successfully obtained the eagle that has the effect of inhibiting tumor/cancer cell proliferation and metastasis and inducing tumor/cancer cell apoptosis. Moored extract.

Accordingly, the present invention provides an extract of Zanthoxylum avicennae (Lam.) DC. which is obtained by a method comprising the steps of: heating a plant to make an eagle The material is subjected to an acid treatment using a first acid, thereby obtaining an acid-treated eagle-free plant material; extracting the acid-treated eagle-free plant material with an alcohol to obtain an alcohol extract; And mixing the alcohol extract with a second acid to form the eagle-free extract.

According to the invention, in the method, the eagle non-parking plant material is selected from the group consisting of roots, stems, leaves and fruits. In a preferred embodiment of the invention, the eagle non-parking plant material is the root skin.

According to the invention, in the method, the first acid is selected from the group consisting of vinegar, acetic acid, formic acid, lactic acid, malic acid, oxalic acid and citric acid. In a preferred embodiment of the invention, the first acid is vinegar.

According to the invention, the vinegar may be fermented vinegar or synthetic vinegar. In a preferred embodiment of the invention, the vinegar is brewed vinegar.

Brewed vinegars suitable for use in the present invention include, but are not limited to, fruit vinegar, grain vinegar, and wine vinegar.

Examples of fruit vinegar according to the present invention include, but are not limited to, banana vinegar, lemon vinegar, apple vinegar, grape vinegar, and diced vinegar. In a preferred embodiment of the invention, the fruit vinegar is banana vinegar.

According to the invention, in the process, the alcohol is selected from the group consisting of methanol, ethanol, propanol, isopropanol, n-butanol and isobutanol. In a preferred embodiment of the invention, the alcohol is ethanol.

According to the invention, in the method, the second acid is selected from the group consisting of vinegar, acetic acid, formic acid, lactic acid, malic acid, oxalic acid and citric acid. In a preferred embodiment of the invention, the second acid is vinegar.

According to the invention, the vinegar can be brewed vinegar or synthetic vinegar. In a preferred embodiment of the invention, the vinegar is brewed vinegar.

Brewed vinegars suitable for use in the present invention include, but are not limited to, fruit vinegar, rice vinegar, and wine vinegar.

Examples of fruit vinegar according to the present invention include, but are not limited to, banana vinegar, lemon vinegar, apple vinegar, grape vinegar, and diced vinegar. In a preferred embodiment of the invention, the fruit vinegar is banana vinegar.

According to the present invention, in the method, the eagle-free extract is further subjected to a refining treatment using a third acid. This refining treatment can be carried out using techniques well known and commonly employed by those skilled in the art [e.g., decompress concentration].

According to the invention, in the method, the third acid is selected from the group consisting of vinegar, acetic acid, formic acid, lactic acid, malic acid, oxalic acid and citric acid. In a preferred embodiment of the invention, the third acid is vinegar.

According to the invention, the vinegar can be brewed vinegar or synthetic vinegar. In a preferred embodiment of the invention, the vinegar is brewed vinegar.

Brewed vinegars suitable for use in the present invention include, but are not limited to, fruit vinegar, rice vinegar, and wine vinegar.

Examples of fruit vinegar according to the present invention include, but are not limited to, banana vinegar, lemon vinegar, apple vinegar, grape vinegar, and diced vinegar. In a preferred embodiment of the invention, the fruit vinegar is banana vinegar.

The eagle-free extract according to the present invention has been confirmed to have an activity of inhibiting proliferation of tumors/cancer cells (especially liver cancer cells). In particular, the extract of the eagle peony of the present invention reduces proliferating cell nuclear antigen (PCNA), cyclin A, cyclin D1, cyclin E, and cyclin B1. The performance of c-Fos and c-Myc proteins, as well as the expression of p21, p27, p-p53 and p53 proteins in cells, thereby arresting the cell cycle in the G ₂ /M phase, thereby inhibiting tumor/cancer cell proliferation utility.

Accordingly, the present invention provides a pharmaceutical composition for inhibiting proliferation of a tumor/cancer cell comprising an extract of eagle-free, as described above.

In addition, the Applicant discovered by the cell migration assay and the cell invasion assay that the extract of the eagle peony according to the present invention has the effect of inhibiting the metastasis of tumors/cancer cells (especially liver cancer cells). active. In particular, the extract of the eagle peony of the present invention inhibits the activation of the urokinase plasminogen activator signal pathway and the β-catenin signal pathway. Thereby, the effect of inhibiting tumor/cancer cell metastasis is achieved.

Accordingly, the present invention also provides a pharmaceutical composition for inhibiting the metastasis of a tumor/cancer cell comprising an extract of eagle peony as described above.

In addition, the applicant found by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) analysis that the extract of the eagle peony according to the present invention has an induced tumor. / Apoptosis activity of cancer cells (especially liver cancer cells). In particular, the eagle-free extract of the present invention activates a death receptor-dependent apoptosis signal pathway and a mitochondria-dependent apoptotic signal transduction pathway (mitochondrial dependent) The apoptosis signal pathway), and causes mitochondrial membrane potential depolarization, thereby achieving the effect of inducing tumor/cancer cell apoptosis.

Accordingly, the present invention also provides a pharmaceutical composition for inducing apoptosis of a tumor/cancer cell comprising an extract of eagle-free, as described above.

The pharmaceutical composition according to the present invention can be manufactured into a dosage form suitable for parenterally, topically or orally, using techniques well known to those skilled in the art, including , but not limited to, an injection [eg, a sterile aqueous solution or dispersion], a sterile powder, a tablet, a tablet, or a troche, Pills, capsules, and the like.

The pharmaceutical composition according to the present invention may further comprise a pharmaceutically acceptable carrier which is widely used in pharmaceutical manufacturing techniques. For example, the pharmaceutically acceptable carrier can comprise one or more agents selected from the group consisting of solvents, emulsifiers, suspending agents, decomposers, and binding agents. Agent), excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, lubricant, Absorption delaying agents, liposomes, and the like.

The dosage and the number of administrations of the pharmaceutical composition according to the present invention vary depending on the severity of the disease to be treated, the route of administration, and the body weight, age, physical condition and response of the individual to be treated. In general, the daily dose of the pharmaceutical composition according to the present invention is usually from 10 mg/kg to 20 mg/kg body weight, in a single dose or divided into several doses, and can be parenterally Administered orally or topically.

Detailed description of the preferred embodiment

The invention is further described in the following examples, but it should be understood that these examples are for illustrative purposes only and are not to be construed as limiting.

Example Experimental Materials: 1. Normal human liver cell line Chang-Liver:

Chang-Liver is available from Cell Lines Service, Germany. Chang-Liver cells were cultured in a medium containing Basal Medium Eagle (BME) (Cat. No. B-9638, Sigma, MO, USA) [added with 10% fetal bovine serum (FBS), 1.5 g/L NaHCO ₃ , 0.1 mM non-essential amino acids, 2.0 mM glutamine, 100 U/mL penicillin G, 100 μg/mL streptomycin In a petri dish of 10 g/L phenol red], the culture was carried out in an incubator in which the culture conditions were set to 37 ° C and 5% CO ₂ . After that, fresh medium was replaced approximately every 2 days. When the cell density reached approximately 80% confluence, the medium was removed and the cells were washed twice with Phosphate Buffered Saline (PBS) followed by 1% trypsin-EDTA (Cat. No. 15400-054, GIBCO, USA) to detach the cells from the bottom of the dish. Thereafter, about 1/5 to 1/10 of the cells were added to a new petri dish containing fresh medium, and cultured in an incubator whose culture condition was set to 37 ° C and 5% CO ₂ .

2. Human hepatoma cell line HA22T (human hepatoma cell line HA22T):

HA22T is a Biosource Collection and Research Center (BCRC) purchased from the Food Industry Research and Development Institute (FIRDI) in Taiwan, and the accession number is BCRC 60168. HA22T cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Cat. No. D5523, Sigma, MO, USA) (with 10% FBS, 1.5 g/L NaHCO _{3 added)} , 0.1 mM non-essential amino acid, 2.0 mM glutamic acid, 100 U/mL penicillin G, 100 μg/mL streptomycin, and 10 g/L phenol red) in a culture dish, and the culture conditions were set to The culture was carried out in an incubator at 37 ° C and 5% CO ₂ . After that, fresh medium was replaced approximately every 2 days. When the cell density reached approximately 80% confluence, the medium was removed and the cells were washed twice with PBS, followed by the addition of 1% trypsin-EDTA to detach the cells from the bottom of the dish. Thereafter, about 1/5 to 1/10 of the cells were added to a new petri dish containing fresh medium, and cultured in an incubator whose culture condition was set to 37 ° C and 5% CO ₂ .

General experimental method: 1. Preparation of total protein samples:

The HA22T cells obtained in the following examples were washed twice with PBS, followed by the addition of 200 μL of lysis buffer [containing 50 mM Tris-base (pH 7.5), 0.5 M NaCl, 1 mM EDTA (pH). 8), 1 mM β-mercaptoethanol, 1% NP40, 1% glycerol, and 5 mg/mL protease inhibitor], and mixed well. The resulting cell mixture was placed in a microcentrifuge tube and allowed to stand on ice for 30 minutes (with shaking once every 5 minutes). Next, after centrifugation at 12,000 rpm for 10 minutes at 4 ° C, the supernatant was collected (as a total protein sample) and the collected supernatant was determined by the Lowry protein assay. Total protein concentration.

2. Western blotting:

This experiment uses a vertical electrophoresis tank (Mini 3 Cell, Bio-Rad, USA) for SDS-PAGE analysis. First, a polypropylene guanamine gel sheet was prepared, which was divided into two layers, which were respectively 12% SDS-PAGE separating gel (determining the separation and condensing according to the molecular size of the protein to be observed). The concentration of the gel) and 3.71% SDS-PAGE stacking gel. Thereafter, 40 μg of the total protein sample, the nuclear protein sample or the cytoplasmic protein sample obtained in the following examples were loaded into a U type well, using an electrophoresis buffer ( The electrophoresis was carried out for about 2.5 hours with a fixed voltage of 75 V containing 0.3% Tris-base, 1.47% glycine and 0.1% SDS.

After electrophoresis, use a transfusion electrophoresis tank (Mini Trans- Cell, Bio-Rad, USA), transfer the separated proteins on the gel sheet to a polyvinylidene difluoride (PVDF) membrane at a fixed voltage of 100 V at 4 ° C. It was 0.45 μm) (IPVH00010, Millipore Corporation, MA, USA) for 2 hours. Next, the transferred PVDF membrane was taken out and 5% (w/v) skimmed milk at room temperature [with TBS (150 mM NaCl, 10 mM Tris-base, 0.05% Tween-20) Blocking was performed for 1 hour, followed by washing 3 times with TBS for 10 minutes each time. Thereafter, a primary antibody (diluted 1,000-fold with TBS) was added, and after standing overnight at 4 ° C, it was washed 3 times with TBS for 10 minutes each time. Next, a secondary antibody (diluted 2,000 times with TBS) was added, and the mixture was allowed to stand at room temperature for 1 hour, and then washed 3 times with TBS for 10 minutes each time. Thereafter, Western Blotting Luminal Reagent (Cat. No. Sc-2048, Santa Cruz Biotechnology, CA, USA) was used for chemiluminescence staining, and LAS-3000 imaging system (Fujifilm) was used. , Tokyo, Japan) to take pictures.

3. Statistical analysis:

In the following examples, Sigma Plot statistical software was used for statistical analysis. Experimental data is expressed as mean ± SEM. All data were analyzed by t-test (t-test) to assess differences between groups. If the resulting statistical alignment result is p < 0.05, it represents statistical significance.

Example 1. Preparation of Eagle No. [ Zanthoxylum avicennae (Lam.) DC] extract

First, 9.5 kg of fresh eagle roots from the traditional Vietnamese market were washed and peeled with water, and then the obtained eagle peper roots were dried or placed in an oven (40 to 45 ° C). ) Drying inside. Thereafter, the dried eagle peper root skin was placed in a pottery pot and heated and stirred while spraying 100 mL of banana vinegar into the pottery pot for processing. The 850 g of the processed eagle-free pelt skin was washed 3 times with water, then immersed in 8 L of 95% ethanol at room temperature and mixed well, and then the resulting mixture was allowed to stand for 2 Day (this is stirred once every 4 hours). Next, the mixture was filtered with gauze to obtain a monoethanol filtrate, followed by 200 mL of banana vinegar to the ethanol filtrate to form a first extract.

The filtered residue of the eagle peper root was immersed in 5 L of 95% ethanol for 2 days (during every 4 hours), then filtered with gauze to obtain a monoethanol filtrate, followed by 200 mL. Banana vinegar is added to the ethanol filtrate. This step is repeated three times to obtain a second extract, a third extract, and a fourth extract, respectively. The first extract, the second extract, the third extract, and the fourth extract are combined, and then subjected to decompress concentrating at 30 ° C (Rotavapor R-114, B) Chi, USA), which lasted 2 days (in this period, 10 mL of banana vinegar was added every 1 hour), and then dried in an oven (40 to 45 ° C) to obtain a powdered eagle-free extract. The obtained eagle-free extract was dissolved in sterilized water to form a stock solution having a concentration of 10 mg/mL for use.

Example 2. High performance liquid chromatography (HPLC) analysis of the extract of eagle peony

In order to understand the main component distribution of the extract of the eagle peony of the present invention, the extract of the eagle peony obtained in the above Example 1 was taken for high performance liquid chromatography.

experimental method:

The HPLC analytical instrument used in this experiment was as follows: Agilent 1100 series liquid chromatography system (Agilent Technologies, Palo Alto, CA, USA) equipped with an online degasser (online degasser) G1379A), binary pump (G1312A) and UV-Vis variable multiwavelength detector (G1314A); analytical column is Xterra RP1 LC column (Waters Corp. , Milford, MA, USA), length: 250 mm x 4.6 mm. The HPLC operating conditions used in this experiment were as follows: UV-Vis variable multi-wavelength detector with a wavelength of 254 nm; mobile phase deionized water/methanol, 20:80 (v/v) The rate of the mobile phase is 0.7 mL/min; the gradient elution of the mobile phase is carried out in the following manner: within 60 minutes, the deionized water is changed from 20% to 50% for 15 minutes, followed by 50% changed to 70% for 25 minutes and then maintained at 70% for 20 minutes.

Further, for comparison, the methanol extract of the eagle-free leaf obtained according to the method disclosed in TT Thuy et al. (1999) (the above) was subjected to the same HPLC analysis.

result:

Figure 1 shows the HPLC elution profile of the eagle-free extract obtained in accordance with Example 1 above. As can be seen from Figure 1, the extract of the eagle peony of the present invention has 13 major elution peaks during the 0 to 60 minute retention period (labeled as peaks a1, a2, a3, a4, a5, a6, respectively). A7, a8, a9, a10, a11, a12, and a13).

Figure 2 shows the HPLC elution profile of the methanol extract of the eagle-free leaf obtained according to the method disclosed in TT Thuy et al . (1999) (supra). As can be seen from Figure 2, the methanol extract of the eagle leaves showed 21 major elution peaks during the 0 to 60 minute retention period (labeled as peaks b1, b2, b3, b4, b5, b6, respectively). B7, b8, b9, b10, b11, b12, b13, b14, b15, b16, b17, b18, b19, b20 and b21).

By comparing the peak positions of Figures 1 and 2, it is clear that the composition of the eagle-free extract obtained according to the present invention is clearly different from that disclosed in TT Thuy et al . (1999) (supra). The methanol extract of the eagle-free leaf obtained by the method.

Example 3. Cell viability analysis of extracts of Acanthopanax and its effect on cell cycle and proliferation of human hepatoma cell line HA22T

To understand the cytotoxicity of the extract of the eagle peony of the present invention and the effect on the cell cycle and proliferation of the human hepatoma cell line HA22T, the extract of the eagle peony obtained according to the above Example 1 was used for the purpose. The experiment below.

A, cell activity analysis:

The HA22T cells were divided into 6 groups including 1 control group and 5 experimental groups (i.e., Eagle non-parking groups 1, 2, 3, 4, and 5). Each group of HA22T cells was cultured in a medium containing 2×10 ⁵ cells/well in DMEM (added with 10% FBS, 1.5 g/L NaHCO ₃ , 0.1 mM non-essential amino acid, 2.0 mM branic acid, 100 U/mL penicillin G, 100 μg/mL streptomycin and 10 g/L phenol red) in 24-well plate and in incubator (37 ° C, 5% CO ₂ ) Cultivate. When the cell density reached approximately 80% confluence, the medium was replaced with fresh DMEM [with 1% enhanced calf serum (CCS), 1.5 g/L NaHCO ₃ , 0.2 mM non-essential amino acid, 2.0 mM branic acid, 100 U/mL penicillin G and 100 μg/mL streptomycin] and continued to culture for 4 hours. Next, an appropriate amount of the stock solution obtained according to the above Example 1 was added to the Eagle Non-Board Groups 1, 2, 3, 4 and 5 so that they had a final concentration of 50, 100, 150, 200 and 250, respectively. Gg/mL of eagle-free extract.

After the cells of each group were cultured in an incubator (37 ° C, 5% CO ₂ ) for 24 hours, the medium was removed and washed twice with PBS. Thereafter, 500 μL of thiazolyl blue tetrazolium bromide (MTT)/DMEM (with 1% reinforced calf serum, 1.5 g/L NaHCO ₃ , 0.2 mM non-essential amino acid, 2.0 mM bran) was added. Proline, 100 U/mL penicillin G and 100 μg/mL streptomycin) (v/v = 1:9), and cultured in an incubator (37 ° C, 5% CO ₂ ) for 4 hours, then Add 500 μL of isopropanol for 10 minutes. 200 μL of the resulting mixture was placed in a 96-well culture dish, and the absorbance (OD ₅₇₀ ) was measured at a wavelength of 570 nm using a spectrophotometer (U-2001, Hitachi). In addition, Chang-Liver cells were also used for the same experiment except that BME was used instead of DMEM.

The percentage of cell viability (%) is calculated by substituting the measured absorbance value (OD ₅₇₀ ) into the following formula (1):

Formula (1): A = (B / C) × 100

Where: A = percentage of cell viability (%)

B = OD ₅₇₀ absorbance measured by each group

C = OD ₅₇₀ absorbance measured in the control group

Thereafter, the obtained experimental data was analyzed in accordance with the method described in the third item "Statistical Analysis" of the "General Experimental Method" above.

B. The performance of proteins related to cell cycle regulation:

The HA22T cells were divided into 6 groups including 1 control group and 5 experimental groups (ie, Eagle non-parking groups 1, 2, 3, 4, and 5). Each group of HA22T cells were cultured in DMEM containing 10% FBS, 1.5 g/L NaHCO ₃ , 0.1 mM non-essential amino acid, 2.0 mM branic acid, 100 U/mL penicillin G, 100 μg/mL. A petri dish (10 cm in diameter) of streptomycin and 10 g/L phenol red) was cultured in an incubator (37 ° C, 5% CO ₂ ). When the cell density reached approximately 80% confluence, the medium was replaced with fresh DMEM (with 1% CCS, 1.5 g/L NaHCO ₃ , 0.2 mM non-essential amino acid, 2.0 mM glutamic acid, 100 U/mL) Penicillin G and 100 μg/mL streptomycin) and continued to culture for 4 hours. Next, an appropriate amount of the stock solution obtained according to the above Example 1 was added to the Eagle Non-Board Groups 1, 2, 3, 4 and 5 so that they had a final concentration of 50, 100, 150, 200 and 250, respectively. Gg/mL of eagle-free extract.

After the cells of each group were cultured in an incubator (37 ° C, 5% CO ₂ ) for 24 hours, the total cells were subjected to the method described in the first item "Preparation of total protein samples" in "General Experimental Methods" above. Preparation of protein samples. The obtained total protein sample of HA22T cells was subjected to proliferating cell nuclear antigen (PCNA) and cyclin A according to the method described in the second item "Western ink dot analysis" of the "General Experimental Method" above. CylinA), cyclin D1, cyclin E, cyclin B1, p21, p27, p-p53, p53, c-Fos, c-Myc, and α-tubulin (α-tubulin) [as internal control Western blot analysis of the group (internal control), and the primary antibody and secondary antibody used for each protein are shown in Table 1 below.

In addition, Chang-Liver cells were also used for the same experiment, except that: (1) BME was substituted for DMEM: and (2) the total protein sample of Chang-Liver cells obtained was according to the above "general experimental method. Western blot analysis of proliferating cell nuclear antigen and alpha-tubulin (as an internal control) was performed as described in the second item "Western Ink Point Analysis".

C, flow cytometry (flow cytometry):

The HA22T cells were divided into 6 groups including 1 control group and 5 experimental groups (ie, Eagle non-parking groups 1, 2, 3, 4, and 5). Each group of HA22T cells were cultured in DMEM containing 10% FBS, 1.5 g/L NaHCO ₃ , 0.1 mM non-essential amino acid, 2.0 mM branic acid, 100 U/mL penicillin G, 100 μg/mL. In a 6-well plate of streptomycin and 10 g/L phenol red), culture was carried out in an incubator (37 ° C, 5% CO ₂ ). When the cell density reached approximately 80% confluence, the medium was replaced with fresh DMEM (with 1% CCS, 1.5 g/L NaHCO ₃ , 0.2 mM non-essential amino acid, 2.0 mM glutamic acid, 100 U/mL) Penicillin G and 100 μg/mL streptomycin) and continued to culture for 4 hours. Next, an appropriate amount of the stock solution obtained according to the above Example 1 was added to the Eagle Non-Board Groups 1, 2, 3, 4 and 5 so that they had a final concentration of 50, 100, 150, 200 and 250, respectively. Gg/mL of eagle-free extract.

Each group of cells was cultured in an incubator (37 ° C, 5% CO ₂ ) for 24 hours, the medium was removed and washed twice with PBS, followed by 0.5 mL of 1% trypsin-EDTA to allow the cells to be self-cultivated The bottom of the detachment. Thereafter, add 0.5 mL of fresh medium (added 1% CCS, 1.5 g/L NaHCO ₃ , 0.2 mM non-essential amino acid, 2.0 mM branic acid, 100 U/mL penicillin G, and 100 μg/mL streptomycin) ), and then centrifuged at 1,500 rpm for 5 minutes. After the supernatant was removed, 1 mL of PBS (4 ° C) was added to suspend the cells, followed by centrifugation at 1,500 rpm for 5 minutes. After the supernatant was removed, 200 μL of PBS was added to suspend the cells, and then 800 μL of absolute alcohol was added dropwise to fix the cells, and allowed to stand at -20 ° C for 1 hour, and then centrifuged at 1,500 rpm for 5 minutes. After removing the supernatant, add 1 mL of Cyde Ntock/Triton X-100 staining solution (containing 1% Triton X-100, 50 μg/mL RNase A, 400 μg/mL Cyde Ntock, and 55% PBS) ), and reacted at room temperature in the dark for 30 minutes. Finally, a flow cytometer ^{(BD FACSCanto TM flow cytometer, BD} Bioscienses, USA) for analysis, 10,000 cells per analysis. The cells are excited by argon ion laser beam excitation at 488 nm. The resulting data was analyzed by the Mod Fit LT software for cell cycle and for statistical analysis.

D, wound healing assay:

The HA22T cells were divided into 6 groups including 1 control group and 5 experimental groups (ie, Eagle non-parking groups 1, 2, 3, 4, and 5). Each group of HA22T cells were cultured in DMEM containing 10% FBS, 1.5 g/L NaHCO ₃ , 0.1 mM non-essential amino acid, 2.0 mM branic acid, 100 U/mL penicillin G, 100 μg/mL. A petri dish (10 cm in diameter) of streptomycin and 10 g/L phenol red) was cultured in an incubator (37 ° C, 5% CO ₂ ). When the cell density reaches about 80% confluence, use a pipette tip to draw a line from the center of the dish to scrape off the HA22T cells attached to the center of the dish, thereby forming a cell-free attachment in the center of the dish. The gap (gap). Thereafter, the medium was removed and washed twice with PBS, followed by fresh DMEM (added with 1% CCS, 1.5 g/L NaHCO ₃ , 0.2 mM non-essential amino acid, 2.0 mM glutamic acid, 100 U/ mL penicillin G and 100 μg/mL streptomycin) and continued to culture for 4 hours. Next, an appropriate amount of the stock solution obtained according to the above Example 1 was added to the Eagle Non-Board Groups 1, 2, 3, 4 and 5 so that they had a final concentration of 50, 100, 150, 200 and 250, respectively. Gg/mL of eagle-free extract.

Each group of cells was cultured in an incubator (37 ° C, 5% CO ₂ ) for 48 hours, the medium was removed and washed twice with PBS, followed by an inverted microscope (CULTURE MICROSCOPES, CKX41, OLYMPUS) , JAPAN) to observe and calculate the number of cells proliferating in the gap. The cell proliferation percentage (%) of each group was calculated by substituting the measured cell number into the following formula (2):

Formula (2): D = (E / F) × 100

Where: D = percentage of cell proliferation (%)

E = number of cells proliferating in each gap

F = number of cells proliferating in the gap in the control group

Thereafter, the obtained experimental data was analyzed in accordance with the method described in the third item "Statistical Analysis" of the "General Experimental Method" above.

result: A, cell activity analysis:

Figure 3A and Figure 3B show the percentage of cell viability as measured by HA22T cells and Chang-Liver cells, respectively, after treatment with different concentrations of extracts of Eagle. As can be seen from Fig. 3A, the percentage of cell viability of the eagle-free group 1 to 5 was significantly reduced as compared with the control group, and in particular, the percentage of cell activity of the eagle-free group 5 was as high as 23.45%. In addition, it can be seen from FIG. 3B that the percentage of cell viability of the eagle-free group 1 to 5 was significantly increased as compared with the control group, in particular, the percentage of cell activity of the eagle-free group 5 was up to 129.55%. . The results of this experiment show that the extract of Eagle's non-pod is cytotoxicity to liver cancer cells, but does not cause damage to normal liver cells.

B. Performance of proteins involved in cell cycle regulation:

Figure 4 is a Western blot analysis showing PCNA, cyclin A, cyclin D1, cyclin E, and cells measured by HA22T cells treated with different concentrations of eagle-free extract. The performance of cyclin B1, p21, p27, p-p53, p53, c-Fos and c-Myc proteins. As can be seen from Fig. 4, compared with the control group, PCNA, cyclin A, cyclin D1, cyclin E, cyclin B1, c-Fos, and c-Myc of the Eagle non-poll group 1 to 5 The expression of protein showed a decrease, while the expression levels of p21, p27, p-p53 and p53 protein all increased, and they all became more obvious with the increase of the concentration of eagle-free extract. .

Figure 5 is a Western blot analysis showing the performance of PCNA protein measured by Chang-Liver cells after treatment with different concentrations of eagle-free extract. As can be seen from Fig. 5, compared with the control group, the expression levels of PCNA protein in the Eagle non-poll group 1 to 5 all increased, and at the same time, the concentration of the extract of the eagle-free group increased. obvious.

The results of this experiment showed that the extract of eagle peony reduced the expression of PCNA, cyclin A, cyclin D1, cyclin E, cyclin B1, c-Fos and c-Myc protein in liver cancer cells and improved The expression levels of p21, p27, p-p53 and p53 proteins in hepatocarcinoma cells have a positive dose-effect relationship, and the extract of eagle-free is increasing the PCNA protein in normal hepatocytes. There is also a positive dose-effect relationship in performance. Applicant's preliminary inference: The extract of the eagle peony of the present invention can inhibit cell cycle progression by regulating the expression of proteins involved in cell cycle regulation in liver cancer cells, thereby inhibiting The utility of HA22T cell proliferation, and the extract of the eagle peony of the present invention can significantly enhance the performance of PCNA protein in normal hepatocytes, and has the effect of promoting normal hepatocyte proliferation.

C, flow cell measurement:

Figure 6 shows cell cycle changes measured by HA22T cells after treatment with different concentrations of extracts from Eagle. As can be seen from Fig. 6, compared with the control group, the number of HA22T cells in the eagle-free group 1 to 5 increased in the G ₂ /M phase (G ₂ /M phase), and the eagle would not move. The concentration of the extract is more pronounced and more pronounced. The results of this experiment show that the eagle-free extract has a positive dose-effect relationship in causing cell cycle arrest. Applicant's preliminary inference: The extract of the eagle peony of the present invention can arrest the cell cycle of HA22T cells in the G ₂ /M phase, thereby achieving the effect of inhibiting the proliferation of HA22T cells.

D, wound healing analysis:

Figure 7 shows the percentage of cell proliferation measured by HA22T cells after treatment with different concentrations of eagle-free extract. As can be seen from Fig. 7, the percentage of cell proliferation of the eagle-free group 1 to 5 was significantly lowered as compared with the control group, and in particular, the percentage of cell proliferation of the eagle-free group 5 was as high as 4.1%. The results of this experiment show that the eagle-free extract has a positive dose-effect relationship in inhibiting the cell proliferation of HA22T.

Example 4. Effect of extract of eagle radix on apoptosis of human hepatoma cell line HA22T

In order to evaluate the effect of the extract of the eagle peony of the present invention on the apoptosis of the human hepatoma cell line HA22T, the extract of the eagle peony obtained according to the above Example 1 was used for the terminal deoxyribonucleoside Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) analysis.

experimental method:

The HA22T cells were divided into 6 groups including 1 control group and 5 experimental groups (ie, Eagle non-parking groups 1, 2, 3, 4, and 5). Each group of HA22T cells were cultured in DMEM containing 10% FBS, 1.5 g/L NaHCO ₃ , 0.1 mM non-essential amino acid, 2.0 mM branic acid, 100 U/mL penicillin G, 100 μg/mL. In a 24-well plate of streptomycin and 10 g/L phenol red), culture was carried out in an incubator (37 ° C, 5% CO ₂ ). When the cell density reached approximately 80% confluence, the medium was replaced with fresh DMEM (with 1% CCS, 1.5 g/L NaHCO ₃ , 0.2 mM non-essential amino acid, 2.0 mM glutamic acid, 100 U/mL) Penicillin G and 100 μg/mL streptomycin) and continued to culture for 4 hours. Next, an appropriate amount of the stock solution obtained according to the above Example 1 was added to the Eagle Non-Board Groups 1, 2, 3, 4 and 5 so that they had a final concentration of 50, 100, 150, 200 and 250, respectively. Gg/mL of eagle-free extract.

Each group of cells was cultured in an incubator (37 ° C, 5% CO ₂ ) for 24 hours, and then subjected to TUNEL staining using In Situ Cell Death Detection Kit, Fluorescein (Roche, Germany). First, the liquid in each well was removed and washed three times with 500 μL of PBS, followed by adding 300 μL of a fixing solution [4% paraformaldehyde in PBS (pH 7.4)] at room temperature. The fixation was carried out for 1 hour and then washed 5 times with 500 μL of PBS to remove the paraformaldehyde. Next, 300 μL of a permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) was added at 4 ° C for 2 minutes, followed by washing with 500 μL of PBS. 5 times. After that, add 4',6-diamidino-2-phenylindole (DAPI) (diluted 10,000 times with PBS), and kept at room temperature in the dark. The effect lasted for 30 minutes and then washed 5 times with 500 μL of PBS. Next, a TUNEL reaction mixture containing terminal deoxyribonucleotidyl transferase and fluorescein [labelling dye] was added and allowed to act in the dark at 37 ° C for 2 hours, and then Wash 5 times with PBS. Finally, the 24-well plate was placed on an inverted microscope (CULTURE MICROSCOPES, CKX41, OLYMPUS, JAPAN) with an excitation wavelength of 488 nm for luciferin and 400 nm for DAPI. It was observed that TUNEL staining [that is, TUNEL positive] indicates that apoptosis occurred, while intact cells were stained blue by DAPI. The number of cells stained by TUNEL and stained with DAPI was calculated using Image J analysis software. The apoptotic percentage (%) of each group was calculated by substituting the measured cell number into the following formula (3):

Formula (3): G=(H/I)×100

Where: G = percentage of apoptosis (%)

H = number of cells stained with TUNEL

I = number of cells stained by DAPI

Thereafter, the obtained experimental data was analyzed in accordance with the method described in the third item "Statistical Analysis" of the "General Experimental Method" above.

result:

Figure 8 shows the percentage of apoptosis measured by HA22T cells after treatment with different concentrations of eagle-free extract. As can be seen from Fig. 8, the percentage of apoptosis of the eagle-free group 1 to 5 was significantly increased as compared with the control group, and in particular, the percentage of apoptosis of the eagle-free group 5 reached 46.66%. The results of this experiment show that the extract of eagle peony has a positive dose-effect relationship in causing apoptosis of human hepatoma cell line HA22T.

Example 5. Effect of extract of eagle radix on apoptosis signal pathway of human hepatoma cell line HA22T

To further investigate the death receptor-dependent apoptosis signal pathway, mitochondrial, caused by the human hepatoma cell line HA22T after being subjected to the extract of the eagle peony of the present invention. The mitochondrial membrane potential and the changes in the mitochondrial dependent apoptosis signal pathway, the following experiments were performed.

A. Performance of proteins associated with death receptor-dependent apoptotic signaling pathways:

This experiment was generally carried out in the manner described in "B, Performance of proteins related to cell cycle regulation" of Example 3 above, except that the primary antibody shown in Table 2 below was used and twice Antibodies for FAS, FAS-L, FADD, TNFα, TNF-R1, caspase-8, t-BID, BID, and caspase-3 Western ink point analysis.

B. Membrane potential analysis of mitochondrial membrane potentialassay:

It is known that the depolarization of the mitochondrial membrane potential is an early marked event in apoptosis, and therefore it is known whether the extract of the eagle peony of the present invention affects human liver cancer. The mitochondrial membrane potential of the cell line HA22T, which in turn induces apoptosis of liver cancer cells, was performed in the following experiment.

First, HA22T cells were divided into 6 groups including 1 control group and 5 experimental groups (i.e., Eagle non-parking groups 1, 2, 3, 4, and 5). Each group of HA22T cells were cultured in DMEM containing 10% FBS, 1.5 g/L NaHCO ₃ , 0.1 mM non-essential amino acid, 2.0 mM branic acid, 100 U/mL penicillin G, 100 μg/mL. A petri dish (4 cm in diameter) of streptomycin and 10 g/L phenol red) was cultured in an incubator (37 ° C, 5% CO ₂ ). When the cell density reached approximately 50% confluence, the medium was replaced with fresh DMEM (added 1% CCS, 1.5 g/L NaHCO ₃ , 0.2 mM non-essential amino acid, 2.0 mM branic acid, 100 U/mL) Penicillin G and 100 μg/mL streptomycin) and continued to culture for 4 hours. Next, an appropriate amount of the stock solution obtained according to the above Example 1 was added to the Eagle Non-Board Groups 1, 2, 3, 4, and 5 so that they had final concentrations of 50, 100, 150, 200, and 250 μg, respectively. /mL of the Eagle's extract.

Each group of cells was cultured in an incubator (37 ° C, 5% CO ₂ ) for 24 hours, using a mitochondrial staining kit (Mitochondria Staining Kit, Sigma-aldrich) (Cat. No. CS0390, Sigma, MO, USA) Dyeing according to the instructions provided by the manufacturer. First, remove the liquid from each well and wash it 3 times with 1 mL of PBS, then add 1 mL of staining solution at room temperature [containing 2.5 μg/mL JC-1, 0.5X JC-1 staining) Buffer and 50% DMEM (added 1% CCS, 1.5 g/L NaHCO ₃ , 0.2 mM non-essential amino acid, 2.0 mM branic acid, 100 U/mL penicillin G, and 100 μg/mL streptomycin) ], and reacted in a cell incubator (37 ° C, 5% CO 2 ) for 20 minutes. After that, the staining solution was removed and washed 3 times with 0.2X JC-1 staining buffer, followed by 3 mL of DMEM (with 10% FBS, 1.5 g/L NaHCO3, 0.1 mM non-essential amino acid, 2.0 mM bran) Proline acid 100, U/mL penicillin G and 100 μg/mL streptomycin). Finally, red fluorescence and green fluorescence were observed at a laser excitation spectral confocal microscope (Leica TCS SP2, Germany) at an excitation wavelength of 577 nm and 488 nm, respectively. The cells showed red fluorescence indicating that the mitochondrial membrane potential was normal, while the cells showed green fluorescence indicating that the mitochondrial membrane potential was depolarized.

C. Performance of proteins associated with mitochondria-dependent apoptotic signaling pathways:

This experiment was generally carried out in the manner described in "B, Performance of proteins related to cell cycle regulation" of Example 3 above, except that the primary antibody shown in Table 3 below was used and twice. Antibodies for Bax, Bak, p-Bad, Bad, Bcl-2, Bcl-x _L , BID, t-BID, cytochrome c, caspase-9, cysteine Western blot analysis of aspartate protease-3 and AIF.

result: A. Performance of proteins associated with death receptor-dependent apoptotic signaling pathways:

Figure 9 is a Western blot analysis showing FAS, FAS-L, FADD, TNFα, TNF-R1, and caspase-day measured after treatment of HA22T cells with different concentrations of eagle-free extract. Pro-form of aspartic acid protease-8 and its active form, t-BID, BID and the original form of caspase-3 and two of them The performance of the activated form. As can be seen from Fig. 9, compared with the control group, the original form of FAS, FAS-L, FADD, TNFα, TNF-R1, and caspase-8 in the Eagle non-poll group 1 to 5 The expression levels of one of the activated forms, t-BID, BID, and the pro-form of caspase-3 and the two activated forms thereof all showed an increase. Therefore, the applicant concludes that the extract of the eagle peony of the present invention can significantly increase the original form of FAS, FAS-L, FADD, TNFα, TNF-R1, caspase-8 and its The activation form, t-BID, BID, and the pro-form of caspase-3 and the expression of its two activated forms, thereby activating the death receptor-dependent apoptotic signaling pathway The effect of inducing apoptosis of HA22T cells is achieved.

B. Membrane potential analysis of mitochondria:

Figure 10 is a JC-1 fluorescent staining analysis showing the results of JC-1 fluorescence staining of HA22T cells treated with extracts of different concentrations of Eagle. As can be seen from Fig. 10, compared with the control group, the depolarization of the mitochondrial membrane potential of the eagle-free group 1 to 5 is increased, and at the same time, the concentration of the eagle-free extract increases. It tends to be obvious. The results of this experiment show that the extract of eagle peony has a positive dose-effect relationship in inducing depolarization of the mitochondrial membrane potential of HA22T cells. Applicant's preliminary inference: The extract of the eagle peony of the present invention can significantly depolarize the mitochondrial membrane potential and induce the occurrence of apoptosis.

C. Performance of proteins associated with mitochondria-dependent apoptotic signaling pathways:

Figure 11 is a Western blot analysis showing Bax, Bak, p-Bad, Bad, Bcl-2, Bcl-x _L measured by HA22T cells after treatment with different concentrations of eagle-free extract. , t-BID, BID, cytochrome c, pro-form of caspase-9 and its activation form, pro-form of caspase-3 and its two The form of activation and the performance of the AIF protein. As can be seen from Fig. 11, compared with the control group, Bax, Bak, Bad, t-BID, BID, cytochrome c, and caspase-9 of the eagle-free group 1 to 5 - The form and its activation form, the pro--form of caspase-3 and its two forms of activation, as well as the expression of AIF protein, all increased, while p-Bad, Bcl-2 and Bcl The amount of -x _L protein expression decreased, and it became more pronounced as the concentration of the eagle-free extract increased. The results of this experiment show that the extract of eagle peony enhances the original form of Bax, Bak, Bad, t-BID, BID, cytochrome c, caspase-9 and its activation form, The pro--form of caspase-3 and its two activated forms and AIF protein expression and a decrease in the expression of p-Bad, Bcl-2 and Bcl-x _L proteins have a positive dose. - effect relationship. Applicant's preliminary inference: The extract of the eagle peony of the present invention can significantly enhance the original form of Bax, Bak, Bad, t-BID, BID, cytochrome c, and caspase-9 in cells. And its activation form, the pro--form of caspase-3 and its two activated forms and the expression of AIF protein, and significantly reduced intracellular p-Bad, Bcl-2 and Bcl-x The expression of _L protein, which in turn activates the mitochondria-dependent apoptotic signaling pathway, thereby achieving the effect of inducing apoptosis of HA22T cells.

Example 6. Effect of extract of eagle peony on cell survival signal pathway of human hepatoma cell line HA22T

To further investigate the changes in the intracellular survival signaling pathway produced by the human hepatoma cell line HA22T after being subjected to the extract of the eagle peony of the present invention, generally refer to "B, related to cell cycle regulation" of Example 3 above. Experiments were carried out in the manner described in the "Performance of Proteins", except that primary antibodies and secondary antibodies shown in Table 4 below were used for p-PI3k, PI3k, p-Akt, Akt, p-Bad, Western blot analysis of Bad, Bcl-2, and Bcl-x _L.

result:

Figure 12 is a Western blot analysis showing p-PI3k, PI3k, p-Akt, Akt, p-Bad, Bad, measured by HA22T cells after treatment with different concentrations of eagle-free extract. The performance of Bcl-2 and Bcl-x _L proteins. As can be seen from Fig. 12, compared with the control group, the expression levels of p-PI3k, PI3k, p-Akt, Akt, p-Bad, Bcl-2, and Bcl-x _L proteins in the Eagle non-poll group 1 to 5 were The situation of decline is present, while the performance of Bad protein is on the rise. Therefore, the Applicant reasoned that the extract of the eagle peony of the present invention can significantly reduce the expression of p-PI3k, PI3k, p-Akt, Akt, p-Bad, Bcl-2 and Bcl-x _L proteins, and significantly It enhances the expression of Bad protein in cells, thereby inhibiting the activation of cell survival signaling pathway, thereby achieving the effect of reducing the survival of HA22T cells.

Example 7. Effect of extract of eagle radix on metastasis of human hepatoma cell line HA22T

In order to understand the effect of the extract of the eagle peony of the present invention on the transfer mechanism of the human hepatoma cell line HA22T, the following experiment was carried out.

A, cell migration assay:

The HA22T cells were divided into 6 groups including 1 control group and 5 experimental groups (ie, Eagle non-parking groups 1, 2, 3, 4, and 5). Each group of HA22T cells were cultured in DMEM containing 10% FBS, 1.5 g/L NaHCO ₃ , 0.1 mM non-essential amino acid, 2.0 mM branic acid, 100 U/mL penicillin G, 100 μg/mL chain Petri dishes (10 g/L phenol red) in a petri dish (6 cm in diameter) and cultured in an incubator (37 ° C, 5% CO ₂ ). When the cell density reached approximately 80% confluence, the medium was replaced with fresh DMEM (with 1% CCS, 1.5 g/L NaHCO ₃ , 0.2 mM non-essential amino acid, 2.0 mM glutamic acid, 100 U/mL) Penicillin G and 100 μg/mL streptomycin) and continued to culture for 4 hours. Thereafter, an appropriate amount of the stock solution obtained according to the above Example 1 was added to the Eagle Non-Board Groups 1, 2, 3, 4 and 5 so that they had a final concentration of 50, 100, 150, 200 and 250, respectively. Gg/mL of eagle-free extract.

Each group of cells was cultured in an incubator (37 ° C, 5% CO ₂ ) for 24 hours, the medium was removed and washed twice with PBS, followed by the addition of 1% trypsin-EDTA to bring the cells from the bottom of the dish. Get rid of. Thereafter, 1 mL of DMEM (with 1% CCS, 1.5 g/L NaHCO ₃ , 0.2 mM non-essential amino acid, 2.0 mM branic acid, 100 U/mL penicillin G, and 100 μg/mL streptomycin) was added. And calculate the total number of cells, followed by DMEM (added 1% CCS, 1.5 g / L NaHCO ₃ , 0.2 mM non-essential amino acid, 2.0 mM branic acid, 100 U / mL penicillin G and 100 μg / mL chain The bacterium is diluted to a cell solution having a concentration of 30 × 10 ⁴ cells/mL.

Thereafter, cell movement analysis was performed using 48 well Boyden chambers (Neuro Probe Inc., MD, USA). First, 32 μL of DMEM (with 10% CCS, 1.5 g/L NaHCO ₃ , 0.2 mM non-essential amino acid, 2.0 mM glutamic acid, 100 U/mL penicillin G, and 100 μg/mL streptomycin) Add to the bottom well of the lower chamber, and then a polycarbonate membrane with a rough surface and a smooth surface (pore size 8 μm) (Cat. No. PFB8, Neuro Probe Inc., USA) placed the rough face down on the lower chamber, then placed the gasket and the upper chamber on top of the polycarbonate membrane and screwed it fixed. Thereafter, 50 μL of the cell solution was separately added to each well of the upper chamber, and the upper chamber was covered with a plastic wrap, and then cultured in an incubator (37 ° C, 5% CO ₂ ) for 4-6 hours. Next, the polycarbonate film was taken out, and the cells on the rough surface of the polycarbonate film were fixed with methanol for 10 minutes, followed by 0.5% Giemsa (Cat. No. GS-500, Sigma, USA). It was dyed for 1 hour. After washing with ddH ₂ O, the smooth surface of the polycarbonate film was wiped clean and attached to a new Petri dish, which was then observed and calculated using an inverted microscope (CULTURE MICROSCOPES, CKX41, OLYMPUS, JAPAN). The number of cells stained with Giemsa on the rough surface of the polycarbonate membrane. The cell migration percentage (%) for each group was calculated by substituting the measured cell number into the following formula (4):

Formula (4): J = (K / L) × 100

Where: J = percentage of cell movement (%)

K = number of cells stained with Giemsa in each group

L = number of cells stained with Giemsa in the control group

B, cell invasion assay:

This experiment is generally carried out in the manner described in the "Cell Movement Analysis" of item A above and the cell invasion assay is performed with 48 well Boyden chambers, except that: (1) the cell solution is added to the upper layer. Prior to each well in the chamber, 10 μL of 0.2 mg/mL matrigel (Cat. No. 354234, BD Biosciences, USA) was added to each well in the upper chamber and allowed to stand for 5 hours to make Matrigel Coagulation; and (2) after covering the upper chamber with plastic wrap, the culture is carried out in an incubator for 7-8 hours. In addition, the cell invasion percentage (%) of each group was calculated by substituting the measured cell number into the following formula (5):

Formula (5): M = (N / O) × 100

Where: M = percentage of cell invasion (%)

N = number of cells stained with Giemsa in each group

O = number of cells stained with Giemsa in the control group

C. Performance of proteins associated with the urokinase plasminogen activator signal pathway:

This experiment was carried out generally in the manner described in "B, Performance of proteins related to cell cycle regulation" of Example 3 above, except that the primary antibody shown in Table 5 below and the second were used. Antibodies for FGF2, p-MEK-1/2, MEK1, p-ERK, ERK1, tPA, uPA, contactin-1, MMP-9, MMP-2, PAI-1, TIMP-1 Western blot analysis of TIMP-2, TIMP-3, and TIMP-4.

D. Performance of proteins associated with mitogen-activated protein kinase (MAPK) signaling pathways:

This experiment was generally carried out in the manner described in "B, Performance of proteins related to cell cycle regulation" of Example 3 above, except that the primary antibody shown in Table 6 below and the second were used. Antibodies were used for Western blot analysis of p-ERK, ERK1, JNK, p-JNK, p-p38, and p38α.

The performance of E, NF-κB p65 and p-NF-κB p65 protein:

This experiment was carried out generally in the manner described in "B, Performance of proteins involved in cell cycle regulation" of Example 3 above, except that the primary antibody shown in Table 7 below and the second were used. Antibodies were used for Western blot analysis of NF-κB p65 and p-NF-κB p65.

result: A. Analysis of cell movement and invasion:

Figure 13A and Figure 13B show the percentage of cell movement and the percentage of cell invasion measured by HA22T cells after treatment with different concentrations of extracts of Eagle. As can be seen from Fig. 13A and Fig. 13B, the percentage of cell migration and the percentage of cell invasion of the eagle-free group 1 to 5 were significantly lowered as compared with the control group, in particular, the percentage of cell migration and cells of the eagle-free group 5 The percentage of intrusion was as high as 7.5% and 3.44%, respectively. The results of this experiment show that the eagle-free extract has a positive dose-effect relationship in inhibiting the movement and invasion of HA22T cells.

B. Performance of proteins associated with the urokinase plasminogen activator signaling pathway:

Figure 14 is a Western blot analysis showing FGF2, p-MEK-1/2, MEK1, p-ERK and ERK1 proteins measured by HA22T cells treated with different concentrations of eagle-free extract. Performance situation. As can be seen from Fig. 14, compared with the control group, the expression levels of FGF2, p-MEK-1/2, MEK1, p-ERK and ERK1 proteins in the Eagle non-poll group 1 to 5 showed a decrease, and It becomes more pronounced as the concentration of the extract of the eagle is increased. The results of this experiment show that the extract of eagle peony has a positive dose-effect relationship in reducing the expression of FGF2, p-MEK-1/2, MEK1, p-ERK and ERK1 proteins. Applicant's preliminary inference: The extract of the eagle peony of the present invention can significantly reduce the expression of FGF2, p-MEK-1/2, MEK1, p-ERK and ERK1 proteins in cells, thereby inhibiting urokinase fibrinolysis Activation of the zymogen activator signaling pathway, thereby reducing the ability of HA22T cells to move and invade.

Figure 15 is a Western blot analysis showing tPA, uPA, contact protein-1, MMP-9 and MMP-2 proteins measured by HA22T cells treated with different concentrations of eagle-free extract. Performance situation. As can be seen from Fig. 15, compared with the control group, the expression levels of tPA, uPA, contact protein-1, MMP-9 and MMP-2 proteins in the Eagle non-poll group 1 to 5 all decreased, and The increase in the concentration of the extract of the eagle is more pronounced. The results of this experiment show that the extract of eagle peony has a positive dose-effect relationship in reducing the expression of tPA, uPA, contact protein-1, MMP-9 and MMP-2 proteins. Applicant's preliminary inference: The extract of the eagle peony of the present invention can significantly reduce the expression of tPA, uPA and contact protein-1 proteins associated with the urokinase plasminogen activator signaling pathway in cells, thereby significantly Reduces the expression of MMP-9 and MMP-2 proteins in cells, thereby reducing the ability of HA22T cells to move and invade.

Figure 16 is a Western blot analysis showing PAI-1, TIMP-1, TIMP-2, TIMP-3, and TIMP- measured by HA22T cells treated with different concentrations of Eagle's extract. 4 protein performance. As can be seen from Fig. 16, compared with the control group, the expression levels of PAI-1, TIMP-1, TIMP-2, TIMP-3 and TIMP-4 proteins in the Eagle non-poll group 1 to 5 increased. At the same time, it will become more obvious as the concentration of the extract of the eagle is increased. The results of this experiment show that the extract of eagle peony has a positive dose-effect relationship in increasing the expression of PAI-1, TIMP-1, TIMP-2, TIMP-3 and TIMP-4 proteins. Applicant's preliminary inference: The extract of the eagle peony of the present invention can significantly enhance the expression of PAI-1, TIMP-1, TIMP-2, TIMP-3 and TIMP-4 proteins in cells, thereby inhibiting MMP-9 and The activity of the MMP-2 protein, thereby reducing the ability of HA22T cells to move and invade.

C. Performance of proteins involved in the MAPK signaling pathway:

Figure 17 is a Western blot analysis showing p-ERK, ERK1, JNK, p-JNK, p-p38 and p38α proteins measured by HA22T cells treated with different concentrations of eagle-free extract. Performance situation. As can be seen from Fig. 17, compared with the control group, the expression levels of p-ERK, ERK1, JNK, p-JNK, p-p38, and p38α proteins in the Eagle non-poll group 1 to 5 were all decreased. Therefore, the Applicant reasoned that the extract of the eagle peony of the present invention can significantly reduce the expression of p-ERK, ERK1, JNK, p-JNK, p-p38 and p38α proteins in the cells, thereby inhibiting the MAPK signaling pathway. Activation, thereby reducing the ability of HA22T cells to move and invade.

D, NF-κB p65 and p-NF-κB p65 protein performance:

Figure 18 is a Western blot analysis showing the expression of NF-κB p65 and p-NF-κB p65 protein measured by HA22T cells after treatment with different concentrations of eagle-free extract. As can be seen from Fig. 18, compared with the control group, the expression levels of NF-κB p65 and p-NF-κB p65 protein in the Eagle non-poll group 1 to 5 were all decreased, and the eagle was not moored. The concentration of the extract is more pronounced and more pronounced. The results of this experiment show that the extract of eagle peony has a positive dose-effect relationship in reducing the expression of NF-κB p65 and p-NF-κB p65 protein. Applicant's preliminary inference: The extract of the eagle peony of the present invention can significantly reduce the expression of NF-κB p65 and p-NF-κB p65 protein in cells, thereby reducing the ability of HA22T cells to move and invade.

Example 8. Effect of extract of eagle peony on β-catenin signal pathway of human hepatoma cell line HA22T

To further investigate whether the extract of the eagle peony of the present invention is related to the β-catenin signaling pathway in the mechanism of inhibition of the metastasis of the human hepatoma cell line HA22T, the following experiment was carried out.

A. Performance of proteins associated with the β-catenin signaling pathway:

This experiment was carried out generally in the manner described in "B, Performance of proteins related to cell cycle regulation" of Example 3 above, except that the primary antibody shown in Table 8 below and the second were used. Antibodies were used for Western blot analysis of β-catenin, GSK-3β, APC, MMP-2, and MMP-9.

B, immunofluorescence staining assay (immunofluorescence staining assay):

The HA22T cells were divided into 6 groups including 1 control group and 5 experimental groups (i.e., Eagle non-parking groups 1, 2, 3, 4, and 5). Each group of HA22T cells were cultured in DMEM containing 10% FBS, 1.5 g/L NaHCO ₃ , 0.1 mM non-essential amino acid, 2.0 mM branic acid, 100 U/mL penicillin G, 100 μg/mL. A petri dish (6 cm in diameter) of streptomycin and 10 g/L phenol red) was cultured in an incubator (37 ° C, 5% CO ₂ ). When the cell density reached approximately 50% confluence, the medium was replaced with fresh DMEM (added 1% CCS, 1.5 g/L NaHCO ₃ , 0.2 mM non-essential amino acid, 2.0 mM branic acid, 100 U/mL) Penicillin G and 100 μg/mL streptomycin) and continued to culture for 4 hours. Next, an appropriate amount of the stock solution obtained according to the above Example 1 was added to the Eagle Non-Board Groups 1, 2, 3, 4 and 5 so that they had a final concentration of 50, 100, 150, 200 and 250, respectively. Gg/mL of eagle-free extract.

After the cells of each group were cultured in an incubator (37 ° C, 5% CO ₂ ) for 24 hours, the medium was removed and washed 5 times with PBS, followed by addition of 2 mL of 4% paraformaldehyde. It took 30 minutes and then washed 5 times with PBS to remove the paraformaldehyde. Next, after adding 2 mL of 2% BSA for 1 hour, the BSA was removed and washed 3 times with PBS for 10 minutes each time. Thereafter, a mouse anti-β-catenin (E-5) monoclonal antibody (Santa Cruz, USA, Cat. No. sc-7963) was added as a primary antibody (Santa Cruz, USA, Cat. No. sc-7963) The mixture was diluted 200-fold with PBS, and the reaction was carried out at 37 ° C for 2 hours, and then washed 3 times with PBS for 10 minutes each time. Next, a goat anti-mouse IgG-HRP antibody (Santa Cruz, USA, Cat. No. sc-2005) labeled with fluorescein was added as a secondary antibody ( The PBS was diluted 400-fold) and allowed to act at 37 ° C for 1 hour, and then washed 3 times with PBS for 10 minutes each time. Thereafter, 2 mL of DAPI (diluted 10,000 times with PBS) was added for 1 minute, then DAPI was removed and washed 3 times with PBS for 10 minutes each time. Finally, 2 mL of PBS was added to the Petri dish, and the Petri dish was placed at a laser scanning spectrum with an excitation wavelength of 488 nm for luciferin and 360 nm for DAPI. Observed on a laser scanning spectral confocal microscope (Leica TCS SP2, Germany).

C, β-catenin performance in the nucleus and cytoplasm:

The HA22T cells were divided into 6 groups including 1 control group and 5 experimental groups (ie, Eagle non-parking groups 1, 2, 3, 4, and 5). Each group of HA22T cells were cultured in DMEM containing 10% FBS, 1.5 g/L NaHCO ₃ , 0.1 mM non-essential amino acid, 2.0 mM branic acid, 100 U/mL penicillin G, 100 μg/mL. A petri dish (10 cm in diameter) of streptomycin and 10 g/L phenol red) was cultured in an incubator (37 ° C, 5% CO ₂ ). When the cell density reached approximately 80% confluence, the medium was replaced with fresh DMEM (with 1% CCS, 1.5 g/L NaHCO ₃ , 0.2 mM non-essential amino acid, 2.0 mM glutamic acid, 100 U/mL) Penicillin G and 100 μg/mL streptomycin) and continued to culture for 4 hours. Next, an appropriate amount of the stock solution obtained according to the above Example 1 was added to the Eagle Non-Board Groups 1, 2, 3, 4 and 5 so that they had a final concentration of 50, 100, 150, 200 and 250, respectively. Gg/mL of eagle-free extract.

Each group of cells was cultured in an incubator (37 ° C, 5% CO ₂ ) for 24 hours, the medium was removed and washed twice with PBS, followed by the addition of 200 μL of buffer I [containing 10 mM Hepes (pH 7.9) 1.5 mM MgCl ₂ , 10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF, 10 μg/mL leupeptin, and 5 mg/mL protease inhibitor] were mixed well. The resulting cell mixture was placed in a microcentrifuge tube and allowed to stand on ice for 5 minutes, followed by the addition of 20 μL of 1% NP40 (Cat. No. I3021, Sigma, MO, USA) for 10 minutes. . Then, after centrifugation at 14,000 rpm for 15 minutes at 4 ° C, the supernatant was used as a cytoplasmic protein sample and the protein concentration was determined by the labor protein analysis method, and the precipitate was taken. The preparation of the nucleus protein sample of the following nuclei was performed.

100 μL of buffer II [containing 20 mM Hepes (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl ₂ , 0.5 mM DTT, 0.2 mM EDTA, 0.2 mM PMSF, 10 μg/mL bright peptide The prime and 5 mg/mL protease inhibitor were added to the collected precipitate and mixed well, and then allowed to stand on ice for 15 minutes. Subsequently, after centrifugation at 14,000 rpm for 15 minutes at 4 ° C, the supernatant was used as a protein sample of the nucleus and the protein concentration was determined by Labor Protein Analysis.

The obtained cytoplasmic protein sample and the nucleus protein sample were subjected to β-catenin and β-actin (β- according to the method described in the second item "Western Ink Point Analysis" of the "General Experimental Method" above. Western blot analysis of actin) (internal control group as cytoplasmic protein) and HDAC1 (internal control group as nuclear protein), and primary antibody and secondary antibody used for each protein are shown in Table 9 below.

D. Performance of proteins associated with β-catenin degradation:

This experiment was carried out generally in the manner described in "B, Performance of proteins related to cell cycle regulation" of Example 3 above, except that the primary antibody shown in Table 10 below and the second time were used. Antibodies were used to perform Western blot analysis of PP2A, PP2B, and β-TrCP/HOS.

E, the effect of proteasome inhibitor on the expression of β-catenin:

HA22T cells were divided into 7 groups, including 1 normal control, 1 blank control group, and 5 experimental groups (ie, proteasome inhibitor groups 1, 2, 3, 4, and 5). ). Each group of HA22T cells were cultured in DMEM containing 10% FBS, 1.5 g/L NaHCO ₃ , 0.1 mM non-essential amino acid, 2.0 mM branic acid, 100 U/mL penicillin G, 100 μg/mL. A petri dish (10 cm in diameter) of streptomycin and 10 g/L phenol red) was cultured in an incubator (37 ° C, 5% CO ₂ ). When the cell density reached approximately 80% confluence, the medium was replaced with fresh DMEM (with 1% CCS, 1.5 g/L NaHCO ₃ , 0.2 mM non-essential amino acid, 2.0 mM glutamic acid, 100 U/mL) Penicillin G and 100 μg/mL streptomycin) and continued to culture for 3 hours. Next, an appropriate amount of proteasome inhibitor was added to the proteasome inhibitor groups 1, 2, 3, 4, and 5 such that they each had a proteasome with a final concentration of 1, 2, 3, 4, 5 μg/mL. Inhibitors were continued and cultured for 1 hour. Thereafter, an appropriate amount of the stock solution obtained according to the above Example 1 was separately added to the normal control group and the five experimental groups (i.e., the proteasome inhibitor groups 1, 2, 3, 4, and 5), so that they were all An extract of eagle peony with a final concentration of 100 μg/mL.

After the cells of each group were cultured in an incubator (37 ° C, 5% CO ₂ ) for 24 hours, the total cells were subjected to the method described in the first item "Preparation of total protein samples" in "General Experimental Methods" above. Extraction of protein. The obtained total protein sample was subjected to the Western ink of β-catenin and α-tubulin (as an internal control group) according to the method described in the second item "Western Ink Point Analysis" of the "General Experimental Method" above. Point analysis, and the primary and secondary antibodies used for these two proteins are shown in Table 8 above.

F, co-immunoprecipitation assay:

The HA22T cells were divided into 3 groups, including 1 control group, and 2 experimental groups (ie, Eagle non-parking groups 1 and 2). Each group of HA22T cells were cultured in DMEM containing 10% FBS, 1.5 g/L NaHCO ₃ , 0.1 mM non-essential amino acid, 2.0 mM branic acid, 100 U/mL penicillin G, 100 μg/mL. A petri dish (10 cm in diameter) of streptomycin and 10 g/L phenol red) was cultured in an incubator (37 ° C, 5% CO ₂ ). When the cell density reached approximately 80% confluence, the medium was replaced with fresh DMEM (with 1% CCS, 1.5 g/L NaHCO ₃ , 0.2 mM non-essential amino acid, 2.0 mM glutamic acid, 100 U/mL) Penicillin G and 100 μg/mL streptomycin) and continued to culture for 3 hours. Next, an appropriate amount of proteasome inhibitor was added to each group so that they all had a proteasome inhibitor at a final concentration of 5 μg/mL, and the culture was continued for 1 hour. Thereafter, an appropriate amount of the stock solution obtained according to the above Example 1 was added to the Eagle Bup Group 1 and 2 so that they each had a final concentration of 100 μg/mL and 250 μg/mL of the extract of the Eagle Things.

Each group of cells was cultured in an incubator (37 ° C, 5% CO ₂ ) for 24 hours, and was generally carried out in accordance with the method described in the first item "Preparation of total protein samples" in "General Experimental Methods" above. The total cell protein sample was prepared by using 200 μL of 1.5 mM MgCl ₂ , 1% Triton X-100, 50 mM HEPES (pH 7.6), 1 mM EDTA, 150 mM NaCl, 10% glycerol, A solution of 1 mM NaVO ₃ , 10 mM NaF, 10 mM β-glycerol phosphate, and 5 mg/mL protease inhibitor was used as a dissolution buffer.

The total protein sample (100 μg) extracted from each group was separately added to 4 microcentrifuge tubes, and a dissolution buffer containing no protease inhibitor was added to a total volume of 500 μL. Thereafter, 7 μL of protein G PLUS-agarose (Santa Cruz, USA, Cat. No. sc-2002) was added and the oscillator was placed at 4 ° C (VORTEX- The rotation was carried out for 1 hour in 2/G-560, Scientific Industries, NY, USA, and then centrifuged at 1,300 rpm for 30 seconds at 4 °C. Next, remove the supernatant and 2.5 μL of mouse anti-β-catenin (E-5) monoclonal antibody (Mouse anti β-catenin (E-5) monoclonal antibody] (Santa Cruz, USA, Cat. No . sc-7963), rabbit anti-GSK-3β (H-76) polyclonal antibody [rabbit anti GSK-3β(H-76) polyclonal antibody] (Santa Cruz, USA, Cat. No. sc-9166), rabbit resistance β-TrCP/HOS (H-300) polyclonal antibody [rabbit anti β-TrCP/HOS (H-309) polyclonal antibody] (Santa Cruz, USA, Cat. No. sc-15354) and mouse anti-APC (F -3) monoclonal antibody [mouse anti APC (F-3) monoclonal antibody] (Santa Cruz, USA, Cat. No. sc-9998) was added to the four microcentrifuge tubes, respectively, and then oscillated at 4 °C. Rotate overnight in the device. Thereafter, 20 μL of protein G PLUS-Sepharose was added and rotated in a shaker at 4 ° C for 2 hours, followed by centrifugation at 1,300 rpm for 30 seconds at 4 ° C. After the supernatant was removed, 1 mL of a lysis buffer containing no protease inhibitor was added to wash the precipitate, followed by centrifugation at 1,300 rpm for 30 seconds at 4 °C. After the supernatant was removed, the cleaning treatment was repeated 5 times. Thereafter, 5 μL of loading dye was added and allowed to act at 100 ° C for 5 minutes, followed by the method described in the second item "Western Ink Point Analysis" of the "General Experimental Methods" above. Western blot analysis of lignin, GSK-3β, β-TrCP/HOS, APC, PP2A, and ubiquitin, and primary and secondary antibodies used for each protein are shown in Table 11 below.

G, β-catenin target genes (β-catenin target genes) performance:

This experiment was carried out generally in the manner described in "B, Performance of proteins related to cell cycle regulation" of Example 3 above, except that the primary antibody shown in Table 12 below was used and twice Antibodies were used for Western blot analysis of TBX3, IL-8, cyclin D1, and c-Myc.

result: A. Performance of proteins associated with the β-catenin signaling pathway:

Figure 19 is a Western blot analysis showing beta-catenin, GSK-3β, APC, MMP-2, and MMP-9 measured by HA22T cells treated with different concentrations of extracts from Eagle. The performance of the protein. As can be seen from Fig. 19, compared with the control group, the expression levels of GSK-3β and APC protein in the Eagle non-poll group 1 to 5 were all increased, while β-catenin, MMP-2 and MMP-9 proteins were observed. The amount of performance is declining, and at the same time it will become more obvious with the increase of the concentration of the extract of the eagle. The results of this experiment show that the extract of eagle peony has a positive dose-effect in increasing the expression of GSK-3β and APC protein and reducing the expression of β-catenin, MMP-9 and MMP-2 protein. relationship. Applicant's preliminary inference: The extract of the eagle peony of the present invention can significantly enhance the expression of intracellular GSK-3β and APC proteins, and significantly reduce the expression of β-catenin, MMP-9 and MMP-2 proteins in cells. In turn, the activation of the β-catenin signaling pathway is inhibited, thereby reducing the metastatic ability of the HA22T cells.

B, immunofluorescence staining analysis:

Figure 20 is a diagram showing immunofluorescence staining analysis showing the expression of β-catenin observed by HA22T cells after treatment with different concentrations of extracts of eagle. As can be seen from Fig. 20, compared with the control group, the expression levels of β-catenin in the eagle-free group 1 to 5 in the nucleus were all decreased, and the concentration of the extract with the eagle was not observed. Increased and more obvious. Therefore, the Applicant reasoned that the extract of the eagle peony of the present invention can significantly reduce the expression of the β-catenin protein in the nucleus, thereby inhibiting the activation of the β-catenin signaling pathway, thereby reducing the transfer ability of the HA22T cell. .

C, β-catenin performance in the nucleus and cytoplasm:

21A and 21B are Western blot analysis charts showing the expression of β-catenin measured in the nucleus and cytoplasm of HA22T cells after treatment with extracts of different concentrations of eagle. As can be seen from Fig. 21A and Fig. 21B, compared with the control group, the expression levels of β-catenin of the eagle-free group 1 to 5 in the nucleus and cytoplasm are both decreased, and the eagle does not move. The concentration of the extract is more pronounced and more pronounced. The results of this experiment show that the eagle-free extract has a positive dose-effect relationship in reducing the expression of β-catenin in the nucleus and cytoplasm. Applicant's preliminary inference: The extract of the eagle peony of the present invention can significantly reduce the expression of β-catenin protein in the nucleus and cytoplasm, thereby inhibiting the activation of the β-catenin signaling pathway, thereby reducing the metastasis of HA22T cells. ability.

D. The performance of proteins involved in the degradation of β-catenin:

Figure 22 is a Western blot analysis showing the performance of PP2A, PP2B and β-TrCP/HOS proteins measured by HA22T cells after treatment with different concentrations of Eagle Bing. As can be seen from Fig. 22, compared with the control group, the expression levels of PP2A and β-TrCP/HOS protein in the Eagle non-poll group 1 to 5 all increased, while the performance of the PP2B protein decreased. At the same time, it will become more obvious with the increase of the concentration of the extract of the eagle. The results of this experiment show that the extract of eagle peony has a positive dose-effect relationship in increasing the amount of PP2A and β-TrCP/HOS protein and reducing the amount of PP2B protein. Applicant's preliminary inference: The extract of the eagle peony of the present invention can significantly enhance the performance of intracellular PP2A and β-TrCP/HOS proteins and significantly reduce the performance of PP2B protein, thereby promoting the phosphorylation and degradation of β-catenin. Thereby, the activation of the β-catenin signaling pathway is inhibited, thereby reducing the metastatic ability of the HA22T cells.

E, the effect of proteasome inhibitors on the expression of β-catenin:

Figure 23 is a Western blot analysis showing the performance of β-catenin measured by HA22T cells after treatment with different concentrations of proteasome inhibitor and the same concentration of eagle-free extract. As can be seen from Fig. 23, compared with the blank control group, the expression level of β-catenin protein in the normal control group was significantly reduced. The results of this experiment showed that the extract of the eagle berber significantly reduced the β-linkage. The performance of the prime. In addition, compared with the normal control group, the expression levels of β-catenin protein in the proteasome inhibitor group 1 to 5 all increased, and the concentration of the proteasome inhibitor increased as the concentration of the proteasome inhibitor increased. obvious. Applicant's preliminary inference: The extract of the eagle peony of the present invention promotes the degradation of β-catenin by the proteasome, thereby inhibiting the activation of the β-catenin signaling pathway, thereby reducing the metastatic ability of HA22T cells.

F, immunoprecipitation analysis:

Figure 24 shows GSH-3β and PP2A, β-catenin, APC, β-TrCP/HOS or HA22T cells measured after treatment with the same concentration of proteasome inhibitor and different concentrations of eagle-free extract. The results of immunoprecipitation analysis of ubiquitin. As can be seen from Fig. 24, in association with the control group, the association between GSK-3β and PP2A, β-catenin, APC, β-TrCP/HOS or ubiquitin was observed in comparison with the control group. Both of them show an increase in the situation, and at the same time, they become more obvious as the concentration of the extract of the eagle is increased.

Figure 25 shows β-catenin and PP2A, GSK-3β, APC, β-TrCP/HOS or HA22T cells measured after treatment with the same concentration of proteasome inhibitor and different concentrations of eagle-free extract. The results of immunoprecipitation analysis of ubiquitin. As can be seen from Fig. 25, compared with the control group, the eagle-free group 1 and 2 exhibited in the association between β-catenin and PP2A, GSK-3β, APC, β-TrCP/HOS or ubiquitin. The increased situation will be more pronounced as the concentration of the eagle-free extract increases.

Figure 26 shows APC and GSK-3β, β-catenin, PP2A, β-TrCP/HOS or as measured by HA22T cells treated with the same concentration of proteasome inhibitor and different concentrations of eagle-free extract. The results of immunoprecipitation analysis of ubiquitin. As can be seen from Fig. 26, compared with the control group, the eagle-free group 1 and 2 exhibited an association between APC and GSK-3β, β-catenin, PP2A, β-TrCP/HOS or ubiquitin. The increased situation will be more pronounced as the concentration of the eagle-free extract increases.

Figure 27 shows β-TrCP/HOS and GSK-3β, β-catenin, PP2A, APC or as measured by HA22T cells treated with the same concentration of proteasome inhibitor and different concentrations of extract of eagle The results of immunoprecipitation analysis of ubiquitin. As can be seen from Fig. 27, compared with the control group, the eagle-free group 1 and 2 exhibited an association between β-TrCP/HOS and GSK-3β, β-catenin, PP2A, APC or ubiquitin. The increased situation is more pronounced as the concentration of the extract of the eagle is increased.

Applicant's preliminary inference: The extract of the eagle peony of the present invention promotes the association between β-catenin, β-TrCP/HOS, GSK-3β, APC and PP2A, and promotes the association of β-catenin and ubiquitin. In combination, the β-catenin is degraded by the proteasome, thereby inhibiting the activation of the β-catenin signaling pathway, thereby reducing the metastatic ability of the HA22T cells.

G, β-catenin target gene performance:

Figure 28 is a Western blot analysis showing the gene products of TBX3, IL-8, cyclin D1 and c-Myc measured by HA22T cells after treatment with different concentrations of extracts of Eagle berber. Performance situation. As can be seen from Fig. 28, compared with the control group, the expression levels of the gene products TBX3, IL-8, cyclin D1, and c-Myc of the β-catenin target in the Eagle non-poll group 1 to 5 decreased. The situation will become more apparent as the concentration of the extract of the eagle is increased. The results of this experiment show that the extract of eagle peony has a positive dose-effect relationship in reducing the expression of TBX3, IL-8, cyclin D1 and c-Myc protein. Applicant's preliminary inference: The extract of the eagle peony of the present invention reduces the expression of β-catenin, thereby significantly reducing the gene products of the β-catenin target genes TBX3, IL-8, cyclin D1 and c-Myc. The performance, thereby achieving the effect of reducing the proliferation and metastasis of HA22T cells.

All of the patents and documents cited in this specification are hereby incorporated by reference in their entirety. In the event of a conflict, the detailed description of the case (including definitions) will prevail.

While the invention has been described with respect to the specific embodiments of the invention, it will be understood that many modifications and changes can be made without departing from the scope and spirit of the invention. It is therefore intended that the invention be limited only by the scope of the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing the elution pattern obtained by HPLC using the extract of the eagle-free pouch produced in Example 1 of the present invention, wherein the peaks a1 to a13 indicate 13 major occurrences during the residence period of 0 to 60 minutes. Ingredient

Figure 2 shows an elution pattern obtained by HPLC using a methanol extract of eagle-free leaves prepared according to the method disclosed in TT Thuy et al. (1999) (supra), wherein the peaks b1 to b21 Indicates the 21 major components that occur during the 0 to 60 minute stay period;

Figure 3A and Figure 3B show the percentage of cell viability measured by HA22T cells and Chang-Liver cells after treatment with different concentrations of eagle-free extracts (data are expressed as mean ± SEM), where The group indicates cells that have not been treated with the extract of the eagle pebbles; the eagle pebbles 1 to 5 represent cells treated with extracts of 50, 100, 150, 200, and 250 μg/mL of eagle pebbles; ",""**" and "***" indicate p < 0.05, p < 0.01 and p < 0.001, respectively;

Figure 4 is a Western blot analysis showing PCNA, cyclin A, cyclin D1, cyclin E, and cells measured by HA22T cells treated with different concentrations of eagle-free extract. The performance of cyclin B1, p21, p27, p-p53, p53, c-Fos, and c-Myc proteins, wherein the control group represents cells that have not been treated with extracts of eagle-free; and the eagle-free group 1 to 5 Cells that were treated with extracts of 50, 100, 150, 200, and 250 μg/mL of eagle peony, respectively;

Figure 5 is a Western blot analysis showing the performance of PCNA protein measured by Chang-Liver cells treated with different concentrations of eagle-free extracts, with the control group indicating no eagle Extract treated cells; and eagle-free groups 1 to 5 represent cells treated with extracts of 50, 100, 150, 200, and 25 μg/mL of eagle pebbles, respectively;

Figure 6 shows cell cycle changes measured after treatment of HA22T cells with different concentrations of eagle-free extracts, with the control group representing cells treated with extracts without eagle pebbles; eagle-free groups 1 to 5 Cells treated with extracts of 50, 100, 150, 200, and 250 μg/mL of eagle peony, respectively; and "*", "**", and "***" indicate p < 0.05, p < 0.01, respectively. With p <0.001;

Figure 7 shows the percentage of cell proliferation measured by HA22T cells treated with extracts of different concentrations of eagle-free (data are expressed as mean ± SEM), with the control group indicating no extract of eagle Treated cells; Eagle non-parking groups 1 to 5 represent cells treated with extracts of 50, 100, 150, 200, and 250 μg/mL of eagle pebbles; and "**" and "***" respectively p <0.01 and p <0.001;

Figure 8 shows the percentage of apoptosis measured by HA22T cells after treatment with different concentrations of eagle-free extract (data are expressed as mean ± SEM), with the control group indicating no extraction by eagle Treated cells; eagle-free groups 1 to 5 represent cells treated with extracts of 50, 100, 150, 200, and 250 μg/mL of eagle pebbles; and "*" and "***" respectively p < 0.05 and p <0.001;

Figure 9 is a Western blot analysis showing FAS, FAS-L, FADD, TNFα, TNF-R1, and caspase-day measured after treatment of HA22T cells with different concentrations of eagle-free extract. Pro-form of aspartic acid protease-8 and its active form, t-BID, BID and the original form of caspase-3 and two of them The performance of the activated form, in which the control group represents cells that have not been treated with the extract of the eagle-free; and the eagle-free groups 1 to 5 represent the eagle at 50, 100, 150, 200, and 250 μg/mL, respectively. Extract treated cells;

Figure 10 is a JC-1 fluorescence staining analysis showing the results of JC-1 fluorescence staining of HA22T cells treated with extracts of different concentrations of eagle peony, wherein the control group showed no eagle Extract-treated cells; eagle-free groups 1 to 5 represent cells treated with extracts of 50, 100, 150, 200, and 250 μg/mL of eagle pebbles; and cells showing red fluorescence indicating mitochondrial membranes The potential is normal, and the cells show green fluorescence indicating that the mitochondrial membrane potential is depolarized;

Figure 11 is a Western blot analysis showing Bax, Bak, p-Bad, Bad, Bcl-2, Bcl-x _L measured by HA22T cells after treatment with different concentrations of eagle-free extract. , t-BID, BID, cytochrome c, pro-form of caspase-9 and its activation form, pro-form of caspase-3 and its two The activated form and the performance of the AIF protein, wherein the control group represents cells that have not been treated with the extract of the eagle, and the eagle-free groups 1 to 5 represent the eagle after 50, 100, 150, 200, and 250 μg/mL, respectively. Non-parked extract treated cells;

Figure 12 is a Western blot analysis showing p-PI3k, PI3k, p-Akt, Akt, p-Bad, Bad, measured by HA22T cells after treatment with different concentrations of eagle-free extract. The performance of Bcl-2 and Bcl-x _L proteins, in which the control group represents cells that have not been treated with extracts of eagle-free; and the eagle-free groups 1 to 5 represent 50, 100, 150, 200, and 250 μg, respectively. /mL of eagle-free extract treated cells;

Figure 13A and Figure 13B show the percentage of cell movement and the percentage of cell invasion measured by HA22T cells after treatment with different concentrations of eagle-free extract, respectively (data are expressed as mean ± SEM), with the control group indicating Cells not treated with eagle-free extracts; eagle-free groups 1 to 5 represent cells treated with extracts of 50, 100, 150, 200, and 250 μg/mL of eagle pebbles; and “**” And "***" indicate p <0.01 and p <0.001, respectively;

Figure 14 is a Western blot analysis showing FGF2, p-MEK-1/2, MEK1, p-ERK and ERK1 proteins measured by HA22T cells treated with different concentrations of eagle-free extract. The performance of the control group, which indicated that the cells were not treated with the extract of the eagle-free, and the eagle-free groups 1 to 5 showed the extracts of the eagle pebbles after 50, 100, 150, 200, and 250 μg/mL, respectively. Treated cells;

Figure 15 is a Western blot analysis showing tPA, uPA, contact protein-1, MMP-9 and MMP-2 proteins measured by HA22T cells treated with different concentrations of eagle-free extract. Performance, in which the control group represents cells that have not been treated with extracts of eagle-free; and the eagle-free groups 1 to 5 represent extracts of 50, 100, 150, 200, and 250 μg/mL of eagle Cell;

Figure 16 is a Western blot analysis showing PAI-1, TIMP-1, TIMP-2, TIMP-3, and TIMP- measured by HA22T cells treated with different concentrations of Eagle's extract. 4 The performance of the protein, in which the control group represents cells that have not been treated with the extract of the eagle, and the eagle-free groups 1 to 5 represent the eagle at 50, 100, 150, 200, and 250 μg/mL, respectively. Extract treated cells;

Figure 17 is a Western blot analysis showing p-ERK, ERK-1, JNK, p-JNK, p-p38 as measured by HA22T cells treated with different concentrations of extracts from E. The performance of the p38α protein, in which the control group represents cells that have not been treated with the extract of the eagle-free, and the eagle-free groups 1 to 5 represent the eagle at 50, 100, 150, 200, and 250 μg/mL, respectively. Extract treated cells;

Figure 18 is a Western blot analysis showing the expression of NF-κB p65 and p-NF-κB p65 protein measured by HA22T cells treated with different concentrations of eagle-free extract. The group represents cells that have not been treated with the extract of the eagle; and the eagle-free groups 1 to 5 represent cells treated with extracts of 50, 100, 150, 200, and 250 μg/mL of eagle pebbles, respectively;

Figure 19 is a Western blot analysis showing beta-catenin, GSK-3β, APC, MMP-2, and MMP-9 measured by HA22T cells treated with different concentrations of extracts from Eagle. The performance of the protein, in which the control group represents cells that have not been treated with the extract of the eagle, and the eagle-free groups 1 to 5 represent the extraction of 50, 100, 150, 200, and 250 μg/mL, respectively. Treated cells;

Figure 20 is an immunofluorescence staining analysis showing the expression of β-catenin observed in HA22T cells treated with different concentrations of eagle-free extract, wherein the control group showed no eagle Extract-treated cells; eagle-free groups 1 to 5 represent cells treated with extracts of 50, 100, 150, 200, and 250 μg/mL of eagle pebbles; and nuclei stained blue by DAPI, --catenin is stained green by luciferin;

21A and 21B are Western blot analysis charts showing the expression of β-catenin in the nucleus and cytoplasm of HA22T cells treated with different concentrations of extracts of eagle pebbles, respectively. The group represents cells that have not been treated with the extract of the eagle; and the eagle-free groups 1 to 5 represent cells treated with extracts of 50, 100, 150, 200, and 250 μg/mL of eagle pebbles, respectively;

Figure 22 is a Western blot analysis showing the performance of PP2A, PP2B and β-TrCP/HOS proteins measured by HA22T cells treated with different concentrations of extracts from Eagle notch, in which the control group indicated Cells that were not treated with eagle-free extracts; and eagle-free groups 1 to 5 represent cells treated with extracts of 50, 100, 150, 200, and 250 μg/mL of eagle pebbles, respectively;

Figure 23 is a Western blot analysis showing the performance of β-catenin measured by HA22T cells treated with different concentrations of proteasome inhibitor and the same concentration of extract of eagle peony. The control group indicated cells that were not treated with proteasome inhibitors and extracts of eagle peony; the normal control group indicated cells treated with 100 μg/mL of extract of eagle peony; and the proteasome inhibitor groups 1 to 5, respectively Means cells treated with 1, 2, 3, 4, and 5 μg/mL proteasome inhibitors and 100 μg/mL of eagle-free extract;

Figure 24 shows GSH-3β and PP2A, β-catenin, APC, β-TrCP/HOS or HA22T cells measured after treatment with the same concentration of proteasome inhibitor and different concentrations of eagle-free extract. The results of immunoprecipitation analysis of ubiquitin, in which the control group indicated cells treated with extracts without eagle pebbles; and eagle peony groups 1 and 2 indicated treatment with 100 and 250 μg/mL extracts of eagle pebbles, respectively. Cell;

Figure 25 shows β-catenin and PP2A, GSK-3β, APC, β-TrCP/HOS or HA22T cells measured after treatment with the same concentration of proteasome inhibitor and different concentrations of eagle-free extract. The results of immunoprecipitation analysis of ubiquitin, in which the control group indicated cells treated with extracts without eagle pebbles; and eagle peony groups 1 and 2 indicated treatment with 100 and 250 μg/mL extracts of eagle pebbles, respectively. Cell;

Figure 26 shows APC and GSK-3β, β-catenin, PP2A, β-TrCP/HOS or as measured by HA22T cells treated with the same concentration of proteasome inhibitor and different concentrations of eagle-free extract. The results of immunoprecipitation analysis of ubiquitin, in which the control group indicated cells treated with extracts without eagle pebbles; and eagle peony groups 1 and 2 indicated treatment with 100 and 250 μg/mL extracts of eagle pebbles, respectively. Cell;

Figure 27 shows β-TrCP/HOS and GSK-3β, β-catenin, PP2A, APC or as measured by HA22T cells treated with the same concentration of proteasome inhibitor and different concentrations of extract of eagle The results of immunoprecipitation analysis of ubiquitin, in which the control group indicated cells treated with extracts without eagle pebbles; and eagle peony groups 1 and 2 indicated treatment with 100 and 250 μg/mL extracts of eagle pebbles, respectively. Cells;

Figure 28 is a Western blot analysis showing the gene products of TBX3, IL-8, cyclin D1 and c-Myc measured by HA22T cells after treatment with different concentrations of extracts of Eagle berber. Performance, in which the control group represents cells that have not been treated with extracts of eagle-free; and the eagle-free groups 1 to 5 represent extracts of 50, 100, 150, 200, and 250 μg/mL of eagle Cell.

Claims (20)

An extract of Zanthoxylum avicennae (Lam.) DC., which is prepared by a method comprising the steps of: heating a plant material that is not anchored by a eagle to use a first acid Acid treatment, thereby obtaining an acid-treated eagle-free plant material; extracting the acid-treated eagle-free plant material with an alcohol to obtain an alcohol extract; and extracting the alcohol extract with The second acid is mixed to form the eagle-free extract. An extract of eagle peony according to claim 1, wherein in the method, the eagle-free plant material is selected from the group consisting of roots, stems, leaves, and fruits. An extract of eagle peony according to claim 1, wherein in the method, the first acid is selected from the group consisting of vinegar, acetic acid, formic acid, lactic acid, malic acid, oxalic acid, and Citric acid. An extract of eagle peony according to claim 1, wherein in the method, the alcohol is selected from the group consisting of methanol, ethanol, propanol, isopropanol, n-butanol, and Isobutanol. An extract of eagle peony according to claim 1, wherein in the method, the second acid is selected from the group consisting of vinegar, acetic acid, formic acid, lactic acid, malic acid, oxalic acid, and Citric acid. An extract of eagle peony, as in claim 1, wherein in the method, the eagle-free extract is further subjected to a refining treatment using a third acid. An extract of eagle peony according to claim 6 wherein, in the method, the third acid is selected from the group consisting of vinegar, acetic acid, formic acid, lactic acid, malic acid, oxalic acid, and Citric acid. A pharmaceutical composition for inhibiting proliferation of a tumor/cancer cell, which comprises an extract of eagle-free, as claimed in any one of claims 1 to 7. The pharmaceutical composition of claim 8, wherein the tumor/cancer cell is a liver cancer cell. A pharmaceutical composition for inhibiting metastasis of a tumor/cancer cell, which comprises an extract of eagle-free, as claimed in any one of claims 1 to 7. A pharmaceutical composition according to claim 10, wherein the tumor/cancer cell is a liver cancer cell. A pharmaceutical composition for inducing apoptosis of a tumor/cancer cell, which comprises an extract of eagle-free, as claimed in any one of claims 1 to 7. The pharmaceutical composition of claim 12, wherein the tumor/cancer cell is a liver cancer cell. A method for preparing an eagle-free extract comprising: heating an eagle-free plant material with an acid treatment using a first acid, thereby obtaining an acid-treated eagle a plant material; extracting the acid-treated eagle-free plant material with an alcohol to obtain an alcohol extract; and mixing the alcohol extract with a second acid to form the eagle-free extract. The method of claim 14, wherein the eagle-free plant material is selected from the group consisting of roots, stems, leaves, and fruits. The method of claim 14, wherein the first acid is selected from the group consisting of vinegar, acetic acid, formic acid, lactic acid, malic acid, oxalic acid, and citric acid. The method of claim 14, wherein the alcohol is selected from the group consisting of methanol, ethanol, propanol, isopropanol, n-butanol, and isobutanol. The method of claim 14, wherein the second acid is selected from the group consisting of vinegar, acetic acid, formic acid, lactic acid, malic acid, oxalic acid, and citric acid. The method of claim 14, wherein the eagle-free extract is further subjected to a refining treatment using a third acid. The method of claim 19, wherein the third acid is selected from the group consisting of vinegar, acetic acid, formic acid, lactic acid, malic acid, oxalic acid, and citric acid.