CN115109135B - Eupolyphaga Seu Steleophaga protein extract with liver cancer resisting and liver fibrosis inhibiting effects and its application - Google Patents
Eupolyphaga Seu Steleophaga protein extract with liver cancer resisting and liver fibrosis inhibiting effects and its application Download PDFInfo
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- CN115109135B CN115109135B CN202210724643.5A CN202210724643A CN115109135B CN 115109135 B CN115109135 B CN 115109135B CN 202210724643 A CN202210724643 A CN 202210724643A CN 115109135 B CN115109135 B CN 115109135B
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- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
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- Peptides Or Proteins (AREA)
Abstract
The invention discloses an Eupolyphaga Seu Steleophaga protein extract with the effects of resisting liver cancer and inhibiting liver fibrosis, which is prepared by the following steps: pulverizing dried Eupolyphaga Seu Steleophaga into powder, adding into ethanol, reflux extracting, mixing extractive solutions, concentrating under reduced pressure, centrifuging, collecting supernatant, and lyophilizing to obtain Eupolyphaga Seu Steleophaga ethanol extract EE powder; dissolving Eupolyphaga Seu Steleophaga ethanol extract EE powder in Tris-HCl buffer solution, adding ammonium sulfate into the solution to saturated state, stirring, standing, centrifuging, collecting precipitate, redissolving, dialyzing to remove salt, and lyophilizing to obtain Eupolyphaga Seu Steleophaga crude protein sample EEP. Through a large number of experimental screening, the invention separates out an active protein part EEPC and an active protein component PC3-III from a ground beetle protein component system, the active protein part EEPC and the active protein component PC3-III exert the anti-liver cancer effect together by inhibiting the growth of tumor cells, inducing the apoptosis of the tumor cells and inhibiting the migration of the tumor cells, and can delay the progress of liver fibrosis by inhibiting the proliferation of activated HSC cells.
Description
Technical Field
The invention relates to an ground beetle protein extract with the effects of resisting liver cancer and inhibiting liver fibrosis and a preparation method thereof.
Background
The Eupolyphaga Seu Steleophaga is dried female Eupolyphaga Seu Steleophaga (Eupolyphaga SINENSIS WALKER) or Eupolyphaga Seu Steleophaga (Steleophaga plancyi (Boleny)) of Eupolyphaga Seu Steleophaga. Cold in nature, salty in taste, with small toxicity, enters liver meridian, belongs to blood-activating and stasis-resolving herbs, and is a rare animal herb with long history and wide use. The animal medicines are blood and meat products and walking channeling products, and have the activities and curative effects which are difficult to reach by plant medicines and mineral medicines in the aspects of activating blood and dissolving stasis, resolving hard mass and removing stasis, dispelling wind and dredging collaterals, eliminating mass and eliminating stagnation, and the like, so that the animal medicines become important animal medicine resources which are indispensable for preventing and treating diseases by traditional Chinese medicines. At present, the Chinese medicinal composition can be used as a Chinese medicine clinic, and also has certain edible health care value. There are many medical books which are recorded in association with them, such as Shen nong Ben Cao Jing, jin Kui Lloyd, ben Cao gang mu. The ground beetle has high medicinal value and wide application. Besides being used as the main component of various Chinese patent medicines, the Chinese medicine composition is also widely used for treating traumatic injury, various liver diseases, post-fracture blood stasis and the like clinically. For example, it is formulated into decoction for removing blood stasis, da Huang Zhe Chong Wan, etc.
Amino acids, polypeptides, proteins are the main components ubiquitous in animal drugs, most of which are also proven by modern researches to be specific components of animal drugs. Ground beetle is an excellent source of high-quality protein, and various active polypeptide and protein components have been separated from ground beetle, and the components have strong anti-tumor, anti-coagulation, anti-thrombosis, anti-oxidation, blood fat reducing, liver protecting and immunoregulation activities. Even so, there are few active ingredients that are currently well-identified, and the mechanism of action of each of the ingredients contained therein is not well-defined. The research difficulty of the ground beetle polypeptide and protein components is separation, purification and structural identification. The combination of the excessively low separation rate and the diversification degree of the components seriously lacks the structural and functional knowledge of the macromolecular components of the ground beetles, so that the functional ground beetle macromolecular components and the action mechanism of pharmacological and medicinal effects are required to be further discovered.
The liver is a metabolic center and is involved in many important physiological functions. Liver injury is caused by hepatopathy, and can be developed into severe diseases such as liver cirrhosis, liver fibrosis, liver cancer, etc., which are the most common pathological process of various liver diseases, and cause harm to human health. Ground beetles are widely used for treating various liver organ diseases from ancient times, whether as single medicines or as meridian formulas. For example, blood stasis decoction in the meridian prescription is used for treating chronic hepatitis and hepatic fibrosis; the pill can be used for treating viral hepatitis and liver cirrhosis.
The invention further researches the medicinal components related to liver diseases (mainly including liver cancer and liver fibrosis) in the traditional Chinese medicine ground beetles, and utilizes a cell evaluation technology to research the parts and components with the effect of inhibiting liver abnormal cell proliferation in the ground beetles, so as to develop new active proteins.
Disclosure of Invention
The invention aims to: the invention aims to separate and purify active protein components in ground beetles through a system, and research the anti-liver cancer and liver fibrosis inhibiting activities of each separated product by adopting an in vitro cell evaluation technology, so as to gradually focus effective parts and components. According to the invention, the salted out ground beetle crude protein product is firstly divided into three different protein parts of EEPA, EEPB and EEPC according to different molecular weights, and the protein part EEPC with the largest molecular weight shows the highest activity of inhibiting the proliferation of liver abnormal cells. Then, through DEAE anion chromatography and Sephadex G-100 gel chromatography, a purified protein PC3-III with a molecular weight of about 10kDa is separated from EEPC, and in vitro experiments prove that PC3-III has the activities of resisting liver cancer and inhibiting liver fibrosis in vitro, which are equivalent to EEPC, plays the role of resisting liver cancer by inhibiting the growth of tumor cells, inducing the apoptosis of tumor cells and inhibiting the migration of tumor cells, and can inhibit the proliferation of activated HSC cells so as to delay the progress of liver fibrosis.
The technical scheme is as follows: in order to achieve the above purpose, the invention adopts the following technical scheme:
An woodlouse protein extract with the effects of resisting liver cancer and inhibiting liver fibrosis is characterized in that the woodlouse protein extract is prepared by the following method:
(1) Pulverizing dried Eupolyphaga Seu Steleophaga into powder, adding into ethanol, reflux extracting, mixing extractive solutions, concentrating under reduced pressure, centrifuging, collecting supernatant, lyophilizing to obtain Eupolyphaga Seu Steleophaga ethanol extract EE powder, and storing at-20deg.C in dark place before use;
(2) Dissolving the ground beetle ethanol extract EE powder prepared in the step (1) in Tris-HCl buffer solution, adding ammonium sulfate into the solution to be saturated, stirring, standing, centrifuging, collecting precipitate, redissolving, dialyzing to remove salt, and freeze-drying to obtain a ground beetle crude protein sample EEP.
As a preferred scheme, the ground beetle protein extract with the effects of resisting liver cancer and inhibiting liver fibrosis is prepared by the following method:
(1) Pulverizing dried ground beetle into powder, adding 20-30% ethanol with the volume of 5-20 times, reflux-extracting at 60-75deg.C for 1-3 times, 1-2 hr each time, discarding residues, mixing the extractive solutions, concentrating under reduced pressure, centrifuging, collecting supernatant, lyophilizing to obtain ground beetle ethanol extract EE powder, and storing at-20deg.C in dark condition before use;
(2) Dissolving the ground beetle ethanol extract EE powder prepared in the step (1) in 30mM Tris-HCl buffer solution, adding ammonium sulfate into the solution to be saturated, stirring, standing, centrifuging, collecting precipitate, redissolving, dialyzing for 36h to remove salt, and freeze-drying to obtain a ground beetle crude protein sample EEP.
An Eupolyphaga Seu Steleophaga protein extract with liver cancer resisting and liver fibrosis inhibiting effects is prepared by the following method:
(1) Pulverizing dried Eupolyphaga Seu Steleophaga into powder, adding into ethanol, reflux extracting, mixing extractive solutions, concentrating under reduced pressure, centrifuging, collecting supernatant, lyophilizing to obtain Eupolyphaga Seu Steleophaga ethanol extract EE powder, and storing at-20deg.C in dark place before use;
(2) Dissolving the ground beetle ethanol extract EE powder prepared in the step (1) in Tris-HCl buffer solution, adding ammonium sulfate into the solution to be saturated, stirring, standing, centrifuging, collecting precipitate, redissolving, dialyzing to remove salt, and freeze-drying to obtain a ground beetle crude protein sample EEP;
(3) The EEP of crude protein ground beetle crude protein sample is redissolved in Tris-HCl buffer solution, and sequentially centrifuged for 2 times by using an ultrafiltration centrifuge tube with the molecular weight cutoff of 10kDa and 3kD, EEP solution is divided into 3 protein part cutoff solutions according to the molecular weight, and the cutoff solutions are collected and freeze-dried and named EEPA with the molecular weight less than 3k, EEPB with the molecular weight of 3-10 k and EEPC with the molecular weight more than 10 k.
As a preferred scheme, the ground beetle protein extract with the effects of resisting liver cancer and inhibiting liver fibrosis is prepared by the following method:
(1) Pulverizing dried ground beetle into powder, adding 20-30% ethanol with the volume of 5-20 times, reflux-extracting at 60-75deg.C for 1-3 times, 1-2 hr each time, mixing the extractive solutions, concentrating under reduced pressure, centrifuging, collecting supernatant, lyophilizing to obtain ground beetle ethanol extract EE powder, and storing at-20deg.C in dark condition before use;
(2) Dissolving the ground beetle ethanol extract EE powder prepared in the step (1) in 30mM Tris-HCl buffer solution, adding ammonium sulfate into the solution to be saturated, stirring, standing, centrifuging, collecting precipitate, redissolving, dialyzing for 36h to remove salt, and freeze-drying to obtain a ground beetle crude protein sample EEP;
(3) The EEP powder of the ground beetle crude protein sample is redissolved in 30mM Tris-HCl buffer solution, and sequentially subjected to centrifugal separation by using an ultrafiltration centrifuge tube with the molecular weight cutoff of 10kDa and 3kD for 2 times, the EEP solution is divided into 3 protein part cutoff solutions according to the molecular weight, and the cutoff solutions are collected and freeze-dried and named as EEPA with the molecular weight less than 3k, EEPB with the molecular weight of 3-10 k and EEPC with the molecular weight more than 10 k.
An Eupolyphaga Seu Steleophaga protein extract with liver cancer resisting and liver fibrosis inhibiting effects is prepared by the following method:
(1) Pulverizing dried ground beetle into powder, adding 20-30% ethanol with the volume of 5-20 times, reflux-extracting at 60-75deg.C for 1-3 times, 1-2 hr each time, mixing the extractive solutions, concentrating under reduced pressure, centrifuging, collecting supernatant, lyophilizing to obtain ground beetle ethanol extract EE powder, and storing at-20deg.C in dark condition before use;
(2) Dissolving the ground beetle ethanol extract EE powder prepared in the step (1) in 30mM Tris-HCl buffer solution, adding ammonium sulfate into the solution to be saturated, stirring, standing, centrifuging, collecting precipitate, redissolving, dialyzing for 36h to remove salt, and freeze-drying to obtain a ground beetle crude protein sample EEP;
(3) Re-dissolving ground beetle crude protein sample EEP powder in 30mM Tris-HCl buffer solution, sequentially performing centrifugal separation for 2 times by using an ultrafiltration centrifuge tube with the molecular weight cutoff of 10kDa and 3kD, dividing the EEP solution into 3 protein part cutoff solutions according to the molecular weight, collecting and freeze-drying the cutoff solutions, wherein the cutoff solutions are named EEPA with the molecular weight less than 3k, EEPB with the molecular weight of 3-10 k and EEPC with the molecular weight more than 10 k;
(4) Dissolving EEPC with molecular weight more than 10k in 30Mm Tris-HCl buffer (pH 8.0) in the step (3), loading on DEAE-52Cellulos anion column chromatography, performing gradient elution with NaCl with concentration of 0-1.0M, detecting the absorbance value of eluent fraction at flow rate of 1ml/min and 280nm, drawing a DEAE elution pattern of EEPC, and collecting a main fraction PC3 separated by EEPC on DEAE column chromatography; further purifying fraction PC3 on Sephadex G-100 chromatographic column, eluting with 30Mm Tris-HCl buffer (pH 8.0), measuring the absorbance value of the eluate fraction at flow rate of 0.75ml/min and 280nm, drawing Sephadex G-100 elution pattern of main fraction PC3 of EEPC, integrating the peak area, calculating the duty ratio of each peak, and collecting main product PC3-III according to the relative content.
An Eupolyphaga Seu Steleophaga protein with effects of resisting liver cancer and inhibiting liver fibrosis comprises amino acid sequence QYSINFISARCNGDSCVCTFR.
Namely: gln-Tyr-Ser-Ile-Asn-Phe-Ile-Ser-Ala-Arg-Cys-Asn-Gly-Asp-Se-rCys-Val-Cys-Thr-Phe-Arg.
The ground beetle protein extract or ground beetle protein can be used for preparing medicines or health products for resisting liver cancer and inhibiting liver fibrosis.
The invention can prepare the ground beetle protein extract or ground beetle protein and pharmaceutically acceptable carriers into tablets, capsules, granules and pills.
When the invention is prepared into tablets, carrier lactose or corn starch is added into ground beetle protein extract or ground beetle protein, and a lubricant magnesium stearate is added when needed, and the mixture is uniformly mixed and then pressed into tablets.
When the capsule is prepared, the ground beetle protein extract or ground beetle protein and carrier lactose or corn starch are mixed uniformly, granulated and then encapsulated to prepare the capsule.
When the invention is prepared into granules, the ground beetle protein extract or ground beetle protein and diluent lactose or corn starch are uniformly mixed, granulated, dried and prepared into granules.
The beneficial effects are that: compared with the prior art, the ground beetle protein extract or ground beetle protein provided by the invention has the following advantages:
The invention separates active protein part EEPC and active protein component PC3-III through a large number of experimental screening, and in vitro cell experiments show that the active protein part EEPC and the active protein component PC3-III have the activities of resisting liver cancer and relieving liver fibrosis progress in vitro, exert anti-liver cancer efficacy jointly by inhibiting tumor cell growth, inducing tumor cell apoptosis and inhibiting tumor cell migration, and delay liver fibrosis progress by inhibiting activated HSC cell proliferation.
Drawings
FIG. 1A is a graph of cytotoxicity of Eupolyphaga Seu Steleophaga isolate (EE, EEP, EEPA, EEPB, EEPC) against human normal stem cell strain L-02; B. c, D, E, F is a histogram of cytotoxicity of EE, EEP, EEPA, EEPB, EEPC against a liver tumor cell line SMMC-7721, respectively.
FIG. 2A is a graph of cytotoxicity of Eupolyphaga isolation product (EE, EEP, EEPA, EEPB, EEPC) on normal HSC; B. c, D, E, F are bar graphs of EE, EEP, EEPA, EEPB, EEPC cytotoxicity of activated HSCs, respectively.
FIG. 3 shows the purification EEPC using DEAE and Sephadex G-100 (FIG. A: EEPC anion exchange chromatography elution profile; B: EEPC quantitative analysis of peak areas of 6 fractions by DEAE elution; C: G-100 gel filtration chromatography elution profile of PC3 fraction; D: quantitative analysis of peak areas of 3 fractions by Sephadex G-100 elution of PC 3).
FIG. 4 shows in vitro antitumor effects (A: EEPC and cytotoxicity of PC3-III against L-02; B: EEPC cytotoxicity against SMMC-7721; C: cytotoxicity of PC3-III against SMMC-7721; D: EEPC inducing morphological changes in apoptosis of SMMC-7721; E: PC3-III inducing morphological changes in apoptosis of SMMC-7721).
FIG. 5 shows the results of flow cytometry for detecting apoptosis of SMMC-7721 (A: EEPC for inducing apoptosis of SMMC-7721; B: PC3-III for inducing apoptosis of SMMC-7721; C: EEPC for quantitative analysis of apoptosis induction; D: PC3-III for quantitative analysis of apoptosis induction).
FIG. 6 shows SMMC-7721 cell migration after EEPC (A) and PC3-III (B) have been treated for various periods of time.
FIG. 7 is a graph showing the effect of EEPC and PC3-III on HSC cell proliferation (A: cytotoxicity of 2 samples on normal HSC; B: cytotoxicity of EEPC on activated HSC; C: cytotoxicity of PC3-III on activated HSC).
FIG. 8 is a characterization of PC3-III protein (A: SDS-PAGE analysis; B: RP-HPLC analysis).
FIG. 9 is a MALDI-TOF-MS plot of a PC3-III sample.
FIG. 10 is a secondary mass spectrum of the main ion of the QE sequence analysis of the PC3-III protein after LC-MS/MS detection.
Detailed Description
The invention will be better understood from the following examples. However, it will be readily understood by those skilled in the art that the specific material ratios, process conditions and results thereof described in the examples are illustrative of the present invention and should not be construed as limiting the invention described in detail in the claims.
Example 1
11. Experimental materials
Dried body raw materials of the ground beetles (Eupolyphaga SINENSIS WALKER) female worms are purchased from a pharmacy in a traditional Chinese medical university affiliated with Zhoushan China. Normal human liver cell strain L-02, hepatic stellate cell HSC-T6, liver cancer cell strain SMMC-7721 (China academy of sciences typical culture Collection Committee cell Bank); fetal bovine serum FBS (ABW brand); TGF-beta (Perprotech, USA); hoechst 33342 fluorescent dye (Biyun biotechnology Co., ltd.); BCA kit, annexin V-FITC apoptosis kit (institute of bioengineering, made by nanjing); preformed glue (Nanjing Philit Biotechnology Co., ltd.); cellulose DEAE-52 (Beijing Soy Bao technology Co., ltd.); sephadex G-100 (Shanghai derived leaf Biotechnology Co., ltd.); chromatographic pure acetonitrile, methanol (Tedia company). Other commercially available chemicals and reagents were all of analytical grade.
2. Extraction and separation of ground beetle active crude protein
(1) Pulverizing dried Eupolyphaga Seu Steleophaga into powder, adding into 20% ethanol with volume of 10 times, reflux extracting at 60-75deg.C for 2 times each for 1 hr, discarding residues, mixing extractive solutions, concentrating under reduced pressure at 60deg.C to specific gravity of about 1.10, centrifuging at 6000rpm for 15min, collecting supernatant, lyophilizing to obtain Eupolyphaga Seu Steleophaga ethanol extract EE powder, and storing at-20deg.C in dark place before use.
(2) 65.19G of ground beetle ethanol extract EE powder is dissolved in 30mM Tris-HCl buffer solution, ammonium sulfate is added into the solution to be saturated (100% (w/v)), the solution is stirred for 1h, then the solution is kept still for 3h, and is centrifuged at 10000rpm for 20min, the precipitate is collected and redissolved and then dialyzed for 36h to remove salt, and 27.59g of ground beetle crude protein sample EEP is obtained through freeze drying.
(3) The ground beetle crude protein EEP powder is redissolved in 30mM Tris-HCl buffer solution, and is sequentially subjected to separation operation by centrifugation (4000 g,15 min) for 2 times by using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10kDa and 3kD, EEP solution is divided into 3 protein part retentate according to the molecular weight, and the retentate is collected and freeze-dried and named EEPA (less than 3 k), EEPB (3-10 k) and EEPC (more than 10 k).
(4) Separation and purification of Eupolyphaga Seu Steleophaga active protein site EEPC
First, carrying out DEAE-52Cellulos anion column chromatography (diameter 1.6cm, height 20 cm) on EEPC parts, dissolving EEPC in 30Mm Tris-HCl buffer (pH 8.0) for sample injection, carrying out gradient elution with different concentrations of (0-1.0M) NaCl, detecting the absorbance value of eluent fractions at a flow rate of 1ml/min and 280nm, drawing a EEPC DEAE elution map, integrating peak areas, and calculating the duty ratio of each peak. As shown in fig. 3A, 3B.
Collecting EEPC main fraction PC3 separated by DEAE column chromatography, further purifying by feeding into Sephadex G-100 chromatographic column (diameter 1.6cm, height 40 cm), eluting with 30MmTris-HCl buffer (pH 8.0), measuring the absorbance value of the eluate fraction at flow rate of 0.75ml/min and 280nm, drawing Sephadex G-100 elution map of main fraction PC3 of EEPC, integrating peak area, calculating peak ratio, separating to obtain 3 components, respectively named PC3-I, PC-II and PC3-III, collecting the abundant components PC3-III (figures 3C and 3D), and freeze drying to obtain protein PC3-III.
(5) Chemical characterization of Eupolyphaga Seu Steleophaga active protein PC3-III
The BCA kit was used for content determination of PC 3-III. Samples PC3-III were analysed by SDS-PAGE using a pre-gel with a separation gel concentration of 12%.
HPLC analysis was performed on PC3-III using a Waters e2695 high performance liquid chromatograph and a Hanbang MEGRESS C (5 μm,4.6 mm. Times.250 mm) column. The eluting solvent systems were water-TFA (solvent A;100:0.1, v/v) and acetonitrile (solvent B). Gradient eluting solvent B from 0% to 20% at flow rate of 1mL/min for 0-30 min; column temperature: 30 ℃; detection wavelength: 280nm.
And (3) identifying the PC3-III by adopting a MALDI-TOF-MS technology to obtain a characteristic map of the PC 3-III.
The identification of PC3-III by LC-MS/MS (QExactive, thermo Fisher Co.) was performed by Shanghai department of life analysis service. PC3-III was digested with endoprotease, followed by LCMS (nanoLC-QE) analysis of the digested sample. And finally, analyzing LCMS data by using mass spectrum matching software such as MASCOT and the like. After reduction and alkylation treatment, PC3-III is added with Trypsin (mass ratio 1:50), and enzymolysis is carried out for 20 hours at 37 ℃. Desalting the enzymolysis product, freeze-drying, re-dissolving in 0.1% FA solution, and preserving at-20deg.C for use. Solution A was an aqueous solution of 0.1% formic acid, and solution B was an aqueous solution of 0.1% formic acid in acetonitrile (84% acetonitrile). After equilibration of the column with 95% solution a, the sample was loaded from the autosampler to the Trap column. The mass-to-charge ratio of the polypeptide and fragments of the polypeptide was collected as follows: 20 fragment patterns were acquired after each full scan. Mass spectrometry the original file was retrieved with mascot2.2 software to the corresponding database (uniprot _ Corydiidae _385_20220425).
The protein content of PC3-III is 96.48% + -2.64% measured by BCA kit method; the results of RP-HPLC (FIG. 8B) showed a symmetrical peak with a purity of more than 97%, indicating that PC3-III is a homogeneous protein; PC3-III appeared as a single band on SDS-PAGE (FIG. 8A), with a molecular weight of about 10kDa; FIG. 9 is a MALDI-TOF-MS of a PC3-III sample, qualitatively analyzing PC3-III using LCMS (nanoLC-QE) technique, resulting in a peptide sequence of QYSINFISARCNGDSCVCTFR. Fig. 10 is a secondary mass spectrum thereof.
EXAMPLE 2 pharmacological Activity screening assay
1. Effect of ground beetle protein isolate on SMMC-7721 cell proliferation
(1) Experimental method
L-02 cells and SMMC-7721 cells were inoculated in DMEM medium containing 10% FBS and 1% chlortetracycline mixed solution, and cultured in a constant temperature incubator at 37℃with 5% CO 2.
Cells L-02 in logarithmic growth phase were inoculated in 96-well plates (100. Mu.L, 6X 104/mL), after 24 hours of inoculation, the medium was discarded, the cells were further cultured for 24 hours with different concentrations of the ground beetle extract (EE, EEP, EEPA, EEPB, EEPC) medium solution prepared in example 1, MTT solution (5 mg/mL, 20. Mu.L/well) was added, and the culture was continued in the dark for 4 hours. Removing supernatant, adding 150 μl DMSO into each well, shaking for 10min to dissolve the crystal, reading light absorption value (absorbance, A) at 490nm of enzyme-labeled instrument, and examining whether Eupolyphaga Seu Steleophaga extract affects proliferation and survival of normal hepatocytes L-02 in a specified concentration range (0-2.0 mg/ml). Cell viability was calculated from the following formula: cell viability= (experimental group a)/(control group a) ×100%. IC 50 values were derived from the response curve of cell viability versus drug concentration.
The effect of varying concentrations (0-2.0 mg/mL) of the ground beetle product (EE, EEP, EEPA, EEPB, EEPC) of example 1 on the viability of liver tumor cells SMMC-7721 was determined by the MTT method described above by inoculating the liver tumor cells SMMC-7721 in the logarithmic growth phase in 96 well plates (100. Mu.L, 6X 104/mL).
(2) Experimental results
Effect of ground beetle protein isolate on SMMC-7721 cell proliferation
The present invention observed that within the concentration range of 2mg/ml, all 5 ground beetle extracts were non-toxic to normal hepatocytes (fig. 1A), so the concentrations of the subsequent experiments were within this range. As can be seen from FIGS. 1B-F, both Eupolyphaga Seu Steleophaga extracts EE, EEP, EEPA, EEPB and EEPC inhibit the growth of liver tumor cells SMMC-7721 in a concentration-dependent manner over the range of action concentrations. EE. The IC 50 values of EEP, EEPA, EEPB and EEPC for SMMC-7721 were >2mg/mL, 1mg/mL, >1mg/mL, 0.93mg/mL, 0.67mg/mL, respectively. The IC 50 value of EE is larger than EEP, which indicates that the EEP of the crude protein sample obtained by salting out separation is the main anti-tumor active ingredient. And 3 samples obtained by ultrafiltration separation of EEP have stronger SMMC-7721 proliferation resistance of EEPC with the largest molecular weight, which shows that EEPC has potential liver cancer resistance in vitro.
2. Effect of Eupolyphaga Seu Steleophaga protein isolate on activated HSC-T6 cell proliferation
(1) Experimental method
HSC-T6 cells were inoculated into DMEM medium containing 10% FBS and 1% green-streptomycin mixed solution, and cultured in a 37℃incubator containing 5% CO 2. The effect of different concentrations (0-2.0 mg/mL) of Eupolyphaga Seu Steleophaga extract (EE, EEP, EEPA, EEPB, EEPC) prepared in example 1 on the viability of normal HSC cells was determined by inoculating logarithmic growth phase cells HSC-T6 in 96 well plates (100. Mu.L, 6X 10 4/mL) using the MTT method described above.
Next, HSC-T6 cells in the logarithmic phase were harvested and plated in 96-well plates (100. Mu.L, 6X 10 4/mL). Each cell hole is set as a blank group, a model group and each ground beetle sample experimental group, and each group is provided with 6 compound holes. Except for the blank, each group of cells was incubated with the cytokine TFG- β (10 ng/mL) for 24h for activating HSC-T6 cells, after which the cells were discarded the supernatant, the blank was incubated with the addition of medium, and the experimental group was incubated with the addition of Eupolyphaga Seu Steleophaga extract (EE, EEP, EEPA, EEPB, EEPC) in a nontoxic concentration range for 24h. Cell viability of each group was determined using the MTT method.
2. Experimental results
The activation and proliferation of rat hepatic stellate cells are currently considered to be a key process leading to liver fibrosis, and under normal conditions, hepatic stellate cells are in a quiescent state, do not express alpha smooth muscle actin (alpha-SMA), and have low proliferation activity. Following HSC activation, the formation of liver fibrosis is involved by proliferation and secretion of extracellular matrix. Continuous activation of hepatic stellate cells is a key element in the development and progression of hepatic fibrosis. It can thus be summarized that liver fibrosis is the result of Hepatic Stellate Cell (HSC) activation followed by deposition of excess extracellular matrix (ECM). Thus, the activation of HSC-T6 cells with cytokines was chosen to investigate the anti-hepatic fibrosis effect of 5 Eupolyphaga extract samples in vitro. The non-activated HSC cells were normal hepatic stellate cells used as a comparison. The present invention observed that within the concentration range of 2mg/ml, all 5 ground beetle extracts were non-toxic to normal HSC cells (fig. 2A), so the subsequent experimental concentrations were within this range. As shown in FIGS. 2B-F, the model control group significantly stimulated the growth of HSC-T6 cells after the addition of TGF-beta, compared to the placebo group, indicating the success of modeling. The 5 ground beetle extract sample solutions all inhibited activated HSC cell viability after administration compared to the model control group. Wherein, the EE and EEP samples are in the investigation concentration range (2 mg/ml), and only EEP sample can obviously inhibit the growth of activated HSC cells and has certain concentration dependence. Samples EEPA, EEPB and EEPC were within the range of action concentrations (1 mg/ml), each of which significantly inhibited the growth of activated HSC cells, the most active of which was the EEPC sample. It follows that EEP and EEPC delay the formation of liver fibrosis, and EEPC is the main active site of EEP to inhibit the progress of liver fibrosis.
3. Evaluation of in vitro anti-tumor and liver fibrosis Activity of Eupolyphaga Seu Steleophaga protein isolate PC3-III
(1) Experimental method
PC3-III prepared in sample example 1 was incubated with L-02 and SMMC-7721 cells, respectively, in vitro at a concentration, the effect of PC3-III samples on SMMC-7721 proliferation in vitro (MTT method) was studied as described in 1 above, and the results were compared with EEPC.
The apoptosis of SMMC-7721 cells after EEPC and PC3-III treatment was observed using Hoechst 33342 staining. SMMC-7712 was inoculated into 24 well plates (500. Mu.L, 16X 10 4/mL) and 24h post-harvest media was dosed (EEPC with PC 3-III). The medium was discarded 48h after dosing, and 200. Mu.L of 4% paraformaldehyde was added for 15min. After the incubation, the paraformaldehyde was discarded, 200. Mu.L of Hoechst 33342 dye was added at a concentration of 5. Mu.g/mL, incubated at 37℃for 30min in the absence of light, and the cell morphology was observed under a fluorescence microscope.
Flow cytometry was used to detect EEPC and PC3-III post-treatment SMMC-7721 apoptosis. SMMC-7712 was inoculated into 6-well plates (2 mL, 25X 10 4/mL) and run according to the instructions for the apoptosis detection kit.
The effect of EEPC and PC3-III on migration of SMMC-7721 cells was examined using a scratch test, SMMC-7712 was inoculated into 12 well plates (1 mL, 1.5X10- 5/mL), scored with a gun after growing, then dosed and photographed under an inverted microscope at 0h,24h,48h, and finally the data were processed using imageJ software.
Samples PC3-III were incubated with normal HSC cells and TGF-beta activated HSC-T6 at a concentration in vitro, respectively, and the effect of PC3-III samples on proliferation in vitro (MTT method) following activation of HSC-T6 was studied as described in 2 above and the results were compared with EEPC samples.
(2) Experimental results
(2.1) PC3-III is a main component of EEPC, and in order to investigate whether PC3-III has an antiproliferative effect on liver cancer cells, MTT assay measures the cell viability of PCSMMC-7721 after PC3-III treatment. The present invention observed that PC3-III was non-toxic to normal hepatocytes L-02 over a concentration range of 2mg/ml (FIG. 4A). As shown in FIG. 4B, C, both sample EEPC and PC3-III significantly inhibited proliferation of tumor cell SMMC-7721 at different concentrations (IC 50: 0.67mg/ml and 0.88mg/ml, respectively). PC3-III is demonstrated to have potential anti-liver cancer effects in vitro and is the main active site of EEPC.
As shown in FIG. 4 at D, E, nuclei of the control group were stained uniformly, nuclei of the group to which the sample solutions of different concentrations were administered were condensed, chromatin was condensed, and apoptotic body formation and fluorescence intensity were enhanced. EEPC and PC3-III were shown to induce apoptosis in SMMC-7721 cells. And meanwhile, annexin V/PI double-staining is used for detecting the apoptosis condition. As shown in FIG. 5, the apoptosis ratio of SMMC-7721 cells was significantly increased and concentration-dependent after treatment of SMMC-7721 cells at different concentrations EEPC and PC3-III compared to the blank. EEPC and PC3-III are shown to exert antitumor activity by inhibiting malignant proliferation by inducing apoptosis of SMMC-7721 cells.
(2.2) EEPC and PC3-III inhibit SMMC-7721 cell migration
Tumor growth and metastasis are the causes of death in most cancer patients. Tumor metastasis is a complex process, and tumor cell migration and invasion are two key steps, so the effect of EEPC and PC3-III on SMMC-7721 migration was observed using scratch test. The cells of the blank group migrated to fill the scratch area at 48h, while EEPC (FIG. 6A) and PC3-III (FIG. 6B) all significantly inhibited SMMC-7721 migration at concentrations of 0.25, 0.5 and 1 mg/ml. It was demonstrated that EEPC and PC3-III also exert an anti-hepatoma effect by inhibiting tumor cell migration.
(2.3) EEPC and PC3-III and Effect on proliferation of activated HSC-T6 cells
The invention also examined the effect of PC3-III on proliferation of activated HSC-T6 cells in vitro. First, the range of non-toxic concentrations for normal HSC cells was determined, and the present invention observed that PC3-III was non-toxic to normal HSC cells within the 2mg/ml concentration range (FIG. 7A), so that the subsequent experimental concentrations were within this range. As shown in FIG. 7B, C, EEPC and PC3-III can obviously inhibit the proliferation of TGF-beta activated HSC-T6 cells at different concentrations, indicating that the effects of the HSC-T6 can delay the progress of hepatic fibrosis. The activity of the extract is slightly stronger than that of PC3-III, and the extract has dose dependency, which shows that the main component of EEPC for delaying the liver fibrosis progress is PC3-III.
The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the present invention and to implement it, but not limit the scope of the present invention, and all equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of the present invention.
Sequence listing
<110> University of Nanjing traditional Chinese medicine
<120> An Eupolyphaga Seu Steleophaga protein extract with liver cancer resisting and hepatic fibrosis inhibiting effects, and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> PRT
<213> Abedus herberti
<400> 1
Gln Tyr Ser Ile Asn Phe Ile Ser Ala Arg Cys Asn Gly Asp Ser Cys
1 5 10 15
Val Cys Thr Phe Arg
20
Claims (4)
1. An Eupolyphaga Seu Steleophaga protein with effects of resisting liver cancer and inhibiting liver fibrosis is characterized in that its amino acid sequence is QYSINFISARCNGDSCVCTFR.
2. The method for preparing ground beetle protein having anti-liver cancer and liver fibrosis inhibiting effects as claimed in claim 1, comprising the steps of:
(1) Pulverizing dried ground beetles into powder, adding 20-30% ethanol with the volume of 5-20 times, reflux-extracting at 60-75 ℃ for 1-3 times, each time for 1-2 hours, discarding residues, combining the extracting solutions, concentrating under reduced pressure, centrifuging, collecting supernatant, freeze-drying to obtain ground beetle ethanol extract EE powder, and storing at-20 ℃ in dark condition before use;
(2) Dissolving the ground beetle ethanol extract EE powder prepared in the step (1) in 30 mM Tris-HCl buffer solution, adding ammonium sulfate into the solution to be saturated, stirring, standing, centrifuging, collecting precipitate, redissolving, dialyzing for 36h to remove salt, and freeze-drying to obtain a ground beetle crude protein sample EEP;
(3) Re-dissolving ground beetle crude protein sample EEP powder in 30 mM Tris-HCl buffer solution, sequentially performing centrifugal separation for 2 times by using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10kDa and 3kD, dividing the EEP solution into 3 protein part cutoff solutions according to the molecular weight, collecting and freeze-drying the cutoff solutions, wherein the cutoff solutions are named as EEPA with the molecular weight less than 3k, EEPB with the molecular weight of 3-10 k and EEPC with the molecular weight more than 10 k;
(4) Dissolving EEPC with molecular weight more than 10 k in the step (3) in 30 Mm Tris-HCl buffer solution with pH of 8.0, loading on DEAE-52 Cellulos anion column chromatography, performing gradient elution with NaCl with concentration of 0-1.0M, detecting the absorbance value of eluent fraction at flow rate of 1 ml/min and 280 nm, drawing a DEAE elution map of EEPC, and collecting a main fraction PC3 separated by EEPC on DEAE column chromatography; further purifying fraction PC3 on Sephadex G-100 chromatographic column, eluting with 30 Mm Tris-HCl buffer solution with pH of 8.0, detecting absorbance value of the eluate fraction at flow rate of 0.75 ml/min and 280 nm, drawing Sephadex G-100 elution pattern of EEPC main fraction PC3, and collecting main product PC 3-III.
3. The use of the ground beetle protein of claim 1 in the preparation of medicaments for treating liver cancer and inhibiting liver fibrosis.
4. Use according to claim 3, wherein the ground beetle protein and a pharmaceutically acceptable carrier are formulated as a tablet, capsule, granule or pill.
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| CN106755256A (en) * | 2016-12-30 | 2017-05-31 | 潍坊医学院 | A kind of preparation method of ground bettle polypeptide, the ground bettle polypeptide for preparing and its application |
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