CN115109135A

CN115109135A - Eupolyphaga sinensis protein extract with effects of resisting liver cancer and inhibiting hepatic fibrosis and application thereof

Info

Publication number: CN115109135A
Application number: CN202210724643.5A
Authority: CN
Inventors: 王令充; 谢佳雨; 刘涛; 虞晓弦; 嵇晶; 程建明; 狄留庆
Original assignee: Nanjing University of Chinese Medicine
Current assignee: Nanjing University of Chinese Medicine
Priority date: 2022-06-23
Filing date: 2022-06-23
Publication date: 2022-09-27
Anticipated expiration: 2042-06-23
Also published as: CN115109135B

Abstract

The invention discloses a ground beetle protein extract with the efficacies of resisting liver cancer and inhibiting hepatic fibrosis, and a preparation method thereof comprises 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 freeze drying 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 saturation state, stirring, standing, centrifuging, collecting precipitate, re-dissolving, dialyzing to remove salt, and freeze drying to obtain Eupolyphaga Seu Steleophaga crude protein sample EEP. According to the invention, through a large number of experimental screenings, the ground beetle protein component system is deeply researched, an active protein part EEPC and an active protein component PC3-III are separated, the active protein part EEPC and the active protein component PC3-III jointly play the anti-liver cancer drug effect by inhibiting the growth of tumor cells, inducing the apoptosis of the tumor cells and inhibiting the migration of the tumor cells, and the process of hepatic fibrosis can be delayed by inhibiting the proliferation of activated HSC cells.

Description

Eupolyphaga sinensis protein extract with effects of resisting liver cancer and inhibiting hepatic fibrosis and application thereof

Technical Field

The invention relates to a ground beetle protein extract with the efficacies of resisting liver cancer and inhibiting liver fibrosis and a preparation method thereof.

Background

The Eupolyphaga Seu Steleophaga is female dry body of Eupolyphaga Seu Steleophaga (Eupolyphaga sinensis Walker) or Ji Eupolyphaga Seu Steleophaga (Boleny) belonging to family Trionychidae. Is cold in nature, salty in taste, slightly toxic and enters liver meridian, belongs to a medicine for promoting blood circulation and removing blood stasis, and is a famous and precious animal medicine with long history and wide use. Animal drugs have the theories of blood and meat products and walking and scurrying substances, have activities and curative effects which are difficult to reach by plant drugs and mineral drugs in the aspects of promoting blood circulation to remove blood stasis, attacking hard masses and breaking masses, searching wind and dredging collaterals, eliminating diseases and dissipating stagnation and the like, and become important animal drug resources which are indispensable to the prevention and treatment of diseases by traditional Chinese medicines. At present, the health food can be used in clinical traditional Chinese medicine and has certain edible health care value. There are many medical records recorded by the book of classical generations, such as Shen nong Ben Cao Jing, jin Kui Yao L ü e, Ben Cao gang mu, etc. The ground beetle has high medicinal value and wide application. Besides being used as the main components of various Chinese patent medicines, the Chinese patent medicine is also widely used for treating traumatic injuries, various liver diseases, blood stasis after fracture and postpartum, and the like in clinic. Such as blood stasis-removing decoction, rhubarb and sting pill, etc.

Amino acids, polypeptides and proteins are the main components commonly existing in animal medicines, and most of the amino acids, polypeptides and proteins are proved to be specific components of the animal medicines by modern researches. The ground beetle is an excellent source of high-quality protein, and various active polypeptides and protein components are separated from the ground beetle at present, and the components have strong anti-tumor, anticoagulation, anti-thrombosis, anti-oxidation, blood fat reduction, liver protection and immunoregulation activities. Even so, the number of active ingredients that have been identified clearly is not so large, and the mechanism of action of each ingredient contained therein is not clearly understood. The difficulty of the research on the polypeptide and protein components of the ground beetle is separation and purification and structural identification. The combination of the low separation rate and the diversity degree of the components seriously lacks the understanding on the structure and the function of the large molecular components of the ground beetles, so that the functional large molecular components of the ground beetles and the action mechanism of pharmacological efficacy are further discovered.

The liver is a metabolic center and is involved in many important physiological functions. Liver damage caused by the change of the liver cell disease can be developed into serious diseases such as liver cirrhosis, hepatic fibrosis and liver cancer as the most common pathological process of various liver diseases, and the liver damage can cause harm to the health of human bodies. From ancient times to present, Eupolyphaga seu Steleophaga, whether used as single medicine or as a meridian, is widely used for treating various liver diseases. For example, decoction for removing blood stasis in channels and collaterals, can be used for treating chronic hepatitis and hepatic fibrosis; the pill can be used for treating viral hepatitis and liver cirrhosis.

The invention deeply researches the pharmacodynamic components related to the treatment of liver diseases (mainly including liver cancer and liver fibrosis) in the Chinese medicinal ground beetle, utilizes the cell evaluation technology to research the parts and components of the ground beetle with the function of inhibiting the proliferation of abnormal liver cells and develops new active protein.

Disclosure of Invention

The invention aims to: the invention aims to systematically separate and purify active protein components in ground beetles, and adopts in vitro cell evaluation technology to research the liver cancer resistance and hepatic fibrosis inhibition activity of each separated product, thereby gradually focusing on effective parts and components. The method firstly divides the salted-out ground beetle crude protein product 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 abnormal liver cell proliferation. Then, a purified protein PC3-III with the molecular weight of about 10kDa is separated from EEPC through DEAE anion chromatography and Sephadex G-100 gel chromatography, and in vitro experiments prove that the PC3-III has the in vitro anti-liver cancer and liver fibrosis inhibition activities equivalent to those of EEPC, plays the anti-liver cancer drug effect by inhibiting the growth of tumor cells, inducing the apoptosis of the tumor cells and inhibiting the migration of the 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 technical scheme that:

a ground beetle protein extract with the efficacies of resisting liver cancer and inhibiting liver fibrosis is characterized in that the ground beetle protein extract is prepared by the following method:

(1) pulverizing dried Eupolyphaga Seu Steleophaga, adding into ethanol, reflux extracting, mixing extractive solutions, concentrating under reduced pressure, centrifuging, collecting supernatant, and freeze drying to obtain Eupolyphaga Seu Steleophaga ethanol extract EE powder, which is stored at-20 deg.C in dark place before use;

(2) dissolving the Eupolyphaga sinensis Walker ethanol extract EE powder prepared in the step (1) in Tris-HCl buffer solution, adding ammonium sulfate into the solution to saturation state, stirring, standing, centrifuging, collecting precipitate, dialyzing after redissolving to remove salt, and freeze-drying to obtain Eupolyphaga sinensis Walker crude protein sample EEP.

Preferably, the woodlouse protein extract with the efficacies of resisting liver cancer and inhibiting liver fibrosis is prepared by the following method:

(1) pulverizing dry Eupolyphaga Seu Steleophaga, adding 5-20 times volume of 20-30% ethanol, reflux-extracting at 60-75 deg.C for 1-3 times (1-2 hr each time), discarding residue, mixing extractive solutions, concentrating under reduced pressure, centrifuging, collecting supernatant, freeze-drying to obtain Eupolyphaga Seu Steleophaga ethanol extract EE powder, and storing at-20 deg.C in dark place before use;

(2) dissolving the Eupolyphaga Seu Steleophaga ethanol extract EE powder prepared in step (1) in 30mM Tris-HCl buffer solution, adding ammonium sulfate into the solution to saturation state, stirring, standing, centrifuging, collecting precipitate, re-dissolving, dialyzing for 36h to remove salt, and freeze-drying to obtain Eupolyphaga Seu Steleophaga crude protein sample EEP.

A ground beetle protein extract with the efficacies of resisting liver cancer and inhibiting liver fibrosis 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, and freeze drying to obtain Eupolyphaga Seu Steleophaga ethanol extract EE powder, which is stored at-20 deg.C in dark place before use;

(2) dissolving the Eupolyphaga sinensis Walker ethanol extract EE powder prepared in the step (1) in Tris-HCl buffer solution, adding ammonium sulfate into the solution to a saturated state, stirring, standing, centrifuging, collecting precipitate, dialyzing after redissolving to remove salt, and freeze-drying to obtain a Eupolyphaga sinensis Walker crude protein sample EEP;

(3) re-dissolving a crude protein sample EEP of the ground beetle crude protein in Tris-HCl buffer solution, continuously centrifuging for 2 times by using ultrafiltration centrifuge tubes with the molecular weight cut-off of 10kDa and 3kD, dividing the EEP solution into 3 protein part cut-off solutions according to the molecular weight, collecting, freezing and drying, and naming 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.

(1) pulverizing dry Eupolyphaga Seu Steleophaga, adding 5-20 times volume of 20-30% ethanol, reflux-extracting at 60-75 deg.C for 1-3 times (1-2 hr each time), mixing extractive solutions, concentrating under reduced pressure, centrifuging, collecting supernatant, freeze-drying to obtain EE powder, and storing at-20 deg.C in dark place before use;

(2) dissolving the Eupolyphaga sinensis Walker ethanol extract EE powder prepared in the step (1) in 30mM Tris-HCl buffer solution, adding ammonium sulfate into the solution to a saturated state, stirring, standing, centrifuging, collecting precipitate, redissolving, dialyzing for 36h to remove salt, and freeze-drying to obtain a Eupolyphaga sinensis Walker crude protein sample EEP;

(3) re-dissolving EEP powder of a ground beetle crude protein sample in 30mM Tris-HCl buffer solution, continuously performing centrifugal separation for 2 times by using ultrafiltration centrifuge tubes with molecular weight cut-off of 10kDa and 3kD, dividing EEP solution into 3 protein part cut-off solutions according to molecular weight, collecting, freezing and drying, and naming EEPA with molecular weight less than 3k, EEPB with molecular weight 3-10 k and EEPC with molecular weight more than 10 k.

(1) pulverizing dry Eupolyphaga Seu Steleophaga, adding 5-20 times volume of 20-30% ethanol, reflux extracting at 60-75 deg.C for 1-3 times, each for 1-2 hr, mixing extractive solutions, concentrating under reduced pressure, centrifuging, collecting supernatant, freeze drying to obtain EE powder, and storing at-20 deg.C in dark place before use;

(3) redissolving EEP powder of a ground beetle crude protein sample in 30mM Tris-HCl buffer solution, sequentially using an ultrafiltration centrifugal tube with the molecular weight cutoff of 10kDa and 3kD for continuous centrifugal separation for 2 times, dividing the EEP solution into 3 protein part trapped solutions according to the molecular weight, collecting, freezing and drying, and naming 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 the molecular weight of more than 10k in the step (3) in 30Mm Tris-HCl buffer solution (pH8.0), performing DEAE-52Cellulos anion column chromatography, performing gradient elution by using NaCl with the concentration of 0-1.0M, detecting the absorbance value of eluent fraction at the flow rate of 1ml/min at 280nm, drawing a DEAE elution map of the EEPC, and collecting main fraction PC3 of the EEPC separated on the DEAE column chromatography; introducing fraction PC3 into Sephadex G-100 chromatographic column for further purification, eluting with 30Mm Tris-HCl buffer solution (pH8.0), detecting absorbance value of eluate fraction at flow rate of 0.75ml/min and 280nm, drawing Sephadex G-100 elution pattern of main fraction PC3 of EEPC, integrating peak areas, calculating peak ratio, and collecting main product PC3-III according to relative content.

A Eupolyphaga Seu Steleophaga protein with effects of resisting hepatocarcinoma and inhibiting hepatic 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 the 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 the ground beetle protein and a pharmaceutically acceptable carrier into tablets, capsules, granules and pills.

When the Chinese medicinal composition is prepared into tablets, lactose or corn starch serving as a carrier is added into a 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 tableted to prepare the tablets.

When the preparation is made into capsules, the ground beetle protein extract or the ground beetle protein and the carrier lactose or the corn starch are mixed evenly, granulated and then encapsulated to prepare the capsules.

When the invention is prepared into granules, the ground beetle protein extract or the ground beetle protein is evenly mixed with the diluent lactose or the corn starch, and the granules are prepared after the granulation and the drying.

Has the advantages that: compared with the prior art, the ground beetle protein extract or the ground beetle protein provided by the invention has the following advantages:

the invention separates the active protein part EEPC and the active protein component PC3-III through a large amount of experimental screening and deep research on a ground beetle protein component system, 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 in vitro and relieving the liver fibrosis process, the active protein part EEPC and the active protein component PC3-III jointly play the anti-liver cancer drug effect by inhibiting the growth of tumor cells, inducing the apoptosis of the tumor cells and inhibiting the migration of the tumor cells, and the process of liver fibrosis can be delayed by inhibiting the proliferation of activated HSC cells.

Drawings

FIG. 1A is the cytotoxicity profile of the isolated product of Eupolyphaga Seu Steleophaga (EE, EEP, EEPA, EEPB, EEPC) against human normal stem cell line L-02; B. c, D, E, F are the cytotoxicity histograms of EE, EEP, EEPA, EEPB and EEPC on the hepatoma cell strain SMMC-7721.

FIG. 2A is a graph showing cytotoxicity of isolated products of Eupolyphaga Seu Steleophaga (EE, EEP, EEPA, EEPB, EEPC) against normal HSC; B. c, D, E, F are histograms of the cytotoxicity of EE, EEP, EEPA, EEPB, EEPC, respectively, on activated HSC.

FIG. 3 shows the EEPC purification using DEAE and Sephadex G-100 (in the figure, A: EEPC anion exchange chromatography elution curve; B: quantitative analysis of 6-component peak area obtained by elution of EEPC with DEAE; C: elution curve of PC3 component by G-100 gel filtration chromatography; D: quantitative analysis of 3-component peak area obtained by elution of PC3 with Sephadex G-100).

FIG. 4 shows the in vitro antitumor effect (A: cytotoxicity of EEPC and PC3-III on L-02, B: cytotoxicity of EEPC on SMMC-7721, C: cytotoxicity of PC3-III on SMMC-7721, D: morphological change of EEPC induced apoptosis of SMMC-7721, and E: morphological change of PC3-III induced apoptosis of SMMC-7721).

FIG. 5 shows flow cytometry for detecting SMMC-7721 apoptosis (A: EEPC induces SMMC-7721 apoptosis; B: PC3-III induces SMMC-7721 apoptosis; C: EEPC induces apoptosis quantification; D: PC3-III induces apoptosis quantification).

FIG. 6 shows migration of SMMC-7721 cells after EEPC (A) and PC3-III (B) treatments at different times.

FIG. 7 is a graph of the effect of EEPC and PC3-III on HSC cell proliferation (A: cytotoxicity of 2 samples on normal HSC; B: cytotoxicity of EEPC on post-activated HSC; C: cytotoxicity of PC3-III on post-activated HSC).

FIG. 8 shows the characterization of the PC3-III protein (A: SDS-PAGE analysis; B: RP-HPLC analysis).

FIG. 9 is a MALDI-TOF-MS chart of a PC3-III sample.

FIG. 10 is a secondary mass spectrum of main ions of the PC3-III protein after LC-MS/MS detection by QE sequence analysis.

Detailed Description

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions and results thereof described in the examples are illustrative only and should not be taken as limiting the invention as detailed in the claims.

Example 1

11. Experimental Material

Raw materials of dried female beetles (Eupolyphaga sinensis Walker) were purchased from pharmacy of Zhoushan Hospital, Shanghai, university of medicine. Normal human liver cell line L-02, hepatic stellate cell HSC-T6, liver cancer cell line SMMC-7721 (cell bank of China academy of sciences type culture Collection); fetal bovine serum FBS (ABW brand); TGF-. beta.s (Perprotech, USA); hoechst 33342 fluorescent dye (bi yun tian biotechnology limited); BCA kit, Annexin V-FITC apoptosis kit (Nanjing institute of bioengineering); pre-made glue (Nanjing Philid Biotechnology, Inc.); cellulose DEAE-52 (Beijing Sorleuba technologies, Inc.); sephadex G-100 (Shanghai-derived leaf Biotech Co., Ltd.); chromatographically pure acetonitrile, methanol (Tedia). Other commercially available chemicals and reagents were of analytical grade.

2. Extraction and separation of ground beetle active crude protein

(1) Pulverizing dried Eupolyphaga Seu Steleophaga into powder, adding 10 times volume of 20% ethanol, reflux extracting at 60-75 deg.C for 2 times, 1 hr each time, discarding residue, mixing extractive solutions, concentrating at 60 deg.C under reduced pressure to specific gravity of about 1.10, centrifuging at 6000rpm for 15min, collecting supernatant, lyophilizing to obtain EE powder, and storing at-20 deg.C in dark place before use.

(2) 65.19g of ethanol extract EE powder of the ground beetle is dissolved in 30mM Tris-HCl buffer solution, ammonium sulfate is added into the solution to be in a saturated (100% (w/v)) state, the solution is stirred for 1h, then is kept stand for 3h, is centrifuged at 10000rpm for 20min, is collected, is redissolved and is dialyzed for 36h to remove salt, and is frozen and dried to obtain 27.59g of crude ground beetle protein sample EEP.

(3) Redissolving crude ground beetle protein EEP powder in 30mM Tris-HCl buffer solution, continuously performing 2 times of centrifugation (4000g, 15min) separation operations by using ultrafiltration centrifuge tubes with the molecular weight cutoff of 10kDa and 3kD, dividing the EEP solution into 3 protein part trapped fluids according to the molecular weight, collecting and freeze-drying, and naming the solution as EEPA (< 3k), EEPB (3-10 k) and EEPC (> 10 k).

(4) Separation and purification of ground beetle active protein part EEPC

Firstly, DEAE-52Cellulos anion column chromatography (diameter is 1.6cm, height is 20cm) is carried out on EEPC part, EEPC is dissolved in 30Mm Tris-HCl buffer solution (pH8.0) for sample injection, gradient elution is carried out by NaCl with different concentrations (0-1.0M), the flow rate is 1ml/min, the absorbance value of eluent fraction is detected at 280nm, DEAE elution pattern of EEPC is drawn, peak area is integrated, and the ratio of each peak is calculated. As shown in fig. 3A, 3B.

Collecting main fraction PC3 separated from EEPC by DEAE column chromatography, introducing Sephadex G-100 chromatographic column (diameter 1.6cm, height 40cm), further purifying, eluting with 30MmTris-HCl buffer solution (pH8.0), flowing at 0.75ml/min, detecting absorbance value of eluate fraction at 280nm, drawing Sephadex G-100 elution pattern of main fraction PC3 of EEPC, integrating peak areas, calculating peak ratio, separating to obtain 3 components, respectively naming the components as PC3-I, PC3-II and PC3-III, collecting abundance components PC3-III (FIGS. 3C and 3D), and freeze drying to obtain protein PC 3-III.

(5) Chemical characterization of Eupolyphaga Seu Steleophaga active protein PC3-III

PC3-III was assayed using the BCA kit. The sample PC3-III was subjected to SDS-PAGE analysis using a pre-prepared gel with a separation gel concentration of 12%.

HPLC analysis of PC3-III was performed using a Waters e2695 high performance liquid chromatograph and a Hanbang MEGRESS C18(5 μm, 4.6 mm. times.250 mm) column. The elution solvent systems were water-TFA (solvent A; 100:0.1, v/v) and acetonitrile (solvent B). Performing gradient elution on the solvent B from 0% to 20% for 0-30min at a flow rate of 1 mL/min; column temperature: 30 ℃; detection wavelength: 280 nm.

And identifying the PC3-III by using a MALDI-TOF-MS technology to obtain a characteristic map of the PC 3-III.

The identification of PC3-III by LC-MS/MS (QOxctive, Thermo Fisher Co., Ltd.) was performed by the New Life analysis service department of Shanghai, science. PC3-III was digested with endoprotease, and the digested sample was analyzed by LCMSMS (nanolC-QE). And finally, analyzing the LCMSMS data by using mass spectrum matching software such as MASCOT and the like. After reduction and alkylation treatment of PC3-III, Trypsin (mass ratio of 1:50) is added, and enzymolysis is carried out for 20 hours at 37 ℃. Desalting the enzymolysis product, freeze-drying, and redissolving in 0.1% FA solution, and storing at-20 deg.C for use. Solution A was a 0.1% formic acid aqueous solution, and solution B was a 0.1% formic acid aqueous acetonitrile solution (84% acetonitrile). After the column was equilibrated with 95% solution A, a sample was applied to the Trap column by an autosampler. 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 test raw files the corresponding database (uniprot _ cordidiae _385_20220425) was retrieved with mascot2.2 software.

The protein content of the PC3-III is 96.48% + -2.64% by a BCA kit method; the results of RP-HPLC (FIG. 8B) showed a symmetrical peak with a purity of over 97%, indicating that PC3-III is a homogeneous protein; PC3-III showed a single band in SDS-PAGE (FIG. 8A) with a molecular weight of about 10 kDa; FIG. 9 is a MALDI-TOF-MS diagram of a PC3-III sample, and a peptide fragment sequence obtained by qualitative analysis of PC3-III by LCMSMS (nano LC-QE) technology is QYSINFISARCNGDSCVCTFR. FIG. 10 is a secondary mass spectrum thereof.

Example 2 pharmacological Activity screening experiment

1. Effects of Eupolyphaga Seu Steleophaga protein isolate on proliferation of SMMC-7721 cell

(1) Experimental methods

L-02 cells and SMMC-7721 cells were inoculated in DMEM medium containing 10% FBS and 1% penicillin-streptomycin mixed solution, and cultured in a 37 ℃ incubator containing 5% CO 2.

Cells in logarithmic growth phase L-02 were seeded in 96-well plates (100. mu.L, 6X104 cells/mL) and 24h after seeding, the medium was discarded and the cells were added to different cellsThe culture medium solution of Eupolyphaga Seu Steleophaga extract (EE, EEP, EEPA, EEPB, EEPC) prepared in example 1 was cultured for another 24h, MTT solution (5mg/mL, 20. mu.L/well) was added, and the culture was continued for 4h in the dark. Discarding the supernatant, adding 150 μ L DMSO into each well, shaking for 10min to dissolve the crystal, reading absorbance (absorbance, A) at 490nm of microplate reader, and examining whether the Eupolyphaga Seu Steleophaga extract affects the proliferation and survival of normal hepatocyte L-02 in a specified concentration range (0-2.0 mg/ml). Cell viability was calculated by the following formula: cell viability ═ (experimental group a)/(control group a) × 100%. IC (integrated circuit) ₅₀ Values were derived from the response curve of cell viability versus drug concentration.

The effect of different concentrations (0-2.0mg/mL) of the ground beetle products (EE, EEP, EEPA, EEPB, EEPC) of example 1 on the viability of the liver tumor cells SMMC-7721 was determined by the MTT method described above by inoculating the liver tumor cells SMMC-7721 in a 96-well plate (100. mu.L, 6X104 cells/mL) in the logarithmic growth phase.

(2) Results of the experiment

Effects of Eupolyphaga Seu Steleophaga protein isolate on SMMC-7721 cell proliferation

It was observed that 5 Eupolyphaga Seu Steleophaga extracts were non-toxic to normal liver cells within the concentration range of 2mg/ml (FIG. 1A), and therefore the concentrations of subsequent experiments were within this range. As can be seen from B-F in FIG. 1, in the concentration range of action, the Eupolyphaga Seu Steleophaga extracts EE, EEP, EEPA, EEPB and EEPC all inhibited the growth of liver tumor cell SMMC-7721 in a concentration-dependent manner. EE. IC of EEP, EEPA, EEPB and EEPC to SMMC-7721 ₅₀ Respectively has a value of>2mg/ml、1mg/ml、>1mg/mL, 0.93mg/mL, 0.67 mg/mL. IC of EE ₅₀ The value is larger than EEP, which indicates that EEP in crude protein sample obtained by salting-out separation is the main anti-tumor active ingredient. In 3 samples obtained by the ultrafiltration separation of EEP, EEPC with the largest molecular weight has stronger anti-SMMC-7721 proliferation effect, which shows that EEPC has potential anti-liver cancer effect in vitro.

2. Effect of ground beetle protein isolate on proliferation of activated HSC-T6 cells

(1) Experimental methods

The HSC-T6 cells were inoculated in a mixed solution containing 10% FBS and 1% penicillin-streptomycinThe cells were cultured in DMEM medium in a 37 ℃ incubator containing 5% CO 2. Logarithmic growth phase cells HSC-T6 were seeded in 96-well plates (100. mu.L, 6X 10) ⁴ one/mL), the effect of different concentrations (0-2.0mg/mL) of the woodlouse extract (EE, EEP, EEPA, EEPB, EEPC) prepared in example 1 on the viability of normal HSC cells was determined using the MTT method described above.

HSC-T6 cells were then harvested in logarithmic growth phase and seeded in 96-well plates (100. mu.L, 6X 10) ⁴ one/mL). Each cell hole is set as a blank group, a model group and each ground beetle sample experiment group, and each group is provided with 6 multiple holes. Except for a blank group, cells of each group are added with a cell factor TFG-beta (10ng/mL) to be cultured for 24h for activating HSC-T6 cells, then the cells are discarded supernatant, the blank group is added with a culture medium to be cultured continuously, and an experimental group is added with ground beetle extracts (EE, EEP, EEPA, EEPB and EEPC) within a non-toxic concentration range to be incubated for 24 h. The viability of each group of cells was determined by the MTT method.

2. Results of the experiment

At present, the activation and proliferation of rat hepatic stellate cells are considered to be key processes causing hepatic fibrosis, and under normal conditions, the hepatic stellate cells are in a static state, do not express alpha smooth muscle actin (alpha-SMA) and have low proliferation activity. HSC, after activation, participate in the formation of liver fibrosis through proliferation and secretion of extracellular matrix. The continuous activation of hepatic stellate cells is a key link in the development process of hepatic fibrosis. It can therefore be concluded that liver fibrosis is the result of Hepatic Stellate Cell (HSC) activation and subsequent excessive extracellular matrix (ECM) deposition. Thus, cytokine-activated HSC-T6 cells were selected to investigate the effect of 5 Eupolyphaga Seu Steleophaga extract samples on anti-liver fibrosis in vitro. The unactivated HSC cells were normal hepatic stellate cells used for comparison. It was observed in the present invention that within the concentration range of 2mg/ml, 5 woodlouse extracts were non-toxic to normal HSC cells (FIG. 2A), and therefore the concentrations of subsequent experiments were within this range. As shown in B-F of FIG. 2, the model control group significantly stimulated the growth of HSC-T6 cells after addition of TGF- β compared to the blank control group, indicating the success of the modeling. Both solutions of 5 ground beetle extract samples inhibited the viability of activated HSC cells after administration compared to the model control group. Wherein, the sample EE and EEP are in the investigation concentration range (2mg/ml), only the sample EEP can obviously inhibit the growth of the activated HSC cells and has certain concentration dependence. The samples EEPA, EEPB and EEPC are in the effective concentration range (1mg/ml), each concentration group can obviously inhibit the growth of the activated HSC cells, and the EEPC sample with the strongest activity is the EEPC sample. Thus, EEP and EEPC can delay the formation of hepatic fibrosis, and EEPC is the main active site of EEP for inhibiting the progress of hepatic fibrosis.

3. In-vitro anti-tumor and hepatic fibrosis activity evaluation of ground beetle isolated protein PC3-III

(1) Experimental method

Samples the PC3-III prepared in example 1 was incubated with L-02 and SMMC-7721 cells in vitro at a concentration, and the effect of the PC3-III samples on the proliferation of SMMC-7721 in vitro (MTT method) was investigated as described in 1 above and the results compared to EEPC.

Apoptosis of SMMC-7721 cells after treatment with EEPC and PC3-III was observed using Hoechst 33342 staining. SMMC-7712 was inoculated into 24-well plates (500. mu.L, 16X 10) ⁴ one/mL), 24h before discarding the medium (EEPC and PC 3-III). After 48h dosing, the medium was discarded and 200. mu.L of 4% paraformaldehyde was added and incubated for 15 min. After the incubation is finished, the paraformaldehyde is discarded, 200 mu L of Hoechst 33342 dye with the concentration of 5 mu g/mL is added, the mixture is incubated at 37 ℃ in a dark place for 30min, and then the cell morphology is observed under a fluorescence microscope.

Flow cytometry is adopted to detect the apoptosis of SMMC-7721 cells after EEPC and PC3-III treatment. SMMC-7712 was plated in 6-well plates (2mL, 25X 10) ⁴ piece/mL) according to the instruction of the apoptosis detection kit.

The influence of EEPC and PC3-III on the migration of SMMC-7721 cells was examined by scratch assay, and SMMC-7712 was plated in 12-well plates (1mL, 1.5X 10) ⁵ one/mL), scored with a tip after overgrowth, then dosed and photographed under an inverted microscope at 0h, 24h, 48h, and finally the data was processed with ImageJ software.

The sample PC3-III was incubated with normal HSC cells and TGF- β activated HSC-T6, respectively, in vitro at a certain concentration, and the effect of the PC3-III sample on HSC-T6 activated in vitro proliferation (MTT method) was studied as described in 2 above and the results were compared to EEPC samples.

(2) Results of the experiment

(2.1) PC3-III is the main component of EEPC, and in order to investigate whether PC3-III has an anti-proliferative effect on hepatoma cells, the MTT assay measured the cell viability of PCSMMC-7721 after PC3-III treatment. It was observed in the present invention that PC3-III is non-toxic to normal liver cells L-02 within a concentration range of 2mg/ml (FIG. 4A). As shown in B, C in FIG. 4, the EEPC and PC3-III significantly inhibited the proliferation of tumor cell SMMC-7721 (IC) at different concentrations ₅₀ Respectively as follows: 0.67mg/ml and 0.88 mg/ml). It shows that PC3-III has potential anti-liver cancer effect in vitro and is the main active site of EEPC.

As shown by D, E in FIG. 4, the nuclei of the blank control group stained uniformly, and the nuclei of the groups administered with the sample solutions of different concentrations shrank, condensed chromatin, and formed apoptotic bodies and increased fluorescence intensity. Indicating that EEPC and PC3-III can induce the apoptosis of SMMC-7721 cells. And simultaneously, Annexin V/PI double staining is used for detecting the apoptosis condition. As shown in FIG. 5, the proportion of apoptosis was significantly increased and concentration-dependent after different concentrations of EEPC and PC3-III treated SMMC-7721 cells compared to the blank control group. It is demonstrated that EEPC and PC3-III exert antitumor activity by inducing apoptosis of SMMC-7721 to inhibit its malignant proliferation.

(2.2) EEPC and PC3-III inhibit SMMC-7721 cell migration

Tumor growth and metastasis are the cause of death in most cancer patients. Tumor metastasis is a complex process, with tumor cell migration and invasion being two critical steps, and therefore the effect of EEPC and PC3-III on SMMC-7721 migration was observed using the scratch test. The cells of the blank control filled the scratched area at 48h migration, while EEPC (FIG. 6A) and PC3-III (FIG. 6B) all significantly inhibited migration of SMMC-7721 at concentrations of 0.25, 0.5 and 1 mg/ml. It is demonstrated that EEPC and PC3-III can also exert anti-liver cancer effect by inhibiting tumor cell migration.

(2.3) the Effect of EEPC and PC3-III on the proliferation of activated HSC-T6 cells

The invention simultaneously examines the influence of PC3-III on the proliferation of activated HSC-T6 cells in vitro. The range of concentrations that are non-toxic to normal HSC cells was first determined, and it was observed that PC3-III is non-toxic to normal HSC cells within the concentration range of 2mg/ml (fig. 7A), and therefore the subsequent experimental concentrations were within this range. As shown in B, C in FIG. 7, EEPC and PC3-III both can significantly inhibit the proliferation of TGF- β activated HSC-T6 cells under different concentrations, indicating that they have the effect of delaying the progression of liver fibrosis. The slightly more active one is PC3-III and has dose dependence, which indicates that the main component of EEPC for delaying the progress of hepatic fibrosis is PC 3-III.

The above embodiments are merely illustrative of the technical concept and features of the present invention, and the present invention is not limited thereto, and equivalent changes and modifications made according to the spirit of the present invention should be covered thereby.

Sequence listing

<110> Nanjing university of traditional Chinese medicine

<120> a ground beetle protein extract with effects of resisting liver cancer and inhibiting liver fibrosis and application thereof

<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

1. A ground beetle protein extract with the efficacies of resisting liver cancer and inhibiting hepatic fibrosis is characterized in that the 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, and freeze drying to obtain Eupolyphaga Seu Steleophaga ethanol extract EE powder;

(2) dissolving the Eupolyphaga Seu Steleophaga ethanol extract EE powder prepared in step (1) in Tris-HCl buffer solution, adding ammonium sulfate into the solution to saturation state, stirring, standing, centrifuging, collecting precipitate, dialyzing after redissolving to remove salt, and freeze-drying to obtain Eupolyphaga Seu Steleophaga crude protein sample EEP.

2. The Eupolyphaga Seu Steleophaga protein extract with anti-hepatocarcinoma and hepatic fibrosis inhibiting effects according to claim 1, which is prepared by the following steps:

(1) pulverizing dry Eupolyphaga Seu Steleophaga into powder, adding 5-20 times volume of 20-30% ethanol, reflux-extracting at 60-75 deg.C for 1-3 times (1-2 hr each time), mixing extractive solutions, concentrating under reduced pressure, centrifuging, collecting supernatant, and freeze-drying to obtain Eupolyphaga Seu Steleophaga ethanol extract EE powder;

(2) dissolving the Eupolyphaga sinensis Walker ethanol extract EE powder prepared in the step (1) in 30mM Tris-HCl buffer solution, adding ammonium sulfate into the solution to a saturated state, stirring, standing, centrifuging, collecting precipitate, redissolving, dialyzing for 36h to remove salt, and freeze-drying to obtain the Eupolyphaga sinensis Walker crude protein sample EEP.

3. A ground beetle protein extract with the efficacies of resisting liver cancer and inhibiting hepatic fibrosis is characterized in that the extract is prepared by the following method:

(3) re-dissolving a crude protein sample EEP of the ground beetle in a Tris-HCl buffer solution, continuously centrifuging for 2 times by using an ultrafiltration centrifugal tube with the molecular weight cutoff of 10kDa and 3kD, dividing the EEP solution into 3 protein part trapped solutions according to the molecular weight, collecting, freezing and drying, and naming the solution 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. The Eupolyphaga Seu Steleophaga protein extract with anti-hepatocarcinoma and hepatic fibrosis inhibiting effects according to claim 3, which is prepared by the following method:

(3) dissolving crude ground beetle protein sample EEP powder in 30mM Tris-HCl buffer solution, sequentially using ultrafiltration centrifuge tubes with cut-off molecular weights of 10kDa and 3kD for continuous 2 times of centrifugal separation, dividing the EEP solution into 3 protein part cut-off solutions according to the molecular weights, collecting, freezing and drying, and naming 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.

5. A ground beetle protein extract with the efficacies of resisting liver cancer and inhibiting liver fibrosis is characterized in that the ground beetle protein extract is prepared by the following method:

(2) dissolving the Eupolyphaga Seu Steleophaga ethanol extract EE powder prepared in step (1) in 30mM Tris-HCl buffer solution, adding ammonium sulfate into the solution to saturation state, stirring, standing, centrifuging, collecting precipitate, re-dissolving, dialyzing for 36h to remove salt, and freeze-drying to obtain Eupolyphaga Seu Steleophaga crude protein sample EEP;

(4) dissolving EEPC with the molecular weight of more than 10k in the step (3) in 30Mm Tris-HCl buffer solution with the pH value of 8.0, performing DEAE-52 cells anion column chromatography, performing gradient elution by using NaCl with the concentration of 0-1.0M, detecting the absorbance value of eluent fraction at the flow rate of 1ml/min and at 280nm, drawing a DEAE elution map of the EEPC, and collecting main fraction PC3 of the EEPC separated on the DEAE column chromatography; and (3) feeding fraction PC3 into a Sephadex G-100 chromatographic column for further purification, wherein an elution solution is 30Mm Tris-HCl buffer solution with the pH of 8.0, the flow rate is 0.75ml/min, the absorbance value of the fraction of the eluent is detected at 280nm, the Sephadex G-100 elution map of main fraction PC3 of EEPC is drawn, and a main product PC3-III is collected.

6. A ground beetle protein with the efficacy of resisting liver cancer and inhibiting liver fibrosis is characterized by comprising an amino acid sequence QYSINFISARCNGDSCVCTFR.

7. The use of the Eupolyphaga Seu Steleophaga protein extract of any one of claims 1-5 in preparing a medicament or health product for resisting liver cancer and inhibiting liver fibrosis.

8. The use of the Eupolyphaga Seu Steleophaga protein of claim 6 in preparing anti-hepatocarcinoma and hepatic fibrosis inhibiting drugs or health products.

9. The use according to claim 7 or 8, wherein the extract of Eupolyphaga Seu Steleophaga protein or Eupolyphaga Seu Steleophaga protein is mixed with pharmaceutically acceptable carrier to make into tablet, capsule, granule, pill.