CN101366733A - Katsutoxin extract, preparation method and application thereof - Google Patents
Katsutoxin extract, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a scorpion venom extract extracted from East Asia scorpions. A preparation method for the scorpion venom extract comprises the following steps: scorpion venom freeze-dried powder is dissolved, centrifuged, filtered by use of 0.45 mu m and 0.22 mu m microporous membranes and freeze-dried; sephadex is added in; III-IV protein peaks of scorpion venom are collected; cation exchange chromatography is carried out; detection is carried out by use of an ultraviolet spectrophotometer; and the protein peaks are collected, so as to obtain the scorpion venom extract. Experiments prove that the scorpion venom extract has definite functions of inhibiting angiogenesis and hepatoma cell reincrease value. The invention successfully extracts effective components inhibiting angiogenesis and hepatoma cell reincrease value from the East Asia scorpions, which provides a foundation for further research and application in the future.
Description
Technical field
The present invention relates to a kind of scorpion venom extract and preparation method thereof, and the application in angiogenesis inhibitor.
Background technology
A lot of to the research of scorpion venom extract in the world, a lot of achievements have also been obtained, at present confirmed that scorpion venom extract has definite inhibitory action to kinds of tumors in clinical practice and basic research, scorpion venom extract can promote apoptosis of tumor cells by mechanism such as downward modulation bcl-2 express, and the human body immunity improving function is with antitumor.
Scorpio is as a Chinese medicine, is commonly used to treat immune diseases such as cardiovascular disease and rheumatoid clinical, but at present and do not know its effective ingredient, also do not have more detailed pharmacological research, and this uses with research it and has been subjected to very big restriction.
Summary of the invention
At above-mentioned prior art, the invention provides a kind of scorpion venom extract that from Scorpio, extracts, and its preparation method and application are provided.
The present invention is achieved by the following technical solutions:
A kind of extracting method of scorpion venom extract may further comprise the steps:
(1) obtains liquid thick poison with low temperature electric pulse stimulation scorpion mouth periproct, with the distilled water mixing, liquid thick by volume poison: distilled water=1:1.5～1:2, then 5000～6000 rev/mins centrifugal 20～30 minutes, obtain the scorpion venom lyophilized powder after the cryogenic vacuum lyophilization.
(2) be the scorpion venom of concentration 1g/40ml, PH7.0～7.6 with above-mentioned scorpion venom lyophilized powder dilute with water, then 5000～6000 rev/mins centrifugal 20～30 minutes, got 11000～13000 rev/mins of supernatant centrifugal 20～30 minutes, get supernatant.
(3) above-mentioned supernatant is used the filtering with microporous membrane one time of aperture 0.45um and 0.22um respectively, collected filtrate, put into freezing tank freezing 12～36 hours, freezing tank is put into freeze dryer vacuum drying 48～72h again in temperature-60～-89 ℃, collects lyophilized powder.
(4) Sephadex G-50 chromatography: chromatographic column is filled with sephadex G-50, the leading absorption of calf serum 50mg/20ml; Learn from else's experience lyophilized powder eluting behind the filtering with microporous membrane, the uv-spectrophotometric instrument detects, and absorbing wavelength 280nm collects scorpion venom III-IV protein peak, and operative temperature is 5 ℃.
(5) cation exchange resin layer is analysed: chromatographic column is filled with cation exchange resin, the scorpion venom III-IV upper prop that will collect through G-50, flow velocity 0.5ml/ minute, gradient elution, the uv-spectrophotometric instrument detects, absorbing wavelength 280nm, collect protein peak, operative temperature is 5 ℃.
(6) filtrate of containing the Buthotoxin polypeptide extract that obtains in the step (5) is inserted freezing 24 hours temperature-70 of freezing tank ℃, again freezing tank is put into the freeze dryer vacuum drying, collect lyophilized powder.
Described scorpion is the Ma Shi Scorpio, and the Ma Shi Scorpio is called Scorpio again, and fecund is in Shandong.
A kind of scorpion venom extract is to use method for preparing.
The application of described scorpion venom extract in angiogenesis inhibitor.
The application of described scorpion venom extract in the inhibition hepatoma carcinoma cell is rised in value again.
The polypeptide of scorpion venom extract of the present invention for extracting from Scorpio is for containing 50～60 amino acid whose mixtures of polypeptides, purity 89.1%, molecular weight 6000～7000 dalton, heat-resisting, pH is stable, show 4 peaks in high performance liquid chromatography, main peak accounts for 41.4% of the gross area.
Through evidence, scorpion venom extract of the present invention has definite inhibition new vessels nucleus formation, comprises suppressing the generation of chick chorioallantoic membrane new vessels, suppress Human umbilical vein endothelial cells propagation and suppressing new vessels generation in the tumor.
Multiple disease exists unusual new vessels to generate, and generating as rheumatoid arthritis patients synovial membrane medium vessels increases, and blood vessel hyperplasia is to cause synovitis, pannus growth, the reason that bone and cartilage destruction and hyperosteogeny form.Therefore suppressing new vessels and generate, is the new method of treatment rheumatoid arthritis; Use scorpion venom extract of the present invention can effectively suppress VEGF, thereby suppress new vessels generation in the synovial membrane.Growth of tumour cell relies on blood vessel required oxygen and nutrient substance is provided, the chemotherapeutic period tumor cell acceleration of breeding more more needs competent blood to supply, use separately or at chemotherapeutic period use in conjunction scorpion venom extract of the present invention, can effectively suppress new vessels generation in the tumor, if use in conjunction chemotherapeutics, as make peroral dosage form or the injection type that contains an amount of pharmaceutic adjuvant, antitumous effect is better.
Scorpion venom extract of the present invention also has the value-added again effect of definite inhibition hepatoma carcinoma cell.After the chemotherapy remaining tumor cell again breeding quicken, be that tumor cell produces one of chemical sproof major reason, therefore suppressing tumor cell, to breed acceleration again in the chemotherapy interval significant to oncotherapy.Experiment confirm, scorpion venom extract of the present invention has lethal effect to kinds of tumor cells.
The present invention has successfully extracted it and has suppressed angiogenesis and suppress the value-added again effective ingredient of hepatoma carcinoma cell from Scorpio, for further research and application provide the foundation later on.
Description of drawings
Fig. 1 is the effect sketch map of scorpion venom extract to Human umbilical vein endothelial cells;
Fig. 2 is that scorpion venom extract generates the inhibition sketch map to the chick chorioallantoic membrane new vessels; Wherein, 1 is the matched group chick chorioallantoic membrane, and 2 is 0.5mg scorpion venom extract intervention group, and 3 is 0.8mg scorpion venom extract intervention group.
Fig. 3 is independent and combined chemotherapy is used the inhibitory action sketch map of scorpion venom extract to new vessels in the tumor; Wherein, 1 is lotus tumor matched group, and 2 is simple scorpion venom extract intervention group, and 3 is scorpion venom extract combined chemotherapy intervention group.
Fig. 4 is that scorpion venom extract suppresses the Human umbilical vein endothelial cells schematic diagram of mechanism; Wherein, 1 for matched group bax expresses, and 2 is that scorpion venom extract group bax expresses, and 3 for matched group bcl-2 expresses, and 4 is that scorpion venom extract group bcl-2 expresses.
Fig. 5 is the schematic diagram of mechanism that scorpion venom extract suppresses new vessels in the tumor, wherein, 1,2,3 is respectively that matched group, simple scorpion venom extract group and scorpion venom extract combined chemotherapy group tumor tissues PDGF express, and 4,5,6 is respectively matched group, simple scorpion venom extract group and scorpion venom extract combined chemotherapy group tumor tissues vegf expression.
Fig. 6 respectively organizes the gross tumor volume sketch map after cultivating for three weeks in the test example 6;
Fig. 7 respectively organizes the tumour inhibiting rate sketch map after cultivating for three weeks in the test example 6;
Fig. 8 respectively organizes H after cultivating for three weeks in the test example 6
22The expression of PCNA (* 200) in the tumor; Wherein, 3a, 3b, 3c are the 1st, 2,3 weeks of model group; 3d is the 3rd week of experimental group; 3e organized for the 3rd week for the PESV treatment; 3f is the 3rd week of lotus tumor matched group.
Fig. 9 respectively organizes H after cultivating for three weeks in the test example 6
22The gray analysis that tumor tissues PCNA expresses; Wherein,
*The 2nd week and model group contrast, P＜0.01; The 3rd week of △ is with model group contrast, P＜0.01;
Figure 10 respectively organizes vegf expression (* 100) after cultivating for three weeks in the test example 6; Wherein, 6A, 6B, 6C are the 1st, 2,3 weeks of model group; 6D is the 3rd week of experimental group; 6E is that PESV organized for the 3rd week; 6F is the 3rd week of lotus tumor matched group.
Figure 11 respectively organizes H after cultivating for three weeks in the test example 6
22The gray analysis of tumor tissues vegf expression; Wherein,
*The 2nd week and model group contrast, P＜0.05;
*The 2nd week and model group contrast, P＜0.01; The 3rd week of △ and model group contrast, P＜0.01; ▲ model group and the contrast of the 3rd week, P＜0.05.
Figure 12 respectively organizes PDGF expression (* 100) after cultivating for three weeks in the test example 6; Wherein, 8A, 8B, 8C are the 1st, 2,3 weeks of model group; 8D is the 3rd week of experimental group; 8E is that PESV organized for the 3rd week; 8F is the 3rd week of lotus tumor matched group.
Figure 13 respectively organizes H after cultivating for three weeks in the test example 6
22The gray analysis that tumor tissues PDGF expresses; Wherein,
*3 weeks and model group contrast, P＜0.05;
*3 weeks and model group contrast, P＜0.01.
The specific embodiment
Below in conjunction with accompanying drawing, embodiment, the present invention is further illustrated for the test example.
Embodiment 1: extract scorpion venom extract from Scorpio, step is as follows:
(1) obtain liquid thick poison with low temperature electric pulse stimulation scorpion mouth periproct, with the distilled water mixing, liquid by volume thick poison: distilled water=1:2, then 5000 rev/mins centrifugal 30 minutes, obtain the scorpion venom lyophilized powder after the cryogenic vacuum lyophilization.
(2) be the scorpion venom of concentration 1g/40ml, PH7.4 with above-mentioned scorpion venom lyophilized powder dilute with water, then 5000 rev/mins centrifugal 30 minutes, got 12000 rev/mins of supernatant centrifugal 30 minutes, get supernatant.
(3) above-mentioned supernatant is used the filtering with microporous membrane one time of aperture 0.45um and 0.22um respectively, collected filtrate, put into freezing tank freezing 24 hours, temperature-79 ℃ is put into freezing tank freeze dryer vacuum drying 72h again, collects lyophilized powder.
(4) Sephadex G-50 chromatography: chromatographic column is filled with sephadex G-50, the leading absorption of calf serum 50mg/20ml; The lyophilized powder 400mg behind the filtering with microporous membrane of learning from else's experience add PH (PH7.4,0.1M) eluting is 36 hours, uv-spectrophotometric instrument (absorbing wavelength 280nm) detects, and collects scorpion venom III-IV protein peak, operative temperature is 5 ℃.
(5) cation exchange resin (SP Sepharose Fast Flow) chromatography: chromatographic column is filled with cation exchange resin, scorpion venom III-IV200ml the upper prop that to collect through G-50, flow velocity 0.5ml/ minute, gradient elution 48 hours, the uv-spectrophotometric instrument detects, absorbing wavelength 280nm collects protein peak, and operative temperature is 5 ℃.
(6) filtrate of containing the Buthotoxin polypeptide extract that obtains in the step (5) is inserted freezing 24 hours temperature-70 of freezing tank ℃, freezing tank was put into the freeze dryer vacuum drying 72 hours again, collect lyophilized powder.
Embodiment 2: extract scorpion venom extract from Scorpio, step is as follows:
(1) obtain liquid thick poison with low temperature electric pulse stimulation scorpion mouth periproct, with the distilled water mixing, liquid by volume thick poison: distilled water=1:1.5, then 5000 rev/mins centrifugal 30 minutes, obtain the scorpion venom lyophilized powder after the cryogenic vacuum lyophilization.
(2) be the scorpion venom of concentration 1g/40ml, PH7.6 with above-mentioned scorpion venom lyophilized powder dilute with water, then 5000 rev/mins centrifugal 30 minutes, got 11000 rev/mins of supernatant centrifugal 30 minutes, get supernatant.
(3) above-mentioned supernatant is used the filtering with microporous membrane one time of aperture 0.45um and 0.22um respectively, collected filtrate, put into freezing tank freezing 12 hours, temperature-89 ℃ is put into freezing tank freeze dryer vacuum drying 56h again, collects lyophilized powder.
(4) Sephadex G-50 chromatography: chromatographic column is filled with sephadex G-50, the leading absorption of calf serum 50mg/20ml; The lyophilized powder 400mg behind the filtering with microporous membrane of learning from else's experience add PH (PH7.4,0.1M) eluting is 36 hours, uv-spectrophotometric instrument (absorbing wavelength 280nm) detects, and collects scorpion venom III-IV protein peak, operative temperature is 5 ℃.
(5) cation exchange resin (SP Sepharose Fast Flow) chromatography: chromatographic column is filled with cation exchange resin, scorpion venom III-IV200ml the upper prop that to collect through G-50, flow velocity 0.5ml/ minute, gradient elution 48 hours, the uv-spectrophotometric instrument detects, absorbing wavelength 280nm collects protein peak, and operative temperature is 5 ℃.
(6) filtrate of containing the Buthotoxin polypeptide extract that obtains in the step (5) is inserted freezing 24 hours temperature-70 of freezing tank ℃, freezing tank was put into the freeze dryer vacuum drying 72 hours again, collect lyophilized powder.
Embodiment 3: extract scorpion venom extract from Scorpio, step is as follows:
(1) obtain liquid thick poison with low temperature electric pulse stimulation scorpion mouth periproct, with the distilled water mixing, liquid by volume thick poison: distilled water=1:22, then 6000 rev/mins centrifugal 20 minutes, obtain the scorpion venom lyophilized powder after the cryogenic vacuum lyophilization.
(2) be the scorpion venom of concentration 1g/40ml, PH7.0 with above-mentioned scorpion venom lyophilized powder dilute with water, then 6000 rev/mins centrifugal 20 minutes, got 13000 rev/mins of supernatant centrifugal 20 minutes, get supernatant.
(3) above-mentioned supernatant is used the filtering with microporous membrane one time of aperture 0.45um and 0.22um respectively, collected filtrate, put into freezing tank freezing 36 hours, temperature-60 ℃ is put into freezing tank freeze dryer vacuum drying 48h again, collects lyophilized powder.
(4) Sephadex G-50 chromatography: chromatographic column is filled with sephadex G-50, the leading absorption of calf serum 50mg/20ml; The lyophilized powder 400mg behind the filtering with microporous membrane of learning from else's experience add PH (PH7.4,0.1M) eluting is 36 hours, uv-spectrophotometric instrument (absorbing wavelength 280nm) detects, and collects scorpion venom III-IV protein peak, operative temperature is 5 ℃.
(5) cation exchange resin (SP Sepharose Fast Flow) chromatography: chromatographic column is filled with cation exchange resin, scorpion venom III-IV200ml the upper prop that to collect through G-50, flow velocity 0.5ml/ minute, gradient elution 48 hours, the uv-spectrophotometric instrument detects, absorbing wavelength 280nm collects protein peak, and operative temperature is 5 ℃.
(6) filtrate of containing the Buthotoxin polypeptide extract that obtains in the step (5) is inserted freezing 24 hours temperature-70 of freezing tank ℃, freezing tank was put into the freeze dryer vacuum drying 72 hours again, collect lyophilized powder.
Test example 1: scorpion venom extract of the present invention is to the active influence of rising in value of HUVEC and MDA-MB-231 cell:
With 1 * 10
5The HUVEC Human umbilical vein endothelial cells joins in 96 orifice plates and cultivates, and culture fluid is that volume fraction is the DMEM culture fluid of 10% calf serum, 37 ℃ of condition of culture, 5% CO
2Cultivate and be replaced by serum-free medium in 24 hours.Processed group adds the scorpion venom extract (4～20 μ g/ml) of variable concentrations, and matched group adds the equivalent normal saline.Act on termination effect in 8 hours, continue to cultivate 24 hours, the ELISA method of mixing with BrdU detects cell-proliferation activity, i.e. D is read in substrate colour developing after cell fixation, degeneration, the effect of anti-BrdU enzyme labelled antibody
450Value.The result as shown in Figure 1, find that scorpion venom extract obviously suppresses the proliferation activity of HUVEC in 8-20 μ g/ml scope, and this inhibitory action shows as the specificity of cell type, because the PESV of each experimental concentration does not have obvious influence to the proliferation activity of breast carcinoma MDA-MB-231 cell.
Test example 2: scorpion venom extract of the present invention is to the influence of chick chorioallantoic membrane (CAM) angiogenesis:
Embryo Gallus domesticus is hatched in 37.5 ℃, and 5d under 60% relative humidity, aseptic condition expose chick chorioallantoic membrane down.Be divided into 3 groups immediately by experimental design, every group 20, to be adsorbed with 5 μ l normal saline respectively, 0.5mg and the glass fiber filter paper of 0.8mg PESV covers on the CAM removal fibrous glass filter paper behind the effect 72h, taking-up chorioallantoic membrane, use 10% formaldehyde fixed, move on the slide, under anatomic microscope, observe new vessels and generate situation, calculate the angiogenesis suppression ratio.The result finds that blood vessel normal saline processed group chick chorioallantoic membrane angiogenesis is vigorous as shown in Figure 2, and branch is obvious, and vessel density is big, and caliber is thick; And PESV processed group vessel density is sparse, and caliber is thin, and branch is few.0.5mg and 0.8mg PESV processed group vessel branch counts and is respectively 12.52 ± 2.90 and 20.42 ± 4.10, is starkly lower than the normal saline group; 0.5mg be respectively 85.0% and 75% with 0.8mg processed group PESV angiogenesis suppression ratio, apparently higher than normal saline matched group 20%, P＜0.01.
Test example 3: independent and combined chemotherapy is used the inhibitory action of scorpion venom extract to new vessels in the tumor:
The subcutaneous plantation 1 * 10 of the right axil of Balb/c mice
6Individual H
22Rat liver cancer tumor cell, lotus tumor matched group give normal saline and irritate stomach, and simple scorpion venom extract group gives scorpion venom extract, dosage is 40mg/kg, the combined chemotherapy group, scorpion venom extract is irritated stomach+5-fluorouracil lumbar injection, and injection volume is injection concentration: 20mg/kg.Experiment was carried out for 3 weeks altogether.Get and respectively organize tumor tissues, detect the tumor tissues VIII factor-microvessel density (MVD) with immunohistochemical method, the result as shown in Figure 3, matched group MVD is 25.2 ± 2.86, simple scorpion venom extract group is 19.8 ± 2.38, uniting group is 11.8 ± 1.92, and scorpion venom extract significantly suppresses new vessels formation in the tumor, and it is more remarkable that combined chemotherapy suppresses.
Test example 4: scorpion venom extract suppresses the Human umbilical vein endothelial cells Study on Mechanism:
Human umbilical vein endothelial cells is by 1 * 10
6Concentration is inoculated in 12 orifice plates and cultivated 12 hours, carries out cell climbing sheet, and processed group adds PESV10 μ g/ml, and matched group adds the equivalent normal saline.Act on 48 hours, after fixing, the expression that detects apoptosis-related genes Bcl-2 and Bax changes.The result as shown in Figure 4, find that SABC shows PESV effect Human umbilical vein endothelial cells 48h after, Bax expresses increase, Bcl-2 expresses reduction.
Test example 5: scorpion venom extract suppresses new vessels Study on Mechanism in the tumor:
Zoopery detects tumor tissues (platelet derived growth factor) PDGF and (VEGF) vegf expression with test example 3 with immunohistochemical method.The result as shown in Figure 5, scorpion venom extract significantly reduces tumor tissues VEGF and PDGF expresses.
In sum, scorpion venom extract of the present invention has tangible blood vessel formation against function, and suppresses growth of tumor by this, illustrates that scorpion venom extract of the present invention contains the anti-angiogenesis activity molecule, can be used for the clinical treatment of malignant tumor.
More than test in the example, antibody: VEGF, bFGF (Santa Cruz, USA), factor-VIII (Dako, USA), Bcl-2, Bax (Beijing Zhong Shan biological reagent company).BrdU EL ISA detection kit: Am ersham Pharmaci company product.
Test example 6: scorpion venom extract suppresses hepatocarcinoma H
22The proliferation experiment research again of cell chemotherapeutic period:
1, material and method:
1.1 material
1.1.1 tumor kind and animal H
22Rat liver cancer ascites tumor kind is so kind as to give by Shandong Province's medical courses in general institute medicine, and the every 7d of ascites tumor kind passes 1 time, and the 2nd substitutes in experiment.Animal is adopted the Balb/c mice, and body weight 18g～20g is male, available from Shandong University's medical college animal center.
1.1.2 medicine, reagent and instrument scorpion venom extract are to extract according to the method for embodiment 1; 5-FU is available from Tianjin gold credit aminoacid company limited, batch number: 0612022.PCNA, VEGF and CD105 one are anti-, available from Bioisystech Co., Ltd of China fir Golden Bridge in Beijing.Germany leicaDM4000B optical microscope.The Germany leica Qwin V3 of company image analysis software.
1.2 method
1.2.1 the aseptic extraction of modelling, grouping and interference method 7d mouse peritoneal tumor cell adds normal saline and adjusts TCD to 4 * 10
6Ml
-1After, in the right oxter injection 0.2ml tumor cell suspension (the 0th day) of every mice, after 5 days, become the mice of tumor to be divided into lotus tumor matched group, model group, experimental group and simple PESV group at random mice, at least 9 every group, be the 5th day the same day of dividing into groups.Model group is carried out chemotherapy with the 5Fu lumbar injection to mice and is set up multiplicative model again, injection concentration: 20mgkg
-1, inject time: 5th, 12,19 days, and irritate stomach, injection volume 10mgkg with normal saline
-1, the 8th, 9,10,16,17,18,23,24,25 days inject time.Experimental group, the same model group of 5-Fu usage.Intervene interference method with PESV: the dilution of PESV normal saline, irritate stomach, dosage is 40mgkg
-1, the time: 8th, 9,10,16,17,18,23,24,25 days.Simple PESV group is only carried out PESV to tumor-bearing mice and is irritated stomach, and the PESV interference method is together with the experimental group usage.Lotus tumor matched group only carries out normal saline to tumor-bearing mice and irritates stomach, irritates the same model group of stomach method.
1.2.2 calculate tumour inhibiting rate and cubing in the 12nd, 19,26 day every group each put to death 3 of mices at random.Get tumor tissues, weigh, fix with neutral formalin.Tumour inhibiting rate calculates: and tumour inhibiting rate (Inhibitory Rate, IR)=lotus tumor matched group tumor heavy (n week)-experimental group tumor heavy (n week)/lotus tumor matched group tumor heavy (n week).Survey gross tumor volume weekly twice, measure tumor major diameter (A) and minor axis (B), calculate as follows: gross tumor volume (V)=AB2/2, and calculate every group of gross tumor volume that on average increases weekly.
1.2.3H
22The detection of tumor tissues PCNA adopts Immunohistochemical method to detect and MVD expresses.3% H
2O
2Eliminate endogenous peroxidase activity, after hot repair is multiple, adds one respectively and resist, working concentration is 1:100, and 4 ℃ are spent the night, DAB colour developing, microscopically observed result.Utilize double-blind method to measure the PDGF staining power, every section is chosen 5 visuals field (* 200) and is carried out the analysis of optics gray value, and numerical value is between 0～255, and it is strong more to dye, and gray value is low more, gets the gray value that its meansigma methods is this sheet.
1.2.4 using SPSS 11.0 softwares, statistical procedures carries out statistical analysis.Measurement data adopts the ANOVA method to detect, and association analysis adopts Pearson to analyze significance level α＜0.05.
2, result
2.1PESV to H
22The model group gross tumor volume that influences of multiplicative model mouse tumor volume has increased by 174% (from 86.9mm3 to 238.7mm3) in the 1st week again, increased by 91% (from 238.7mm3 to 458.3mm3) in the 2nd week, the 3rd week increased by 122% (from 458.3mm3 to 1022mm3).The model group tumor propagation develops with the chemotherapy cycles progress with the rule of " accelerating-slow-accelerate ".The simple PESV group percentage ratio that gross tumor volume increases in 3 weeks is 196% (from 77.2mm3 to 228.6mm3), 22% (from 228.6mm3 to 279.1mm3), 65% (from 279.1mm3 to 463mm3) successively, and rule be " accelerating-very slow-slow ".The the 19th and 26 day (being respectively the 2nd and 3 weekends), simple PESV group growth of tumor was effectively suppressed, as shown in Figure 6.
2.2PESV to H
22The model group tumor tumour inhibiting rate that influences of tumor tumour inhibiting rate is respectively 37%, 47%, 38% in the 1st, 2,3 weeks, and model group is the highest at second all tumor tumour inhibiting rates, but in the 3rd week, the tumor tumour inhibiting rate significantly reduces.Experimental group tumor in 3 weeks is respectively 45%, 54%, 57% at the 1st, 2,3 all tumour inhibiting rates, and tumour inhibiting rate raises gradually.Compare with model group, the experimental group growth of tumor is effectively suppressed, as shown in Figure 7.
2.3PESV to H
22Fig. 8 and Fig. 9 (gray value analysis) are seen in the influence that tumor tissues PCNA expresses, and model group and PESV group PCNA expression all were the 1st week from high to low successively〉the 3rd week〉the 2nd week.Model group tumor tissues the 1st week and have significant difference (P＜0.01) the 2nd week.Show that generation obviously kills and wounds and inhibitory action 5-Fu to the H22 tumor cell in the 2nd week.In the 3rd week, expression raises (having significant difference with the 2nd week, P＜0.01), shows that tumor cell proliferation quickens after the 3rd all chemotherapy.In the 2nd week, there is significant difference (P＜0.01) in the PESV group with model group, shows that PESV strengthens the 5-Fu antitumor action.In the 3rd week, the model group expression is higher than PESV group (P＜0.01), show PESV suppressed chemotherapy after tumor proliferation quicken.
2.4PESV to H
22Tumor tissues PDGF expresses influences model group and experimental group vegf expression level all was the 1st week from high to low successively〉the 3rd week〉the 2nd week.Compare with model group, there is significant difference (P＜0.01) the 2nd week in lotus tumor matched group, shows that 5-Fu has certain inhibition VEGF secretory action.Compare with model group, experimental group is in the 2nd week and all have significant difference (P＜0.01) the 3rd week, proves associating 5-Fu, and PESV further reduces vegf expression.Correlation analysis shows that there are positive correlation (r=0.669) in vegf expression level and CD105-MVD, see Figure 10 and Figure 11 (gray value analysis), and model group PDGF expresses then and descends gradually, and experimental group PDGF expresses then in raise in the 3rd week (P＜0.05).See Figure 12, Figure 13.
3, discuss
This experiment adopts 5-Fu to intervene the subcutaneous tumor-bearing mice of H22.Discover that volume is temporary to be dwindled though model group does not have, increase slow phenomenon the 2nd week occurring volume, occur again increasing sharply in the 3rd week.Roughly the same the acceleration model of propagation again of professor's Tannock proposition proves that 5-Fu intervenes the subcutaneous bearing mouse model existence of H22 and breeds hastening phenomenon again.Find that simultaneously experimental group is in the 2nd week, the experimental group gross tumor volume is significantly less than model group, illustrate that PESV has strengthened the 5-Fu antitumor action, equally significantly less than model group, illustrate and the 3rd week that PESV has effectively suppressed to occur after the chemotherapy breed hastening phenomenon at the 3rd all experimental grouies.Immunohistochemistry detects PCNA and observes the tumor cell demonstration that is in the propagation phase, model group is lower than the 1st week in the 2nd all expression levels, the 3rd week was significantly higher than for the 2nd week, showed be in the proliferating cells minimizing the 2nd week, increased sharply again but the 3rd all model group are in the tumor cell of propagation.Change and the PCNA differential expression in conjunction with gross tumor volume, prove chemotherapy after tumor breed hastening phenomenon again.
Indication PESV is a scorpion venom extract of the present invention in the literary composition.
Claims (4)
1. the extracting method of a scorpion venom extract is characterized in that, may further comprise the steps:
(1) obtains liquid thick poison with low temperature electric pulse stimulation scorpion mouth periproct, with the distilled water mixing, liquid thick by volume poison: distilled water=1:1.5～1:2, then 5000～6000 rev/mins centrifugal 20～30 minutes, obtain the scorpion venom lyophilized powder after the cryogenic vacuum lyophilization;
(2) be the scorpion venom of concentration 1g/40ml, PH7.0～7.6 with above-mentioned scorpion venom lyophilized powder dilute with water, then 5000～6000 rev/mins centrifugal 20～30 minutes, got 11000～13000 rev/mins of supernatant centrifugal 20～30 minutes, get supernatant;
(3) above-mentioned supernatant is used the filtering with microporous membrane one time of aperture 0.45um and 0.22um respectively, collected filtrate, put into freezing tank freezing 12～36 hours, freezing tank is put into freeze dryer vacuum drying 48～72h again in temperature-60～-89 ℃, collects lyophilized powder;
(4) Sephadex G-50 chromatography: chromatographic column is filled with sephadex G-50, the leading absorption of calf serum 50mg/20ml; Learn from else's experience lyophilized powder eluting behind the filtering with microporous membrane, the uv-spectrophotometric instrument detects, and absorbing wavelength 280nm collects scorpion venom III-IV protein peak, and operative temperature is 5 ℃;
(5) cation exchange resin layer is analysed: chromatographic column is filled with cation exchange resin, the scorpion venom III-IV upper prop that will collect through G-50, flow velocity 0.5ml/ minute, gradient elution, the uv-spectrophotometric instrument detects, absorbing wavelength 280nm, collect protein peak, operative temperature is 5 ℃;
(6) filtrate of containing the Buthotoxin polypeptide extract that obtains in the step (5) is inserted freezing 24 hours temperature-70 of freezing tank ℃, again freezing tank is put into the freeze dryer vacuum drying, collect lyophilized powder.
2. scorpion venom extract according to the preparation of the described method of claim 1.
3. the application of the described scorpion venom extract of claim 3 in angiogenesis inhibitor.
4. the application of the described scorpion venom extract of claim 3 in the inhibition hepatoma carcinoma cell is rised in value again.
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|---|---|---|---|
| CNA200810157559XA CN101366733A (en) | 2008-10-07 | 2008-10-07 | Katsutoxin extract, preparation method and application thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102119937A (en) * | 2011-03-01 | 2011-07-13 | 通化卫京药业股份有限公司 | Application of scorpion venom injection in treatment of cancers |
| CN104306406A (en) * | 2014-11-07 | 2015-01-28 | 山东中医药大学 | Scorpion ferment and ultra-filtrated object, and preparation procedure and usage thereof |
| CN105061574A (en) * | 2015-09-17 | 2015-11-18 | 北京化工大学 | Anti-tumor peptide |
| CN104193813B (en) * | 2014-09-05 | 2017-01-25 | 四川省中医药科学院 | Separation and purification method of scorpion venom polypeptide and use thereof |
| CN111821252A (en) * | 2020-08-12 | 2020-10-27 | 辽宁盛京干细胞科技有限公司 | Anti-wrinkle essence containing adipose-derived stem cell extract and preparation method thereof |
| CN113817024A (en) * | 2021-07-30 | 2021-12-21 | 山东中医药大学附属医院 | Scorpion active peptide, pharmaceutical composition and application thereof in preparation of anti-tumor products |
| CN114031678A (en) * | 2021-12-08 | 2022-02-11 | 中国科学院新疆理化技术研究所 | Preparation method and application of polypeptide component of scorpion venom polypeptide |
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2008
- 2008-10-07 CN CNA200810157559XA patent/CN101366733A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102119937A (en) * | 2011-03-01 | 2011-07-13 | 通化卫京药业股份有限公司 | Application of scorpion venom injection in treatment of cancers |
| CN102119937B (en) * | 2011-03-01 | 2012-11-21 | 通化卫京药业股份有限公司 | Application of scorpion venom injection in treatment of cancers |
| CN104193813B (en) * | 2014-09-05 | 2017-01-25 | 四川省中医药科学院 | Separation and purification method of scorpion venom polypeptide and use thereof |
| CN104306406A (en) * | 2014-11-07 | 2015-01-28 | 山东中医药大学 | Scorpion ferment and ultra-filtrated object, and preparation procedure and usage thereof |
| CN105061574A (en) * | 2015-09-17 | 2015-11-18 | 北京化工大学 | Anti-tumor peptide |
| CN105061574B (en) * | 2015-09-17 | 2018-03-30 | 北京化工大学 | Natineoplaston |
| CN111821252A (en) * | 2020-08-12 | 2020-10-27 | 辽宁盛京干细胞科技有限公司 | Anti-wrinkle essence containing adipose-derived stem cell extract and preparation method thereof |
| CN113817024A (en) * | 2021-07-30 | 2021-12-21 | 山东中医药大学附属医院 | Scorpion active peptide, pharmaceutical composition and application thereof in preparation of anti-tumor products |
| CN114031678A (en) * | 2021-12-08 | 2022-02-11 | 中国科学院新疆理化技术研究所 | Preparation method and application of polypeptide component of scorpion venom polypeptide |
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