CN114832017B - Application of placenta caprae seu ovis extract with molecular weight greater than 50KD in preparation of antitumor drugs - Google Patents
Application of placenta caprae seu ovis extract with molecular weight greater than 50KD in preparation of antitumor drugs Download PDFInfo
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Abstract
The invention belongs to the technical field of anti-tumor, and in particular relates to application of a sheep placenta extract with a molecular weight of more than 50KD in preparation of an anti-tumor medicament. The invention surprisingly found that, compared with the whole molecular weight section placenta caprae seu ovis extract (placenta caprae seu ovis whole extract) and other molecular weight section placenta caprae seu ovis extracts, the molecular weight section placenta caprae seu ovis extract with more than 50KD has remarkable inhibitory activity on colorectal cancer; wherein the inhibition effect is more remarkable when the molecular weight is 50-100KD; compared with other tumor cells, the sheep placenta extract with the molecular weight of more than 50KD has specificity to colorectal cancer cells and can specifically inhibit proliferation of colorectal cancer cells; meanwhile, the sheep placenta extract with the molecular weight of more than 50KD has good safety, can be used for preparing antitumor drugs, and has wide application prospects.
Description
Technical Field
The invention belongs to the technical field of anti-tumor, and in particular relates to application of a sheep placenta extract with a molecular weight of more than 50KD in preparation of an anti-tumor medicine.
Background
Malignant tumors are a large group of diseases that endanger human health, 800 tens of thousands of people die from cancer annually worldwide, while china has nearly 200 tens of thousands of people die from cancer annually. Tumor generation and development are dynamic processes, involve interaction of a plurality of signal molecules, and form a complex molecular regulation network. Despite the great progress made in the drug treatment of tumors, it is an essential major measure for the current clinical treatment. However, the problems of high toxic and side effects, drug resistance and the like are still the main obstacle faced by the clinical tumor drug treatment.
Among them, colorectal cancer (CRC) is the third most common cancer worldwide and is also the second leading cause of cancer-related death. The chemotherapy for patients with advanced colorectal cancer aims at reducing the tumor volume, reducing the tumor growth and inhibiting the tumor metastasis. Wherein the common drugs comprise 5-fluorouracil (5-FU), capecitabine, oxaliplatin, cisplatin, irinotecan and the like, and wherein the 5-fluorouracil (5-FU) and capecitabine are used for inhibiting the activity of thymidylate synthase in the process of DNA replication; oxaliplatin inhibits tumor cell growth and causes cell G2 arrest by covalently binding DNA and forming platinum-DNA adducts. Combinations of these chemotherapeutics are widely used in the treatment of CRC, but most patients with advanced colorectal cancer initially respond to combination chemotherapy. However, due to drug resistance, patients eventually experience tumor recurrence. Moreover, most cancer chemotherapeutics are currently toxic to the host.
Therefore, providing a natural drug with good specificity and safety is of great importance for the treatment of cancers, especially liver cancer and breast cancer. The sheep placenta extract is a general term of various active substances extracted from sheep placenta, and comprises various proteins, essential amino acids, nucleic acids, hormones, collagen, immune factors, calcium, iron, zinc and other nutritional ingredients, and has various health care functions of supplementing qi, nourishing blood, supplementing essence, tonifying kidney, regulating organism immunity, delaying aging, whitening skin, keeping body shape and the like. Can be used for preventing liver cirrhosis, improving immunity, and treating viral hepatitis, leukopenia, myasthenia gravis, etc. The invention surprisingly discovers that compared with the whole placenta caprae seu ovis extract powder, the placenta caprae seu ovis extract with molecular weight larger than 50KD has remarkable effect of inhibiting proliferation of liver cancer and breast cancer cells, has good safety, and can be used for preparing antitumor drugs.
Disclosure of Invention
Aiming at the technical problems, the invention unexpectedly discovers that compared with the whole placenta caprae seu ovis extract, the placenta caprae seu ovis extract with the molecular weight of more than 50KD has remarkable effect of inhibiting the proliferation of colon cancer cells, has good safety and can be used for preparing antitumor drugs. Therefore, the invention aims to provide an application of the sheep placenta extract with the molecular weight of more than 50KD in preparing an anti-tumor medicament. The method specifically comprises the following steps:
in a first aspect, the present invention provides the use of a sheep placenta extract having a molecular weight of greater than 50KD in the manufacture of an antitumor drug.
Preferably, the tumor is colon cancer.
Preferably, the molecular weight of the sheep placenta extract is 50-100KD.
Preferably, the preparation method of the sheep placenta extract comprises the following steps: and (3) selecting an ultrafiltration membrane with the molecular weight of 50KD to carry out ultrafiltration on the fresh placenta caprae seu ovis extract to obtain the placenta caprae seu ovis extract with the molecular weight of more than 50KD.
Preferably, the preparation method of the fresh sheep placenta extract comprises the following steps:
(1) Pretreatment: removing surface lipid, connective tissue and blood clot of placenta caprae seu ovis, and mincing to obtain placenta tissue;
(2) Extraction: adding an extracting solution into the placenta tissue in the step (1) for homogenate extraction, wherein the extracting solution consists of normal saline, PMSF and a protein phosphatase inhibitor;
(3) Centrifuging and filtering to obtain crude extract;
(4) Carrying out ultrasonic treatment on the crude extract obtained in the step (3), and then carrying out microfiltration to obtain a microfiltration liquid;
(5) Ultrafiltration: and (3) selecting a 50KD ultrafiltration membrane to carry out ultrafiltration on the micro-filtrate obtained in the step (4) to obtain the sheep placenta extract with the molecular weight of more than 50KD.
Preferably, the ultrafiltration step in the step (5) is as follows: and (3) selecting ultrafiltration membranes with 50KD and 100KD to carry out ultrafiltration on the micro-filtrate obtained in the step (4) to obtain the sheep placenta extract with the molecular weight of 50-100KD.
Preferably, the composition of the extract in the step (2) is as follows: 0.86% (m/m) physiological saline, 1% (v/v) PMSF, 1% (v/v) protein phosphatase inhibitor.
Preferably, the ratio of placenta tissue to extract in the step (2) is 1g:5ml.
Preferably, the homogenization parameters in the step (2) are: 60Hz,30 s/time, 30s gap, 3-5 times continuously; homogenizing, and stirring at 4deg.C for 30-60min.
Preferably, the centrifugation parameters in the step (3) are: 4 ℃,10000r,20min.
Preferably, the ultrasonic parameter in the step (4) is 200Hz, and the ultrasonic is performed for 5min; the microfiltration was performed using a 0.45 μm aqueous microfiltration membrane and a microfiltration membrane filter.
Preferably, the sheep placenta extract is added with pharmaceutically acceptable carriers/auxiliary materials to prepare any pharmaceutically acceptable dosage form.
The beneficial effects of the invention are as follows: the invention surprisingly discovers that compared with the whole extract of sheep placenta and the extract of sheep placenta with other molecular weight segments, the molecular weight of the extract of sheep placenta with molecular weight of more than 50KD, especially the extract of sheep placenta with molecular weight of 50-100KD has remarkable effect of inhibiting the proliferation of colon cancer cells; compared with other tumor cells, the sheep placenta extract with the molecular weight larger than 50KD, especially the molecular weight of 50-100KD, has specificity to colon cancer and can specifically inhibit proliferation of colon cancer cells; meanwhile, the placenta caprae seu ovis extract with the molecular weight of more than 150KD has good safety and can be used for preparing antitumor drugs.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. Specific conditions are not noted in the examples, and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The human cancer cell lines used in the following examples include human colon cancer cell HCT116, human gastric cancer cell SGC7901, human pancreatic cancer cell PANC-1, human lung cancer cell A549 and human normal intestinal epithelial cell HIEC; cell lines were cultured in 1640 medium (1640,Solarbio Invitrogen Corp, beijin, china); the culture conditions are as follows: 37 DEG C、5％CO 2 All cells were supplemented with 10% Fetal Bovine Serum (FBS), 100U/mL penicillin and 100mg/mL streptomycin under conditions.
Example 1 preparation of sheep placenta extract
1. Materials and methods
Experimental materials and instrument preparation: fresh sheep placenta, sodium chloride, 1L Lan Gaiping ×2, tissue shears, cutter, meat grinder, triangular funnel, absorbent paper, beaker, centrifuge tube (sterilized in advance);
sterile 1L of 0.86% physiological saline was prepared: 7.74g of sodium chloride is weighed by an analytical balance and dissolved in 900mL of distilled water, and the solution is obtained after dissolution, sterilized at 121 ℃ and cooled for standby;
sterilizing the centrifuge tube at 121 ℃ in advance for standby;
precooling: all the used equipment and normal saline are put into 4 ℃ for precooling, and the subsequent experiment is carried out under ice bath;
sterilizing experimental equipment such as tissue shears, cutters, meat grinder, etc. with ethanol, and sterilizing by irradiating with ultraviolet for 30 min.
2. Experimental procedure
Sterilizing experimental equipment such as tissue shears, cutters, meat grinder and the like by alcohol, and then putting the experimental equipment into ultraviolet radiation for 30min in advance for sterilization; opening the ice maker to prepare sufficient ice;
taking out aseptically preserved placenta caprae seu ovis, thawing, washing with pre-cooled normal saline to meat color, removing surface lipid, connective tissue and blood clot, and drying with absorbent paper;
tissue sample pretreatment and consumable preparation: mincing placenta tissue with a meat mincer, mincing into minced meat, transferring to a 2mL centrifuge tube, each tube about 0.3-0.4g, sub-packaging, and transferring to-80deg.C for pre-freezing overnight;
preparing an extract: 0.86% physiological saline +1% pmsf +1% protein phosphatase inhibitor, ready to use as prepared, on ice;
homogenizing: taking out the pre-frozen placenta tissue from a refrigerator at-80 ℃, adding the extract according to the weight (g) of volume (mL) =1:5, and homogenizing by using a low-temperature tissue homogenizer (frequency 60Hz, time 30 s/time, gap 30s, 3-5 times continuously);
uniformly pouring the homogenate into a beaker, placing the beaker on ice, and gently stirring the homogenate for 30-60min at 4 ℃ so as to fully extract active substances, wherein bubbling is avoided in the homogenate process;
and (3) centrifuging: stirring, collecting homogenate, centrifuging at 4deg.C for 20min at 10000r, and removing cell debris and other substances;
and (3) filtering: eight layers of gauze (or a layer of glass wool) are laid in a filter funnel to filter the supernatant, remove lipid substances floating on the surface, and the gauze is carefully wrung out so as to obtain the maximum amount of filtrate, which is called crude extract;
ultrasonic: performing ultrasonic treatment on the obtained crude extract by using an ultrasonic breaker, performing ultrasonic treatment for 5min at 200Hz, and performing operation on ice;
microfiltration: microfiltration is carried out by using a water system microporous filter membrane with the diameter of 0.45 mu m and a microporous filter membrane filter to obtain a microfiltration liquid;
ultrafiltration segmentation: carrying out ultrafiltration segmentation by adopting an ultrafiltration device pressurized by nitrogen, respectively selecting ultrafiltration membranes of 50KD and 100KD, and carrying out ultrafiltration to obtain placenta caprae seu ovis extract with molecular weight of more than 100KD and placenta caprae seu ovis extract with molecular weight of 50-100KD;
and (3) freeze-drying: lyophilizing the active substance of placenta caprae seu ovis extract to obtain lyophilized powder.
Example 2 anti-tumor Activity assay
The whole extract of sheep placenta extracted from natural sheep placenta was evaluated for antitumor activity and toxicity by the sheep placenta extract prepared in example 1 above.
In vitro anticancer activity on different human cancer cell lines by MTT method. The specific method comprises the following steps: human colon cancer cell HCT116, human gastric cancer cell SGC7901, human pancreatic cancer cell PANC-1, human lung cancer cell A549 and human normal intestinal epithelial cell HIEC were taken in 96-well plates (1×10) 4 Individual cells/100 μl), preincubated for 12h at 37 ℃ to allow adherence. Incubating the cells with sheep placenta extracts of different molecular weights for 48h; after the incubation, 10. Mu.L of MTT solution (5 mg/mL) was added to each well and incubation was continued for 4h at 37 ℃; the medium was discarded, 100. Mu.L of DMSO was added to each well, and the mixture was then passed through a microplate reader (SpectraMax190, usa) determines the IC of the derivative by reading the OD value of each well at a wavelength of 490nm 50 Values.
The results show that:
(1) whole extract of placenta caprae seu ovis extracted from placenta caprae seu ovis of natural product, and above extract of placenta caprae seu ovis with molecular weight greater than 50KD prepared in example 1, IC of extract of placenta caprae seu ovis with molecular weight of 50-100KD on cancer cells 50 The values are shown in Table 1: IC of sheep placenta total extract for colon cancer cell 50 11726.69. Mu.g/ml; IC of sheep placenta extract with molecular weight less than 50KD for colon cancer cells 50 8456.54. Mu.g/ml; IC of sheep placenta extract with molecular weight greater than 100KD for colon cancer cells 50 2118.37. Mu.g/ml; IC of 50-100KD molecular weight sheep placenta extract for colon cancer cells 50 555.38. Mu.g/ml; compared with the whole extract of the sheep placenta, only the sheep placenta extract with the molecular weight larger than 50KD, especially with the molecular weight of 50-100KD, can obviously inhibit the proliferation of colon cancer cells, and the sheep placenta extract with the molecular weight larger than 50KD, especially with the molecular weight of 50-100KD, can obviously inhibit the proliferation of colon cancer cells, has no inhibition effect on normal cells and has good safety.
TABLE 1 IC of sheep placenta extracts of different molecular weight segments for colon cancer cells 50 Value of
| Cells | Sheep placenta extract | Greater than 100KD | 50-100KD | Less than 50KD |
| HCT116 | 11726.69μg/ml | 2118.37±56.83μg/ml | 555.38±254.73μg/ml | 8456.54±2369.83μg/ml |
| HIEC | - | - | - | - |
(2) Placenta Caprae Seu Ovis extract with molecular weight greater than 50KD and IC of placenta Caprae Seu Ovis extract with molecular weight of 50-100KD for different cancer cells 50 The values are shown in Table 2: the invention provides an IC of a sheep placenta extract with molecular weight of more than 100KD to human colon cancer cells HCT116, human gastric cancer cells SGC7901, human pancreatic cancer cells PANC-1 and human lung cancer cells A549 50 2118.37 μg/ml, 5079.39 μg/ml, 24746.03 μg/ml and 14870.17 μg/ml, respectively; the invention provides an IC of 50-100KD molecular weight section sheep placenta extract to human colon cancer cell HCT116 and human gastric cancer cell SGC7901 50 555.38. Mu.g/ml, 4524.69. Mu.g/ml, respectively, without detection of IC of human pancreatic cancer cells PANC-1 and human lung cancer cells A549 50 The method comprises the steps of carrying out a first treatment on the surface of the Compared with other cancer cells, the molecular weight range of more than 50KD, especially the molecular weight range of 50-100KD, of the sheep placenta extract provided by the invention can specifically inhibit proliferation of colon cancer cells, and has a remarkable inhibition effect.
TABLE 2 IC of placenta Caprae Seu Ovis extract with molecular weight of more than 50KD for different cancer cells 50 Value of
| Greater than 100KD | 50-100KD | |
| HCT116 | 2118.37±56.83μg/ml | 555.38±254.73μg/ml |
| SGC7901 | 5079.39±2792.86μg/ml | 4524.69±2890.89μg/ml |
| PANC-1 | 24746.03μg/ml | - |
| A549 | 14870.17μg/ml | - |
In conclusion, the sheep placenta extract with the molecular weight of more than 50KD, especially 50-100KD, has remarkable inhibitory activity on liver cancer and breast cancer cells; and the safety is good, and the medicine for treating liver cancer or breast cancer cells can be prepared.
Claims (1)
1. The application of the sheep placenta extract in preparing the anti-colon cancer medicament is characterized in that the molecular weight of the sheep placenta extract is 50-100KD; the preparation method of the sheep placenta extract comprises the following steps:
(1) Pretreatment: removing surface lipid, connective tissue and blood clot of placenta caprae seu ovis, and mincing to obtain placenta tissue;
(2) Extraction: adding an extracting solution into the placenta tissue in the step (1) according to the weight (g): volume (mL) =1:5 for homogenization; the extract consists of 0.86% (m/m) physiological saline, 1% (v/v) PMSF and 1% (v/v) protein phosphatase inhibitor; the homogenization parameters are as follows: 60Hz,30 s/time, 30s gap, 3-5 times continuously; homogenizing, and stirring at 4deg.C for 30-60min;
(3) Collecting homogenate, and centrifuging at 4deg.C for 20min at 10000 r; filtering to obtain a crude extract;
(4) Carrying out 200Hz ultrasonic treatment on the crude extract obtained in the step (3) for 5min, and then carrying out microfiltration by using a 0.45 mu m water system microporous filter membrane and a microporous filter membrane filter to obtain a microfiltration liquid;
(5) Ultrafiltration: and (3) respectively selecting ultrafiltration membranes of 50KD and 100KD to carry out ultrafiltration on the micro-filtrate obtained in the step (4) to obtain the sheep placenta extract with the molecular weight of 50-100KD.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107118257A (en) * | 2017-05-17 | 2017-09-01 | 兰州名德农牧科技有限公司 | A kind of industrialized Goat Placenta-peptide extracting method |
| CN107254502A (en) * | 2017-07-26 | 2017-10-17 | 兰州名德农牧科技有限公司 | A kind of method that animal protease prepares Goat Placenta-peptide |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107118257A (en) * | 2017-05-17 | 2017-09-01 | 兰州名德农牧科技有限公司 | A kind of industrialized Goat Placenta-peptide extracting method |
| CN107254502A (en) * | 2017-07-26 | 2017-10-17 | 兰州名德农牧科技有限公司 | A kind of method that animal protease prepares Goat Placenta-peptide |
Non-Patent Citations (3)
| Title |
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| Placenta Peptide Can Protect Mitochondrial Dysfunction through Inhibiting ROS and TNF-α Generation, by Maintaining Mitochondrial Dynamic Network and by Increasing IL-6 Level during Chronic Fatigue;Rekik A Muluye等;Front Pharmacol.;第7卷;328 * |
| 山羊胎盘肽的分离和初步鉴定及营养、免疫、抗氧化作用的研究;王炳祥;中国优秀硕士学位论文全文数据库农业科技辑;第2011年卷(第08期);D050-37 * |
| 牛羊胎盘免疫调节因子的提取及对PANC-Ⅰ增殖和迁移影响的比较;王晶等;农业生物技术学报;第24卷(第12期);1863-1871 * |
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