CN114848676A - Application of sheep placenta extract with molecular weight of more than 100KD in preparation of antitumor drugs - Google Patents
Application of sheep placenta extract with molecular weight of more than 100KD in preparation of antitumor drugs Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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Abstract
The invention belongs to the technical field of anti-tumor, and particularly relates to application of a sheep placenta extract with a molecular weight of more than 100KD in preparation of an anti-tumor medicament. The invention unexpectedly discovers that compared with the sheep placenta extract with the full molecular weight segment (sheep placenta full extract) and other sheep placenta extracts with the molecular weight segments, the sheep placenta extract with the molecular weight segment of more than 100KD has the obvious effect of inhibiting the proliferation of liver cancer and breast cancer cells; compared with other tumor cells, the sheep placenta extract with the molecular weight of more than 100KD has specificity on liver cancer and breast cancer, and can specifically inhibit the proliferation of liver cancer and breast cancer cells; meanwhile, the sheep placenta extract with the molecular weight of more than 100KD has good safety, can be used for preparing antitumor drugs, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of anti-tumor, and particularly relates to application of a sheep placenta extract with a molecular weight of more than 100KD in preparation of an anti-tumor medicament.
Background
Malignant tumors are a large group of diseases that endanger human health, and 800 million people die of cancer every year worldwide. The generation and development of tumors are a dynamic process involving the interaction of a plurality of signal molecules, and a complex molecular regulation network is formed. Despite the great progress made in the medical treatment of tumors, it is becoming an indispensable main measure for current clinical treatment. However, the problems of high toxic and side effects, drug resistance and the like are still the main obstacles in clinical tumor drug treatment.
Among them, liver cancer is one of the clinically common malignant tumors of the digestive system, of which 90% is hepatocellular carcinoma, and the incidence rate of which tends to increase. China is a big liver cancer country and is also the country with the highest incidence rate of liver cancer worldwide, and liver cancer is called as 'king of cancer' in China, and poses serious threat to the health of people in China. Liver cancer is a heterogeneous disease derived from chronic liver diseases such as chronic inflammation and cirrhosis. To date, therapeutic strategies for liver cancer have primarily included resection, transplantation, local ablation, and chemotherapy. Since early symptoms of liver cancer are not obvious, most liver cancer patients are diagnosed at a late stage, the chance of operation is lost, and most of the patients who have undergone the operation in time still have relapse and metastasis. In the past decades, despite significant advances in conventional treatment regimens for liver cancer patients, the treatment is limited, the prognosis is poor, and the recurrence rate is high, which remains one of the most fatal malignancies worldwide. New effective and promising therapeutic strategies still need to be explored extensively on a global scale.
The breast cancer is a main cause of cancer-related death of women worldwide, the prevalence rate of the breast cancer is on the rise, the breast cancer becomes a common disease and a frequently encountered disease which seriously affect the physical and mental health of women and even endanger life, and the breast cancer becomes a malignant tumor with the highest morbidity of women in recent years and is the first killer of the health of women. Chemotherapy is one of the three main basic means of breast cancer treatment, and although many advances have been made in the current chemotherapy of breast cancer, the current anticancer drugs have large toxic and side effects, poor selectivity and drug resistance, so that the expected curative effect of breast cancer treatment cannot be achieved. Therefore, the search for new medicines with small toxic and side effects and low treatment dosage has important significance.
Moreover, most cancer chemotherapeutic drugs are currently toxic to the host. Therefore, the provision of specific and safe natural drugs is of great significance for the treatment of cancer, especially liver cancer and breast cancer. The sheep placenta extract is a general term for various active substances extracted from sheep placenta, 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 tonifying qi and blood, replenishing essence and tonifying the kidney, regulating the immunity of the organism, delaying aging, whitening and tendering the skin, keeping the body shape and the like. Can be used for preventing liver cirrhosis, improving immunity, and treating immune diseases such as viral hepatitis, leukopenia, myasthenia gravis, etc. Compared with the whole sheep placenta powder, the sheep placenta extract with the molecular weight of more than 100KD has the obvious effect of inhibiting the proliferation of liver cancer cells and breast cancer cells, has good safety and can be used for preparing anti-tumor medicaments.
Disclosure of Invention
Aiming at the technical problems, the invention unexpectedly discovers that compared with a sheep placenta whole extract, the sheep placenta extract with the molecular weight of more than 100KD has the obvious effect of inhibiting the proliferation of liver cancer cells and breast cancer cells, has good safety and can be used for preparing anti-tumor drugs. Therefore, the invention aims to provide the application of the sheep placenta extract with the molecular weight of more than 100KD in preparing the anti-tumor medicament. The method specifically comprises the following steps:
in a first aspect, the invention provides an application of a sheep placenta extract in preparing an anti-tumor medicament, wherein the molecular weight of the sheep placenta extract is more than 100 KD.
Preferably, the tumor is liver cancer and breast cancer.
Preferably, the preparation method of the sheep placenta extract comprises the following steps: ultrafiltering the fresh sheep placenta extractive solution with ultrafiltration membrane of 100KD to obtain sheep placenta extract with molecular weight of more than 100 KD.
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 extract into the placenta tissue in the step (1) for homogenate extraction, wherein the extract consists of normal saline, PMSF and a protein phosphatase inhibitor;
(3) centrifuging and filtering to obtain a crude extract;
(4) performing ultrasonic treatment on the crude extract obtained in the step (3), and then performing microfiltration to obtain a micro-filtrate;
(5) and (3) ultrafiltration: ultrafiltering the micro filtrate obtained in step (4) with 100KD ultrafiltration membrane to obtain sheep placenta extract with molecular weight of more than 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 (phenyl methionine fluoride, hereinafter referred to as "PMSF").
Preferably, the ratio of the placenta tissue to the extract in the step (2) is 1 g: 5 ml.
Preferably, the homogenization parameters in step (2) are: 60Hz, 30 s/time, and 3-5 times continuously; homogenizing, and stirring the homogenate at 4 deg.C for 30-60 min.
Preferably, the centrifugation parameters in the step (3) are as follows: 4 ℃, 10000r, 20 min.
Preferably, the ultrasonic parameters in the step (4) are 200Hz, and the ultrasonic time is 5 min; the microfiltration is performed using a 0.45 μm water system 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 preparation.
The invention has the beneficial effects that: the invention unexpectedly discovers that compared with the whole sheep placenta extract and sheep placenta extracts with other molecular weight segments, the sheep placenta extract with the molecular weight segment of more than 100KD has obvious effect of inhibiting the proliferation of liver cancer and breast cancer cells; compared with other tumor cells, the sheep placenta extract with the molecular weight of more than 100KD has specificity on liver cancer and breast cancer, and can specifically inhibit the proliferation of liver cancer and breast cancer cells; meanwhile, the sheep placenta extract with the molecular weight of more than 100KD 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 clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The specific conditions are not specified in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The human cancer cell lines used in the following examples include breast cancer cell line MCF-7, liver cancer SMMC-7721, human colon cancer cell HCT116, human stomach cancer cell SGC7901 and AGC, 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., Beijing, China); the culture conditions were: 37 ℃ and 5% CO 2 Conditions, all cells were supplemented with 10% Fetal Bovine Serum (FBS), 100U/mL penicillin and 100mg/mL streptomycin.
Example 1 preparation of sheep placenta extract having molecular weight of greater than 100KD
1. Materials and methods
Experimental materials and equipment preparation: fresh sheep placenta, sodium chloride, 1L blue cap bottle multiplied by 2, tissue scissors, a cutter, a meat grinder, a triangular funnel, absorbent paper, a beaker and a centrifuge tube (pre-sterilization);
sterile 1L of 0.86% physiological saline was prepared: weighing 7.74g of sodium chloride by using an analytical balance, dissolving in 900mL of distilled water, sterilizing at 121 ℃, and cooling for later use;
sterilizing the centrifugal tube at 121 ℃ in advance for later use;
pre-cooling: all the used equipment and normal saline are placed into 4 ℃ for precooling, and subsequent experiments are carried out in an ice bath;
sterilizing experimental instruments such as tissue scissors, cutter, meat grinder, etc. with ethanol, and sterilizing by irradiating with ultraviolet for 30 min.
2. Procedure of experiment
Sterilizing experimental instruments such as tissue scissors, cutter, meat grinder, etc. with ethanol, and sterilizing by irradiating with ultraviolet for 30 min; opening the ice maker to prepare sufficient ice;
taking out aseptically preserved placenta caprae seu ovis, thawing, cleaning with pre-cooled normal saline to flesh color, removing surface lipid, connective tissue and blood clot, and drying with absorbent paper;
tissue sample pretreatment and consumable preparation: mincing placenta tissue with meat mincer, transferring to 2mL centrifuge tube with each tube about 0.3-0.4g, packaging, and pre-freezing at-80 deg.C overnight;
preparing an extraction solution: 0.86% of normal saline, 1% of PMSF and 1% of protein phosphatase inhibitor, wherein the preparation is prepared immediately before use and is operated on ice;
homogenizing: taking out the pre-frozen placenta tissue from a refrigerator at-80 deg.C, adding the extractive solution according to weight (g) volume (mL) of 1:5, and homogenizing with a low temperature tissue homogenizer (frequency 60Hz, time 30 s/time, interval 30s, continuous 3-5 times);
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 no bubbles are generated in the homogenate process;
centrifuging: stirring, collecting homogenate, centrifuging at 4 deg.C at 10000r for 20min, and removing cell debris and other substances in homogenate;
and (3) filtering: filtering the supernatant by spreading eight layers of gauze (or adding one layer of glass wool) in the filter funnel, removing lipid floating on the surface, and carefully wringing out the gauze to obtain maximum amount of filtrate, which is called coarse extract;
ultrasonic: carrying out ultrasonic treatment on the obtained crude extract by using an ultrasonic crusher at 200Hz for 5min, and carrying out ice operation;
and (3) microfiltration: performing microfiltration by using a 0.45-micron water system microfiltration membrane and a microfiltration membrane filter to obtain a microfiltration solution;
and (3) ultrafiltration segmentation: carrying out ultrafiltration segmentation by adopting an ultrafiltration device pressurized by nitrogen, selecting an ultrafiltration membrane with the molecular weight of 100KD, and carrying out ultrafiltration to obtain a sheep placenta extract with the molecular weight of more than 100 KD;
freeze-drying: freeze-drying the active substance of sheep placenta extract with molecular weight greater than 100KD obtained by ultrafiltration to obtain freeze-dried powder.
Example 2 antitumor Activity assay
The sheep placenta whole extract extracted from natural product sheep placenta and the sheep placenta extract with molecular weight of more than 100KD prepared in the above example 1 were evaluated for anti-tumor activity and toxicity.
In vitro anti-cancer activity on different human cancer cell lines by the MTT method. The specific method comprises the following steps: respectively taking breast cancer cell line MCF-7, liver cancer SMMC-7721, human colon cancer cell HCT116, human stomach cancer cell SGC7901 and AGC, human pancreatic cancer cell PANC-1, human lung cancer cell A549 and human normal intestinal epithelial cell HIEC in 96-well plate (1 × 10) 4 Individual cells/100 μ L) and preincubated at 37 ℃ for 12h to allow attachment. Incubating the cells with sheep placenta extract of different doses and different molecular weights for 48 h; after the incubation is finished, 10 mu L of MTT solution (5mg/mL) is added into each well, and the incubation is continued for 4h at 37 ℃; the medium was discarded, 100. mu.L of DMSO was added to each well, and the IC of the derivative was determined by reading the OD of each well at 490nm using a microplate reader (SpectraMax190, USA) 50 The value is obtained.
The results show that:
the whole extract of sheep placenta extracted from natural product sheep placenta and the IC of the sheep placenta extract with molecular weight of more than 100KD prepared in the above example 1 to cancer cells 50 The values are shown in Table 1: IC of sheep placenta whole extract on liver cancer and breast cancer cells 50 185.98 mug/ml and 970.172 mug/ml respectively; IC of sheep placenta extract with molecular weight less than 50KD on liver cancer and breast cancer cells 50 474.86 mug/ml and 1297.72.172 mug/ml respectively; IC of 50-100KD molecular weight segment sheep placenta extract on liver cancer and breast cancer cells 50 227.79 mug/ml and 1021.35 mug/ml respectively; compared with the whole sheep placenta extract, only the sheep placenta extract with the molecular weight of more than 100KD provided by the invention can obviously inhibit the proliferation of liver cancer and breast cancer cells, and the IC of the sheep placenta extract 50 179.36 μ g/ml and 783.50 μ g/ml, respectively. The sheep placenta extract with the molecular weight of more than 100KD provided by the invention can obviously inhibit the liverProliferation of cancer and breast cancer cells, no inhibition to normal cells, and good safety.
TABLE 1 IC of sheep placenta extract on cancer cells with different molecular weights 50 Value of
| Cells | Sheep placenta whole extract | Greater than 100KD | 50-100KD | Less than 50KD |
| SMMC-7721 | 185.98±14.17μg/ml | 179.36±12.13μg/ml | 227.79±103.10μg/ml | 474.86±43.38μg/ml |
| MCF-7 | 970.172μg/ml | 783.50μg/ml | 1021.35μg/ml | 1297.72.172μg/ml |
| HIEC | - | - | - | - |
② IC of sheep placenta extract with molecular weight of more than 100KD for different cancer cells 50 The values are shown in Table 2: the sheep placenta extract with molecular weight of more than 100KD provided by the invention has IC of human colon cancer cells HCT116, human gastric cancer cells SGC7901 and AGC, human pancreatic cancer cells PANC-1 and human lung cancer cells A549 50 2118.37 μ g/ml, 5079.39 μ g/ml, 3398.09 μ g/ml, 24746.03 μ g/ml and 14870.17 μ g/ml, respectively; compared with other cancer cells, the sheep placenta extract with the molecular weight of more than 100KD provided by the invention can specifically inhibit the proliferation of liver cancer and breast cancer cells and has a remarkable inhibiting effect.
TABLE 2 IC of sheep placenta extract with molecular weight of more than 100KD for different cancer cells 50 Value of
| Cancer cells | IC 50 Value of |
| SMMC-7721 | 179.36±12.13μg/ml |
| MCF-7 | 783.50μg/ml |
| HCT116 | 2118.37±56.83μg/ml |
| SGC7901 | 5079.39±2792.86μg/ml |
| AGS | 3398.09±789.43μg/ml |
| PANC-1 | 24746.03μg/ml |
| A549 | 14870.17μg/ml |
In conclusion, the sheep placenta extract with the molecular weight section of more than 100KD has obvious inhibitory activity on liver cancer cells and breast cancer cells; and has good safety, and can be used for preparing medicines for treating hepatocarcinoma or breast cancer.
Claims (10)
1. The application of the sheep placenta extract in preparing the antitumor drugs is characterized in that the molecular weight of the sheep placenta extract is more than 100 KD.
2. The use of claim 1, wherein the tumor is liver cancer and breast cancer.
3. The use of claim 1, wherein said sheep placenta extract is prepared by a method comprising: ultrafiltering the fresh sheep placenta extractive solution with ultrafiltration membrane of 100KD to obtain sheep placenta extract with molecular weight of more than 100 KD.
4. The use as claimed in claim 3, wherein said fresh sheep placenta extract is prepared by a method comprising the steps of:
(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 extract into the placenta tissue in the step (1) for homogenate extraction, wherein the extract consists of normal saline, a protease inhibitor and a protein phosphatase inhibitor;
(3) centrifuging and filtering to obtain a crude extract;
(4) performing ultrasonic treatment on the crude extract obtained in the step (3), and then performing microfiltration to obtain a micro-filtrate;
(5) and (3) ultrafiltration: and (4) selecting an ultrafiltration membrane with the molecular weight of 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 more than 100 KD.
5. The use according to claim 4, wherein the composition of the extract in step (2) is: 0.86% (m/m) physiological saline, 1% (v/v) PMSF, 1% (v/v) protein phosphatase inhibitor.
6. The use of claim 5, wherein the ratio of placental tissue to extract in step (2) is 1 g: 5 ml.
7. The use according to claim 6, wherein the homogenization parameters in step (2) are: 60Hz, 30 s/time, and 3-5 times continuously; homogenizing, and stirring the homogenate at 4 deg.C for 30-60 min.
8. The use of claim 7, wherein the centrifugation parameters in step (3) are: 4 ℃, 10000r, 20 min.
9. The use of claim 8, wherein the ultrasound parameters in step (4) are 200Hz, ultrasound 5 min; the microfiltration is carried out using a 0.45 μm water system microfiltration membrane and a microfiltration membrane filter.
10. The use of any one of claims 1 to 9, wherein said sheep placenta extract is formulated with pharmaceutically acceptable carriers/excipients into any pharmaceutically acceptable dosage form.
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Citations (4)
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|---|---|---|---|---|
| CN101134050A (en) * | 2006-08-29 | 2008-03-05 | 江卫世 | Pharmaceutical composition for reinforcing organism immunity and technique of preparing the same |
| CN101856496A (en) * | 2010-06-07 | 2010-10-13 | 四川大学 | Placenta stem-cell anti-tumor vaccine, preparation method and application thereof |
| CN107216366A (en) * | 2017-07-26 | 2017-09-29 | 西藏天慈生物科技有限公司 | A kind of preparation method and applications of Goat Placenta small-molecular peptides |
| CN108753876A (en) * | 2018-05-25 | 2018-11-06 | 华子昂 | Utilize the method for sheep placenta extracts enhancing stem cell surface receptor CXCR4 expression |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101134050A (en) * | 2006-08-29 | 2008-03-05 | 江卫世 | Pharmaceutical composition for reinforcing organism immunity and technique of preparing the same |
| CN101856496A (en) * | 2010-06-07 | 2010-10-13 | 四川大学 | Placenta stem-cell anti-tumor vaccine, preparation method and application thereof |
| CN107216366A (en) * | 2017-07-26 | 2017-09-29 | 西藏天慈生物科技有限公司 | A kind of preparation method and applications of Goat Placenta small-molecular peptides |
| CN108753876A (en) * | 2018-05-25 | 2018-11-06 | 华子昂 | Utilize the method for sheep placenta extracts enhancing stem cell surface receptor CXCR4 expression |
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| Title |
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| LIU, J.W.等: "The Protective Effect of Sheep Placental Extract on Concanavalin A-induced Liver Injury in Mice", MOLECULES, pages 1 - 13 * |
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