CN101991608B - Fly maggot extractive as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses a fly maggot extract, the main component of which is water-soluble protein, and the mass percentage of the water-soluble protein in the fly maggot extract is 50%-70%; the molecular weight of the water-soluble protein is It is 2-16KDa. The fly maggot extract not only inhibits human myeloid leukemia HL-60 cells, human erythroleukemia K562 cells, human liver cancer SMMC-7721, mouse leukemia P388 cells, human lung adenocarcinoma A549 cells, and human nasopharyngeal carcinoma CNE cells in vitro. , human prostate cancer PC3 cells, and human cervical cancer HeLa have obvious inhibitory effects; and it has a significant inhibitory effect on mouse S ₁₈₀ sarcoma and mouse Heps liver cancer solid tumors, and it also has the effect of enhancing the body's humoral immunity. The invention also discloses a preparation method of the fly maggot extract, which is simple to operate, easy to control, low in cost and suitable for industrial production.

Description

Fly maggot extract and its preparation method and application

technical field

The invention relates to the field of medicinal extracts, in particular to a fly maggot extract and its preparation method and application. the

Background technique

The housefly larva is Chrysomiamegacephala (Fab.). As a medicine, it is recorded in "Compendium of Materia Medica" written by Li Shizhen in the Ming Dynasty: "Maggots, also known as 'grain worms', 'Luo Xianzi', and 'water fairy', can cure malnutrition fever in children and are good medicine for malnutrition. "Fly maggots are cold and non-toxic, and are a high-quality animal protein source. Not only is the protein content rich, but the amino acid components in the protein are also very reasonable and complete. The mass percentage of protein in fly maggot dry powder is generally about 62%. The mass percentage of fat is generally 10%-15%. It is reported that no pathogenic bacteria of any kind can survive in the fly maggot for more than seven days. Therefore, scientists believe that it most likely has a unique immune defense mechanism against pathogenic infection. the

Malignant tumor is a frequently-occurring and common disease that seriously threatens people's life and health. Every year, 7 million people in the world suffer from tumors, 5 million people are killed by tumors, and now there are more than 10 million cancer patients. At present, it is estimated that the annual number of cancer patients in the country is about 1.6 million, and the number of people who die from cancer diseases has reached 1.3 million, and there is still an upward trend. Malignant tumor diseases are surpassing cardiovascular diseases and becoming the number one cause of death in the population. In addition, the pain caused by tumors to patients, the mental and economic burdens on families, and the impact on social productivity are even more difficult to estimate. Therefore, all countries attach great importance to the prevention and treatment of tumors. the

Today, surgical treatment, radiotherapy, and chemical drug therapy are still the three most commonly used and most important treatments for tumor diseases. Although the development history of chemical drug therapy is relatively short, it is playing an increasingly important role in the comprehensive treatment of tumors. . However, there are great disadvantages in chemotherapeutics. This is mainly because the selective inhibition of tumor cells by existing antineoplastic drugs is not strong, the toxicity of systemic drugs is large, and most of them have immunosuppressive effects, which limits the its use. With my country's accession to WTO, western medicine will face severe challenges. Due to the protection of intellectual property rights, the imitation of western medicine will be restricted, and the research and development of traditional Chinese medicine products with independent intellectual property rights will be the key to the development of my country's pharmaceutical products. At the same time, the research and development of natural anti-tumor drugs with high efficiency and low toxicity is also an urgent and important global topic. the

In 2003, Qiu Xiaoyan from Jimei University studied the extraction of the house fly antimicrobial peptide and its inhibitory effect on tumor cell growth, showing that the extracted house fly antimicrobial peptide can effectively inhibit human gastric cancer cells MGC80-3 and BGC-823, human breast cancer cells The in vitro proliferation of MCF-7 and human lung cancer cell SPC-A-1 is in a concentration-dependent relationship (China Sanitary Insecticide, 2003, 9(1): 13-16). the

In 2006, Cao Xiaohong, Tianjin University of Science and Technology, etc. conducted research on the active substances in the engineering fly maggots and the anti-tumor effect in vitro. 1): 316-319). the

In 2008, Xu Shuixiang from the Zhejiang Academy of Medical Sciences and others studied the inhibitory effect of the extract of Chrysalis macrocephala on leukemia and lung cancer cells. The unsaturated fatty acids and crude fats of Chrysophalis macrocephala dry powder extracts were used for tumor inhibition tests in vitro respectively, and it was observed that unsaturated fatty acids and crude fats had obvious proliferation inhibitory effects on human myeloid leukemia HL-60 (Pharmaceutical Biotechnology, 2008 , 15(4):284-286). the

The studies on fly maggot extracts in the above literatures all stay at the in vitro cell level, and most of them are extracting antimicrobial peptides and fatty acids in fly maggots, and no further research has been carried out on other components in fly maggots. the

Contents of the invention

The invention provides a fly maggot extract which has significant inhibitory effect on various cancer cells. the

The invention also provides a preparation method of the fly maggot extract, which is simple to operate, easy to control, low in cost and suitable for industrial production. the

A fly maggot extract, the main component of which is water-soluble protein, the mass percentage of water-soluble protein in the fly maggot extract is 50%-70%; the molecular weight of the water-soluble protein is 2-16 Kilodaltons (KDa). the

In order to achieve a better invention effect, preferably:

The water-soluble protein is mainly composed of the following amino acids in mass percentage: aspartic acid 6.3%-7.2%, threonine 3.6%-4.0%, serine 3.6%-3.9%, glutamic acid 14.5%-15.5%, Glycine 5.5%-6.0%, Alanine 17.8%-19.6%, Cystine 1.6%-1.8%, Valine 5.6%-6.5%, Methionine 2.3%-2.4%, Isoleucine 3.1% -3.8%, Leucine 4.2%-5.2%, Tyrosine 2.5%-2.7%, Phenylalanine 2.6%-3.1%, Lysine 8.4%-8.9%, Histidine 3.4%-3.8%, Arginine 1.1%-1.3% and Proline 4.2%-4.8%. the

The mass percent content of sugar in the fly maggot extract is 0.5-2.0%. the

The preparation method of described fly maggot extract comprises steps:

(1) Pulverization: the fly maggots are rinsed, dried and crushed into fly maggot powder;

(2) degreasing: in the fly maggot powder that step (1) obtains, add the sherwood oil of 1-5 times volume of fly maggot powder, room temperature soaks 6-12 hour, remove sherwood oil, obtain the fly maggot powder after degreasing;

(3) Ultrasonic water extraction: in the degreased fly maggot powder obtained in step (2), add water ultrasonic extraction of 5-10 times the volume of the defatted fly maggot powder for 1-3 times, each ultrasonic 10-30 minutes , filtered; the filter residue was ultrasonically extracted 1-3 times with 4-8 times the volume of the filter residue, and filtered for 10-30 minutes each time; the secondary filtrate was combined; through centrifugation, ultrafiltration and freeze-drying, the fly maggot extraction was obtained. thing. the

The fly maggot extract can be used to prepare one or more medicines for treating leukemia, liver cancer, lung adenocarcinoma, nasopharyngeal cancer, prostate cancer, cervical cancer, etc., such as can be prepared for inhibiting human myeloid leukemia HL-60 cells, human erythroleukemia K562 cells, human liver cancer SMMC-7721 cells, mouse leukemia P388 cells, human lung adenocarcinoma A549 cells, human nasopharyngeal carcinoma CNE cells, human prostate cancer PC3 cells, human cervical cancer HeLa cells, Drugs for one or more of mouse S ₁₈₀ sarcoma and mouse Heps liver cancer solid tumor. The dosage form of the drug is preferably an injection, and the best route of administration is intravenous injection.

The fly maggot extract can also be used to prepare medicines for enhancing the humoral immunity of the body. The dosage form of the drug is preferably an injection, and the best route of administration is intravenous injection. the

The fly maggot extract can also be used to prepare a pharmaceutical composition containing the fly maggot extract. The dosage form of the pharmaceutical composition is preferably an injection, and the best route of administration is intravenous injection. the

The fly maggots described in the present invention can generally be selected from the fly maggots cultured by adopting the breeding method of the fly maggots with anti-tumor biological activity in Chinese invention patent ZL200510060277.4. the

Compared with prior art, the advantage of the present invention is:

The fly maggot extract of the invention has the effects of high efficiency, low toxicity, anti-tumor and enhancing body fluid immunity. This fly maggot extract not only inhibits human myeloid leukemia HL-60 cells, human erythroleukemia K562 cells, human liver cancer SMMC-7721, mouse leukemia P388 cells, human lung adenocarcinoma A549 cells, and human nasopharyngeal carcinoma CNE in vitro. Cells, human prostate cancer PC3 cells, and human cervical cancer HeLa have obvious inhibitory effects; and it has a significant inhibitory effect on mouse S180 sarcoma and mouse Heps liver cancer solid tumors, and it can also enhance the humoral immunity of the body. the

The preparation method of the invention is easy to operate, easy to control, low in cost, suitable for industrial production, and the obtained fly maggot extract is highly efficient and non-toxic, can be used to treat tumor patients, and has great potential for development. the

Description of drawings

Fig. 1 is the SDS-PAGE electrophoresis figure of the molecular weight of the fly maggot extract, fly maggot powder and protein molecular weight standard item (Mark) prepared by embodiment 1;

Fig. 2 is the morphological change of human myeloid leukemia HL-60 cell apoptosis caused by the fly maggot extract prepared by different concentrations of embodiment 1 under optical microscope; wherein, A represents the control group cells; B represents the fly maggot extract 0.056mg/ ml treatment group cells; C represents the fly maggot extract 0.167mg/ml treatment group cells; D represents the fly maggot extract 0.500mg/ml treatment group cells; scale bar (Bar) = 50μm;

Fig. 3 is the morphological change of human liver cancer SMMC-7721 cell apoptosis caused by the fly maggot extract prepared by different concentrations of embodiment 1 under optical microscope; wherein, A represents the control group cells; B represents the fly maggot extract 0.056mg/ml treatment group Cells; C represents the cells of the fly maggot extract 0.167mg/ml treatment group; D represents the fly maggot extract 0.500mg/ml treatment group cells; Bar=50μm;

Fig. 4 is the morphological changes of human myeloid leukemia HL-60 cells caused by the fly maggot extracts prepared in different concentrations of Example 1 under a fluorescence microscope after AO/EB fluorescence double staining; wherein, A and B represent control group cells; C and D represent the cells of the fly maggot extract 0.056mg/ml treatment group; E and F represent the fly maggot extract 0.167mg/ml treatment group cells; G and H represent the fly maggot extract 0.500mg/ml treatment group cells; A, C, E and G are cell morphology under common light source, B, D, F and H are intracellular AO/EB fluorescence under excitation light; Bar=50μm;

Fig. 5 is the morphological changes of human liver cancer SMMC-7721 cells caused by the fly maggot extract prepared by different concentrations of embodiment 1 under a fluorescent microscope after AO/EB fluorescent double staining; wherein, A and B represent control group cells; C and D Indicates the cells of the fly maggot extract 0.056mg/ml treatment group; E and F indicate the fly maggot extract 0.167mg/ml treatment group cells; G and H indicate the fly maggot extract 0.500mg/ml treatment group cells; A, C, E and G are cell morphology under ordinary light source, B, D, F and H are intracellular AO/EB fluorescence under excitation light; Bar=50μm;

Fig. 6 is Hoechst 33342/PI fluorescent double staining and observing the morphological changes of human myeloid leukemia HL-60 cells caused by the fly maggot extract prepared in different concentrations in Example 1 under a fluorescence microscope; wherein, A, B and C represent controls Group cells; D, E and F represent the cells of the fly maggot extract 0.056mg/ml treatment group; G, H and I represent the fly maggot extract 0.167mg/ml treatment group cells; J, K and L represent the fly maggot extract 0.500 The cells in the mg/ml treatment group; A, D, G, and J are cell morphology under normal light; B, E, H, and K are Hoechst fluorescence in cells under excitation light; C, F, I, and L are cells in cells under excitation light PI fluorescence; Bar=25μm;

Fig. 7 is Hoechst 33342/PI fluorescence double staining and observes under the fluorescence microscope the morphological changes of human liver cancer SMMC-7721 cells caused by the fly maggot extract prepared in different concentrations embodiment 1; wherein, A, B and C represent the control group cells; D, E and F represent the cells of the fly maggot extract 0.056mg/ml treatment group; G, H and I represent the fly maggot extract 0.167mg/ml treatment group cells; J, K and L represent the fly maggot extract 0.500mg/ml Cells in treatment group; A, D, G and J are cell morphology under common light source; B, E, H and K are intracellular Hoechst fluorescence under excitation light; C, F, I and L are intracellular PI fluorescence under excitation light; Bar=25μm;

Fig. 8 is the case of phosphatidylserine externalization in human myeloid leukemia HL-60 cells caused by the fly maggot extracts prepared in different concentrations in Example 1 under a fluorescence microscope after Annexin V/PI fluorescent double staining; wherein, A, B and C Indicates the cells of the control group; D, E and F indicate the cells of the fly maggot extract 0.056mg/ml treatment group; G, H and I indicate the fly maggot extract 0.167mg/ml treatment group cells; J, K and L indicate the fly maggot extract 0.500mg/ml treatment group cells; A, D, G and J are cell morphology under common light source; B, E, H and K are cell Annexin V fluorescence under excitation light; C, F, I and L are cells under excitation light Intracellular PI fluorescence; Bar=50μm;

Figure 9 is the observation under a fluorescence microscope of different concentrations of fly maggot extracts prepared in Example 1 after Annexin V/PI fluorescent double staining causes human liver cancer SMMC-7721 cell phosphatidylserine eversion; wherein, A, B and C represent the control group Cells; D, E and F represent the cells of the fly maggot extract 0.056mg/ml treatment group; G, H and I represent the fly maggot extract 0.167mg/ml treatment group cells; J, K and L represent the fly maggot extract 0.500mg /ml treatment group cells; A, D, G and J are cell morphology under normal light source; B, E, H and K are cells Annexin V fluorescence under excitation light; C, F, I and L are intracellular PI under excitation light Fluorescence; Bar=50μm;

Figure 10 is the observation of the cell morphology changes of human myeloid leukemia HL-60 caused by the fly maggot extract prepared in Example 1 of the test substance under a transmission electron microscope; wherein, A represents the cells of the control group; B represents 0.500 mg of the fly maggot extract /ml treatment group cells; Bar=5μm;

Figure 11 is the observation of the cell morphology changes of human liver cancer SMMC-7721 cells caused by the fly maggot extract prepared in Example 1 of the test substance under a transmission electron microscope; wherein, A represents the cells of the control group; B represents the fly maggot extract 0.500mg/ml Treatment group cells; Bar=5 μm. the

Detailed ways

Embodiment 1: Preparation of fly maggot extract

(1) Special cultured fly maggots

Chrysocephalus chinensis takes animal protein 35%, 40%, 45%, brown sugar 20%, 25%, 30%, fruit 30%, 35%, 40% as food, wherein animal protein is maggot pulp. The traditional Chinese medicine Epimedium 20%, 25%, 30%, plus lysine 15%, 20%, 25%, methionine 15%, 20%, 25%, vitamin B2 25%, 30%, 35% are used as "ovum urging". "Prime", collect fly eggs with egg collection trays. Use a ventilated fly maggot room, place multiple fly maggot ponds, put fly maggot bait in the maggot breeding pond, and put the above-mentioned fly eggs at the same time, control the temperature at 28°C-60°C, use the bait that has been crushed and fermented for 4-6 days Feed maggots. Feed selection A: Frog 15%, 23%, 30%, freshwater fish 15%, 23%, 30%, Cordyceps 0.1%, 0.15%, 0.2%, Ganoderma lucidum powder 1%, 1.5%, 2%, chicken 20%, 25%, 30%, fruit 25%, 30%, 35%, the rest is plant feed; bait selection B: pig lung 25%, 30%, 35%, frog 15%, 23%, 30%, freshwater fish 15%, 23%, 30%, 1%, 1.5%, 2% of ganoderma powder, 0.1%, 0.15%, 0.2% of ginseng powder, 20%, 25%, 30% of fruit, and the rest are plant feed; bait C is frog, fresh water Fish or pork lungs. the

The fly maggots from the above-mentioned special breeding are rinsed, freeze-dried and crushed to obtain the fly maggot powder. the

(2) Extraction of fly maggot powder

50g of fly maggot powder was added with 5 times the volume of petroleum ether of fly maggot powder, soaked at room temperature for 6 hours, and the petroleum ether was removed to obtain degreased fly maggot powder. the

Add 10 times the volume of the degreased fly maggot powder to the degreased fly maggot powder for ultrasonic extraction 3 times, each ultrasonic extraction for 10 minutes, and filter; the filter residue is then ultrasonically extracted 3 times with 8 times the volume of the filter residue, Ultrasonic extraction for 10 minutes each time, filtering, and combining the secondary filtrates; centrifuging at 5°C at 16,000 rpm for 30 minutes, and freeze-drying the ultrafiltrate after ultrafiltration with an ultrafiltration membrane to obtain a brown-yellow fly maggot extract 9.6g. the

The mass percentage of water-soluble protein in the fly maggot extract was determined by Lowry method to be 59.3%; the mass percentage of sugar in the fly maggot extract was determined to be 1.0% by phenol sulfuric acid method; the SDS-PAGE electrophoresis technique was used to identify the The molecular weight of fly maggot extract is 2-16KDa, see Fig. 1; Measure the amino acid composition of this fly maggot extract with amino acid analyzer, detect the concentration of fly maggot extract in sample and be 1.1320 mg/ml (solvent is water), detect The results are shown in Table 1. the

Table 1

Embodiment 2: Preparation of fly maggot extract

Add the petroleum ether of 2 times the volume of the fly maggot powder to the 50 g of the fly maggot powder prepared by special cultured fly maggot in Example 1, soak at room temperature for 12 hours, remove the petroleum ether, and obtain the fly maggot powder after degreasing. the

Add 10 times the volume of the degreased fly maggot powder to the degreased fly maggot powder for 2 times, ultrasonic extraction for 10 minutes each time, filter; Ultrasonic extraction for 10 minutes, filtered, and combined secondary filtrate; centrifuged at 5°C at 16,000 rpm for 30 minutes, and freeze-dried the ultrafiltrate after ultrafiltration with an ultrafiltration membrane to obtain a brown-yellow fly maggot extract 7.4 g. the

The mass percentage of water-soluble protein in the fly maggot extract was determined by Lowry method to be 62.7%; the mass percentage of sugar in the fly maggot extract was determined to be 1.8% by phenol sulfuric acid method; the SDS-PAGE electrophoresis technique was used to identify the The molecular weight of the fly maggot extract is 2-16KDa; Measure the amino acid composition of this fly maggot extract with an amino acid analyzer, and the concentration of the fly maggot extract in the detection sample is 1.7940 mg/ml (solvent is water), and the test results are shown in Table 2 . the

Table 2

Example 3

Preparation of fly maggot extract injection

Prescription: the fly maggot extract obtained in Example 1 125g

Add water for injection to 1000mL. the

Preparation method: add water for injection to 1000 mL to 125 g of the fly maggot extract prepared in Example 1, maintain isotonic and isotonic pH 7.0 to 7.2, filter, fill and seal in an ampoule bottle to prepare the fly maggot extract injection. The whole process is sterile, depyrogenated, and allergen-free. the

1. The fly maggot extract obtained in Example 1 was used for broad-spectrum screening of human tumor cell lines cultured in vitro by the thiazolium blue (MTT) method, and the broad-spectrum anti-tumor effect of the fly maggot extract was detected in vitro. the

1 cell line:

Human myeloid leukemia HL-60, human liver cancer SMMC-7721, human erythroleukemia K562, mouse leukemia P388, human lung adenocarcinoma A549, human prostate cancer PC3, human nasopharyngeal carcinoma CNE, and human cervical cancer Hela. the

2 methods

Take the tumor cells in the logarithmic growth phase, adjust the concentration of the cell suspension, inoculate 100 μl of the cell suspension in each well in a 96-well cell culture plate, administer the drug (100 μl/well) 24 hours after inoculation, set up a blank control group, a cell control group and 5 doses of test drug groups. After continuing to cultivate for 72 hours, add 100 μl MTT (1mg/ml, dissolved in DMEM culture solution) to each well, incubate at 37°C for 2 hours, discard the liquid in each well, add 150 μl acidified isopropanol (containing 0.04mol/L HCl), and store in the dark After 30 minutes, the DG3022A enzyme-linked immunosorbent assay instrument measured the absorbance at 570nm, and calculated the inhibition rate of the tested drug on the proliferation of various tumor cells. the

3 results:

Table 3 Fly maggot extract inhibits tumor cell proliferation in vitro (%)

The fly maggot extract prepared in Example 1 has a proliferation inhibitory effect on various tumor cells in vitro, among which it is effective on leukemia cells (including human myeloid leukemia HL-60 cells, human erythroleukemia K562 cells and mouse leukemia P388 cells) And the inhibitory effect on human liver cancer SMMC-7721 cell proliferation is relatively strong. the

Two, investigate the inhibitory action of fly maggot extract of the present invention to mouse _S180 sarcoma and mouse Heps liver cancer solid tumor

1 method

Aseptically extract ascites tumor fluid from S ₁₈₀ mice or Heps mice, count under a microscope, adjust the concentration of the cell suspension, inoculate 0.2ml of the above ascites tumor fluid subcutaneously in the right axilla of the mice, and randomly group them into groups according to body weight the next day. It is the tumor-bearing model group, the positive control group (cisplatin) and the test drug groups of different doses, namely 0.400g/kg, 0.160g/kg and 0.064g/kg (expressing the grams of fly maggot extract injected per kilogram of mice) , using the fly maggot extract prepared in Example 1). Each group was administered once a day for 9 consecutive days. Animals were sacrificed 24 hours after the last administration, body weight was weighed, tumor mass was dissected and weighed, tumor inhibition rate was calculated, thymus and spleen were weighed, and thymus index and spleen index were calculated.

Data statistics: Calculate the mean and standard deviation of body weight, tumor weight, and visceral index of animals in each group, and use t test for statistical analysis. the

2 result

The inhibitory effect of table 4 fly maggot extract on mouse _S180 sarcoma

Mean±SD, * means P<0.05, ** means P<0.01, *** means P<0.001 vs. tumor-bearing model, t-test. the

Table 5 Inhibitory effect of fly maggot extract on mouse Heps liver cancer solid tumor

Mean±SD, *P means <0.05, ** means P<0.01, *** means P<0.001 vs. tumor-bearing model, t-test. the

As can be seen from the results of table 4 and table 5, the injection dose of fly maggot extract of the present invention is that 0.064-0.400g/kg has significant inhibitory effect on mouse _S180 sarcoma (P＜0.05); injection dose is 0.026-0.160g /kg has significant inhibitory effect on mouse Heps liver cancer solid tumor (P<0.05).

3. Investigate the effect of the fly maggot extract of the present invention on inducing tumor cell apoptosis in vitro

1 cell line

Human myeloid leukemia HL-60 cells and human liver cancer SMMC-7721 cells

2 Morphological detection of apoptosis

Take the tumor cells in the logarithmic growth phase, adjust the concentration of the cell suspension, inoculate 3ml of the cell suspension in each hole in a 6-well cell culture plate, administer the drug (3ml/well) 24 hours after inoculation, and set up a cell control group and different concentrations of the test In the drug treatment group, after 24 hours of continuous culture, optical microscope, fluorescence microscope observation (AO/EB fluorescence double staining, Hoechst 33342/PI fluorescence double staining), transmission electron microscope observation, AnnexinV detection, and flow cytometry detection of cell apoptosis. the

3 results

The results show that the fly maggot extract prepared in Example 1 of the test substance can significantly inhibit the proliferation of human myeloid leukemia HL-60 cells and human liver cancer SMMC-7721 cells in a concentration-dependent manner, with IC50 being 0.384±0.029 respectively mg/ml and 0.349±0.019mg/ml; optical microscope and transmission electron microscope observation showed significant changes in cell morphology, see Figure 2, Figure 3, Figure 10 and Figure 11; AO/EB fluorescence double staining and Hoechst 33342/PI fluorescence double Observation under the fluorescent microscope after staining showed that HL-60 cells and SMMC-7721 cells in the fly maggot extract 0.500mg/ml treatment group showed typical apoptosis changes, and even cell necrosis, as shown in Figure 4, Figure 5, and Figure 5. 6 and Figure 7; after AnnexinV/PI fluorescent double staining, it was observed under a fluorescence microscope that the membrane phosphatidylserine of HL-60 cells and SMMC-7721 cells were everted, which further proved that the cells in the fly maggot extract 0.500mg/ml treatment group undergo apoptosis, see Figure 8 and Figure 9; Quantitative detection by flow cytometry shows that the apoptosis rate is above 30%. the

4. Screening of the effect of fly maggot extract on immune enhancement in vivo

1 grouping: tumor-bearing model group, positive control group (thymosin) and different doses of test drug groups, namely 0.400g/kg, 0.160g/kg and 0.064g/kg (representing the grams of fly maggot extract injected per kilogram of mice) number, using the fly maggot extract prepared in Example 1)

2 methods:

(1) Immunoenhancement test of mice bearing S ₁₈₀ sarcoma in vivo: grouping and administration: aseptically extract ascites from S ₁₈₀ mice, count under a microscope, adjust the concentration of cell suspension to 4×10 ⁷ /ml, and place on the right side of the mouse Aseptically inoculate 0.2ml of ascites tumor fluid subcutaneously in the armpits (the number of cells inoculated per mouse is 8×10 ⁶ ), and randomly divide them into tumor-bearing model group, positive control group (thymosin) and different doses of test drugs on the next day according to body weight. Group. Each group was administered once a day for 9 consecutive days. The animals were sacrificed 24 hours after the last administration, weighed, and the thymus and spleen were weighed, and the thymus index and spleen index were calculated.

(2) Dinitrochlorobenzene (DNCB)-induced mouse ear swelling test: on the 4th day of administration, the abdominal skin of the mice was depilated with sodium sulfide, with a range of about 3cm×3cm, and 50 μl of dinitrochlorobenzene solution was evenly applied to the depilated area After 5 days of sensitization (i.e., the 9th day of administration), 10 μl of DNCB solution was evenly applied to the right ear (both sides) of the mouse for challenge. After 24 hours, the animal was sacrificed, the left and right ear shells were cut off, and the diameter of the ear was removed with a punch. 8mm ear pieces were weighed to calculate ear swelling rate. the

(3) Serum hemolysin test by hemagglutination method: on the 5th day of administration, mice were injected intraperitoneally with 2% sheep red blood cells 0.2ml/only for immunization. Bloodletting, after standing at room temperature for 1 hour, the coagulated blood was peeled off from the tube wall to allow the serum to be fully separated, centrifuged at 2000r/min for 10min, and the serum was collected. The serum was diluted with normal saline, and 0.4ml of the diluted serum was drawn and added to another test tube, and 0.4ml of 0.5% sheep red blood cells were added, mixed well, added to the blood coagulation plate, covered and placed in a 37°C incubator for 3 hours . Observe the degree of erythrocyte agglutination, record it in 5 grades, and calculate the anti-volume number according to the formula: anti-volume number=ε(S ₁ +2S ₂ +3S ₃ +4S ₄ +...nS _n ). In the formula, 1, 2, 3...n represents the index of doubling dilution, S represents the degree of agglutination, and the larger the number of anti-volumes, the higher the serum antibody.

Data statistics: Calculate the average and standard deviation of body weight, wet weight of organs, organ index, serum antibody volume, and ear swelling rate of animals in each group, and use t test for statistical analysis.

3 results

The effect of table 6 fly maggot extract on bearing S ₁₈₀ sarcoma mouse thymus, spleen

Mean±SD, * means P<0.05, ** means P<0.01, *** means P<0.001 vs. tumor-bearing model, t-test. the

The result of table 6 shows, the fly maggot extract 0.064-0.400g/kg that embodiment 1 prepares can significantly increase bearing _S180 sarcoma mouse spleen wet weight and spleen index (P＜0.01), to bearing _S180 sarcoma mouse thymus Wet weight and thymus index had no significant effect (P>0.05).

The effect of table 7 fly maggot extract on bearing S ₁₈₀ sarcoma mice serum antibody volume number

Mean±SD, * means P<0.05, ** means P<0.01, *** means P<0.001 vs. tumor-bearing model, t-test. the

The results in Table 7 show that the fly maggot extract prepared in Example 1 has no significant effect on ear swelling of mice bearing _S180 sarcoma (P>0.05).

The effect of table 8 fly maggot extract on bearing S ₁₈₀ sarcoma mouse ear swelling

Mean±SD, * means P<0.05, ** means P<0.01, *** means P<0.001 vs. tumor-bearing model, t-test. the

The results in Table 8 show that 0.160-0.400 g/kg of the fly maggot extract prepared in Example 1 can significantly increase the serum antibody volume of mice bearing _S180 sarcoma, indicating that the fly maggot extract of the present invention has the effect of enhancing the humoral immunity of the body.

The fly maggot extract prepared in Example 1 has no significant effect on the ear swelling of mice bearing _S180 sarcoma, indicating that the fly maggot extract of the present invention has no effect on enhancing the body's cellular immunity.

5. Acute toxicity test of intraperitoneal injection in mice

1 method:

The maximum tolerated dose test method was used. ICR mice, half male and half female, were intraperitoneally injected with the drug administration route of the pharmacodynamic test, given to the animals once at the highest concentration and largest volume that the animals could tolerate, observed continuously for 14 days, and recorded the animal reactions in detail. The maximum dose that does not cause death is the maximum tolerated dose. the

2 results

The fly maggot extract injection prepared in Example 3 was administered intraperitoneally to mice at a maximum dose of 1.5 g/kg fly maggot extract, and no animal died within 14 days. The maximum tolerated dose is 1.5g/kg. the

Claims (10)

1. a fly maggot extract is characterized in that, the main component of described fly maggot extract is a water soluble protein, and the quality percentage composition of water soluble protein is 50%-70% in the described fly maggot extract; The molecular weight of described water soluble protein is the 2-16 kilodalton.

2. fly maggot extract according to claim 1; It is characterized in that described water soluble protein mainly is made up of the aminoacid of following mass percent: aspartic acid 6.3%-7.2%, threonine 3.6%-4.0%, serine 3.6%-3.9%, glutamic acid 14.5%-15.5%, glycine 5.5%-6.0%, alanine 17.8%-19.6%, cystine 1.6%-1.8%, valine 5.6%-6.5%, methionine 2.3%-2.4%, isoleucine 3.1%-3.8%, leucine 4.2%-5.2%, tyrosine 2.5%-2.7%, phenylalanine 2.6%-3.1%, lysine 8.4%-8.9%, histidine 3.4%-3.8%, arginine 1.1%-1.3% and proline-4 .2%-4.8%.

3. fly maggot extract according to claim 1 is characterized in that, the quality percentage composition of sugar is 0.5%-2.0% in the described fly maggot extract.

4. according to the method for preparing of each described fly maggot extract of claim 1-3, comprise step:

(1) pulverizes: with the clean after drying of fly larvae rinsing, be ground into fly maggot powder;

(2) defat: in the fly maggot powder that step (1) obtains, add the petroleum ether of 1-5 times of volume of fly maggot powder, soaking at room temperature 6-12 hour, remove petroleum ether, obtain the fly maggot powder after the defat;

(3) ultrasonic water is carried: the water supersound extraction of the fly maggot powder 5-10 times volume in the fly maggot powder after the defat that step (2) obtains after adding defat 1-3 time, each ultrasonic 10-30 minute, filter; The water supersound extraction of 4-8 times of volume of filtering residue reuse filtering residue 1-3 time each ultrasonic 10-30 minute, is filtered; Merge secondary filtrating; Through centrifugal, ultrafiltration and lyophilization, obtain fly maggot extract.

5. be used for treating the application in one or more the medicine of medicine, the effect of preparation enhancing body humoral immunization of leukemia, hepatocarcinoma, adenocarcinoma of lung, nasopharyngeal carcinoma, carcinoma of prostate, cervical cancer according to each described fly maggot extract of claim 1-3 in preparation.

6. application according to claim 5 is characterized in that, the dosage form of described medicine is an injection.

7. be used to suppress people's promyelocytic leukemia HL-60 cell, HEL K562 cell, people's hepatocarcinoma SMMC-7721 cell, mouse leukemia P388 cell, human lung adenocarcinoma A549 cell, human nasopharyngeal carcinoma CNE cell, human prostata cancer PC3 cell, HeLa Cells, mice S according to each described fly maggot extract of claim 1-3 in preparation ₁₈₀Application in the medicine of one or more in sarcoma and the mice Heps liver-cancer solid tumor.

8. application according to claim 7 is characterized in that, the dosage form of described medicine is an injection.

9. pharmaceutical composition, it comprises each described fly maggot extract of claim 1-3 and/or pharmaceutically acceptable carrier.

10. pharmaceutical composition according to claim 9 is characterized in that, the dosage form of described pharmaceutical composition is an injection.

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