CN101991608B - Fly maggot extractive as well as preparation method and application thereof - Google Patents
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Abstract
The invention discloses a fly maggot extractive containing the principal components of water-soluble proteins, and the water-soluble proteins contained in the fly maggot extractive have the mass percentage of 50-70 percent and the molecular weight of 2-16 KDa. The fly maggot extractive not only has obvious inhibiting effect on human promyelocytic leukemia HL-60 cells, human erythroleukemia K562 cells, human liver cancers SMMC-7721, mouse leukemia P388 cells, human lung adenocarcinoma A549 cells, human nasopharyngeal darcinoma CNE cells, human prostatic carcinoma PC3 cells, human cervical carcinoma HeLa in vitro, but also has outstanding inhibiting effect on mouse S180 sarcomas and mouse Heps liver cancer solid tumors, and also has the effect on enhancing the humoral immunity of organisms. The invention also discloses a preparation method of the fly maggot extractive. The preparation method is easy and convenient for operation and control and low in cost and is suitable for industrialized production.
Description
Technical field
The present invention relates to the medicinal extract field, be specifically related to a kind of fly maggot extract.
Background technology
Musca domestica larva, for the Calliphoridae chrysomyia megacephala (big head golden fly) (Chrysomiamegacephala, Fab.).As medicinal, just on the books in the Compendium of Material Medica of Ming Dynasty's Li Shizhen (1518-1593 A.D.): " maggot is claimed ' Chrysomyiame gacephala (Fab.) ', ' Luo Xianzi ', ' Semen Narcissi Chinensis ' again, can control children's's infantile malnutrition with fever, for controlling the good medicine of infantile malnutrition." fly larvae is cold in nature nontoxic; be a kind of fine animal protein source, not only rich in protein, and also amino acid whose component is also very rationally various in the protein; and generally about 62%, the quality percentage composition of fat is generally at 10%-15% for proteinic quality percentage composition in the fly larvae dry powder.It is reported that the pathogen of any kind of all is no more than seven days in the intravital time-to-live of fly larvae.Therefore, scientist thinks that it has a kind of immune epidemic preventing mechanism of resisting the uniqueness of pathogenic bacterial infection most probably.
Malignant tumor is serious threat people's life and healthy frequently-occurring disease and commonly encountered diseases, and the whole world has 7,000,000 people that tumor takes place every year, and 5,000,000 people are devitalized by tumor, and existing tumor patient is more than 10,000,000 people.Estimate that at present national annual tumor invasion number is about 1,600,000, the number that oncosis is died from the whole nation every year has reached 1,300,000 people, and still on the rise, and malignancy disease is surpassing cardiovascular disease and forming first reason into the human mortality.In addition, tumor misery that patient is caused, the spirit that family is brought and financial burden, the influence that social productive forces are caused are difficult to estimate especially.Therefore, each state all pays much attention to the study on prevention of tumor.
Now, operative treatment, radiotherapy, chemotherapy remain treatment tumor disease the most frequently used most important three big treatment meanss, though wherein chemotherapy developing history is shorter, on the Comprehensive Treatment of tumor, play an increasingly important role.But chemotherapy exists very big shortcoming, and this mainly is that existing antitumor drug is strong to the selectivity inhibition of tumor cell, systemic administration toxicity is big and major part has immunosuppressive action, and this has limited its use to a certain extent.Along with China joined WTO, Western medicine will be faced with stern challenge, and because of receiving protection of Intellectual Property Rights, Western medicine is imitated will to be restricted, and will be China's medical product development key and research and develop the tcm product that has independent intellectual property right.Simultaneously, the natural antitumor medicine of research and development high-efficiency low-toxicity also is urgent and important global problem.
The Qiu Xiao of Collects The American University swallows in 2003 etc. have been studied the extraction of house diptercin and to the inhibitory action of growth of tumour cell; Show that the house diptercin of extraction can suppress the in-vitro multiplication of gastric carcinoma cells MGC80-3 and BGC-823, human breast cancer cell MCF-7 and human lung carcinoma cell SPC-A-1 effectively; And be concentration dependence (Chinese hygienic biocide medical instruments; 2003,9 (1): 13-16).
The little red grade of Cao of University Of Science and Technology Of Tianjin was studied engineering fly larvae activity in vivo material and extracorporeal anti-tumor function in 2006; The result shows that engineering fly larvae activity in vivo material is at external effect (the Chinese food journal that the K562 cell is had the inhibition growth; 2006,6 (1): 316-319).
The auspicious grade in Xushui, Zhejiang Academy of Medical Sciences in 2008 has been studied the inhibitory action of Chrysomya megacephala maggot extract to leukemia and lung carcinoma cell.Chrysomya megacephala maggot powder extract unsaturated fatty acid, crude fat are made external inhibition test respectively; Observed unsaturated fatty acid, crude fat has tangible inhibited proliferation (medicine biotechnology to people's promyelocytic leukemia HL-60; 2008,15 (4): 284-286).
Above document all rests on the cell in vitro level to the research of fly maggot extract, and is to extract antibacterial peptide in the fly larvae, fatty acid etc. mostly, other compositions in the fly larvae is not further studied.
Summary of the invention
The invention provides and a kind of multiple cancerous cell is had remarkable inhibiting fly maggot extract.
The present invention also provides a kind of method for preparing of fly maggot extract, and this method is simple to operate, be easy to control, cost is low, is suitable for suitability for industrialized production.
A kind of fly maggot extract, its main component are 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 2-16 kilodalton (KDa).
In order to reach better invention effect, preferably:
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%.
The quality percentage composition of sugar is 0.5-2.0% in the described fly maggot extract.
The method for preparing of described fly maggot extract comprises 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.
Described fly maggot extract can be used for preparing one or more the medicine in treatment leukemia, hepatocarcinoma, adenocarcinoma of lung, nasopharyngeal carcinoma, carcinoma of prostate, the cervical cancer etc., is 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 as preparing
180The medicine of one or more in sarcoma and the mice Heps liver-cancer solid tumor etc.The dosage form of described medicine is preferably injection, and route of administration is best to be intravenous injection.
Described fly maggot extract can also prepare the medicine that is used for the effect of enhancing body humoral immunization.The dosage form of described medicine is preferably injection, and route of administration is best to be intravenous injection.
Described fly maggot extract can also be used to prepare the pharmaceutical composition that contains fly maggot extract.The dosage form of described pharmaceutical composition is preferably injection, and route of administration is best to be intravenous injection.
Fly larvae of the present invention generally can be selected the fly larvae that adopts the cultural method breed that has the anti-tumor biological fly larvae among the Chinese invention patent ZL200510060277.4 for use.
Compared with prior art, the invention has the advantages that:
Fly maggot extract of the present invention, the effect with high-efficiency low-toxicity, antitumor, enhancing body humoral immunization.This fly maggot extract not only has the obvious suppression effect external to people's promyelocytic leukemia HL-60 cell, HEL K562 cell, people's hepatocarcinoma SMMC-7721, mouse leukemia P388 cell, human lung adenocarcinoma A549 cell, human nasopharyngeal carcinoma CNE cell, human prostata cancer PC3 cell, human cervical carcinoma HeLa; And mice S180 sarcoma and mice Heps liver-cancer solid tumor had remarkable inhibitory action, it can also the effect of enhancing body humoral immunization simultaneously.
Method for preparing of the present invention is easy and simple to handle, be easy to control, cost is low, is suitable for suitability for industrialized production, extracts the fly maggot extract high effect nontoxic that obtains, and can be used for treating tumor patient, great exploitation potential for its.
Description of drawings
Fig. 1 is the SDS-PAGE electrophoretogram of molecular weight of fly maggot extract, fly maggot powder and the molecular weight of albumen standard substance (Mark) of embodiment 1 preparation;
Fig. 2 causes people's promyelocytic leukemia HL-60 apoptosis morphological change for the fly maggot extract of observation by light microscope variable concentrations embodiment 1 preparation; Wherein, A representes cellular control unit; B representes fly maggot extract 0.056mg/ml processed group cell; C representes fly maggot extract 0.167mg/ml processed group cell; D representes fly maggot extract 0.500mg/ml processed group cell; Scale (Bar)=50 μ m;
Fig. 3 causes people's hepatocarcinoma SMMC-7721 apoptosis morphological change for the fly maggot extract of observation by light microscope variable concentrations embodiment 1 preparation; Wherein, A representes cellular control unit; B representes fly maggot extract 0.056mg/ml processed group cell; C representes fly maggot extract 0.167mg/ml processed group cell; D representes fly maggot extract 0.500mg/ml processed group cell; Bar=50 μ m;
Fig. 4 dyes the back fluorescence microscope and observes the morphological change that the fly maggot extract of variable concentrations embodiment 1 preparation causes people's promyelocytic leukemia HL-60 cell down for AO/EB fluorescence is two; Wherein, A and B represent cellular control unit; C and D represent fly maggot extract 0.056mg/ml processed group cell; E and F represent fly maggot extract 0.167mg/ml processed group cell; G and H represent fly maggot extract 0.500mg/ml processed group cell; A, C, E and G are cellular morphology under the ordinary light source, and B, D, F and H are the interior AO/EB fluorescence of cell under the exciting light; Bar=50 μ m;
Fig. 5 dyes the back fluorescence microscope and observes the morphological change that the fly maggot extract of variable concentrations embodiment 1 preparation causes people's hepatocarcinoma SMMC-7721 cell down for AO/EB fluorescence is two; Wherein, A and B represent cellular control unit; C and D represent fly maggot extract 0.056mg/ml processed group cell; E and F represent fly maggot extract 0.167mg/ml processed group cell; G and H represent fly maggot extract 0.500mg/ml processed group cell; A, C, E and G are cellular morphology under the ordinary light source, and B, D, F and H are the interior AO/EB fluorescence of cell under the exciting light; Bar=50 μ m;
Fig. 6 dyes the back fluorescence microscope and observes the morphological change that the fly maggot extract of variable concentrations embodiment 1 preparation causes people's promyelocytic leukemia HL-60 cell down for Hoechst 33342/PI fluorescence is two; Wherein, A, B and C represent cellular control unit; D, E and F represent fly maggot extract 0.056mg/ml processed group cell; G, H and I represent fly maggot extract 0.167mg/ml processed group cell; J, K and L represent fly maggot extract 0.500mg/ml processed group cell; A, D, G and J are cellular morphology under the ordinary light source; B, E, H and K are the interior Hoechst fluorescence of cell under the exciting light; C, F, I and L are the interior PI fluorescence of cell under the exciting light; Bar=25 μ m;
Fig. 7 dyes the back fluorescence microscope and observes the morphological change that the fly maggot extract of variable concentrations embodiment 1 preparation causes people's hepatocarcinoma SMMC-7721 cell down for Hoechst 33342/PI fluorescence is two; Wherein, A, B and C represent cellular control unit; D, E and F represent fly maggot extract 0.056mg/ml processed group cell; G, H and I represent fly maggot extract 0.167mg/ml processed group cell; J, K and L represent fly maggot extract 0.500mg/ml processed group cell; A, D, G and J are cellular morphology under the ordinary light source; B, E, H and K are the interior Hoechst fluorescence of cell under the exciting light; C, F, I and L are the interior PI fluorescence of cell under the exciting light; Bar=25 μ m;
Fig. 8 dyes the back fluorescence microscope to be observed the fly maggot extract of variable concentrations embodiment 1 preparation down and causes people's promyelocytic leukemia HL-60 cell Phosphatidylserine situation of turning up for Annexin V/PI fluorescence is two; Wherein, A, B and C represent cellular control unit; D, E and F represent fly maggot extract 0.056mg/ml processed group cell; G, H and I represent fly maggot extract 0.167mg/ml processed group cell; J, K and L represent fly maggot extract 0.500mg/ml processed group cell; A, D, G and J are cellular morphology under the ordinary light source; B, E, H and K are cell Annexin V fluorescence under the exciting light; C, F, I and L are the interior PI fluorescence of cell under the exciting light; Bar=50 μ m;
Fig. 9 dyes the back fluorescence microscope to be observed the fly maggot extract of variable concentrations embodiment 1 preparation down and causes people's hepatocarcinoma SMMC-7721 cell Phosphatidylserine situation of turning up for Annexin V/PI fluorescence is two; Wherein, A, B and C represent cellular control unit; D, E and F represent fly maggot extract 0.056mg/ml processed group cell; G, H and I represent fly maggot extract 0.167mg/ml processed group cell; J, K and L represent fly maggot extract 0.500mg/ml processed group cell; A, D, G and J are cellular morphology under the ordinary light source; B, E, H and K are cell Annexin V fluorescence under the exciting light; C, F, I and L are the interior PI fluorescence of cell under the exciting light; Bar=50 μ m;
Figure 10 observes the fly maggot extract that is tried thing embodiment 1 preparation down for transmission electron microscope and causes that the morphocytology of people's promyelocytic leukemia HL-60 changes; Wherein, A representes cellular control unit; B representes fly maggot extract 0.500mg/ml processed group cell; Bar=5 μ m;
Figure 11 observes the fly maggot extract that is tried thing embodiment 1 preparation down for transmission electron microscope and causes that the morphocytology of people's hepatocarcinoma SMMC-7721 cell changes; Wherein, A representes cellular control unit; B representes fly maggot extract 0.500mg/ml processed group cell; Bar=5 μ m.
The specific embodiment
Embodiment 1: the preparation fly maggot extract
(1) special cultivation fly larvae
Chrysomyia megacephala (big head golden fly) is a food with animal proteinum 35%, 40%, 45%, brown sugar 20%, 25%, 30%, fruit 30%, 35%, 40%, and wherein animal proteinum is the maggot slurry.With Chinese medicine Herba Epimedii 20%, 25%, 30%, add lysine 15%, 20%, 25%, methionine 15%, 20%, 25%, vitamin B2 25%, 30%, 35% as " urge ovum plain ", collect fly blow with collection ovum plate.Adopt airy fly larvae room, place a plurality of fly larvae pond, in maggot culturing pool, put into maggot bait, put into above-mentioned fly blow simultaneously, temperature is controlled at 28 ℃-60 ℃, with feeding fly larvae through pulverizing 4-6 days bait of fermentation.Bait is selected A: Rana nigromaculata 15%, 23%, 30%, fresh-water fishes 15%, 23%, 30%, Cordyceps 0.1%, 0.15%, 0.2%, Ganoderma powder 1%, 1.5%, 2%, chicken 20%, 25%, 30%, fruit 25%, 30%, 35%, and all the other are plant feed; Bait is selected B: Pulmonis Sus domestica 25%, 30%, 35% Rana nigromaculata 15%, 23%, 30%, fresh-water fishes 15%, 23%, 30%, Ganoderma powder are 1%, 1.5%, 2%, Radix Ginseng powder 0.1%, 0.15%, 0.2%, fruit 20%, 25%, 30%, and all the other are plant feed; Bait C is Rana nigromaculata, fresh-water fishes or Pulmonis Sus domestica.
The fly larvae of above-mentioned special cultivation is obtained fly maggot powder through rinsing, lyophilizing and pulverizing.
(2) extraction of fly maggot powder
Add the petroleum ether of 5 times of volumes of fly maggot powder in the 50g fly maggot powder, soaking at room temperature 6 hours is removed petroleum ether, obtains the fly maggot powder after the defat.
The water supersound extraction of the 10 times of volumes of fly maggot powder in the fly maggot powder after defat after the adding defat 3 times, each supersound extraction 10 minutes is filtered; The water supersound extraction of 8 times of volumes of filtering residue reuse filtering residue 3 times, each supersound extraction 10 minutes is filtered, and merges secondary filtrating; Under 5 ℃ in 16000 rev/mins rotating speed centrifugal 30 minutes, the ultrafiltrate lyophilization after the ultrafilter membrane ultrafiltration obtained pale brown color fly maggot extract 9.6g.
The quality percentage composition of measuring water-solubility protein in this fly maggot extract through the Lowry method is 59.3%; The quality percentage composition that the phenol sulfuric acid process is measured sugar in this fly maggot extract is 1.0%; Adopt the SDS-PAGE electrophoretic techniques to identify that the molecular weight of this fly maggot extract is 2-16KDa, see Fig. 1; Measure the aminoacid of this fly maggot extract with amino-acid analyzer and form, the concentration of fly maggot extract is 1.1320 mg/ml (solvent is a water) in the test sample, and testing result is seen table 1.
Table 1
Embodiment 2: the preparation fly maggot extract
In embodiment 1, adopt the petroleum ether that adds 2 times of volumes of fly maggot powder in the 50g fly maggot powder of special cultivation fly larvae preparation, soaking at room temperature 12 hours is removed petroleum ether, obtains the fly maggot powder after the defat.
The water supersound extraction of the 10 times of volumes of fly maggot powder in the fly maggot powder after the defat after the adding defat 2 times, each supersound extraction 10 minutes is filtered; The water supersound extraction of 8 times of volumes of filtering residue reuse filtering residue 2 times, each supersound extraction 10 minutes is filtered, and merges secondary filtrating; Under 5 ℃ in 16000 rev/mins rotating speed centrifugal 30 minutes, the ultrafiltrate lyophilization after the ultrafilter membrane ultrafiltration obtained pale brown color fly maggot extract 7.4g.
The quality percentage composition of measuring water-solubility protein in this fly maggot extract through the Lowry method is 62.7%; The quality percentage composition that the phenol sulfuric acid process is measured sugar in this fly maggot extract is 1.8%; Adopt the SDS-PAGE electrophoretic techniques to identify that the molecular weight of this fly maggot extract is 2-16KDa; Measure the aminoacid of this fly maggot extract with amino-acid analyzer and form, the concentration of fly maggot extract is 1.7940 mg/ml (solvent is a water) in the test sample, and testing result is seen table 2.
Table 2
Embodiment 3
The preparation of fly maggot extract injection
Prescription: the fly maggot extract 125g that embodiment 1 makes
Water for injection adds to 1000mL.
Method for making: in the fly maggot extract 125g that embodiment 1 makes, add the injection water to 1000mL, keep to wait to ooze and wait pH7.0～7.2, filtration promptly makes the fly maggot extract injection in the embedding ampoule bottle.Omnidistance aseptic, the source of reducing phlegm and internal heat, disallergization is former.
One, the fly maggot extract that embodiment 1 is made, employing tetrazolium bromide (MTT) method is carried out the wide spectrum screening to the human tumor cell line of In vitro culture, detects the external broad-spectrum anti-tumor effect of fly maggot extract.
1 cell strain:
People's promyelocytic leukemia HL-60, people's hepatocarcinoma SMMC-7721, HEL K562, mouse leukemia P388, human lung adenocarcinoma A549, human prostata cancer PC3, human nasopharyngeal carcinoma CNE, human cervical carcinoma Hela.
2 methods
The trophophase tumor cell of taking the logarithm, adjustment concentration of cell suspension, every hole 100 μ l cell suspension inoculations are in 96 porocyte culture plates, and administration (100 μ l/ hole) behind the inoculation 24h is established blank group, cell matched group and 5 dosage respectively and tried drug group.Every hole adds 100 μ lMTT (1mg/ml after continuing to cultivate 72h; With the dissolving of DMEM culture fluid); Cultivate 2h for 37 ℃, discard and add 150 μ l acidify isopropyl alcohols (containing 0.04mol/L HCl) each hole in behind the liquid, lucifuge placement 30min; DG3022A type enzyme-linked immunosorbent assay instrument is measured 570nm place absorbance, calculates and receives the proliferation inhibition rate of reagent thing to kinds of tumor cells.
3 results:
Table 3 fly maggot extract is to tumor cell in vitro inhibited proliferation (%)
The fly maggot extract that embodiment 1 makes has inhibited proliferation external to kinds of tumor cells, and is wherein stronger relatively to leukaemia's (comprising people's promyelocytic leukemia HL-60 cell, HEL K562 cell and mouse leukemia P388 cell) and the effect of people's hepatocarcinoma SMMC-7721 cell inhibitory effect.
Two, investigate fly maggot extract of the present invention to mice S
180The inhibitory action of sarcoma and mice Heps liver-cancer solid tumor
1 method
Aseptic extraction S
180Mice or Heps mouse ascites tumor liquid; The microscopically counting; The adjustment concentration of cell suspension, in the above-mentioned ascitic tumor fluid 0.2ml of the subcutaneous aseptic inoculation of right side of mice axillary fossa, next day is by the body weight random packet; Being respectively lotus tumor model group, positive controls (cisplatin) and various dose, to be tried drug group be 0.400g/kg, 0.160g/kg and 0.064g/kg (the gram number of representing the fly maggot extract of every kilogram of injected in mice adopts the fly maggot extract of embodiment 1 preparation).Each organizes administration every day 1 time, continuous 9 days.The last administration was put to death animal after 24 hours, weighed, and dissected the tumor piece and weighed, and calculated tumour inhibiting rate, and got thymus with spleen is weighed, calculating thymus index and index and spleen index.
Data statistics: body weight, the tumor of calculating each treated animal weighs, the meansigma methods and the standard deviation of organ index, adopts t check carrying out statistical analysis.
2 results
Table 4 fly maggot extract is to mice S
180The inhibitory action of sarcoma
Mean ± SD, * represent P＜0.05, and * * representes P＜0.01, and * * * representes P＜0.001vs. lotus tumor model, t-test.
Table 5 fly maggot extract is to the inhibitory action of mice Heps liver-cancer solid tumor
Mean ± SD, * P represent＜0.05, and * * representes P＜0.01, * * * representes P＜0.001vs. lotus tumor model, t-test.
Can find out that from the result of table 4 and table 5 fly maggot extract ID of the present invention is that 0.064-0.400g/kg is to mice S
180Sarcoma has remarkable inhibitory action (P＜0.05); ID is that 0.026-0.160g/kg has remarkable inhibitory action (P＜0.05) to mice Heps liver-cancer solid tumor.
Three, investigate the external evoked apoptosis of tumor cells effect of fly maggot extract of the present invention
1 cell strain
People's promyelocytic leukemia HL-60 cell and people's hepatocarcinoma SMMC-7721 cell
2 apoptosis morphologic detection
The trophophase tumor cell of taking the logarithm; The adjustment concentration of cell suspension; Every hole 3ml cell suspension inoculation is in 6 porocyte culture plates; Administration (3ml/ hole) behind the inoculation 24h is established cell matched group and variable concentrations respectively and is received reagent thing processed group, continue to cultivate that optical microscope behind the 24h, fluorescence microscope (AO/EB fluorescence is two to be dyed, Hoechst 33342/PI fluorescence is two dyes), transmission electron microscope observing, AnnexinV detect, flow cytometer detects apoptosis.
3 results
The result shows; Tried the fly maggot extract that thing embodiment 1 makes and all can significantly be suppressed people's promyelocytic leukemia HL-60 cell and people's hepatocarcinoma SMMC-7721 cell proliferation; And have concentration dependent, IC50 is respectively 0.384 ± 0.029mg/ml and 0.349 ± 0.019mg/ml; Optical microscope and transmission electron microscope observing are found cellular morphology generation significant change, see Fig. 2, Fig. 3, Figure 10 and Figure 11; AO/EB fluorescence is two to be dyed and the two back fluorescence microscopies that dye of Hoechst 33342/PI fluorescence are observed down; Show that all fly maggot extract 0.500mg/ml processed group HL-60 cell and SMMC-7721 cell show typical changes of cell apoptosis; Even necrocytosis appears, see Fig. 4, Fig. 5, Fig. 6 and Fig. 7; AnnexinV/PI fluorescence is two to be dyed the back fluorescence microscope and observes down and find that HL-60 cell and SMMC-7721 cell membrane phospholipid acyl serine turn up, and further proves fly maggot extract 0.500mg/ml processed group cell generation apoptosis, sees Fig. 8 and Fig. 9; Flow cytometer detection by quantitative showed cell apoptosis rate is more than 30%.
Four, fly maggot extract is to the screening of immunological enhancement in the body
1 divides into groups: it is 0.400g/kg, 0.160g/kg and 0.064g/kg (the gram number of representing the fly maggot extract of every kilogram of injected in mice, the fly maggot extract that adopts embodiment 1 to prepare) that lotus tumor model group, positive controls (thymosin) and various dose are tried drug group
2 methods:
(1) lotus S in the body
180The sarcoma mouse immune strengthens test: divide into groups and administration: aseptic extraction S
180Mouse ascites, the microscopically counting, the adjustment concentration of cell suspension is 4 * 10
7/ ml, (every Mus inoculating cell number is 8 * 10 in the subcutaneous aseptic inoculation ascitic tumor fluid of right side of mice axillary fossa 0.2ml
6Individual), be respectively lotus tumor model group, positive controls (thymosin) and various dose and tried drug group by the body weight random packet next day.Each organizes administration every day 1 time, continuous 9 days.The last administration was put to death animal after 24 hours, weighed, and got thymus and spleen is weighed, and calculated thymus index and index and spleen index.
(2) dinitrochlorobenzene (DNCB) inducing mouse ear swelling test: the 4th day mouse part skin of administration loses hair or feathers with sodium sulfide, the about 3cm * 3cm of scope, and 50 μ l evenly smear depilation place sensitization with the dinitro-chlorine benzole soln; (being administration the 9th day) evenly is applied in mouse right ear (two sides) with DNCB solution 10 μ l and attacks after 5 days, puts to death animal after 24 hours; Cut left and right sides auricular concha; Take off the auricle of diameter 8mm with card punch, weigh, calculate the ear swelling rate.
(3) the blood clotting method is surveyed the serum hemolysin test: the 5th day mouse peritoneal of administration injected 2% sheep red blood cell 0.2ml/ and only carried out immunity; Immunity back (being that the last administration is after 24 hours) mice femoral artery blood-letting in the 5th day; Room temperature was placed after 1 hour, and solidification blood and tube wall are peeled off, and serum is fully separated out; The centrifugal 10min of 2000r/min collects serum.Serum is made doubling dilution with normal saline, and the serum 0.4ml that draws dilution adds in another test tube, adds 0.5% sheep red blood cell 0.4ml, and mixing adds on the blood coagulation plate, adds a cover and place 37 ℃ of incubator incubations 3 hours.Observe the RCA degree, divide 5 grades of records, and calculating antibody product by formula: antibody product=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 rank of coagulation degree, the antibody product is big more, the expression serum antibody is high more.
Data statistics: calculate body weight, internal organs weight in wet base, organ index, the serum antibody product of each treated animal, the meansigma methods and the standard deviation of ear swelling rate, adopt t check carrying out statistical analysis
3 results
Table 6 fly maggot extract is to lotus S
180The influence of sarcoma mouse thymus, spleen
Mean ± SD, * represent P＜0.05, and * * representes P＜0.01, and * * * representes P＜0.001vs. lotus tumor model, t-test.
The result of table 6 shows that the fly maggot extract 0.064-0.400g/kg of embodiment 1 preparation can significantly increase lotus S
180Sarcoma mouse spleen weight in wet base and index and spleen index (P＜0.01) are to lotus S
180Sarcoma mouse thymus weight in wet base and thymus index do not make significant difference (P＞0.05).
Table 7 fly maggot extract is to lotus S
180The influence of sarcoma mice serum antibody product
Mean ± SD, * represent P＜0.05, and * * representes P＜0.01, and * * * representes P＜0.001vs. lotus tumor model, t-test.
The result of table 7 shows that the fly maggot extract of embodiment 1 preparation is to lotus S
180Sarcoma mice ear do not make significant difference (P＞0.05).
Table 8 fly maggot extract is to lotus S
180The influence of sarcoma mice ear
Mean ± SD, * represent P＜0.05, and * * representes P＜0.01, and * * * representes P＜0.001vs. lotus tumor model, t-test.
The result of table 8 shows that the fly maggot extract 0.160-0.400g/kg of embodiment 1 preparation can significantly increase lotus S
180Sarcoma mice serum antibody product shows that fly maggot extract of the present invention has the effect of enhancing body humoral immunization.
The fly maggot extract of embodiment 1 preparation is to lotus S
180The sarcoma mice ear does not make significant difference, and shows that fly maggot extract of the present invention does not have the enhancing body immunization of cell.
Five, mouse peritoneal injection acute toxicity test
1 method:
Adopt the mtd test method.The ICR mice, male and female half and half are with the route of administration lumbar injection of pharmacodynamics test; Dosage with the tolerant maximum concentration of animal, maximum volume gives animal 1 time; Observed 14 days continuously, itemized record reaction of animals situation is a maximum tolerated dose not produce dead maximal dose.
2 results
Give mice with the fly maggot extract injection of embodiment 3 preparations with fly maggot extract maximal dose 1.5g/kg lumbar injection, do not have animal dead in 14 days.Maximum tolerated dose is 1.5g/kg.
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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|---|---|---|---|
| CN2010105517711A CN101991608B (en) | 2010-11-19 | 2010-11-19 | Fly maggot extractive as well as preparation method and application thereof |
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| CN2010105517711A CN101991608B (en) | 2010-11-19 | 2010-11-19 | Fly maggot extractive as well as preparation method and application thereof |
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| CN101991608B true CN101991608B (en) | 2012-06-06 |
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Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103271947A (en) * | 2013-06-06 | 2013-09-04 | 四川科伦新光生物科技开发有限公司 | Novel antitumor application of maggot |
| CN104163847B (en) * | 2014-05-03 | 2018-09-21 | 吉林金梓源生物科技股份有限公司 | The preparation method of fly maggot active protein peptide and prepared fly maggot active protein peptide and application thereof |
| CN106420827A (en) * | 2016-02-26 | 2017-02-22 | 浙江佰科堂生物科技股份有限公司 | Application of Larva ChrysomyiamegacephalaFab. product in preparing dugs, health care products or foods for treating colon cancer |
| CN106265752A (en) * | 2016-02-26 | 2017-01-04 | 浙江佰科堂生物科技股份有限公司 | Chrysomyiame gacephala (Fab.) goods are for preparing the application in the treatment medicine of carcinoma of prostate, health product or food |
| CN106265753A (en) * | 2016-09-08 | 2017-01-04 | 浙江佰科堂生物科技股份有限公司 | Chrysomyiame gacephala (Fab.) goods are for preparing the application in the treatment medicine of Leydig's cell tumor, health product or food |
| CN106420829A (en) * | 2016-09-08 | 2017-02-22 | 浙江佰科堂生物科技股份有限公司 | Application of maggot product to preparation of medicine, health care product or food for treating gliocytoma |
| CN108741056A (en) * | 2018-06-08 | 2018-11-06 | 四川理工学院 | A kind of preparation method for climbing sandworm extract adjusting immune function |
| CN108853119A (en) * | 2018-09-20 | 2018-11-23 | 广西壮族自治区药用植物园 | For treating the compound combination and purposes of tumour |
| CN110327374B (en) * | 2019-07-31 | 2020-07-28 | 浙江佰科堂生物科技股份有限公司 | A pharmaceutical composition prepared from maggot for preventing and/or treating convulsion |
| CN112322681B (en) * | 2019-08-01 | 2022-09-09 | 大连麦琪克生物技术有限公司 | Traditional Chinese medicine maggot active peptide and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1077409C (en) * | 1998-04-08 | 2002-01-09 | 上海野生源高科技有限公司 | Insects protein capsule or powder medicine and preparation process thereof |
| CN1170853C (en) * | 2002-04-06 | 2004-10-13 | 李长茂 | Method for extracting chitin and biological protein powder |
| CN1298735C (en) * | 2005-06-02 | 2007-02-07 | 上海交通大学 | Process for preparing low-molecular insect protein by pressure hydrolysis of fly maggot protein |
| CN101032525B (en) * | 2007-04-10 | 2010-12-08 | 浙江省医学科学院 | Extracting active principles having extracorporeal anti-tumor function from housefly larva |
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