CN111518701A - Pholiota adipose fungus extract and preparation method and application thereof - Google Patents
Pholiota adipose fungus extract and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of preparation of clitocybe maxima extracts, in particular to a clitocybe maxima fungus extract and a preparation method and application thereof, wherein the clitocybe maxima fungus extract is obtained according to the following steps: culturing clitocybe strain on PDA plate, inoculating to rice culture medium, culturing, extracting the culture solution with ethyl acetate for 3 times, mixing extractive solutions, and concentrating under reduced pressure to obtain clitocybe fungus extract. The invention discloses the application of the clitocybe parviflora fungus extract in preparing medicaments for preventing tumors, antitumor medicaments and health-care products for preventing and treating tumors, wherein the clitocybe parviflora fungus extract has a good inhibiting effect on SGC-7901 cells and Hela cells for the first time.
Description
Technical Field
The invention relates to the technical field of preparation of clitocybe maxima extracts, and discloses a clitocybe maxima fungus extract and a preparation method and application thereof.
Background
The Clitocybe cl (Clitocybe cl) Clitocybe fungus extract is a high-grade fungus of Clitocybe of the family white mushroom, scattered growth or clumped growth is carried out in the forest in summer and autumn, the fact that Clitocybe is deeply loved by common people in the local is found in the fourth national resource general investigation and the field investigation and visit process shows that the Clitocybe is popular among people as an edible mushroom, the mushroom seems to be particularly effective in treating liver cancer and stomach cancer in folk circulation, the general application principle can be used for treating acute stomachache and antianaphylaxis, but the severe headache, dizziness, confusion and exhaustion can be caused by a large amount of wine drunk within 1 to 2 days after the mushroom is eaten. The clitocybe maxima is taken as a higher fungus, grows out only when the environment is suitable, the persistence time is short, and the research reports on the chemical components and the pharmacological activity of the clitocybe maxima are not seen at home and abroad so far, so that the development and the utilization of the clitocybe maxima are of great significance.
Disclosure of Invention
The invention provides an clitocybe corymbosa fungus extract, and a preparation method and application thereof, and discloses the application of the clitocybe corymbosa fungus extract in preparing medicines for preventing and treating tumors, antitumor medicines and health-care products for preventing and treating tumors, wherein the clitocybe corymbosa fungus extract has a good inhibition effect on SGC-7901 cells and Hela cells for the first time.
One of the technical schemes of the invention is realized by the following measures: an extract of clitocybe armandii fungus is obtained by the following method: firstly, mixing required amount of rice and distilled water, sterilizing and cooling to obtain a rice culture medium, wherein the weight ratio of the rice to the distilled water is 3: 4; secondly, growing the clitocybe fungus strains on a PDA flat plate for 30 days, dividing the clitocybe fungus strains into small blocks, inoculating the small blocks into a rice culture medium, and standing and culturing for 30 days to obtain the clitocybe fungus flora; thirdly, soaking a rice culture medium containing the clitocybe armata fungus flora in ethyl acetate at room temperature, performing ultrasonic treatment, standing for 7 days, filtering to obtain a primary extracting solution and primary filter residue, adding ethyl acetate into the primary filter residue, soaking and performing ultrasonic treatment, standing for 7 days, filtering to obtain a secondary extracting solution and secondary filter residue, adding ethyl acetate into the secondary filter residue, soaking and performing ultrasonic treatment, standing for 7 days, and filtering to obtain a tertiary extracting solution; and fourthly, combining the primary extract, the secondary extract and the tertiary extract to obtain a mixed extract, and performing reduced pressure recovery and concentration on the mixed extract to obtain the clitocybe fungus extract.
The following is further optimization or/and improvement of the technical scheme of the invention:
in the first step, the sterilization temperature is 115 ℃ to 120 ℃, and the sterilization time is 30min to 35 min.
In the second step, the culture temperature of the rice medium is 23 ℃ to 27 ℃.
In the third step, the ultrasonic time is 2 to 3 hours.
The second technical scheme of the invention is realized by the following measures: a preparation method of an extract of clitocybe corymbosa fungi comprises the following steps: firstly, mixing required amount of rice and distilled water, sterilizing and cooling to obtain a rice culture medium, wherein the weight ratio of the rice to the distilled water is 3: 4; secondly, growing the clitocybe fungus strains on a PDA flat plate for 30 days, dividing the clitocybe fungus strains into small blocks, inoculating the small blocks into a rice culture medium, and standing and culturing for 30 days to obtain the clitocybe fungus flora; thirdly, soaking a rice culture medium containing the clitocybe armata fungus flora in ethyl acetate at room temperature, performing ultrasonic treatment, standing for 7 days, filtering to obtain a primary extracting solution and primary filter residue, adding ethyl acetate into the primary filter residue, soaking and performing ultrasonic treatment, standing for 7 days, filtering to obtain a secondary extracting solution and secondary filter residue, adding ethyl acetate into the secondary filter residue, soaking and performing ultrasonic treatment, standing for 7 days, and filtering to obtain a tertiary extracting solution; and fourthly, combining the primary extract, the secondary extract and the tertiary extract to obtain a mixed extract, and performing reduced pressure recovery and concentration on the mixed extract to obtain the clitocybe fungus extract.
The following is further optimization or/and improvement of the technical scheme of the invention:
in the first step, the sterilization temperature is 115 ℃ to 120 ℃, and the sterilization time is 30min to 35 min.
In the second step, the culture temperature of the rice medium is 23 ℃ to 27 ℃.
In the third step, the ultrasonic time is 2 to 3 hours.
The third technical scheme of the invention is realized by the following measures: an application of Pholiota adipose fungus extract in preparing medicine for preventing tumor is provided.
The fourth technical scheme of the invention is realized by the following measures: an application of Pholiota adipose fungus extract in preparing antitumor drugs is provided.
The fifth technical scheme of the invention is realized by the following measures: an application of Pholiota adipose fungus extract in preparing health product for preventing and treating tumor is provided.
The invention discloses the application of the clitocybe parviflora fungus extract in preparing medicaments for preventing tumors, antitumor medicaments and health-care products for preventing and treating tumors, wherein the clitocybe parviflora fungus extract has a good inhibiting effect on SGC-7901 cells and Hela cells for the first time.
Drawings
FIG. 1 is a bar graph showing the effect of P.clavuligerum fungus extract on the volume inhibition rate of a gastric orthotopic tumor graft according to the present invention.
FIG. 2 is a bar graph showing the effect of P.clavuligerum fungus extract on the inhibition rate of transplanted tumor mass in gastric orthotopic tumor.
FIG. 3 is a morphological diagram of the effect of Pholiota adipose fungus extract on gastric carcinoma cell nude mouse stomach in-situ transplantation tumor.
Detailed Description
The present invention is not limited by the following examples, and specific embodiments may be determined according to the technical solutions and practical situations of the present invention. The various chemical reagents and chemical articles mentioned in the invention are all the chemical reagents and chemical articles which are well known and commonly used in the prior art, unless otherwise specified; the percentages in the invention are mass percentages unless otherwise specified; the solution in the present invention is an aqueous solution in which the solvent is water, for example, a hydrochloric acid solution is an aqueous hydrochloric acid solution, unless otherwise specified; the normal temperature and room temperature in the present invention generally mean a temperature of 15 ℃ to 25 ℃, and are generally defined as 25 ℃.
The invention is further described below with reference to the following examples:
example 1: the clitocybe corymbosa fungus extract is obtained by the following method: firstly, mixing required amount of rice and distilled water, sterilizing and cooling to obtain a rice culture medium, wherein the weight ratio of the rice to the distilled water is 3: 4; secondly, growing the clitocybe fungus strains on a PDA flat plate for 30 days, dividing the clitocybe fungus strains into small blocks, inoculating the small blocks into a rice culture medium, and standing and culturing for 30 days to obtain the clitocybe fungus flora; thirdly, soaking a rice culture medium containing the clitocybe armata fungus flora in ethyl acetate at room temperature, performing ultrasonic treatment, standing for 7 days, filtering to obtain a primary extracting solution and primary filter residue, adding ethyl acetate into the primary filter residue, soaking and performing ultrasonic treatment, standing for 7 days, filtering to obtain a secondary extracting solution and secondary filter residue, adding ethyl acetate into the secondary filter residue, soaking and performing ultrasonic treatment, standing for 7 days, and filtering to obtain a tertiary extracting solution; and fourthly, combining the primary extract, the secondary extract and the tertiary extract to obtain a mixed extract, and performing reduced pressure recovery and concentration on the mixed extract to obtain the clitocybe fungus extract.
The PDA plate is prepared from PDA agar medium provided by Shanghai Liaobao for Biotech Co.
Example 2: as the optimization of the embodiment, in the first step, the sterilization temperature is 115 ℃ to 120 ℃, and the sterilization time is 30min to 35 min.
Example 3: as an optimization of the above embodiment, in the second step, the culture temperature of the rice medium is 23 ℃ to 27 ℃.
Example 4: as an optimization of the above embodiment, in the third step, the sonication time is 2h to 3 h.
Example 5: the clitocybe corymbosa fungus extract is obtained by the following method: step one, mixing required amount of rice and distilled water, sterilizing and cooling to obtain a rice culture medium, wherein the weight ratio of the rice to the distilled water is 3:4, the sterilization temperature is 115 ℃, and the sterilization time is 30 min; secondly, growing the clitocybe fungus strains on a PDA flat plate for 30 days, dividing the clitocybe fungus strains into small blocks, inoculating the small blocks into a rice culture medium, standing and culturing for 30 days to obtain the clitocybe fungus flora, wherein the culture temperature is 23 ℃; thirdly, soaking a rice culture medium containing the clitocybe armata fungus flora in ethyl acetate at room temperature, performing ultrasonic treatment, standing for 7 days, filtering to obtain a primary extracting solution and a primary filter residue, adding ethyl acetate into the primary filter residue, soaking and performing ultrasonic treatment, standing for 7 days, filtering to obtain a secondary extracting solution and a secondary filter residue, adding ethyl acetate into the secondary filter residue, soaking and performing ultrasonic treatment, standing for 7 days, and filtering to obtain a tertiary extracting solution, wherein the ultrasonic treatment time is 2 hours; and fourthly, combining the primary extract, the secondary extract and the tertiary extract to obtain a mixed extract, and performing reduced pressure recovery and concentration on the mixed extract to obtain the clitocybe fungus extract.
Example 6: the clitocybe corymbosa fungus extract is obtained by the following method: step one, mixing required amount of rice and distilled water, sterilizing and cooling to obtain a rice culture medium, wherein the weight ratio of the rice to the distilled water is 3:4, the sterilization temperature is 120 ℃, and the sterilization time is 35 min; secondly, growing the clitocybe fungus strains on a PDA flat plate for 30 days, dividing the clitocybe fungus strains into small blocks, inoculating the small blocks into a rice culture medium, standing and culturing for 30 days to obtain the clitocybe fungus flora, wherein the culture temperature is 27 ℃; thirdly, soaking a rice culture medium containing the clitocybe armata fungus flora in ethyl acetate at room temperature, performing ultrasonic treatment, standing for 7 days, filtering to obtain a primary extracting solution and a primary filter residue, adding ethyl acetate into the primary filter residue, soaking and performing ultrasonic treatment, standing for 7 days, filtering to obtain a secondary extracting solution and a secondary filter residue, adding ethyl acetate into the secondary filter residue, soaking and performing ultrasonic treatment, standing for 7 days, and filtering to obtain a tertiary extracting solution, wherein the ultrasonic treatment time is 3 hours; and fourthly, combining the primary extract, the secondary extract and the tertiary extract to obtain a mixed extract, and performing reduced pressure recovery and concentration on the mixed extract to obtain the clitocybe fungus extract.
Example 7: the clitocybe corymbosa fungus extract is obtained by the following method: step one, mixing required amount of rice and distilled water, sterilizing and cooling to obtain a rice culture medium, wherein the weight ratio of the rice to the distilled water is 3:4, the sterilization temperature is 118 ℃, and the sterilization time is 32 min; secondly, growing the clitocybe fungus strains on a PDA flat plate for 30 days, dividing the clitocybe fungus strains into small blocks, inoculating the small blocks into a rice culture medium, standing and culturing for 30 days to obtain the clitocybe fungus flora, wherein the culture temperature is 25 ℃; thirdly, soaking a rice culture medium containing the clitocybe armata fungus flora in ethyl acetate at room temperature, performing ultrasonic treatment, standing for 7 days, filtering to obtain a primary extracting solution and a primary filter residue, adding ethyl acetate into the primary filter residue, soaking and performing ultrasonic treatment, standing for 7 days, filtering to obtain a secondary extracting solution and a secondary filter residue, adding ethyl acetate into the secondary filter residue, soaking and performing ultrasonic treatment, standing for 7 days, and filtering to obtain a tertiary extracting solution, wherein the ultrasonic treatment time is 2.5 hours; and fourthly, combining the primary extract, the secondary extract and the tertiary extract to obtain a mixed extract, and performing reduced pressure recovery and concentration on the mixed extract to obtain the clitocybe fungus extract.
The following are experiments of in vitro antitumor drug efficacy and in vivo activity of the prepared clitocybe corymbosa fungus extract.
In vitro antitumor drug efficacy experiments: the in vitro antitumor drug efficacy experiment adopts MTT colorimetric method for investigation. Taking the clitocybe corymbosa fungus extract prepared in the embodiment 7 of the invention as an experimental group, taking 5-FU (5-fluorouracil) as a control group, simultaneously setting a blank group, selecting SGC-7901 (human gastric cancer) cells and Hela (human cervical cancer) cells as experimental objects from the experimental group, the control group and the blank group, diluting the experimental group, the control group and the blank group by using a culture medium, inoculating the experimental objects into a 96-well plate at the density of 6 x 104/ml, wherein each well is 100 mu l, and after normally culturing for 24 hours in an incubator, corresponding drugs are added into each group, so that the final concentration of each group of drugs is respectively 2.5 mu g/ml (1 group), 5 mu g/ml (2 group), 10 mu g/ml (3 group), 20 mu g/ml (4 group) and 40 mu g/ml (5 group), and 5 concentrations are totally set, and each concentration is 3 duplicate wells; after 48 hours of incubation, each well was stained with MTT 10. mu.l; after further culturing for four hours, the original culture solution is aspirated and discarded, 150 μ l of DMSO (dimethyl sulfoxide) is added to each well, the culture solution is placed on a shaking table and shaken at a low speed for 10min to fully dissolve crystals, the optical density value is detected at a wavelength of 570nm of an enzyme linked immunosorbent assay (ELISA) detector, the 50% inhibition concentration (IC 50, μ g/mL) is calculated according to the optical density value, and the calculation method for calculating the IC50 according to the optical density value is the prior known technology. IC50 of SGC-7901 cells and Hela cells in experimental groups and control groups is shown in Table 1, and it can be known from Table 1 that the P.clavuligerum fungus extract of the present invention has a good inhibitory effect on SGC-7901 cells and Hela cells.
In vivo activity assay: the in vivo anti-gastric cancer activity of the clitocybe maxima fungus extract is researched by adopting a nude mouse transplantation tumor model. Male BALB/C nude mice (20. + -.2 g) were taken and were adapted for tumor inoculation after 3 days of acclimation. Inoculating a tumor mass with a size of about 1mm × 1mm × 1mm in a good state into a gastric cavity under sterile conditions, fixing OB biological gel, judging that a model is successful when a solid tumor grows up after two weeks, measuring the tumor volume, dividing nude mice successfully modeled after the tumor volume is uniform into three groups, namely a blank control group, a positive control group and an administration group, wherein the administration group is a low, medium and high concentration group (low, medium and high concentrations are respectively 20, 40 and 80 mg/kg) of the Pholiota adipose fungal extract prepared in example 7 of the invention, the administration dose is 10 mg/kg, the positive control group is administered with 5-fluorouracil (concentration is 10 mg/kg) in an equal amount, and the negative control group (PBS and 10 ml/kg) is administered with physiological saline in an equal amount instead of the blank control group, and each group is continuously administered intraperitoneally for 4 weeks, during the test, tumor volumes and body weights were measured every 3 days, possible toxic side effects were observed, mice were sacrificed after weighing on the last dosing day, tumor masses were stripped and weighed, and inhibition rates were calculated. The results of in vivo anti-gastric cancer activity are shown in FIGS. 1 to 3, in which the abscissa 1, 2 and 3 in FIGS. 1 and 2 are the administration groups to which the low, medium and high doses of the fungus extract of P.clavuligerus of the present invention were administered, respectively, 4 is a positive control group, 5 is a blank control group, in FIG. 3, 1, 2 and 3 are administration groups to which the low, medium and high doses of the fungus extract of P.clavuligerus of the present invention are administered, 5, 6 and 7 are administration groups to which the low, medium and high doses of the fungus extract of P.clavuligerus of the present invention are administered, 8 is a positive control group, 4 is a blank control group, as can be seen from FIGS. 1 to 3, the volume and weight of the orthotopic transplantation tumor of the nude mice of the administration group and the positive control group are reduced compared with the blank control group, and the difference is statistically significant (P < 0.05), and with the increase of the administration concentration of the administration group, the volume inhibition rate and the weight inhibition rate of the gastric orthotopic tumor transplantation tumor are correspondingly enhanced.
In conclusion, the invention discloses that the clitocybe for the first time has stronger inhibition effect on SGC-7901 cells and Hela cells, so that the clitocybe extract can be applied to the preparation of tumor prevention medicines and antitumor medicines.
The technical characteristics form an embodiment of the invention, which has strong adaptability and implementation effect, and unnecessary technical characteristics can be increased or decreased according to actual needs to meet the requirements of different situations.
Claims (10)
1. An extract of clitocybe armandii fungus, which is characterized by being obtained by the following method: firstly, mixing required amount of rice and distilled water, sterilizing and cooling to obtain a rice culture medium, wherein the weight ratio of the rice to the distilled water is 3: 4; secondly, growing the clitocybe fungus strains on a PDA flat plate for 30 days, dividing the clitocybe fungus strains into small blocks, inoculating the small blocks into a rice culture medium, and standing and culturing for 30 days to obtain the clitocybe fungus flora; thirdly, soaking a rice culture medium containing the clitocybe armata fungus flora in ethyl acetate at room temperature, performing ultrasonic treatment, standing for 7 days, filtering to obtain a primary extracting solution and primary filter residue, adding ethyl acetate into the primary filter residue, soaking and performing ultrasonic treatment, standing for 7 days, filtering to obtain a secondary extracting solution and secondary filter residue, adding ethyl acetate into the secondary filter residue, soaking and performing ultrasonic treatment, standing for 7 days, and filtering to obtain a tertiary extracting solution; and fourthly, combining the primary extract, the secondary extract and the tertiary extract to obtain a mixed extract, and performing reduced pressure recovery and concentration on the mixed extract to obtain the clitocybe fungus extract.
2. The P.clavuligerus fungal extract according to claim 1, wherein the sterilization temperature in the first step is 115 ℃ to 120 ℃ and the sterilization time is 30min to 35 min.
3. The P.clavuligerus fungal extract according to claim 1 or 2, wherein the culture temperature of the rice medium in the second step is 23 ℃ to 27 ℃.
4. An P.clavuligerus fungal extract according to claim 1 or 2 or 3, characterized in that in the third step the sonication time is 2 to 3 h.
5. A preparation method of an extract of clitocybe corymbosa fungi is characterized by comprising the following steps: firstly, mixing required amount of rice and distilled water, sterilizing and cooling to obtain a rice culture medium, wherein the weight ratio of the rice to the distilled water is 3: 4; secondly, growing the clitocybe fungus strains on a PDA flat plate for 30 days, dividing the clitocybe fungus strains into small blocks, inoculating the small blocks into a rice culture medium, and standing and culturing for 30 days to obtain the clitocybe fungus flora; thirdly, soaking a rice culture medium containing the clitocybe armata fungus flora in ethyl acetate at room temperature, performing ultrasonic treatment, standing for 7 days, filtering to obtain a primary extracting solution and primary filter residue, adding ethyl acetate into the primary filter residue, soaking and performing ultrasonic treatment, standing for 7 days, filtering to obtain a secondary extracting solution and secondary filter residue, adding ethyl acetate into the secondary filter residue, soaking and performing ultrasonic treatment, standing for 7 days, and filtering to obtain a tertiary extracting solution; and fourthly, combining the primary extract, the secondary extract and the tertiary extract to obtain a mixed extract, and performing reduced pressure recovery and concentration on the mixed extract to obtain the clitocybe fungus extract.
6. The method for preparing P.clavuligerum fungal extract according to claim 5, wherein the sterilization temperature is 115 ℃ to 120 ℃ and the sterilization time is 30min to 35min in the first step.
7. The method for preparing P.clavuligerum fungal extract according to claim 5 or 6, wherein in the second step, the culture temperature of the rice medium is 23 ℃ to 27 ℃; or/and in the third step, the ultrasonic time is 2h to 3 h.
8. Use of an extract of clitocybe armandii according to 1 to 4 for the preparation of a medicament for the prevention of tumors.
9. Use of an extract of Pholiota adipose fungus according to 1 to 4 for the preparation of an antitumor medicament.
10. An application of the clitocybe corymbosa fungus extract according to 1 to 4 in preparing health care products for preventing and treating tumors.
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Application publication date: 20200811 |