CN107080759B - Ganoderma lucidum spore wall breaking method, wall-broken ganoderma lucidum spore powder prepared by same and application of wall-broken ganoderma lucidum spore powder - Google Patents
Ganoderma lucidum spore wall breaking method, wall-broken ganoderma lucidum spore powder prepared by same and application of wall-broken ganoderma lucidum spore powder Download PDFInfo
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- CN107080759B CN107080759B CN201710296519.2A CN201710296519A CN107080759B CN 107080759 B CN107080759 B CN 107080759B CN 201710296519 A CN201710296519 A CN 201710296519A CN 107080759 B CN107080759 B CN 107080759B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
- A61K36/074—Ganoderma
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B02—CRUSHING, PULVERISING, OR DISINTEGRATING; PREPARATORY TREATMENT OF GRAIN FOR MILLING
- B02C—CRUSHING, PULVERISING, OR DISINTEGRATING IN GENERAL; MILLING GRAIN
- B02C17/00—Disintegrating by tumbling mills, i.e. mills having a container charged with the material to be disintegrated with or without special disintegrating members such as pebbles or balls
- B02C17/10—Disintegrating by tumbling mills, i.e. mills having a container charged with the material to be disintegrated with or without special disintegrating members such as pebbles or balls with one or a few disintegrating members arranged in the container
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
Abstract
The invention discloses a ganoderma lucidum spore wall breaking method, which is characterized in that ganoderma lucidum spore powder and fullerene powder are mixed and pretreated, cluster particles in the fullerene powder are crushed into micron and/or nanometer particles, and the micron and/or nanometer particles enter the shell wall and the interior of the ganoderma lucidum spore; then, the fullerene is irradiated by an external energy source to generate phase change and volume expansion, so that the outer wall of the spore is expanded, and the wall breaking of the ganoderma lucidum spore is realized. The invention also discloses a preparation method of the wall-broken ganoderma lucidum spore powder and the wall-broken ganoderma lucidum spore powder prepared by the method; the method does not involve high temperature and high pressure, the content and the activity of active ingredients of the ganoderma lucidum spores are retained to the greatest extent, the tumor inhibition rate is high, and the prepared wall-broken ganoderma lucidum spore powder has obvious curative effect on liver injury caused by chemotherapy. The invention also discloses a preparation method of the ganoderma lucidum spore oil and the ganoderma lucidum spore oil prepared by the method, the ganoderma lucidum spore oil is extracted from the wall-broken ganoderma lucidum spore powder prepared by the method, and the extraction rate is high.
Description
Technical Field
The invention belongs to the technical field of ganoderma lucidum spore wall breaking, and particularly relates to a ganoderma lucidum spore wall breaking method, wall-broken ganoderma lucidum spore powder prepared by the method and application of the wall-broken ganoderma lucidum spore powder.
Background
Ganoderma lucidum is a substance rich in physiological activity, and comprises ganoderan, triterpenes, nucleosides, sterols, alkaloids, furan derivatives, amino acids, polypeptides, mineral elements, fatty acids, etc. The pharmacological actions of the medicine mainly comprise anti-tumor, immunoregulation, anti-radiation, sedation, tranquilization, cardiotonic, blood pressure reduction, cough relief, asthma relief, blood sugar reduction and the like. Most of the active ingredients of ganoderma lucidum exist in ganoderma lucidum spores, so that the ganoderma lucidum spores need to be subjected to wall breaking treatment to fully utilize the active ingredients of the ganoderma lucidum spores, and more active substances can be released. However, the outer wall of the ganoderma lucidum spore is thicker, so the ganoderma lucidum spore wall breaking technology becomes the key for the utilization of the ganoderma lucidum spore.
At present, the wall breaking technology for ganoderma lucidum spores includes various methods, such as micronization wall breaking method, homogenization wall breaking method, ultrasonic wall breaking method, germination wall breaking method, biological enzyme wall breaking method, CO2Supercritical wall breaking method, high-energy ball milling impact method, etc. However, the superfine grinding wall breaking method, the homogenate wall breaking method, the ultrasonic wall breaking method and the high-energy ball milling impact method have low wall breaking rate, and the superfine grinding wall breaking method, the high-energy ball milling impact method and the ultrasonic wall breaking method have large destructive power on active substances, so that the active substances are easy to deteriorate. The germination wall-breaking method has a long wall-breaking process, is complicated to operate, is not suitable for large-scale treatment, and has a low breaking rate. The biological enzyme wall breaking method has too many influencing factors, wherein the enzyme dosage, temperature, pH value, time and the like all influence the wall breaking efficiency, so the practicability is not strong. CO 22The wall breaking process is carried out under ultrahigh pressure by supercritical wall breaking method, and CO is2Under critical liquid conditions, a large amount of fatty acid active substances are released, and the active substances are lost.
Therefore, developing a method which can fully retain the active substances of the ganoderma lucidum spore powder and can realize higher wall breaking rate is the key point for optimally exerting the efficacy of the ganoderma lucidum spore powder.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a ganoderma lucidum spore wall-breaking method, wall-broken ganoderma lucidum spore powder prepared by the method and application of the wall-broken ganoderma lucidum spore powder.
The inventor finds that the ganoderma lucidum spore powder and the fullerene powder are mixed and pretreated, so that cluster particles in the fullerene powder are crushed into micron and/or nanometer particles and enter the shell wall and the interior of the ganoderma lucidum spore; then, the fullerene is irradiated by an external energy source to generate phase change and volume expansion, so that the outer wall of the ganoderma lucidum spore is expanded, and the wall breaking of the ganoderma lucidum spore is realized. The fullerene phase change process induced by the external energy source does not generate high heat and high pressure, can keep the stability of active ingredients in the ganoderma lucidum spores, and can realize at least 98% of wall breaking rate of the ganoderma lucidum spores. Meanwhile, the adopted fullerene can be recycled and reused, and the production cost is greatly saved from the aspect of raw material utilization. The present invention has been completed based on such a concept.
The purpose of the invention is realized by the following technical scheme:
a wall breaking method of ganoderma lucidum spores comprises the following steps:
1) mixing fullerene powder and ganoderma lucidum spore powder and carrying out pretreatment to make the fullerene powder enter the ganoderma lucidum spore;
2) and irradiating the pretreated mixed powder.
Those skilled in the art will appreciate that various fullerenes may be used in accordance with the present invention. Preferably, the fullerene is C60，C70，C76More preferably C60，C70Most preferably C or a mixture thereof60。
According to the present invention, the particle size of the fullerene powder is preferably 10nm to 50 μm, more preferably 10nm to 5000nm, and most preferably 100nm to 1000 nm; preferably, the particle size of the fullerene powder after pretreatment is 50nm to 500nm, most preferably 100nm to 200 nm.
According to the present invention, those skilled in the art will understand that the ganoderma lucidum spore powder is not limited, and any one of the prior art may be used.
According to the invention, the mass ratio of the fullerene powder to the ganoderma lucidum spore powder is preferably 1: 5-10000, more preferably 1: 10-1000, and most preferably 1: 100-500.
According to the invention, in step 1), the temperature of the pretreatment is preferably ≦ 50 ℃.
According to the present invention, in step 1), it is preferable that the pretreatment includes, but is not limited to, low-temperature high-energy ball milling, high-pressure homogenization, high-speed homogenization, etc.; most preferably, the pretreatment is low temperature high energy ball milling.
According to the invention, the ball milling temperature of the low-temperature high-energy ball milling is preferably 20-40 ℃, more preferably 25-35 ℃, and most preferably 30 ℃; preferably, the ball milling time is 1-20 h, more preferably 2-16 h, more preferably 4-12 h, and most preferably 8 h; the ball milling frequency is preferably 200 to 2000r/min, more preferably 300 to 1500r/min, even more preferably 500 to 1000r/min, and most preferably 700 r/min.
According to the invention, through pretreatment (such as low-temperature high-energy ball milling, high-pressure homogenization treatment, high-speed homogenization treatment and the like), on one hand, cluster particles in the fullerene powder can be crushed into micron and/or nanometer particles, and meanwhile, certain cracks can be generated on the outer wall of the ganoderma lucidum spore, so that the fullerene particles can better enter the shell wall and the interior of the ganoderma lucidum spore.
According to the invention, the irradiation is realized by an external energy source, and can be one or more of a magnetic field, radio frequency and rays; preferably radio frequency.
According to the invention, it is preferred that the energy of the magnetic field is 0.1-10T; the time of the magnetic field irradiation is preferably 1 to 12 hours.
According to the invention, the energy of the radio frequency is preferably 100-1000 MHz, most preferably 300-500 MHz; preferably, the time of the radio frequency irradiation is 0.5-20 h, more preferably 1-12h, and most preferably 3-9 h.
According to the invention, it is preferred that the energy of the radiation is between 1 and 1000 eV; the time for the radiation irradiation is preferably 0.5 to 12 hours.
In the invention, the fullerene in the ganoderma lucidum spores generates phase change and volume expansion through irradiation, and the outer walls of the spores are expanded, thereby realizing the wall breaking treatment of the ganoderma lucidum spores.
The invention also provides a preparation method of the wall-broken ganoderma lucidum spore powder, which comprises the following steps:
1) mixing fullerene powder and ganoderma lucidum spore powder and carrying out pretreatment to make the fullerene powder enter the ganoderma lucidum spore;
2) irradiating the pretreated mixed powder to obtain wall-broken ganoderma lucidum spore fullerene mixed powder;
3) adding water into the wall-broken ganoderma lucidum spore and fullerene mixed powder obtained in the step 2), stirring, centrifuging, removing precipitates, taking supernate, and drying to obtain the wall-broken ganoderma lucidum spore powder.
According to the invention, the temperature of the water is preferably 50-100 ℃, and most preferably 60-80 ℃.
According to the invention, the mass-volume ratio of the wall-broken ganoderma lucidum spore-fullerene mixed powder to water is preferably 1-1000 mg/mL; most preferably 50-500 mg/mL.
According to the invention, the adding of water, stirring and centrifugation are preferably repeated 3-5 times.
In accordance with the present invention, one skilled in the art will appreciate that various dry forms can be used. Preferably, the drying is vacuum drying or normal pressure air drying; preferably the temperature of the drying is 20-50 ℃, more preferably 30-45 ℃, most preferably 40 ℃; preferably the drying time is from 1 to 48 hours, more preferably from 10 to 24 hours, most preferably 12 hours.
In the invention, as the fullerene is insoluble in water, the wall-broken or non-wall-broken ganoderma lucidum spore powder is insoluble in water at room temperature, while the wall-broken or non-wall-broken ganoderma lucidum spore powder is soluble in water at 50-100 ℃ (preferably 60-80 ℃), and the ganoderma lucidum spore powder is separated and extracted from the fullerene powder by utilizing the solubility of the ganoderma lucidum spore powder; and the fullerene may be removed by centrifugation.
The invention also provides wall-broken ganoderma lucidum spore powder which is prepared by the method.
The invention also provides a preparation method of the ganoderma lucidum spore oil, which comprises the following steps:
1) mixing fullerene powder and ganoderma lucidum spore powder and carrying out pretreatment to make the fullerene powder enter the ganoderma lucidum spore;
2) irradiating the pretreated mixed powder to obtain wall-broken ganoderma lucidum spore fullerene mixed powder;
3) adding water into the wall-broken ganoderma lucidum spore and fullerene mixed powder obtained in the step 2), stirring, centrifuging, removing precipitates, taking supernate, and drying to prepare wall-broken ganoderma lucidum spore powder;
4) extracting ganoderma lucidum spore oil from the wall-broken ganoderma lucidum spore powder obtained in the step 3).
Those skilled in the art will appreciate that various methods known in the art can be used to extract ganoderma lucidum spore oil from wall-broken ganoderma lucidum spore powder.
The invention also provides ganoderma lucidum spore oil which is prepared by the method.
The invention further provides application of the wall-broken ganoderma lucidum spore powder or ganoderma lucidum spore oil in preparing a medicament for preventing and/or treating tumors or application in preparing a medicament for preventing and/or treating liver injury caused by chemotherapy medicaments.
The invention has the beneficial effects that:
1. the invention provides a ganoderma lucidum spore wall breaking method, which is characterized in that ganoderma lucidum spore powder and fullerene powder are mixed and pretreated, cluster particles in the fullerene powder are crushed into micron and/or nanometer particles, and the micron and/or nanometer particles enter the shell wall and the interior of the ganoderma lucidum spore; and then, the fullerene is irradiated by an external energy source to generate phase change and volume expansion, so that the outer wall of the ganoderma lucidum spore is expanded, the wall breaking of the ganoderma lucidum spore is realized, and the wall breaking rate of the ganoderma lucidum spore is at least 98%.
2. The invention also provides a preparation method of the wall-broken ganoderma lucidum spore powder and the wall-broken ganoderma lucidum spore powder prepared by the method; the method does not involve high temperature and high pressure, maximally retains the active ingredient content and activity of Ganoderma spore, has high tumor inhibition rate, and has obvious protective effect on liver injury caused by chemotherapy drugs.
3. The invention also provides a preparation method of the ganoderma lucidum spore oil and the ganoderma lucidum spore oil prepared by the preparation method, wherein the ganoderma lucidum spore oil is extracted from the wall-broken ganoderma lucidum spore powder prepared by the invention, and the extraction rate is high.
Drawings
FIG. 1 shows the condition of different groups of liver cells in example 9 of the present invention.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes or modifications can be made by those skilled in the art after reading the description of the present invention, and such equivalents also fall within the scope of the invention.
The ball milling in the embodiment of the invention is carried out in an XQM-2L type ball mill, and the ball milling temperature is 30 ℃; the ball milling time is 8 h; the ball milling frequency was 700 r/min.
In the invention, the calculation method of the wall breaking rate comprises the following steps:
1) weighing 2.0mg of wall-broken ganoderma spore powder by using an analytical balance (precision 0.lmg) at room temperature, putting the wall-broken ganoderma spore powder into 10mL test tubes, adding 2mL of distilled water into each test tube by using a precision handheld dropper, and shaking for about 5min, wherein the ganoderma spore powder is insoluble in water at room temperature, so that suspension of the ganoderma spore powder is prepared;
2) sucking 6 mu L of suspension liquid by a micro-syringe (10 mu L), dripping the suspension liquid into a counting disc at one time, covering a glass sheet, standing for 2-3 min to ensure that the suspension liquid is uniformly distributed in the counting disc, and counting after the ganoderma lucidum spore powder does not move any more after sinking. Whether the wall of the spore is broken or not is identified by an optical microscope, the spore wall which is not broken is smooth, complete and plump, the double-wall structure is clear, and the individual forms are consistent; after wall breaking, the spore wall is incomplete, and the content is in an irregular block shape;
3) calculating the wall breaking rate of the ganoderma lucidum spores:
the formula for calculating the wall-breaking rate of the ganoderma lucidum spores is as follows: the wall breaking rate is [ (A-B)/A ] × 100%;
wherein A is the number of complete spores before wall breaking; b is the number of complete spores after wall breaking; the number of intact spores before wall breaking is the sum of the number of intact spores after wall breaking and the number of incomplete spores after wall breaking.
In the invention, the extraction method of the ganoderma lucidum spore oil is supercritical CO2An extraction method; the method for calculating the extraction rate of the ganoderma lucidum spore oil comprises the step of judging the content of fatty acid in the ganoderma lucidum spore oil by detectingThe extraction rate of the ganoderma lucidum spore oil is determined as follows:
weighing 20mg of the obtained spore oil in L0mL in a stoppered test tube, adding 2mL of 0.4mol/L KOH-methanol solution, heating in 70 ℃ water bath for 30min, adding 2mL of distilled water after the reaction is finished, and adding concentrated hydrochloric acid to neutralize until the pH value is neutral. And then adding 2mL of n-hexane for extraction, taking supernatant, and repeatedly washing for 2-3 times. And combining the supernatants, concentrating the combined supernatants under reduced pressure until the combined supernatants are dry, adding 2mL of 1% sulfuric acid-methanol solution, carrying out water bath at 70 ℃ for 30min, extracting the combined supernatants with n-hexane after the reaction is finished, fully oscillating the combined supernatants with distilled water, standing the combined supernatants for layering, taking the supernatants, repeatedly washing the supernatants for 2-3 times, combining the supernatants, drying the combined supernatants with anhydrous sodium sulfate, finally taking 1 mu L of the supernatants for GC-MS analysis, calculating the content of fatty acid in the ganoderma spore oil through the esterified substances obtained by the analysis, and further determining the extraction rate.
Example 1
1g of fullerene C60And fully mixing and ball-milling the ganoderma lucidum spore powder and 100g of ganoderma lucidum spore powder in a ball mill, then placing the mixed powder in a glass bottle, connecting the glass bottle with a radio frequency instrument through a coil, turning on the radio frequency instrument, adjusting the power to 300MHz, continuously irradiating for 2 hours, and then turning off the radio frequency instrument. Taking out the mixed powder, adding 80 ℃ water, fully stirring, performing centrifugal operation, and repeating for 3-5 times; due to the fact that fullerene is insoluble in water and can be deposited at the bottom, soluble substance, namely supernatant is collected and dried (at the temperature of 40 ℃ for 12 hours), and the wall-broken ganoderma lucidum spore powder is obtained.
Example 2
1g of fullerene C60And fully mixing and ball-milling the ganoderma lucidum spore powder and 500g of ganoderma lucidum spore powder in a ball mill, then placing the mixed powder in a glass bottle, connecting the glass bottle with a radio frequency instrument through a coil, turning on the radio frequency instrument, adjusting the power to 400MHz, continuously irradiating for 4 hours, and then turning off the radio frequency instrument. Taking out the mixed powder, adding 80 ℃ water, fully stirring, performing centrifugal operation, and repeating for 3-5 times; due to the fact that fullerene is insoluble in water and can be deposited at the bottom, soluble substance, namely supernatant is collected and dried (at the temperature of 40 ℃ for 12 hours), and the wall-broken ganoderma lucidum spore powder is obtained.
Example 3
Mixing 1g of fullereneAlkene C60And fully mixing and ball-milling the ganoderma lucidum spore powder and 300g of ganoderma lucidum spore powder in a ball mill, then placing the mixed powder in a glass bottle, connecting the glass bottle with a radio frequency instrument through a coil, turning on the radio frequency instrument, adjusting the power to 300MHz, continuously irradiating for 3 hours, and then turning off the radio frequency instrument. Taking out the mixed powder, adding 80 ℃ water, fully stirring, performing centrifugal operation, and repeating for 3-5 times; due to the fact that fullerene is insoluble in water and can be deposited at the bottom, soluble substance, namely supernatant is collected and dried (at the temperature of 40 ℃ for 12 hours), and the wall-broken ganoderma lucidum spore powder is obtained.
Example 4
1g of fullerene C70And fully mixing and ball-milling the ganoderma lucidum spore powder and 100g of ganoderma lucidum spore powder in a ball mill, then placing the mixed powder in a glass bottle, connecting the glass bottle with a radio frequency instrument through a coil, turning on the radio frequency instrument, adjusting the power to 300MHz, continuously irradiating for 3 hours, and then turning off the radio frequency instrument. Taking out the mixed powder, adding 80 ℃ water, fully stirring, performing centrifugal operation, and repeating for 3-5 times; due to the fact that fullerene is insoluble in water and can be deposited at the bottom, soluble substance, namely supernatant is collected and dried (at the temperature of 40 ℃ for 12 hours), and the wall-broken ganoderma lucidum spore powder is obtained.
Example 5
1g of fullerene C76Mixing with 100g Ganoderma spore powder in a ball mill, ball milling, placing the mixed powder in a glass bottle, linking the glass bottle with a radio frequency instrument via a coil, turning on the radio frequency instrument, adjusting power to 300MHz, continuously irradiating for 3.5h, and turning off the radio frequency instrument. Taking out the mixed powder, adding 80 ℃ water, fully stirring, performing centrifugal operation, and repeating for 3-5 times; due to the fact that fullerene is insoluble in water and can be deposited at the bottom, soluble substance, namely supernatant is collected and dried (at the temperature of 40 ℃ for 12 hours), and the wall-broken ganoderma lucidum spore powder is obtained.
Comparative example 1 high energy ball milling impact method
And (3) taking 300g of ganoderma lucidum spore powder, fully mixing and ball-milling in a ball mill, wherein the rotating speed of the ball mill is 700r/min, and the ball-milling time is 10 h. And after the ball milling is finished, taking out the powder in the ball milling tank to obtain the wall-broken ganoderma lucidum spore powder.
Comparative example 2-CO2Supercritical wall breaking method
Adding 300g Ganoderma spore powder into 1LCO2In the supercritical extraction kettle, the temperature and pressure of the extraction kettle and the separation kettle are respectively set to 35MPa and 40 ℃, the operation is carried out for 3h, and then the supercritical CO gas is rapidly released after the supercritical CO gas is stood for 12h under the high-pressure condition2. Critical gas CO2And after the release is finished, collecting a sample, namely the wall-broken ganoderma lucidum spore powder.
Example 6-wall-breaking ratio of wall-broken Ganoderma spore powder prepared in example 3, comparative example 1 and comparative example 2
Since in the preparation method of the present invention, when fullerene is removed by centrifugation, there will be very little amount of non-wall-broken ganoderma lucidum spores removed together with fullerene, for more scientific comparison, the precipitate of example 3 is slightly treated: adding excessive toluene into the precipitate, centrifuging the supernatant to obtain purple solution with fullerene dissolved therein, repeatedly cleaning the precipitate with ethanol for several times, drying, mixing the dried powder with the wall-broken Ganoderma spore powder obtained in example 3, and comparing the wall-broken rate of the mixed sample.
The wall-broken ganoderma lucidum spore powder obtained in the comparative example 1 and the comparative example 2 does not need to be treated.
The following results are obtained by calculating the wall breaking rate through the calculation method:
| case(s) | Wall breaking rate |
| Example 3 | 98％ |
| Comparative example 1 | 75％ |
| Comparative example 2 | 80％ |
As can be seen from the comparison in the table above, the wall-breaking method of the invention can greatly improve the wall-breaking rate of the ganoderma lucidum spores.
Example 7 extraction efficiency of Ganoderma spore oil from wall-broken Ganoderma spore powder prepared in example 3, comparative example 1 and comparative example 2
The following results are obtained by calculation through the extraction method and the calculation method of the extraction rate:
| case(s) | Extraction rate of ganoderma spore oil |
| Example 3 | 45％ |
| Comparative example 1 | 23％ |
| Comparative example 2 | 22％ |
As can be seen from the comparison of the table above, the wall-breaking method of the invention can greatly improve the utilization rate of the active substances of the ganoderma lucidum spores.
Example 8-comparison of anticancer Properties of wall-broken Ganoderma spore powder prepared in example 3, comparative example 1, and comparative example 2
The present example investigates the growth inhibition effect of the sporoderm-broken ganoderma lucidum spore powder on mouse liver cancer tumors, wherein,
animal strain: balb/c female mouse, 5 weeks, the weight between 16-20 g;
tumor model: mouse liver cancer H22 tumor strain;
the administration mode comprises the following steps: orally taking;
administration dose: 400mg/kg, 10 times in total, 1 time per day;
grouping experiments: and (4) grouping randomly, and 6 in each group.
Drug group a: the wall-broken ganoderma lucidum spore powder prepared in the embodiment 3;
drug group B: the wall-broken ganoderma lucidum spore powder prepared in the comparative example 1;
drug group C: the wall-broken ganoderma lucidum spore powder prepared in the comparative example 2;
control group: physiological saline;
the experimental method comprises the following steps: subcutaneous inoculation at a concentration of 5X 10 of 100. mu.L7/mL H22 hepatoma cells; the administration is started 24 hours after the inoculation, and the administration is continuously carried out for 10 times; and weighing the weight of the mouse every other day during the experiment, observing the growth condition of the tumor, finishing the experiment by 15 days after inoculation, weighing the tumor of the mouse, measuring the volume of the tumor, and calculating the tumor inhibition rate.
The tumor inhibition rate is calculated according to the formula:
tumor inhibition rate (mean tumor weight in control group-mean tumor weight in treatment group)/mean tumor weight in control group × 100%
The following results are obtained by the anti-cancer method and the tumor inhibition rate calculation:
| case(s) | Average tumor weight (g) | Tumor inhibition rate |
| Example 3 | 0.52±0.13 | 65.5％ |
| Comparative example 1 | 0.78±0.08 | 47.9％ |
| Comparative example 2 | 0.67±0.15 | 55.3％ |
| Control group | 1.5±0.17 | -- |
As can be seen from the comparison in the table above, the wall-broken ganoderma lucidum spore powder prepared by the wall-breaking method has the highest tumor inhibition rate.
Example 9 protective Effect of wall-broken Ganoderma spore powder on liver injury caused by chemotherapeutic drugs
Animal model: ICR 5-week-old female mice were selected and randomly divided into 5 groups of 6 mice each, corresponding to a control group, a model group and an experimental group: comparative example 1 group, comparative example 2 group and example 3 group.
Experimental groups: the chemotherapy drugs are administered to the mice by intragastric administration, and the specific doses are as follows: cyclophosphamide 80mg/kg body weight/time, busulfan 20mg/kg body weight/time; the wall-broken ganoderma lucidum spore powder in the example 3, the comparative example 1 and the comparative example 2 is administered to mice in a gastric lavage mode, and the specific dosage is as follows: 100 μ L/time, 10 mg/mL.
Control group: the same volume of physiological saline is used for replacing chemotherapeutic drugs and wall-broken ganoderma lucidum spore powder of the experimental group, and other treatment modes are consistent with those of the experimental group.
Model group: the chemotherapy drugs cyclophosphamide and busulfan are administrated to the mice in an intragastric manner, the dosage of the chemotherapy drugs is the same as that of an experimental group, the normal saline with the same volume is used for replacing the wall-broken ganoderma lucidum spore powder in the example 3, the comparative example 1 and the comparative example 2, the chemotherapy drugs are administrated to the mice in an intragastric manner, and other treatment modes are consistent with those of the experimental group.
The mice were cultured for 2-3 days, and the state of the mice was observed and taken as the 1 st day of the experiment after the state was stabilized. Then, the mice were administered with chemotherapy drugs or physiological saline of the same volume by gavage on days 2, 5, 9 and 12, respectively, and the mice were observed for effects on days 19, 20, 21, 24 and 25 after administration of the cell-wall broken ganoderma lucidum spore powder of example 3, comparative example 1 and comparative example 2 or physiological saline of the same volume by gavage.
As can be seen from FIG. 1, the model group showed that the liver of the mice had more obvious damage after the administration of the chemotherapeutic agent by gavage; experimental groups show that the wall-broken ganoderma lucidum spore powder in the example 3 is obviously improved in liver damage of mice by adopting the intragastric administration mode, the liver cell condition of the mice is closer to that of a control group, and the wall-broken ganoderma lucidum spore powder in the comparative example 1 and the comparative example 2 is weaker in protection effect on the liver damage of the mice by adopting the intragastric administration mode, and the liver cell condition of the mice is closer to that of a model group.
The wall-broken ganoderma lucidum spore powder prepared by the invention has good protection effect on liver injury caused by chemotherapy drugs.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (17)
1. A wall breaking method of ganoderma lucidum spores is characterized by comprising the following steps:
1) mixing fullerene powder and ganoderma lucidum spore powder and carrying out pretreatment to make the fullerene powder enter the ganoderma lucidum spore; wherein the fullerene powder is C60，C70，C76One or a mixture of several of them;
2) irradiating the pretreated mixed powder;
in the step 1), the temperature of the pretreatment is less than or equal to 50 ℃; the pretreatment comprises low-temperature high-energy ball milling, high-pressure homogenization and high-speed homogenization.
2. A preparation method of wall-broken ganoderma lucidum spore powder is characterized by comprising the following steps:
1) mixing fullerene powder and ganoderma lucidum spore powder and carrying out pretreatment to make the fullerene powder enter the ganoderma lucidum spore; wherein the fullerene powder is C60，C70，C76One or a mixture of several of them;
2) irradiating the pretreated mixed powder to obtain wall-broken ganoderma lucidum spore fullerene mixed powder;
3) adding water into the wall-broken ganoderma lucidum spore and fullerene mixed powder obtained in the step 2), stirring, centrifuging, removing precipitates, taking supernate and drying to obtain wall-broken ganoderma lucidum spore powder;
in the step 1), the temperature of the pretreatment is less than or equal to 50 ℃; the pretreatment comprises low-temperature high-energy ball milling, high-pressure homogenization and high-speed homogenization;
in the step 3), the temperature of the water is 50-100 ℃; the drying is vacuum drying or normal pressure air drying; the drying temperature is 20-50 ℃, and the drying time is 1-48 h.
3. A preparation method of ganoderma lucidum spore oil is characterized by comprising the following steps:
1) mixing fullerene powder and ganoderma lucidum spore powder and carrying out pretreatment to make the fullerene powder enter the ganoderma lucidum spore; wherein the fullerene powder is C60，C70，C76One or a mixture of several of them;
2) irradiating the pretreated mixed powder to obtain wall-broken ganoderma lucidum spore fullerene mixed powder;
3) adding water into the wall-broken ganoderma lucidum spore and fullerene mixed powder obtained in the step 2), stirring, centrifuging, removing precipitates, taking supernate, and drying to prepare wall-broken ganoderma lucidum spore powder;
4) extracting ganoderma lucidum spore oil from the wall-broken ganoderma lucidum spore powder obtained in the step 3);
in the step 1), the temperature of the pretreatment is less than or equal to 50 ℃; the pretreatment comprises low-temperature high-energy ball milling, high-pressure homogenization and high-speed homogenization;
in the step 3), the temperature of the water is 50-100 ℃; the drying is vacuum drying or normal pressure air drying; the drying temperature is 20-50 ℃, and the drying time is 1-48 h.
4. The method according to any of claims 1-3, wherein the fullerene powder is C60，C70Or a mixture thereof.
5. The method of claim 4, wherein the fullerene powder is C60。
6. The method according to any one of claims 1-3, wherein the particle size of the fullerene powder is 10 nm-50 μm.
7. The method according to any of claims 1-3, wherein the particle size of the pre-treated fullerene powder is between 50nm and 500 nm.
8. The method according to any one of claims 1-3, wherein the mass ratio of the fullerene powder to the ganoderma lucidum spore powder is 1: 5-10000.
9. The method according to any one of claims 1 to 3, wherein the ball milling temperature of the low-temperature high-energy ball milling is 20 to 40 ℃; the ball milling time is 1-20 h; the ball milling frequency is 200 to 2000 r/min.
10. A method according to any of claims 1-3, wherein said irradiating is by means of an external energy source, said external energy source being one or more of a magnetic field, radio frequency, radiation.
11. The method of claim 10, wherein the magnetic field has an energy of 0.1-10T; the irradiation time of the magnetic field is 1-12 h.
12. The method of claim 10, wherein the energy of the radio frequency is 100-1000 MHz; the radio frequency irradiation time is 0.5-20 h.
13. The method of claim 10, wherein the radiation has an energy of 1-1000 eV; the irradiation time of the rays is 0.5-12 h.
14. The method according to claim 2 or 3, wherein the mass-to-volume ratio of the wall-broken ganoderma lucidum spore-fullerene mixed powder to water is 1-1000 mg/mL.
15. A wall-broken ganoderma lucidum spore powder, which is prepared by the method of any one of claims 2 and 4-14.
16. A ganoderma lucidum spore oil, wherein the ganoderma lucidum spore oil is prepared by the method of any one of claims 3 to 14.
17. Use of the wall-broken ganoderma lucidum spore powder of claim 15 or the ganoderma lucidum spore oil of claim 16 in the preparation of a medicament for preventing and/or treating tumors or for preventing and/or treating liver injury caused by chemotherapeutic drugs.
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| CN101002805A (en) * | 2006-12-27 | 2007-07-25 | 宜兴市鹏鹞天然保健营养素有限公司 | Method for breaking trachytectum of glossy ganoderma |
| CN102793728A (en) * | 2012-08-24 | 2012-11-28 | 安徽哈博药业有限公司 | High-frequency oscillation wall-broken ganoderma lucidum spore powder |
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| CN104127872A (en) * | 2014-07-29 | 2014-11-05 | 中国科学院化学研究所 | Application of metal fullerene monocrystal nanoparticles in preparation of specific tumor vascular disrupting agent |
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| CN101002805A (en) * | 2006-12-27 | 2007-07-25 | 宜兴市鹏鹞天然保健营养素有限公司 | Method for breaking trachytectum of glossy ganoderma |
| CN102793728A (en) * | 2012-08-24 | 2012-11-28 | 安徽哈博药业有限公司 | High-frequency oscillation wall-broken ganoderma lucidum spore powder |
| CN104042642A (en) * | 2014-07-03 | 2014-09-17 | 绵阳三利农业科技有限公司 | Ganoderma spore wall breaking process |
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