CN116139179A - Preparation method of ganoderma lucidum spore oil with auxiliary enhancement and tumor metastasis inhibition effects - Google Patents
Preparation method of ganoderma lucidum spore oil with auxiliary enhancement and tumor metastasis inhibition effects Download PDFInfo
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- CN116139179A CN116139179A CN202310136613.7A CN202310136613A CN116139179A CN 116139179 A CN116139179 A CN 116139179A CN 202310136613 A CN202310136613 A CN 202310136613A CN 116139179 A CN116139179 A CN 116139179A
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- ganoderma lucidum
- lucidum spore
- spore oil
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- 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
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P35/04—Antineoplastic agents specific for metastasis
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
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- 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
- A61K2236/30—Extraction of the material
- A61K2236/37—Extraction at elevated pressure or temperature, e.g. pressurized solvent extraction [PSE], supercritical carbon dioxide extraction or subcritical water extraction
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Abstract
The invention provides a preparation method of ganoderma lucidum spore oil with auxiliary enhancement of tumor metastasis inhibition effect, belonging to the technical field of biology. The preparation method mainly comprises the following steps: 1) Treating the non-broken ganoderma lucidum spore powder with ethanol, water, centrifuging, and performing spray drying and low-temperature physical wall breaking on the ganoderma lucidum spore powder obtained by centrifugal separation to obtain purified broken ganoderma lucidum spore powder; 2) Granulating and drying the purified wall-broken ganoderma lucidum spore powder, and extracting by using supercritical carbon dioxide to obtain ganoderma lucidum spore oil. The method removes residual harmful substances and liposoluble impurities in the wall shell of Ganoderma spore powder, and the obtained Ganoderma spore oil has no plasticizer residue and pesticide residue, has high content of total triterpene, ergosterol, etc., and low acid value and peroxide value, and improves quality of Ganoderma spore oil. Animal experiments prove that the ganoderma lucidum spore oil prepared by the method has the effects of assisting in enhancing and inhibiting tumor metastasis and improving the immunity of organisms.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method of ganoderma lucidum spore oil with auxiliary enhancement of tumor metastasis inhibition effect.
Background
Ganoderma lucidum (Ganoderma lucidum) has been specially used for more than 2000 years as a Chinese herbal medicine, and is listed as "medicine-feeding" in Shennong Ben Cao Jing "in the eastern Han of China. The ganoderma lucidum spore oil has very rich components, comprises more than ten effective compounds such as ganoderma lucidum triterpene, triglyceride, ergosterol and the like, and has attracted wide attention in the aspects of antioxidation, anticancer, immunoregulation, anti-arthritis, blood sugar reduction, heart protection and the like.
The preparation of ganoderma lucidum spore oil generally comprises the steps of directly breaking the wall of ganoderma lucidum spore powder, and extracting the spore oil from the wall-broken ganoderma lucidum spore powder by using a supercritical carbon dioxide technology, wherein the ganoderma lucidum spore oil prepared by the method is easy to cause pesticide residues and excessive plasticizers, and contains fat-soluble components in the spore wall shells. The patent 'a method for removing fat-soluble harmful components in ganoderma lucidum spore oil' (application number 202210675602.1) proposes that ethanol is adopted as entrainer for the non-broken ganoderma lucidum spore powder, supercritical carbon dioxide is used for extraction to remove the harmful substances in the spore wall shell, the method is low in efficiency, and ethanol is also adopted as entrainer for the subsequent extraction of ganoderma lucidum spore oil, and ethanol residues are unavoidable in the spore oil.
The long-term contact or eating of the product with pesticide residues can lead the pesticide to be continuously accumulated in the body, and can form a potential threat to the body health, namely chronic poisoning, which can affect the nervous system, destroy the liver function, cause physiological disorder, affect the reproductive system, generate malformed strange embryo, cause cancer and the like; plasticizers can cause reduced immunity and reproductive ability in humans, with di (2-ethyl) hexylphthalate (DEHP) being 20 times more toxic than melamine; ethanol reacts with various drugs to produce adverse effects such as cephalosporins, hypoglycemic drugs including glibenclamide, metformin, insulin and the like. Ganoderma spore oil is a natural healthy product with auxiliary tumor metastasis inhibiting effect, is expensive, is usually used by tumor patients, and is often taken together with medicines, so that if the ganoderma spore oil contains more pesticide, plasticizer and ethanol residue, the treatment is unfavorable.
Disclosure of Invention
The invention aims to overcome the defect that the conventional ganoderma lucidum spore oil preparation method is easy to cause pesticide residue, excess plasticizer and ethanol residue, and provides a high-quality ganoderma lucidum spore oil preparation method with auxiliary enhancement and tumor metastasis inhibition effects without pesticide, plasticizer and ethanol residue.
The technical scheme adopted by the invention is a preparation method of ganoderma lucidum spore oil with auxiliary enhancement of tumor metastasis inhibition effect, which is characterized in that: breaking the wall of the purified ganoderma lucidum spore powder, granulating, drying, and extracting by supercritical carbon dioxide to obtain ganoderma lucidum spore oil.
The invention creatively proposes that after the wall of the purified ganoderma lucidum spore powder is broken, supercritical carbon dioxide extraction is adopted to prepare ganoderma lucidum spore oil, after pesticide residues, plasticizer residues and ethanol residues in the ganoderma lucidum spore powder wall shell are removed, supercritical carbon dioxide extraction is carried out, and the obtained high-quality ganoderma lucidum spore oil has high content of ganoderma lucidum total triterpene and ergosterol and low acid value and peroxide value.
Specifically: the preparation process of the purified ganoderma lucidum spore powder comprises the following steps:
1) Treating non-broken Ganoderma spore powder with 8 times of 95% ethanol, keeping micro boiling, treating for 2 hr, and centrifuging; filtering, adding 8 times of water, keeping micro boiling, treating for 1 hr, and centrifuging;
2) Spray drying the spore powder obtained by centrifugal filtration, wherein the air inlet temperature of spray drying is 190+/-10 ℃, and the air outlet temperature is 90+/-10 ℃, so as to obtain purified ganoderma lucidum spore powder;
3) The purified ganoderma lucidum spore powder is subjected to low-temperature physical wall breaking to obtain the purified wall-broken ganoderma lucidum spore powder.
The 95% ethanol is adsorbed by activated carbon or rectified to remove the residual plasticizer.
Ethanol is an organic solvent, and a trace amount of plasticizer may remain in the production process. When the spore powder is treated by ethanol, the dosage of the ethanol is relatively large and is 10 times of that of the spore powder. For this purpose, before the spore powder is treated with ethanol, the plasticizer which may be left in the spore powder is removed, so that the plasticizer left in the ethanol is not transferred to the treated spore powder, and the plasticizer which is left in the ethanol is removed by an activated carbon adsorption or rectification method.
The method comprises the steps of removing trace plasticizer which may be remained by the method of adsorbing or rectifying the used 95% ethanol by using activated carbon, purifying the non-broken ganoderma lucidum spore powder by using 95% ethanol and water to remove harmful substances such as pesticide residues, plasticizers and the like in the spore powder wall shells respectively, and finally removing the residual ethanol by using water. The spore powder is pretreated by ethanol and water and dried, so that the spore wall shell structure becomes more loose, after wall breaking, the spore powder is favorable for dissolving out spore oil and active ingredients during the extraction of the spore oil, the total triterpene and ergosterol content in the spore oil is high, the extraction efficiency of the ganoderma lucidum spore oil is improved, and the extraction time is saved by about 50 percent compared with that of the conventional method. Animal experiments prove that the ganoderma lucidum spore oil product prepared by the method has the effects of assisting in enhancing and inhibiting tumor metastasis and improving the immunity of organisms. In the process of removing pesticide, plasticizer and ethanol residue, the method also removes fat-soluble components in the spore powder wall shell, and further improves the concentration of total triterpene and ergosterol in the obtained ganoderma lucidum spore oil.
The ganoderma lucidum triterpene and ergosterol are main active components of ganoderma lucidum spore oil, have auxiliary anti-tumor pharmacological activity, and according to the research of Li Xiangmin, xieyzhen and the like, the total triterpene content in ganoderma lucidum spore oil has a positive correlation with an anti-tumor effect Cheng Xian, namely, the higher the total triterpene content is, the stronger the tumor-resisting effect is. According to the research of Guo Wei, luo Qiong and the like, ergosterol has a tumor inhibiting effect on S180 tumor-bearing mice, and the higher the dosage, the better the effect. The spore oil prepared by the invention has higher content of total triterpene and ergosterol, and has stronger auxiliary effect of inhibiting tumor metastasis.
The specific process of granulating, drying and supercritical carbon dioxide extraction comprises the following steps:
1) Granulating the purified wall-broken ganoderma lucidum spore powder with water, sieving with a 10-mesh sieve, boiling and drying at 60-70 ℃ until the water content is less than or equal to 6.0%.
2) Supercritical carbon dioxide extraction is carried out at 40-50 ℃ and 25-30 Mpa, the 1 st separation kettle is at 8-14 Mpa, the temperature is 45-50 ℃, the 2 nd separation kettle is at 6-7 Mpa, and the temperature is 40-50 ℃.
The total triterpene of the ganoderma lucidum spore oil obtained by the invention is more than or equal to 30 percent, and the ergosterol is more than or equal to 350mg/100g.
Animal experiments prove that the prepared ganoderma lucidum spore oil has the effects of assisting in enhancing and inhibiting tumor metastasis and improving the immunity of organisms.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the method firstly removes the trace plasticizer possibly remained in the ethanol, then removes pesticide residues and plasticizer residues in the spore powder wall shells by using the ethanol, removes fat-soluble components in the wall shells, and the prepared ganoderma lucidum spore oil has no pesticide and plasticizer residues, no fat-soluble components in the spore wall shells and good quality.
2. As the spore powder is pretreated by ethanol and water and dried, the spore wall shell structure becomes more loose, and the leaching of the spore oil is facilitated during the extraction of the spore oil, the extraction efficiency of the ganoderma lucidum spore oil is improved, and the extraction time is saved by about 50 percent compared with that of the conventional method.
3. The ganoderma lucidum spore oil prepared by the invention has high total triterpene and ergosterol content, the total triterpene content is more than or equal to 30%, and the ergosterol content is more than or equal to 350mg/100g. Triterpenes have cytotoxicity on tumor cells, induce apoptosis of tumor cells by regulating cell cycle, and can directly inhibit growth and proliferation. Ergosterol not only can enhance resistance, but also has the effects of resisting oxidation, resisting tumor, inhibiting bacteria and promoting urination. The spore oil effective component has the effects of inhibiting tumor growth and increasing value, and promoting immunity, and can fully exert the anti-tumor effect of the ganoderma lucidum spore oil on human body.
4. The method has high efficiency, can remove pesticide residues, harmful substances and fat-soluble impurities in the spore powder wall shell, can be carried out in a common traditional Chinese medicine multifunctional extraction tank, can treat hundreds of kilograms at a time, and has short time as long as about 3 hours; supercritical carbon dioxide extraction is adopted, and only a few kilograms to tens of kilograms can be processed in the same time.
5. According to the technical scheme, the ganoderma lucidum spore oil can obviously assist cyclophosphamide to inhibit breast cancer tumors from forming metastasis in the lung, relieve weight reduction caused by cyclophosphamide, promote generation of CD8+T cells in a mouse blood sample, and obviously enhance the activity of NK cells in vivo, so that an effect of assisting in preventing tumor metastasis is effectively exerted. The activity of the spore oil of the invention is higher than that of the spore oil prepared by the conventional method.
Drawings
Fig. 1A is the body weight numbers of the blank group, the control group, the cyclophosphamide group, the ganoderma lucidum spore oil group of cyclophosphamide binding sample 1 and the ganoderma lucidum spore oil group of cyclophosphamide binding sample 2 on day 21 after the mice were inoculated with tumors.
FIG. 1B shows the number of breast cancer metastases in mice lung formed by the control group, cyclophosphamide combined sample 1 Ganoderma lucidum spore oil group and cyclophosphamide combined sample 2 Ganoderma lucidum spore oil group.
Fig. 2A is the lung weight to body weight ratio of the blank, control, cyclophosphamide combined sample 1 and cyclophosphamide combined sample 2.
Fig. 2B is a ratio of spleen weight to body weight in mice in the blank, control, cyclophosphamide combined sample 1 and cyclophosphamide combined sample 2.
Fig. 3A is the percentage of cd8+ T cells in blood samples of mice in the blank, control, cyclophosphamide combined sample 1, and cyclophosphamide combined sample 2.
FIG. 3B shows NK cell activity in mice in the blank, control, cyclophosphamide-bound sample 1, and cyclophosphamide-bound sample 2.
Detailed Description
In order to fully disclose the present invention, the following description will be given with reference to examples, but the scope of the present invention is not limited to these examples.
The ganoderma lucidum spore oil can enhance the activity of immune cells and assist chemotherapeutics to inhibit tumors from forming metastasis in lung.
In the present invention, the tumor is preferably breast cancer, or other cancers such as colon cancer, etc., which are prone to form lung metastasis. The ganoderma lucidum spore oil can obviously assist cyclophosphamide to inhibit breast cancer from forming and transferring in lung, reduce the ratio of lung weight to body weight of mice, reduce the ratio of spleen weight to body weight of mice, promote the generation of CD8+ T cells in blood samples of mice, and remarkably enhance the activity of NK cells in vivo.
The technical scheme of the invention is further described by the specific examples.
Preparation of ganoderma lucidum spore oil sample 1:
1. 35kg of non-broken ganoderma lucidum spore powder is subjected to low-temperature physical wall breaking to obtain 33.3kg of broken ganoderma lucidum spore powder.
2. Granulating 33.3kg of wall-broken Ganoderma spore powder with water, sieving with 10 mesh sieve, boiling, drying at 70deg.C until the water content is less than or equal to 6.0%.
3. Extracting the prepared wall-broken ganoderma lucidum spore powder particles with supercritical carbon dioxide at 45 ℃ under 25Mpa, 8Mpa in a 1 st separation kettle at 45 ℃ under 6Mpa in a 2 nd separation kettle at 40 ℃ for 8 hours to obtain 11.2kg of ganoderma lucidum spore oil (marked as sample 1).
Preparation of Ganoderma lucidum spore oil sample 2
1. 300kg of 95% ethanol is taken and placed in a storage tank, 6kg of active carbon is added, and the mixture is stirred and adsorbed for 2 hours and filtered for standby.
2. 35kg of non-broken ganoderma lucidum spore powder is treated by 280kg of 95% ethanol for 2 hours after micro boiling, and is centrifugally filtered; the filtered spore powder is treated by micro boiling for 1 hour by using 280kg of drinking water, and is centrifugally filtered.
3. Spray drying the spore powder obtained by centrifugal filtration, wherein the air inlet temperature of spray drying is 190+/-10 ℃, and the air outlet temperature is 80+/-10 ℃, thus obtaining 32.8kg of purified ganoderma lucidum spore powder.
4. 32.8kg of purified ganoderma lucidum spore powder is subjected to low-temperature physical wall breaking to obtain 31.5kg of wall-broken ganoderma lucidum spore powder.
5. Granulating 31.5kg of purified wall-broken ganoderma lucidum spore powder with water, sieving with a 10-mesh sieve, boiling and drying, setting the temperature to 60 ℃, and drying until the water content is less than or equal to 6.0%.
6. Extracting the prepared wall-broken ganoderma lucidum spore powder particles with supercritical carbon dioxide at 45 ℃ under 25Mpa, 8Mpa in a 1 st separation kettle at 45 ℃ under 6Mpa in a 2 nd separation kettle at 40 ℃ for 4h to obtain 9.8kg of purified ganoderma lucidum spore oil (marked as sample 2).
Preparation of Ganoderma lucidum spore oil sample 3
1. 300kg of 95% ethanol is taken and placed in a storage tank, 6kg of active carbon is added, and the mixture is stirred and adsorbed for 2 hours and filtered for standby.
2. 35kg of non-broken ganoderma lucidum spore powder is treated by 280kg of 95% ethanol for 2 hours after micro boiling, and is centrifugally filtered; the filtered spore powder is kept micro-boiling by 280kg of drinking water, treated for 1 hour and centrifugally filtered.
3. Spray drying the spore powder obtained by centrifugal filtration, wherein the air inlet temperature of spray drying is 190+/-10 ℃, and the air outlet temperature is 80+/-10 ℃, thus obtaining 32.7kg of purified ganoderma lucidum spore powder.
4. 32.7kg of purified ganoderma lucidum spore powder is subjected to low-temperature physical wall breaking to obtain 31.1kg of wall-broken ganoderma lucidum spore powder.
5. Granulating 31.1kg of purified wall-broken ganoderma lucidum spore powder with water, sieving with a 10-mesh sieve, boiling and drying, setting the temperature to 70 ℃, and drying until the water content is less than or equal to 6.0%.
6. Extracting the prepared wall-broken ganoderma lucidum spore powder particles with supercritical carbon dioxide at 50 ℃ under 30Mpa, 11Mpa in a 1 st separation kettle at 50 ℃ under 7Mpa in a 2 nd separation kettle at 45 ℃ for 4h to obtain 9.4kg of purified ganoderma lucidum spore oil (marked as sample 3).
Preparation of Ganoderma lucidum spore oil sample 4
1. And (3) taking 300kg of 95% ethanol, rectifying the ethanol by using an alcohol rectifying tower, and adding a proper amount of purified water into the rectified ethanol to prepare the ethanol with the concentration of 95% for later use.
2. 35kg of non-broken ganoderma lucidum spore powder is treated by 280kg of 95% ethanol for 2 hours after micro boiling, and is centrifugally filtered; the filtered spore powder is kept micro-boiling by 280kg of drinking water, treated for 1 hour and centrifugally filtered.
3. Spray drying the spore powder obtained by centrifugal filtration, wherein the air inlet temperature of spray drying is 190+/-10 ℃, and the air outlet temperature is 80+/-10 ℃, thus obtaining 32.5kg of purified ganoderma lucidum spore powder.
4. 32.5kg of purified ganoderma lucidum spore powder is subjected to low-temperature physical wall breaking to obtain 31.2kg of wall-broken ganoderma lucidum spore powder.
5. Granulating 31.2kg of purified wall-broken ganoderma lucidum spore powder with water, sieving with a 10-mesh sieve, boiling and drying, setting the temperature to 70 ℃, and drying until the water content is less than or equal to 6.0%.
6. Extracting the prepared wall-broken ganoderma lucidum spore powder particles with supercritical carbon dioxide at 50 ℃ under 30Mpa, 14Mpa in a 1 st separation kettle at 50 ℃ under 6Mpa in a 2 nd separation kettle at 45 ℃ for 4 hours to obtain 9.2kg of purified ganoderma lucidum spore oil (marked as sample 4).
The samples prepared in the above examples were tested
TABLE 1 detection results of total triterpenes and ergosterol in Ganoderma lucidum spore oil samples
Note that: 1. sample 1 is a conventional spore oil preparation method at present, and samples 2, 3 and 4 are preparation methods of the invention;
2. the standard specification of the peroxide value is less than or equal to 0.25g/100g, and the standard specification of the acid value is less than or equal to 12mgKOH/g;
3. the plasticizer is selected from dibutyl phthalate (DBP) and di (2-ethyl) hexyl phthalate (DEHP) which are most easily detected, and the standard rule is respectively less than or equal to 0.3mg/kg and less than or equal to 1.5mg/kg;
4. undetected, indicated below the detection limit.
TABLE 2 detection results of pesticide residues in Ganoderma lucidum spore oil samples
As can be seen from the detection results in tables 1 and 2, according to the preparation method of the invention, the total triterpene content and the ergosterol content are higher than those of the conventional preparation method, the peroxide value and the acid value are lower than those of the conventional preparation method, and the pesticide residue and the plasticizer in the ganoderma lucidum spore powder can be effectively removed.
Experimental example 1
1. Experimental materials
1.1 reagents
The ganoderma lucidum spore oil and the soybean oil of the samples 1 and 2 are respectively prepared into liquid medicine of 60mg ganoderma lucidum spore oil/mL.
10% fetal bovine serum (ExCell Bio, uyerba); 1640 medium (Hyclone); 0.25% pancreatin (GENVIEW, usa); soybean oil was purchased from Shanghai Ala Biochemical technologies Co., ltd; perCP-Cy5.5-CD8 streaming antibodies were purchased from Hangzhou Union Biotechnology Co.
1.2 cell lines
Mouse breast cancer cell line 4T1 was purchased from the Shanghai cell bank; YAC-1 cells were purchased from the company of biological technology limited liability, prosperous in the ancient cooking vessel, beijing.
1.3 laboratory animals
The experimental animals were female BALB/C mice 6 weeks old, weighing 18+ -2 g, purchased from Jiangsu Wuku Biotech Co. The treatment and study of experimental animals met the standards of the ethical committee of animals. The environment for experimental animal feeding management meets the requirements of SPF-level animal experimental facilities, and experimental animals drink water and eat food freely.
1.3 measurement index
Pulmonary transfer junction: tumor cells in mice form metastases in the lungs and form markers of node tumor metastasis. CD8 + Percentage of T lymphocytes: CD8 + T lymphocytes can differentiate into cytotoxic cells, can resist invasion of external pathogens, and have a certain protection effect on organisms. NK fineCell activity: the main activity of natural killer NK cells is killing tumor, and the enhancement of NK cell activity has important significance in the aspects of immunoregulation and anti-tumor.
2. Animal model building
2.1 Culture of 4T1 cells
4T1 cells were placed in 1640 medium containing 10% fetal bovine serum, 100U/mL penicillin sodium and 100U/mL streptomycin under culture conditions of 5% CO 2 Is placed in a incubator at 37 ℃; culturing 4T1 cells in logarithmic growth phase, digesting the cells with 0.25% pancreatin for 10min when the cells grow to about 80% abundance, stopping digestion with serum-containing medium, centrifuging, and counting for use.
2.2 building of a model for breast cancer lung metastasis
30 female BALB/C mice were selected, and 100uL concentration was 5X 10 by tail vein injection into each mouse 5 The 4T1 breast cancer suspension cells with the volume of one mL are subjected to normal diet, after 21 days of modeling, one control group of mice is randomly dissected to take the lung, and the formation of lung nodes is observed, so that the success of the breast cancer mice metastasis model modeling is indicated.
2.3 grouping of laboratory animals and tissue treatment
Mice were divided into 5 groups, wherein the mice injected with breast cancer cells were randomly divided into 4 groups, namely, a control group, a cyclophosphamide group, a ganoderma lucidum spore oil group of cyclophosphamide binding sample 1 and a ganoderma lucidum spore oil group of cyclophosphamide binding sample 2, and a blank group (i.e., mice not injected with breast cancer cells) was established. The blank group was given physiological saline daily, the control group was given soybean oil daily, the cyclophosphamide group was given cyclophosphamide abdominal cavity at 0.05g/kg once every 6 days (total 3 times), the cyclophosphamide-conjugated ganoderma lucidum spore oil group of sample 1 was given ganoderma lucidum spore oil of sample 1 daily at 0.3g/kg, and cyclophosphamide abdominal cavity at 0.05g/kg once every 6 days (total 3 times), the ganoderma lucidum spore oil of sample 2 was given cyclophosphamide-conjugated ganoderma lucidum spore oil of sample 2 daily at 0.3g/kg, and cyclophosphamide abdominal cavity at 0.05g/kg once every 6 days (total 3 times). Mice were dosed with ganoderma lucidum spore oil three days prior to modeling, and dosing was continued for 21 days. After 4h of last dose, the mice were weighed, and after orbital bleeding, the mice of each group were sacrificed and the lungs and spleen were taken and weighed separately.
3. Experimental method
3.1 recording of weight changes in mice
Body weight was recorded for each group of mice from day 1 to day 21 of modeling, once every 2 days.
3.2 recording of pulmonary metastasis in mice
Lungs of each group of mice after collection and sacrifice were placed in brussels dye solution and after 24 hours, node formation of the lungs of the mice was counted.
3.3 ratio of pulmonary weight to weight of mice
The mice were weighed prior to sacrifice, lung tissue from freshly sacrificed mice was collected and weighed, and the value of the mouse lung weight was divided by the value of the mouse body weight to give the ratio of the mouse lung weight to the body weight.
3.4 ratio of spleen weight to weight of mice
The mice were weighed prior to sacrifice, spleen tissue from freshly sacrificed mice was collected and weighed, and the value of the spleen weight of the mice was divided by the value of the body weight of the mice to obtain the ratio of the spleen weight of the mice to the body weight.
3.5 CD8 in mouse blood samples + Determination of the percentage of T cells
Mouse blood samples were collected from the orbit prior to sacrifice, placed in heparin sodium anticoagulation tubes, and mixed at 1:10 adding erythrocyte lysate, lysing at 37deg.C for 10min, centrifuging at 400g level for 10min, collecting cell precipitate to obtain mononuclear cells in blood sample, and adjusting cell concentration to 1×10 6 mu.L/100 uL of mouse PerCP-Cy5.5-CD8 antibody was added, incubated at 4℃for 30 minutes, blood samples were centrifuged horizontally in 400g for 10 minutes, cell pellet was resuspended in 1mL of flow cell dilution, and cells positive for PerCP-Cy5.5 expression were detected by flow cytometry and the percentage of PerCP-Cy5.5 positive cells was determined as the percentage of CD8 positive.
3.6 determination of NK cell Activity in spleen of mouse (LDH method)
YAC-1 cell culture (target cells): YAC-1 cells were placed in 1640 medium containing 10% fetal bovine serum, 100U/mL penicillin sodium and 100U/mL streptomycin, culture stripsThe parts are of 5% CO 2 Is placed in a incubator at 37 ℃; culturing 4T1 cells in logarithmic growth phase, sucking 2-3mL of cell suspension when the cells grow to about 80% abundance, centrifuging 400g for 5min, collecting cell precipitate, and counting for later use.
Preparation of spleen cell suspension (effector cells): the spleen was aseptically removed, washed 3 times with an appropriate amount of asepsis Hank's solution, centrifuged for 10min (1000 r/min) each time, and the cell suspension was then removed. Filtering with 200 mesh sieve, washing with Hank's solution for 3 times, centrifuging for 10min (1000 r/min), suspending the cells in 2ml of complete culture, counting spleen cells with cell counter, and adjusting cell concentration to 2×10 with RPMI1640 complete culture solution 7 And each mL. NK cell activity assay: the concentration is 4 multiplied by 10 5 100uL of YAC-1 target cells and effector cells per mL are added into a U-shaped 96-well plate; target cells and culture solution of the target cell natural release hole are 100uL each, and target cells naturally release Kong Jiaba cells and 1% NP40 are 100uL each; all the above-mentioned materials are equipped with three compound holes, at 37 deg.C, 5% CO 2 The cells were cultured in an incubator for 4 hours, and then the 96-well plate was centrifuged at 1500r/min for 5 minutes. 100uL of the supernatant is sucked up from each well and placed in a 96-well flat-bottomed culture plate, 100uL of newly prepared LDH matrix liquid is added for reaction for 3min, 30uL of HCL of 1mol/L is added into each well, and the optical density value is measured at 492nm/490nm of an enzyme-labeled instrument.
3.7 statistical analysis
All data are expressed by mean positive and negative SD, and P < 0.05 is taken as a significance difference, wherein significance marks are respectively * P＜0.05， ** P＜0.01， # P is less than 0.05. All data were analyzed using SPSS software, more than three groups were analyzed using one-way ANOVA, and comparisons between the two groups were made using unpaired t-tests.
4. Experimental results
The invention establishes a breast cancer lung metastasis mouse model by using breast cancer cells 4T1, and evaluates whether the ganoderma lucidum spore oil according to the sample 2 has the functions of enhancing immune cell function and assisting a chemotherapeutic drug to inhibit tumor metastasis compared with the ganoderma lucidum spore oil according to the sample 1 through the experiment. The experimental results are shown in FIGS. 1-3.
(1) Monitoring of body weight and recording of lung metastasis nodules in tumor vaccinated day 21 mice
As shown in fig. 1A, on day 21 of tumor inoculation of mice, the ganoderma lucidum spore oil of sample 2 was more effective in alleviating weight loss in mice caused by cyclophosphamide as a chemotherapeutic agent (P < 0.05VS nsP > 0.05 and #p < 0.05) than the ganoderma lucidum spore oil of sample 1. As shown in fig. 1B, the ganoderma lucidum spore oil group of cyclophosphamide-bound sample 2 significantly inhibited the metastasis node of breast cancer in the lung of mice (P < 0.01) compared to the control group, and the ganoderma lucidum spore oil of sample 2 inhibited tumor lung metastasis more significantly (#p < 0.05) compared to the ganoderma lucidum spore oil group of cyclophosphamide-bound sample 1.
Table 1 effect on mouse body weight from day 1 to day 21 in mouse lung metastasis model (n=6, x±s)
Table 2 effect on the number of mouse lung metastasis nodes in mouse lung metastasis model (n=6, x±s)
| Group of | Number of mouse lung metastasis node |
| 4T1 control group | 29.00±14.53 |
| Cyclophosphamide group | 9.83±5.34 |
| Cyclophosphamide binding sample group 1 | 8.67±2.94 |
| Cyclophosphamide bound |
4.67±1.86 |
As shown in fig. 2A-B, the ganoderma lucidum spore oil of sample 2 was effective in reducing the lung weight, spleen weight to body weight ratio of mice, which was closer to the lung weight, spleen weight to body weight ratio of mice in the placebo group, than the ganoderma lucidum spore oil of sample 1. (. Times.P < 0.05VS nsP > 0.05)
TABLE 3 influence on the weight-to-weight ratio of mouse lung weights in mouse lung metastasis model (n=6, x+ -s)
| Group of | Ratio of pulmonary weight to weight of mice |
| Blank group | 0.0043±0.0006 |
| 4T1 control group | 0.0119±0.0052 |
| Cyclophosphamide group | 0.0080±0.0010 |
| Cyclophosphamide binding sample group 1 | 0.0078±0.0013 |
| Cyclophosphamide bound |
0.0070±0.0010 |
TABLE 4 influence on the weight ratio of mouse spleen re-occupation in a mouse lung metastasis model (n=6, x+ -s)
| Group of | Spleen weight ratio of mice |
| Blank group | 0.0061±0.0008 |
| 4T1 control group | 0.0156±0.0043 |
| Cyclophosphamide group | 0.0099±0.0026 |
| Cyclophosphamide binding sample group 1 | 0.0096±0.0021 |
| Cyclophosphamide bound |
0.0087±0.0022 |
As shown in FIG. 3A, the Ganoderma lucidum spore oil of sample 2 combined with cyclophosphamide was more effective in increasing the percentage of CD8+ T cells (#P < 0.05) than the Ganoderma lucidum spore oil of sample 1. As shown in FIG. 3B, the ganoderma lucidum spore oil of sample 2 combined with cyclophosphamide increased NK cell activity (#P < 0.05) more effectively than the ganoderma lucidum spore oil of sample 1.
TABLE 5 CD8 on mice in a mouse lung metastasis model + Effect of T cell activity (n=6,x±s)
| Group of | Mouse CD8 + T cell Activity (%) |
| Blank group | 37.42±5.45 |
| 4T1 control group | 3.01±1.20 |
| Cyclophosphamide group | 13.58±2.45 |
| Cyclophosphamide binding sample group 1 | 14.03±2.18 |
| Cyclophosphamide bound |
16.87±2.16 |
TABLE 6 influence on mouse NK cell activity in mouse lung metastasis model (n=6, x.+ -. S)
| Group of | Mouse NK cell Activity (%) |
| Blank group | 1088.17±167.61 |
| 4T1 control group | 1578.66±31.24 |
| Cyclophosphamide group | 1261.74±257.85 |
| Cyclophosphamide binding sample group 1 | 1333.50±200.61 |
| Cyclophosphamide bound |
1627.47±74.84 |
In combination with the above embodiments, the product has the function of improving the immunity of the organism, can assist the chemotherapeutic drugs to inhibit the lung metastasis of tumors, and assists cancer patients to fight against the cancers.
Claims (6)
1. A preparation method of ganoderma lucidum spore oil with auxiliary enhancement of tumor metastasis inhibition effect is characterized by comprising the following steps: breaking the wall of the purified ganoderma lucidum spore powder, granulating, drying, and extracting by supercritical carbon dioxide to obtain ganoderma lucidum spore oil.
2. The method for preparing ganoderma lucidum spore oil with auxiliary enhancement of tumor metastasis inhibition effect according to claim 1, which is characterized in that: the preparation process of the purified ganoderma lucidum spore powder comprises the following steps:
1) Treating non-broken Ganoderma spore powder with 8 times of 95% ethanol, keeping micro boiling, treating for 2 hr, and centrifuging; filtering, adding 8 times of water, keeping micro boiling, treating for 1 hr, and centrifuging;
2) Spray drying the spore powder obtained by centrifugal filtration, wherein the air inlet temperature of spray drying is 190+/-10 ℃, and the air outlet temperature is 90+/-10 ℃, so as to obtain purified ganoderma lucidum spore powder;
3) The purified ganoderma lucidum spore powder is subjected to low-temperature physical wall breaking to obtain the purified wall-broken ganoderma lucidum spore powder.
3. The method for preparing ganoderma lucidum spore oil with auxiliary enhancement of tumor metastasis inhibition effect according to claim 2, wherein the 95% ethanol is used for removing the plasticizer which remains by the method of activated carbon adsorption or rectification.
4. The method for preparing ganoderma lucidum spore oil with auxiliary enhancement of tumor metastasis inhibition effect according to claim 2, which is characterized in that:
1) Granulating the purified wall-broken ganoderma lucidum spore powder with water, sieving with a 10-mesh sieve, and boiling and drying at 60-70 ℃ until the water content is less than or equal to 6.0%;
2) Supercritical carbon dioxide extraction is carried out at 40-50 ℃ and 25-30 Mpa, the 1 st separation kettle is at 8-14 Mpa, the temperature is 45-50 ℃, the 2 nd separation kettle is at 6-7 Mpa, and the temperature is 40-50 ℃.
5. The method for preparing ganoderma lucidum spore oil with auxiliary enhancement of tumor metastasis inhibition effect according to claim 1, wherein the total triterpene of the obtained ganoderma lucidum spore oil is more than or equal to 30%, and ergosterol is more than or equal to 350mg/100g.
6. The ganoderma lucidum spore oil with auxiliary enhancement of tumor metastasis inhibition effect prepared by any one of the methods of claims 1-4, wherein the prepared ganoderma lucidum spore oil has the effects of auxiliary enhancement of tumor metastasis inhibition effect and improvement of body immunity proved by animal experiments.
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| CN1883590A (en) * | 2006-05-24 | 2006-12-27 | 广东粤微食用菌技术有限公司 | Method for preparing ganoderma spore oil |
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| CN110051708A (en) * | 2019-05-08 | 2019-07-26 | 浙江寿仙谷医药股份有限公司 | A kind of ganoderma lucidum spore oil and its preparation method and application |
| CN115125059A (en) * | 2022-06-15 | 2022-09-30 | 中科健康产业集团股份有限公司 | Method for removing fat-soluble harmful ingredients in ganoderma lucidum spore oil |
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| CN1883590A (en) * | 2006-05-24 | 2006-12-27 | 广东粤微食用菌技术有限公司 | Method for preparing ganoderma spore oil |
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