CN111826416A - Ganoderma lucidum quality evaluation method based on in-vitro tumor cell culture model - Google Patents
Ganoderma lucidum quality evaluation method based on in-vitro tumor cell culture model Download PDFInfo
- Publication number
- CN111826416A CN111826416A CN202010668871.6A CN202010668871A CN111826416A CN 111826416 A CN111826416 A CN 111826416A CN 202010668871 A CN202010668871 A CN 202010668871A CN 111826416 A CN111826416 A CN 111826416A
- Authority
- CN
- China
- Prior art keywords
- ganoderma lucidum
- solution
- sample
- tumor cells
- ganoderma
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Cell Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Toxicology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to a ganoderma lucidum quality evaluation method based on an in-vitro tumor cell culture model, which comprises the following steps: preparing a sample solution and a positive control solution from a ganoderma lucidum sample to be detected and a positive control, diluting, and mixing or respectively co-incubating with 2D or 3D cultured tumor cells; respectively adding cell activity indicators into the incubated solution for reaction, detecting the absorbance or fluorescence intensity of the reaction solution, calculating the inhibition rate of the reaction solution on the proliferation of tumor cells, and calculating the corresponding IC50 or growth inhibition rate to evaluate the quality of the ganoderma lucidum according to the inhibition rate or the growth inhibition rate. The quality of the ganoderma lucidum is evaluated by measuring the inhibition effect of the ganoderma lucidum on the tumor cells based on the tumor cells cultured in vitro, and the detection index is directly related to the anti-tumor activity of the ganoderma lucidum and can reflect the clinical curative effect of the ganoderma lucidum, so that the quality of the ganoderma lucidum can be comprehensively evaluated; the 3D culture of the tumor cells is closer to in-vivo in-situ tumors, the evaluation result is more accurate, animal ethics is not involved, the analysis process is short, and the analysis cost is low.
Description
(I) technical field
The invention relates to a ganoderma lucidum quality evaluation method based on an in-vitro tumor cell culture model.
(II) background of the invention
The quality control of traditional Chinese medicine has important significance for ensuring the safety, effectiveness and consistency of traditional Chinese medicine products, and is the key of modernization and internationalization of traditional Chinese medicine. With the continuous development of related subjects such as analytical chemistry, pharmaceutical analysis and the like, the quality evaluation technology of traditional Chinese medicines is greatly improved, a quality inspection technology system taking chemical detection as a core is gradually formed, and the quality of traditional Chinese medicine products is evaluated by detecting main chemical components, index components or active components and the like. However, the chemical composition of the traditional Chinese medicine is complex and the effective components are often unknown, and the determination of one or a few index components is difficult to truly reflect the quality of most traditional Chinese medicine products, which also leads to the fact that the chemical detection method is often disjointed from the efficacy and clinical curative effect of the traditional Chinese medicine. In comparison, the detection of biological activity is more directly related to the curative effect of traditional Chinese medicine. Therefore, the establishment of a quality evaluation method related to the drug effect by detecting the biological activity of the traditional Chinese medicine has great practical significance for improving the quality of traditional Chinese medicine products, and becomes the leading edge of research in the field of quality control of traditional Chinese medicines.
Ganoderma lucidum is a dried fruiting body of Ganoderma lucidum or Ganoderma sinensis of Polyporaceae, and is a traditional rare and rare medicinal material in China. The compendium of materia Medica records: glossy ganoderma is neutral in nature, bitter in taste and non-toxic, mainly binds to the interior of the chest, benefits heart-qi, supplements middle-jiao, increases wisdom, does not forget, lightens body for long-term taking, does not age and prolongs the life of the immortal spirit. Modern researches show that ganoderma lucidum has various pharmacological effects of resisting tumor, regulating immunity, strengthening heart, regulating blood fat, reducing blood sugar, protecting liver and the like, and is mainly used for treating diseases such as coronary heart disease, hyperlipidemia, hypertension, diabetes, tumor and the like clinically. Ganoderma lucidum has a variety of chemical components including polysaccharides, triterpenes, alkaloids, fatty acids, etc., wherein polysaccharides and triterpenes are the main active ingredients.
At present, the quality evaluation of the lucid ganoderma mainly adopts the content determination of chemical components. For example, 2015 edition of Chinese pharmacopoeia specifies the contents of polysaccharides, triterpenes and sterols, which are main active ingredients of ganoderma lucidum, wherein the content of polysaccharides should not be less than 0.9%, and the content of triterpenes and sterols should not be less than 0.5%. According to pharmacopoeia standards, the sum of the two components is less than 1.5% of the dry weight of the ganoderma lucidum, and the overall quality of the ganoderma lucidum is difficult to objectively evaluate.
In the journal paper' Ganoderma quality evaluation based on UPLC-MS/MS multi-index component determination (Chinese pharmacist, 2019,22 (05): 844-. The detection method comprises the following steps: and detecting the contents of ganoderic acid C2, ganoderic acid A, ganoderic acid G, ganoderic acid B, ganoderic acid A and ganoderic acid D in different batches of ganoderma samples by using a UPLC-MS/MS technology by using a mixed solution of ganoderic acid C2, ganoderic acid A, ganoderic acid G, ganoderic acid B, ganoderic acid A and ganoderic acid D as a reference substance. The disadvantages of this method are: although the contents of ganoderic acid C2, ganoderic acid A, ganoderic acid G, ganoderic acid B, ganoderic acid A and ganoderic acid D in a ganoderma lucidum sample can be rapidly measured by using the UPLC-MS/MS technology, the contents cannot directly reflect the biological activity of each component and cannot comprehensively reflect the quality of a ganoderma lucidum medicinal material.
In the 'quality evaluation method of ganoderma lucidum medicinal material' with the patent application number of CN201610821304.3, the content of total ganoderma triterpene in a ganoderma lucidum medicinal material sample is determined by adopting a liquid chromatography, and the content of the total ganoderma triterpene is taken as a quality standard for evaluating the quality of the ganoderma lucidum medicinal material. The evaluation method comprises the following steps: and (3) taking the ganoderma lucidum extract reference product as a reference, measuring the ganoderma lucidum medicinal material sample to be measured at any wavelength of 210-280 nm by using a liquid chromatography, summing all ultraviolet absorption peak areas with the retention time of 15-65 min in the obtained liquid chromatogram, comparing the sum with the ultraviolet absorption peak area corresponding to ganoderic acid A in the ganoderma lucidum extract reference product, and calculating the total ganoderma lucidum triterpene content in the ganoderma lucidum medicinal material sample to be measured. The disadvantages of this method are: although the contents of ganoderic acid A, ganoderic acid B, ganoderic acid C2 and ganoderic acid G in the ganoderma lucidum medicinal material can be rapidly measured by using the high performance liquid chromatography, and the content of the total triterpene of the ganoderma lucidum is calculated, the content cannot directly reflect the biological activity of the total triterpene of the ganoderma lucidum, and cannot comprehensively reflect the quality of the ganoderma lucidum medicinal material.
In "a new method for evaluating the quality of ganoderma lucidum extract" with patent application number CN201210382193.2, the in vitro antioxidant activity of ganoderma lucidum extract is used as the physicochemical index for evaluating the quality of ganoderma lucidum. The evaluation method comprises the following steps: determination of DPPH cation free radical and ABTS removal of Ganoderma extract sample+The capacity of cation free radicals, superoxide anion free radicals and hydroxyl anion free radicals, the total oxidation resistance, the capacity of reducing iron ions and the capacity of chelating ferrous ions, and the half effective concentration of oxidation resistance is used as a qualitative preliminary evaluation index of the quality of the ganoderma lucidum extract. The disadvantages of this method are: the antioxidant activity of the ganoderma lucidum extract is detected, the quality of the ganoderma lucidum medicinal material can be reflected to a certain degree, but the correlation between the antioxidant activity and the clinical application and the curative effect of the ganoderma lucidum is not strong.
In journal paper "Ganoderma lucidum Capsule quality Standard research and quality evaluation (2005 (09): 88-89)", the content of adenosine is used as the quality evaluation index of Ganoderma lucidum capsules. The detection method comprises the following steps: taking adenosine solution as reference substance, and determining adenosine content in the Ganoderma capsule by high performance liquid chromatography. The disadvantages of this method are: although the high performance liquid chromatography can accurately detect the content of adenosine in the ganoderma lucidum capsules, the content of adenosine cannot comprehensively reflect the activity and the quality of the ganoderma lucidum capsules.
Due to the defects of the chemical detection method, it is necessary to establish a method based on the pharmacodynamic action or biological activity to more comprehensively evaluate the quality of the ganoderma lucidum and the product thereof.
Modern researches show that the ganoderma lucidum has good anti-tumor activity and can be clinically used for preventing and treating tumors. Therefore, the antitumor activity can be used as a more ideal ganoderma lucidum quality evaluation index. In vitro experiments, tumor cell models are often used to evaluate the tumor-inhibiting effect of drugs. A two-Dimensional (2-Dimensional,2D) culture mode of tumor cell monolayer adherent growth is a main method for evaluating the activity of an anti-tumor medicament at present, the culture mode is convenient, economic and easy to operate, but lacks a microenvironment for in-vivo cell growth and differentiation, cannot well simulate the interaction between in-vivo tumor cells and a tumor microenvironment, and possibly has a certain difference with the results of animal experiments and clinical experiments. The in vitro three-Dimensional (3-Dimensional,3D) culture mode of tumor cells is a new cell culture technology developed in recent years, compared with 2D cell culture and in vivo research of animal models, the 3D cell culture mode can simulate the growth environment of cells in human bodies, and formed multicellular spheroids (MCS) have similar proliferation curve, gradient physiological environment and drug resistance characteristics to solid tumors in vivo, have short research period and no animal ethical dispute, and can be better used for antitumor activity evaluation. Therefore, both 2D and 3D tumor cell culture models can be developed into an ideal method for evaluating the quality of lucid ganoderma.
Disclosure of the invention
Aiming at the defects, in order to better evaluate the quality of the lucid ganoderma and the products thereof, the invention constructs 2D and 3D in-vitro culture models of tumor cells and provides a lucid ganoderma quality evaluation method based on the in-vitro tumor cell culture models.
The technical scheme adopted by the invention is as follows:
a method for evaluating the quality of lucid ganoderma based on an in-vitro tumor cell culture model comprises the following steps:
(1) preparing a ganoderma lucidum sample to be detected into a sample solution by using a solvent;
(2) preparing a positive control substance into a positive control substance solution by using a solvent;
(3) constructing a 2D or 3D culture model of the tumor cells, diluting the sample solution and the positive reference substance solution into different concentrations by using a culture solution, and incubating the sample solution and the positive reference substance solution together with the tumor cells;
(4) respectively sucking the solutions incubated in the step (3), adding a fresh culture solution, adding a cell activity indicator, reacting for 1-5 hours, detecting the absorbance or fluorescence intensity of the reaction solution by using an enzyme-linked immunosorbent assay, calculating the inhibition rate of the ganoderma sample solution and the positive control solution on the proliferation of tumor cells, calculating corresponding IC50, and evaluating the quality of the ganoderma by using the inhibition rate of cell proliferation; the method for calculating the cell proliferation inhibition rate comprises the following steps:
cell proliferation inhibition rate (%) ([ 1- (OD)Sample group or positive control group-ODBlank group/ODBlank control group-ODBlank group)]×100％。
Alternatively, the method is as follows:
(1) preparing a ganoderma lucidum sample to be detected into a sample solution by using a solvent;
(2) preparing a positive control substance into a positive control solution by using a solvent; the final concentration of the positive control solution is 1-800 ug/mL;
(3) constructing a 2D or 3D culture model of the tumor cells, diluting the sample solution and the reference solution into different concentrations by using a culture solution (conventional culture solution, such as DMEM and the like), and respectively incubating with the tumor cells; the final concentration of the sample solution is 0.001-60 mg/mL;
(4) constructing a 3D culture model of tumor cells, diluting a sample solution and a reference solution into different concentrations by using a culture solution (conventional culture solution, such as DMEM and the like), mixing the sample solution and the reference solution, and incubating the obtained mixed solution and the tumor cells; the final concentration of the sample solution is 0.001-10 mg/mL;
(5) respectively sucking and removing the lucid ganoderma sample solution and the positive control solution incubated in the steps (3) and (4), adding a fresh culture solution (conventional culture solution, such as DMEM and the like), adding a cell viability indicator for reacting for 1-5 hours, detecting the absorbance or fluorescence intensity of the reaction solution by using an enzyme linked immunosorbent assay detector, calculating the inhibition rate of the lucid ganoderma sample solution and the positive control solution on the proliferation of tumor cells, calculating the corresponding IC50 or growth inhibition rate, and evaluating the quality of lucid ganoderma by using the cell proliferation inhibition rate or growth inhibition rate; the method for calculating the cell proliferation inhibition rate comprises the following steps:
cell proliferation inhibition rate (%) ([ 1- (OD)Sample group or positive control group-ODBlank group/ODBlank control group-ODBlank group)]×100％；
Percent (%) increase inhibition ratioMixed solutionInhibition RatePositive control solution；
The OD value is the absorbance or fluorescence intensity measured at the detection wavelength corresponding to the cell viability indicator.
The tumor cell may be one of: liver cancer cells (e.g., HepG2 and HCCC9810), breast cancer cells (e.g., MDA-MB-231 and MCF-7), colon cancer cells (e.g., HCT116, HT-29 and Lovo), lung cancer cells (e.g., A549 and H460), stomach cancer cells (e.g., SGC-7901 and MGC-803), or pancreatic cancer cells (e.g., BxPC-3 and Panc-1).
The positive control is solid powder, and can be one of the following: fluorouracil, cisplatin, irinotecan, or docetaxel.
The concentration of the ganoderma lucidum sample in the mixed solution in the step (4) is 0.001-10 mg/mL.
The cell viability indicator is one of the following: acridine orange/ethidium bromide, DAPI, Hoechst, MTT, XTT, SRB, MTS, CCK-8, CTG, or Calcein/PI.
When the cell viability indicator is CCK-8, the reaction time is 3.5h, and the cell proliferation inhibition rate (%) is [1- (OD)Sample group or positive control group-ODCulture medium/ODBlank control group-ODCulture medium)]×100％。
When the cell viability indicator is MTT, the reaction time is 4h, and the cell proliferation inhibition rate (%) is [1- (OD)Sample group or positive control group-ODDimethyl sulfoxide/ODBlank control group-ODDimethyl sulfoxide)]×100％。
After CCK-8 and MTT enter cells, they can be reduced to blue-purple formazan crystal by succinate dehydrogenase in mitochondria of living cells, after formazan crystal is dissolved in dimethyl sulfoxide or culture medium, the absorbance value is measured by enzyme linked immune detector at 490 or 450nm wavelength, which can indirectly reflect the number of living cells and the proliferation inhibition rate of sample solution to tumor cells.
The 2D cultured tumor cells were obtained by the following method: after the tumor cells are digested by pancreatin, the tumor cells are diluted by a culture medium and resuspended, the cells are uniformly mixed to obtain cell suspension, and the cell suspension is uniformly dispersed in a micropore plate in the same volume to obtain a tumor cell 2D culture model.
The 3D cultured tumor cells are obtained by the following method: digesting the tumor cells by pancreatin, diluting the tumor cells by using a culture medium, resuspending the tumor cells, uniformly mixing to obtain a cell suspension, dissolving a matrix material, uniformly dispersing the matrix material in a microporous plate in an equal volume manner, and after the matrix material is gelatinized, uniformly dispersing the cell suspension in the microporous plate in an equal volume manner to obtain the tumor cell 3D culture model.
The matrix material is one of I-type rat tail collagen, agarose gel, Matrigel hydrogel, Geltrex matrix gel and Col-Tgle matrix gel, and the dilution ratio of the matrix material is 1:1, 1:2 or 1: 3.
Preferably, the tumor cells in the step (4) are colon cancer cells HCT116 and breast cancer cells MDA-MB-231; the matrix material is Matrigel hydrogel. Further preferably, when the matrix material is Matrigel hydrogel, the dilution ratio is 1: 2.
The ganoderma lucidum sample comprises a ganoderma lucidum medicinal material, ganoderma lucidum decoction pieces, a ganoderma lucidum extract, a traditional Chinese medicine compound product containing ganoderma lucidum, a wall-broken ganoderma lucidum spore powder extract, a traditional Chinese medicine compound product containing wall-broken ganoderma lucidum spore powder, wall-removed ganoderma lucidum spore powder extract, a traditional Chinese medicine compound product containing wall-removed ganoderma lucidum spore powder and the like; the Ganoderma lucidum includes Ganoderma lucidum (Ganoderma lucidum) and Ganoderma sinense (Ganoderma sinense).
The ganoderma lucidum sample to be detected in the step (1) is obtained by the following method: adding Ganoderma into solvent, extracting by reflux extraction, ultrasonic extraction, Soxhlet extraction, leaching or pressure countercurrent extraction to obtain sample solution, and drying by nitrogen blowing, evaporating, concentrating under reduced pressure, centrifuging, or lyophilizing to obtain sample to be tested; the solvent is one of the following: water, ethanol, 5-95% ethanol solution, methanol, 5-95% methanol solution, ethyl acetate and trichloromethane. Preferably, the solvent is water or ethanol; the extraction method is reflux extraction or ultrasonic extraction; the drying method is freeze drying or reduced pressure drying. When the extraction solvent is water, the sample powder to be detected takes the culture medium as the solvent; when the extraction solvent is ethanol, the sample powder to be detected uses absolute ethanol as the solvent, the final concentration of the water extraction sample is 0.001-40 mg/mL, the final concentration of the ethanol extraction sample is 1-600 ug/mL, and when the sample to be detected is a ganoderma lucidum slice, the final concentration of the ganoderma lucidum slice sample solution is 1-60 mg/mL. Preferably, when the sample preparation solvent is water, the final concentration of the sample is 0.625-30 mg/mL; when the sample preparation solvent is ethanol, the final concentration of the sample is 12.5-400 mug/mL, and when the sample is a ganoderma lucidum tablet, the final concentration of the sample is 3.125-60 mg/mL.
In step (1) or (2), the solvent is one of the following independently: purified water, culture medium (conventional culture medium such as DMEM and the like), phosphate buffer, methanol, absolute ethanol or dimethyl sulfoxide. Wherein, the fluorouracil and cisplatin use purified water as a solvent; irinotecan takes dimethyl sulfoxide as a solvent; docetaxel uses absolute ethyl alcohol as a solvent.
The invention has the following beneficial effects:
(1) the quality of the ganoderma lucidum is evaluated by measuring the inhibition effect of the ganoderma lucidum on the tumor cells based on the tumor cells cultured in vitro, and the detection index is directly related to the anti-tumor activity of the ganoderma lucidum and can reflect the clinical curative effect of the ganoderma lucidum, so that the quality of the ganoderma lucidum can be comprehensively evaluated.
(2) According to the invention, 2D and 3D cultured tumor cells are adopted to carry out research on the anti-tumor effect of the ganoderma lucidum, the quality of the ganoderma lucidum can be comprehensively evaluated, the 3D culture of the tumor cells is closer to in-vivo in-situ tumor, the evaluation result is more accurate, animal ethics is not involved, the analysis flow is short, and the analysis cost is low.
(IV) description of the drawings
FIG. 1 shows that water extracts of various batches of Ganoderma lucidum inhibited the proliferation of 2D cultured HCT116 cells by IC50(Mean + -SD, n-3);
FIG. 2 shows that water extraction of various Ganoderma lucidum extracts shows IC50(Mean + -SD, n-3) that inhibits proliferation of HCT116 cells cultured in 3D;
FIG. 3 shows the inhibition rate of different batches of Ganoderma lucidum combined with fluorouracil on the proliferation of HCT116 cells cultured in 3D (Mean + -SD, n-3);
FIG. 4 shows that IC50(Mean + -SD, n-3) of the ethanol extract of the wall-broken Ganoderma lucidum spore powder of different batches inhibits the proliferation of MDA-MB-231 cells cultured in 2D;
FIG. 5 shows that different batches of Ganoderma lucidum spore powder extract has IC50(Mean + -SD, n is 3) for inhibiting the proliferation of MDA-MB-231 cells cultured in 3D;
FIG. 6 different batches of aqueous extracts of Ganoderma lucidum flakes IC50(Mean + -SD, n-3) showing that the samples inhibited proliferation of 2D cultured HCT116 cells;
FIG. 7 different batches of aqueous extracts of Ganoderma lucidum flakes show IC50(Mean + -SD, n-3) that inhibits proliferation of 3D cultured HCT116 cells.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
in the examples, Matrigel was purchased from BD, type I rat tail collagen from Melam Biotechnology Inc., DMEM medium, Sepharose and Geltrex Matrigel from Sammer Feishel technology Inc., Col-Tgle Matrigel from Huaya regenerative medicine Biotechnology Inc., MTT solution from Sigma, CCK-8, DAPI, Hoechst, Calcein/PI solution from Byunnan Biotechnology Inc., SRB, MTS solution from Abcam, acridine orange/ethidium bromide from Solebao Biotechnology Inc., CTG solution from Shengshikang Biomedicine cisplatin Inc., Fluorouracil, irinotecan, docetaxel from Aladdin.
Example 1: ganoderma lucidum medicinal material quality evaluation based on chemical detection
5 batches of Ganoderma lucidum (Ganoderma lucidum) medicinal materials and 5 batches of Ganoderma sinense (Ganoderma sinense) medicinal materials are provided by Wuyi shouxian valley traditional Chinese medicine decoction pieces company Limited. The contents of polysaccharide, triterpene and sterol in the 10 batches of ganoderma lucidum samples are determined according to a method specified in ganoderma lucidum item of China pharmacopoeia 2015 edition: the polysaccharide is measured by adopting an anthrone-sulfuric acid method, and anhydrous glucose is used as a reference substance; the determination of triterpene and sterol adopts vanillin-glacial acetic acid method, and oleanolic acid as reference. The results are shown in Table 1.
As can be seen from Table 1, the polysaccharide content of 5 batches of Ganoderma lucidum is 1.19% -1.91%, and the triterpene and sterol content is 0.85% -1.58%; the content of ganoderma polysaccharide in 5 batches is 1.20-1.77%, and the content of triterpene and sterol is 0.84-1.17%. The contents of polysaccharide, triterpene and sterol of Ganoderma lucidum and Ganoderma sinense of the 10 batches have no obvious difference. According to the regulations in pharmacopoeia that polysaccharide is not less than 0.90% and triterpene and sterol are not less than 0.50%, the samples are all qualified medicinal materials.
Table 1: content of polysaccharide, triterpene and sterol (%) (of Ganoderma lucidum) in different batches
Example 2: ganoderma lucidum medicinal material quality evaluation based on 2D tumor cell culture model
The same ganoderma lucidum and ganoderma sinensis medicinal materials as in example 1 are taken, and the quality of the ganoderma lucidum and ganoderma sinensis medicinal materials is evaluated according to the following steps:
(1) taking 2g of ganoderma lucidum medicinal material powder (sieved by a 40-mesh sieve) of different batches, adding 50mL of water, heating and refluxing in a water bath for 1h, filtering, and evaporating filtrate on the water bath to dryness to obtain ganoderma lucidum samples to be detected;
(2) preparing a sample solution (with the concentration of 40mg/mL) from each ganoderma lucidum sample to be detected by using a DMEM medium;
(3) taking 10mg of fluorouracil, and preparing a positive control solution (with the concentration of 10mg/mL) by using purified water;
(4) colon cancer cell HCT116 was digested with 0.25% pancreatic enzymes, diluted with DMEM medium, and tumor cells were resuspended (cell concentration about 1X 10)5Per mL) to obtain a cell suspension, and uniformly dispersing the cell suspension in a microplate in an equal volume to obtain a 2D culture model of the tumor cell HCT 116; and (3) uniformly dispersing the colon cancer cells HCT116 cultured in 2D into a 96-well plate, diluting the ganoderma lucidum sample solution and the fluorouracil solution obtained in the steps (2) and (3) into different concentrations by using a DMEM medium (ganoderma lucidum sample solution: 40, 35, 30, 25, 20, 15, 12.5, 10, 7.5, 5, 2.5, 1.25, 0.625 and 0.3125 mg/mL; fluorouracil solution: 200, 175, 150, 125, 100, 75, 50, 25, 12.5, 6.25, 3.125, 1.5625 and 0.78125 mu g/mL), and respectively incubating with the HCT116 cells for 48h, wherein each concentration is 3 duplicate wells.
(5) Absorbing and removing the ganoderma lucidum sample solution and the fluorouracil solution, adding 90 mu L of fresh DMEM culture medium, adding 10 mu L of CCK-8 solution, reacting for 3.5h, detecting the absorbance of the reaction solution by adopting an enzyme-linked immunosorbent assay detector under the wavelength of 450nm, calculating the inhibition rate of the ganoderma lucidum sample solution and the positive control solution on the proliferation of tumor cells, calculating corresponding IC50, and evaluating the quality of the ganoderma lucidum by adopting IC 50. The results of the detection are shown in FIG. 1.
As can be seen from FIG. 1, the IC50 of fluorouracil for inhibiting HCT116 cell proliferation is 2.40. + -. 0.53 ug/mL; the IC50 of 5 batches of ganoderma lucidum water extract samples for inhibiting the proliferation of HCT116 cells cultured in 2D has no obvious difference, and the IC50 value is between 1.58 +/-0.05 mg/mL and 1.69 +/-0.12 mg/mL; the IC50 of 5 batches of Ganoderma sinensis water extract samples for inhibiting the proliferation of HCT116 cells cultured in 2D has no obvious difference, and the IC50 value is between 2.84 + -0.17 mg/mL and 3.02 + -0.18 mg/mL. The detection result shows that the antitumor activity of the ganoderma lucidum in the test batch is higher than that of ganoderma lucidum, and the 2D tumor cell culture model can better distinguish and evaluate the quality of different ganoderma lucidum medicinal materials.
In order to ensure that the method is suitable for quality evaluation, the precision, reproducibility and stability of the method were examined. Taking the ganoderma lucidum medicinal material of batch 1 as a quality control sample. The precision is that after preparing the sample, the sample is continuously measured for 6 times by an enzyme-labeling instrument; the repeatability is that 6 samples of the ganoderma lucidum medicinal material of batch 1 are prepared in parallel for detection; the stability is that after preparing the sample to be detected of the lucid ganoderma, the lucid ganoderma is placed for 0, 2, 4, 6, 8, 12 and 24 hours, and then the detection is carried out according to the subsequent steps. The result shows that the precision RSD is less than 5%, the repeatability RSD is less than 15%, and the stability RSD is less than 8%, which shows that the method has good performance and is suitable for the quality evaluation of the ganoderma lucidum.
Example 3: ganoderma lucidum sample quality evaluation based on 3D tumor cell culture model
The quality of ganoderma lucidum and ganoderma sinense medicinal materials in the same batch as in example 1 is evaluated according to the following steps:
steps (1) to (3) are the same as in example 2.
(4) And (3) taking the colon cancer cells HCT116 cultured in 2D (the construction method of the 2D culture model is the same as that in the example 1) and taking Matrigel hydrogel diluted in 1:2 as a matrix material to construct a 3D culture model, diluting the ganoderma lucidum sample solution and the fluorouracil solution obtained in the steps (2) and (3) into different concentrations (the final concentrations are the same as that in the step (4) in the example 2) by using a DMEM culture medium, and incubating the diluted ganoderma lucidum sample solution and the fluorouracil solution with the HCT116 cells for 48h respectively, wherein each concentration is 3 duplicate wells.
(5) Absorbing and removing the ganoderma lucidum sample solution and the fluorouracil solution, adding 90 mu L of fresh DMEM culture medium, adding 10 mu L of CCK-8 solution, reacting for 3.5h, absorbing the isovolumetric reaction solution in a new 96-well plate, detecting the absorbance of the reaction solution at the wavelength of 450nm by using an enzyme linked immunosorbent assay detector, calculating the inhibition rate of the ganoderma lucidum sample solution and the fluorouracil solution on the proliferation of tumor cells, calculating corresponding IC50, and evaluating the quality of the ganoderma lucidum by using IC 50. The results of the detection are shown in FIG. 2.
As can be seen from FIG. 2, the IC50 of fluorouracil inhibiting the proliferation of 3D cultured HCT116 cells was 113.78. + -. 7.52 ug/mL; the IC50 of 5 batches of ganoderma lucidum water extract samples for inhibiting the proliferation of HCT116 cells cultured in 3D has no obvious difference, and the IC50 value is between 9.64 +/-0.42 mg/mL and 10.20 +/-0.98 mg/mL; the IC50 of 5 batches of Ganoderma sinensis water extract samples for inhibiting the proliferation of HCT116 cells cultured in 3D has no obvious difference, and the IC50 value is between 21.29 +/-1.68 mg/mL and 23.37 +/-1.12 mg/mL. The detection result shows that the antitumor activity of the ganoderma lucidum in the test batch is higher than that of ganoderma lucidum, and the 3D tumor culture model can better distinguish and evaluate the quality of different ganoderma lucidum medicinal materials.
In order to ensure that the method is suitable for quality evaluation, the precision, reproducibility and stability of the method were examined. Taking the ganoderma lucidum medicinal material of batch 1 as a quality control sample. The precision is that after preparing the sample, the sample is continuously measured for 6 times by an enzyme-labeling instrument; the repeatability is that 6 samples of the ganoderma lucidum medicinal material of batch 1 are prepared in parallel for detection; the stability is that after preparing the sample to be detected of the lucid ganoderma, the lucid ganoderma is placed for 0, 2, 4, 6, 8, 12 and 24 hours, and then the detection is carried out according to the subsequent steps. The result shows that the precision RSD is less than 8%, the repeatability RSD is less than 13%, and the stability RSD is less than 10%, which shows that the method has good performance and is suitable for the quality evaluation of ganoderma lucidum.
The results show that both the 2D and 3D in vitro culture models of the tumor cells can be used for quality evaluation of the ganoderma lucidum, wherein the IC50 value measured by the tumor 3D in vitro culture model is larger than that of the 2D model due to the drug resistance, the interaction between the tumor cells and the microenvironment and the like, and the clinical practical situation is more met. Therefore, the quality of the ganoderma lucidum can be evaluated more objectively by adopting a tumor cell 3D in-vitro culture model.
Example 4: ganoderma lucidum sample quality evaluation based on 3D tumor cell culture model-growth inhibition rate
The quality of the ganoderma lucidum (ganoderma lucidum) medicinal material with the same batch as that in the example 1 is evaluated according to the following steps:
steps (1) to (3) are the same as in example 2.
(4) Mixing the sample solution obtained in the step (2) with the fluorouracil solution obtained in the step (3) according to the volume ratio of 1: 1.
(5) And (3) taking the colon cancer cells HCT116 cultured in 2D (the construction method of the 2D culture model is the same as that of the embodiment 1) and taking Matrigel hydrogel diluted by 1:2 as a matrix material to construct a 3D culture model, and incubating the solutions obtained in the steps (2) to (4) with the HCT116 cells for 48 hours respectively by using a DMEM culture medium, wherein each concentration is 3 multiple wells.
(6) Absorbing the solution in the 96-well plate, adding 90 mu L of fresh DMEM culture medium, adding 10 mu L of CCK-8 solution, reacting for 3.5h, absorbing the reaction solution with the same volume in a new 96-well plate, detecting the absorbance of the reaction solution at the wavelength of 450nm by using an enzyme linked immunosorbent assay detector, and calculating the inhibition rate of the mixed solution on the proliferation of the tumor cells cultured in 3D and the growth inhibition rate of the relative positive drugs. The results of the detection are shown in FIG. 3.
As can be seen from fig. 3, 5 batches of ganoderma lucidum aqueous extract samples had no significant effect on the proliferation of 3D-cultured HCT116 cells, and the inhibition rate of fluorouracil on the proliferation of 3D-cultured HCT116 cells was 35.82 ± 4.50%; compared with the single fluorouracil treatment, the mixed solution of the 5 batches of ganoderma lucidum water extract samples and fluorouracil has the inhibition rates of 65.53 +/-1.66%, 59.51 +/-2.84%, 55.93 +/-2.88%, 63.85 +/-4.29% and 58.13 +/-3.98% on the proliferation of HCT116 cells cultured in 3D, and the increase inhibition rates are respectively about 29.71%, 23.69%, 20.11%, 28.03% and 22.31%. The detection result shows that the ganoderma lucidum can strengthen the anti-tumor effect of the fluorouracil. Therefore, in addition to IC50, the quality of Ganoderma lucidum can also be evaluated by using the growth inhibition rate as an index. And the fluorouracil is used as a positive control, so that the detection result is more reliable.
Example 5: quality evaluation of wall-broken ganoderma lucidum spore powder sample based on chemical detection
Wall-broken Ganoderma lucidum spore powder 5 batches provided by Wuyi shouxian grain Chinese medicinal decoction pieces Co. The polysaccharide and the glycerol trioleate of the 5 batches of the wall-broken ganoderma lucidum spore powder are determined according to a method specified under the item of ganoderma lucidum spore powder of 2015 edition of Zhejiang Chinese medicine processing Specification: the polysaccharide is measured by adopting an anthrone-sulfuric acid method, and anhydrous glucose is used as a reference substance; the triolein is measured by high performance liquid chromatography using triolein as a control. The results are shown in Table 2.
As can be seen from Table 2, the content of polysaccharides in the wall-broken Ganoderma lucidum spore powder of 5 batches is 1.88-2.75%, the content of triolein is 6.02-6.87%, and the Relative Standard Deviation (RSD) between the batches is 12.1% and 4.7%, respectively, which shows that the consistency between the 5 batches is better. According to the regulations of the processing specifications that the polysaccharide is not less than 0.80% and the glycerol trioleate is not less than 3.0%, the samples are all qualified traditional Chinese medicine decoction pieces.
Table 2: content of wall-broken ganoderma lucidum spore powder polysaccharide and glycerol trioleate (%)
Example 6: quality evaluation of wall-broken ganoderma lucidum spore powder sample based on 2D tumor cell culture model
The quality of the wall-broken ganoderma lucidum spore powder with the same batch as that in the example 5 is evaluated according to the following steps:
(1) taking 2g of wall-broken ganoderma lucidum spore powder of different batches, adding 50mL of ethanol, carrying out ultrasonic extraction for 1h, filtering, and volatilizing the filtrate at 50 ℃ by using a centrifugal concentrator to obtain a sample to be detected;
(2) taking 60mg of each sample to be detected of the wall-broken ganoderma lucidum spore powder, and preparing a sample solution (with the concentration of 60mg/mL) by using absolute ethyl alcohol;
(3) taking 10mg of docetaxel, and preparing a positive control solution (with the concentration of 10mg/mL) by using absolute ethyl alcohol;
(4) and (3) uniformly dispersing the breast cancer cells MDA-MB-231 cultured in 2D (the construction method of the 2D culture model is the same as that in example 1) in a 96-well plate, diluting the ganoderma lucidum spore powder sample solution and the docetaxel solution obtained in the steps (2) and (3) into different concentrations by using a DMEM culture medium (the ganoderma lucidum spore powder sample is 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 75, 50, 25, 12.5 and 6.25 mu g/mL, the docetaxel is 100, 75, 50, 25, 12.5, 6.25, 3.125, 1.5625 and 0.78125 mu g/mL), and respectively incubating with the MDA-MB-231 cells for 48h, wherein each concentration is 3 multiple wells.
(5) Adding 20 mu L (5mg/mL) of MTT solution into each well for reaction for 4h, sucking out supernatant, adding 200 mu L of dimethyl sulfoxide for reaction for 10min, sucking 150 mu L of reaction solution into a new 96-well plate, detecting the absorbance of the reaction solution at 490nm by using an enzyme linked immunosorbent assay detector, calculating the inhibition rate of the ganoderma lucidum spore powder sample solution and the docetaxel solution on the proliferation of tumor cells, and calculating corresponding IC 50. The results of the detection are shown in FIG. 4.
As can be seen in FIG. 4, the IC50 of docetaxel for inhibiting the proliferation of 2D cultured MDA-MB-231 cells was 1.72. + -. 0.19. mu.g/mL; the IC50 that 5 batches of ganoderma lucidum spore powder ethanol extraction samples inhibit the proliferation of MDA-MB-231 cells cultured in 2D has no obvious difference, the IC50 value is between 45.61 +/-3.12 mu g/mL and 54.28 +/-2.35 mu g/mL, and the detection result shows that the 2D tumor cell culture model can better evaluate the quality of the wall-broken ganoderma lucidum spore powder.
In order to ensure that the method is suitable for quality evaluation, the precision, reproducibility and stability of the method were examined. And taking the ganoderma lucidum spore powder of batch 1 as a quality control sample. The precision is that after preparing the sample, the sample is continuously measured for 6 times by an enzyme-labeling instrument; the repeatability is that the ganoderma lucidum spore powder of batch 1 is taken, and 6 samples are prepared in parallel for detection; the stability is that after preparing the sample to be detected of the ganoderma lucidum spore powder, the sample is placed for 0, 2, 4, 6, 8, 12 and 24 hours, and then the detection is carried out according to the subsequent steps. The result shows that the precision RSD is less than 4%, the repeatability RSD is less than 17%, and the stability RSD is less than 7%, which shows that the method has good performance and is suitable for quality evaluation of ganoderma lucidum spore powder.
Example 7: quality evaluation of wall-broken ganoderma lucidum spore powder sample based on 3D tumor cell culture model
The same wall-broken ganoderma lucidum spore powder as in example 6 was taken, and the quality thereof was evaluated according to the following steps:
steps (1) to (3) are the same as in example 5.
(4) Taking colon cancer cells MDA-MB-231 cultured in 2D (the construction method of the 2D culture model is the same as that of the embodiment 1) and taking Matrigel hydrogel diluted by 1:2 as a matrix material to construct a 3D culture model, diluting the ganoderma lucidum spore powder sample solution and the docetaxel solution obtained in the steps (2) and (3) into different concentrations (the final concentration is the same as that of the step 4 of the embodiment 2) by using a DMEM culture medium, and co-incubating the ganoderma lucidum spore powder sample solution and the docetaxel solution with MDA-MB-231 cells for 48h respectively, wherein each concentration is 3 multiple wells.
(5) Adding 20 mu L (5mg/mL) of MTT solution into each well for reaction for 4h, sucking out supernatant, adding 200 mu L of dimethyl sulfoxide for reaction for 10min, sucking 150 mu L of reaction solution into a new 96-well plate, detecting the absorbance of the reaction solution at 490nm by using an enzyme linked immunosorbent assay detector, calculating the inhibition rate of the ganoderma lucidum spore powder sample solution and the docetaxel solution on the proliferation of tumor cells, and calculating corresponding IC 50. The results of the detection are shown in FIG. 5.
As can be seen in FIG. 5, the IC50 of docetaxel for inhibiting the proliferation of MDA-MB-231 cells cultured in 3D was 63.29. + -. 1.53. mu.g/mL; the IC50 of the 5 batches of ganoderma lucidum spore powder ethanol extraction samples for inhibiting the proliferation of HCT116 cells cultured in 3D has no obvious difference, and the IC50 value is between 159.61 +/-15.28 mu g/mL and 178.36 +/-18.16 mg/mL, which shows that the tolerance of the MDA-MB-231 cells cultured in 3D to docetaxel and the ganoderma lucidum spore powder ethanol extraction samples is improved; compared with 2D culture, the tolerance index of MDA-MB-231 cells cultured by 3D culture to docetaxel is about 36.79, and the tolerance indexes to 5 batches of ganoderma lucidum spore powder ethanol extraction samples are respectively 3.72, 3.55, 3.66, 3.10 and 3.11, and the detection result shows that the ganoderma lucidum spore powder ethanol extraction samples are not easy to cause the tolerance of tumor cells compared with docetaxel.
In order to ensure that the method is suitable for quality evaluation, the precision, reproducibility and stability of the method were examined. And taking the ganoderma lucidum spore powder of batch 1 as a quality control sample. The precision is that after preparing the sample, the sample is continuously measured for 6 times by an enzyme-labeling instrument; the repeatability is that the ganoderma lucidum spore powder of batch 1 is taken, and 6 samples are prepared in parallel for detection; the stability is that after preparing the sample to be detected of the ganoderma lucidum spore powder, the sample is placed for 0, 2, 4, 6, 8, 12 and 24 hours, and then the detection is carried out according to the subsequent steps. The result shows that the precision RSD is less than 6%, the repeatability RSD is less than 12%, and the stability RSD is less than 13%, which shows that the method has good performance and is suitable for quality evaluation of ganoderma lucidum spore powder.
The results show that the 2D and 3D in vitro culture models of the tumor cells can be used for quality evaluation of the wall-broken ganoderma lucidum spore powder.
Example 8: ganoderma lucidum sample quality evaluation based on chemical detection
The Chinese patent medicine ganoderma lucidum tablets are 5 batches and are provided by Jinhua city life-recovering pharmaceutical limited company. The polysaccharide content of the 10 batches of ganoderma lucidum slice samples is measured according to a method specified under the item of ganoderma lucidum in the first part of Chinese pharmacopoeia 2015 edition: the polysaccharide was measured by anthrone-sulfuric acid method using anhydrous glucose as a control. The detection result shows that the polysaccharide content of the 5 batches of the ganoderma lucidum tablets is 75%, 86%, 82%, 71% and 80% respectively. The Ganoderma lucidum tablet has high polysaccharide content because it contains high content of adjuvants including starch, dextrin, sucrose, etc. These auxiliary materials also react with sulfuric acid and anthrone to develop color, so that if the anthrone-sulfuric acid method is adopted, a very large error exists, and the content of the ganoderma lucidum slices is difficult to objectively evaluate.
Example 9: ganoderma lucidum tablet quality evaluation based on 2D tumor cell culture model
The quality of the ganoderma lucidum slices in the same batch as in example 8 is evaluated according to the following steps:
(1) taking 2g of ganoderma lucidum slice powder (sieved by a 40-mesh sieve) of different batches, adding 50mL of water, heating and refluxing in a water bath for 1h, filtering, and evaporating the filtrate on the water bath to dryness to obtain a ganoderma lucidum sample to be detected;
(2) preparing 60mg of each ganoderma lucidum sample to be detected into sample solution (with the concentration of 60mg/mL) by using a DMEM medium;
(3) taking 40mg of irinotecan, and preparing a positive control solution (with the concentration of 80mg/mL) by using dimethyl sulfoxide;
(4) colon cancer cells HCT116 cultured in 2D (2D culture model construction method is same as example 1) are uniformly dispersed in a 96-well plate, the ganoderma lucidum slice sample solution and irinotecan solution obtained in steps (2) and (3) are diluted to different concentrations by DMEM culture medium (ganoderma lucidum slice sample solution: 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 12.5, 6.25, 3.125, 1.5625 mg/mL; irinotecan solution: 800, 600, 400, 300, 200, 100, 75, 50, 25, 12.5, 6.25, 3.125. mu.g/mL), and are respectively incubated with HCT116 cells for 48h, and each concentration is 3 multiple wells.
(5) Absorbing the ganoderma lucidum sample solution and the irinotecan solution, adding 90 mu L of fresh DMEM culture medium, adding 10 mu L of CCK-8 solution, reacting for 3.5h, detecting the absorbance of the reaction solution by adopting an enzyme-linked immunosorbent assay detector at the wavelength of 450nm, calculating the inhibition rate of the ganoderma lucidum sample solution and the irinotecan solution on the proliferation of tumor cells, and calculating the corresponding IC 50. The results of the detection are shown in FIG. 6.
As can be seen in FIG. 6, IC50 for irinotecan to inhibit HCT116 cell proliferation was 9.83. + -. 0.87. mu.g/mL; the IC50 of the water extraction samples of the 5 batches of the ganoderma lucidum slices for inhibiting the proliferation of the HCT116 cells cultured in the 2D mode has no obvious difference, and the IC50 value is between 6.21 +/-0.40 mg/mL and 6.64 +/-0.58 mg/mL. The detection result shows that the 2D tumor cell culture model can better evaluate the quality of the ganoderma lucidum tablets.
In order to ensure that the method is suitable for quality evaluation, the precision, reproducibility and stability of the method were examined. And taking the ganoderma lucidum slices of the batch 1 as a quality control sample. The precision is that after preparing the sample, the sample is continuously measured for 6 times by an enzyme-labeling instrument; the repeatability is that the ganoderma lucidum tablets of batch 1 are taken, and 6 samples are prepared in parallel for detection; the stability is that after preparing the sample to be tested of the ganoderma lucidum tablet, the sample is placed for 0, 2, 4, 6, 8, 12 and 24 hours, and then the detection is carried out according to the subsequent steps. The result shows that the precision RSD is less than 4%, the repeatability RSD is less than 16%, and the stability RSD is less than 9%, which shows that the method has good performance and is suitable for the quality evaluation of the ganoderma lucidum tablets.
Example 10: ganoderma lucidum sample quality evaluation based on 3D tumor cell culture model
The quality of the ganoderma lucidum slices in the same batch as in example 8 is evaluated according to the following steps:
steps (1) to (3) are the same as in example 9.
(4) Taking colon cancer cells HCT116 cultured in 2D (the construction method of the 2D culture model is the same as that of the embodiment 1) and taking Matrigel hydrogel diluted by 1:2 as a matrix material to construct a 3D culture model, diluting the ganoderma lucidum slice sample solution and the irinotecan solution obtained in the steps (2) and (3) into different concentrations (the final concentrations are the same as that of the step 4 of the embodiment 9) by using a DMEM culture medium, and incubating the ganoderma lucidum slice sample solution and the irinotecan solution with the HCT116 cells for 48h respectively, wherein each concentration is 3 multiple wells.
(5) Absorbing the ganoderma lucidum sample solution and the irinotecan solution, adding 90 mu L of fresh DMEM culture medium, adding 10 mu L of CCK-8 solution, reacting for 3.5h, absorbing the equal volume of reaction solution into a new 96-well plate, detecting the absorbance of the reaction solution at the wavelength of 450nm by using an enzyme linked immunosorbent assay detector, calculating the inhibition rate of the ganoderma lucidum sample solution and the irinotecan solution on the proliferation of tumor cells, and calculating the corresponding IC 50. The results of the detection are shown in FIG. 7.
As can be seen in fig. 7, IC50 for irinotecan to inhibit proliferation of 3D cultured HCT116 cells was 473.62 ± 18.51 ug/mL; the water extraction samples of 5 batches of ganoderma lucidum sheets have no obvious difference in IC50 for inhibiting the proliferation of HCT116 cells cultured in 3D, and the IC50 value is between 43.22 +/-1.80 mg/mL and 47.35 +/-1.37 mg/mL. The detection result shows that the 3D tumor cell culture model can better evaluate the quality of the ganoderma lucidum tablets.
In order to ensure that the method is suitable for quality evaluation, the precision, reproducibility and stability of the method were examined. And taking the ganoderma lucidum slices of the batch 1 as a quality control sample. The precision is that after preparing the sample, the sample is continuously measured for 6 times by an enzyme-labeling instrument; the repeatability is that the ganoderma lucidum tablets of batch 1 are taken, and 6 samples are prepared in parallel for detection; the stability is that after preparing the sample to be tested of the ganoderma lucidum tablet, the sample is placed for 0, 2, 4, 6, 8, 12 and 24 hours, and then the detection is carried out according to the subsequent steps. The result shows that the precision RSD is less than 8%, the repeatability RSD is less than 17%, and the stability RSD is less than 5%, which shows that the method has good performance and is suitable for the quality evaluation of the ganoderma lucidum tablets.
Example 11:
the optimization process of the parameters involved in the embodiments 1 to 10 is as follows:
optimization of tumor cell lines and matrix materials
(1) Tumor cell line optimization in 2D culture: taking liver cancer cells HepG2 and HCCC810 cultured by 2D, breast cancer cells MDA-MB-231 and MCF-7, colon cancer cells HCT116, HT-29 and Lovo, lung cancer cells A549 and H460, stomach cancer cells SGC-7901 and MGC-803 (the construction method of the 2D culture model is the same as that in example 1), incubating the ganoderma lucidum sample solutions with different concentrations and the tumor cells for 48H, and calculating the IC50 of the ganoderma lucidum sample solution for inhibiting the proliferation of the tumor cells.
The results show that the IC50 values of the ganoderma lucidum water extract sample and the ethanol extract sample for inhibiting the proliferation of breast cancer cells MDA-MB-231 and MCF-7, colon cancer cells HCT116 and stomach cancer cells MGC-803 are lower. Therefore, the preferred cell lines are breast cancer cells MDA-MB-231 and MCF-7, colon cancer cells HCT116 and stomach cancer cells MGC-803.
(2) Optimization of tumor cell lines and matrix materials in 3D culture: and (3) taking the optimized cell line in the step (1) and respectively taking I-type rat tail collagen, agarose gel, Matrigel hydrogel, Geltrex Matrigel and Col-Tgle Matrigel as matrix materials to construct a 3D culture model, and observing the growth form of tumor cells in the matrix materials.
The results show that breast cancer cells MDA-MB-231 and colon cancer cells HCT116 form well-defined, dense multicellular spheroids in Matrigel hydrogel. Therefore, preferred cell lines are breast cancer cell MDA-MB-231 and colon cancer cell HCT 116; the matrix material is Matrigel hydrogel.
(3) Optimization of dilution ratio of matrix material: diluting the optimized matrix material in the step (2) by using a DMEM culture medium according to the proportion of 1:1, 1:2 and 1:3, taking the optimized cell line in the step (1) and respectively using stock solution and diluent as matrix materials to construct a 3D model, and observing the growth form of tumor cells in the matrix materials.
The results show that breast cancer cells MDA-MB-231 and colon cancer cells HCT116 formed well-defined, dense multicellular spheroids in Matrigel hydraulic collagen solution and 1:2 diluted dilution, whereas the multicellular spheroids formed in 1:2 diluted dilution were larger in volume. Therefore, a 1:2 diluted Matrigel hydrogel is preferred as the matrix material.
Second, optimization of final concentration of ganoderma lucidum sample solution and positive control sample solution
(1) Optimizing the final concentration of ganoderma water extract samples: extracting ganoderma lucidum water to obtain sample powder, preparing a sample solution of 0.001-40 mg/mL by using a DMEM (DMEM) culture medium, incubating the sample solution with tumor cells for 48 hours, adding a cell activity indicator to react, and measuring the absorbance or fluorescence intensity of a reaction solution.
The result shows that when the final concentration of the ganoderma aqueous extract sample solution is lower than 0.625mg/mL, the absorbance or fluorescence intensity of the reaction solution tends to be flat; when the concentration is more than 30mg/mL, the absorbance or fluorescence intensity of the reaction solution is too low to facilitate detection. Therefore, the final concentration of the ganoderma lucidum aqueous extract sample solution is preferably 0.625-30 mg/mL.
(2) Optimizing the final concentration of the ganoderma lucidum ethanol extraction sample: extracting sample powder from ganoderma lucidum by using ethanol, preparing a sample solution with the concentration of 1-600 mu g/mL by using absolute ethanol, incubating the sample solution with tumor cells for 48 hours, adding a cell activity indicator for reaction, and measuring the absorbance or fluorescence intensity of a reaction solution.
The result shows that when the final concentration of the ganoderma lucidum ethanol extraction sample solution is lower than 12.5ug/mL, the absorbance or fluorescence intensity of the reaction solution tends to be flat; when the concentration is more than 500ug/mL, the absorbance or fluorescence intensity of the reaction solution is too low to facilitate detection. Therefore, the final concentration of the ganoderma lucidum ethanol extraction sample solution is preferably 12.5-500 ug/mL.
(3) Optimization of final concentration of positive control sample: and taking the positive control sample powder, preparing a sample solution of 0.3125-800 ug/mL by using purified water, incubating with the tumor cells for 48 hours, adding a cell activity indicator for reaction, and determining the absorbance or fluorescence intensity of the reaction solution.
The result shows that when the final concentration of the positive control sample solution is lower than 0.625ug/mL, the absorbance or fluorescence intensity of the reaction solution tends to be flat; when the concentration is more than 600ug/mL, the absorbance or fluorescence intensity of the reaction solution is too low to facilitate detection. Therefore, the final concentration of the positive control sample solution is preferably 0.625-600 ug/mL.
(4) Optimizing the final concentration of the ganoderma lucidum extract sample in the mixed solution: taking a ganoderma lucidum water extraction sample, and preparing a 0.625-5 mg/mL ganoderma lucidum water extraction sample solution by using a DMEM culture medium; preparing a ganoderma lucidum ethanol extraction sample solution with the concentration of 12.5-75 ug/mL by using absolute ethanol; incubating the Ganoderma water extract sample solution and Ganoderma ethanol extract sample solution with 3D cultured tumor cells for 48 hr, adding cell activity indicator, measuring absorbance or fluorescence intensity of reaction solution, and calculating the inhibition rate of Ganoderma water extract sample solution and Ganoderma ethanol extract sample solution on tumor cell proliferation.
The results showed that 1.25mg/mL aqueous Ganoderma extract sample solution and 50ug/mL ethanol Ganoderma extract sample solution had minimal effect on tumor cell proliferation. Therefore, the final concentration of the ganoderma lucidum water extraction sample is preferably 1.25 mg/mL; the final concentration of the ganoderma lucidum ethanol extract sample is 50 ug/mL.
(5) Optimizing the final concentration of the ganoderma lucidum slice sample: taking ganoderma lucidum slice sample powder, preparing 1-60 mg/mL sample solution by using a DMEM (DMEM) culture medium, incubating the sample solution with tumor cells for 48 hours, adding a cell activity indicator for reaction, and determining the absorbance or fluorescence intensity of reaction liquid.
The results show that the absorbance or fluorescence intensity of the reaction solution tends to be flat when the final concentration of the ganoderma lucidum slice sample solution is less than 3.125 mg/mL. Therefore, the final concentration of the ganoderma lucidum aqueous extract sample solution is preferably 3.125-60 mg/mL.
Cell viability indicator optimization
(1) Indicator solution optimization: extracting a ganoderma water sample solution, an ethanol sample solution and a positive control sample solution, incubating the solutions and tumor cells for 48 hours, respectively adding acridine orange/ethidium bromide, DAPI, Hoechst, MTT, SRB, MTS, CCK-8, CTG and Calcein/PI for reaction, measuring the absorbance or fluorescence intensity of a reaction solution, and calculating the inhibition rate of each solution on the proliferation of the tumor cells or IC 50.
The results show that when SRB, CCK-8, CTG and MTT are used as cell viability indicators, the inhibition rates of the solutions on the proliferation of tumor cells are not obviously different. Therefore, for economic and convenience, CCK-8 and MTT are preferred as indicators of cell viability.
(2) Indicator reaction time optimization: extracting Ganoderma water sample solution, ethanol sample solution and positive control sample solution, incubating the solutions with tumor cells for 48 hr, adding CCK-8 or MTT for reaction for 2, 2.5, 3, 3.5, 4, 4.5 and 5 hr, and measuring absorbance of the reaction solution.
The result shows that after the CCK-8 is reacted for 3.5 hours, the absorbance of the reaction solution reaches the maximum and does not change significantly; after 4h of MTT reaction, the absorbance of the reaction solution reached the maximum and did not change significantly. Therefore, the CCK-8 reaction time is preferably 3.5 h; the reaction time of MTT was 4 h.
Claims (10)
1. A method for evaluating the quality of lucid ganoderma based on an in-vitro tumor cell culture model comprises the following steps:
(1) preparing a ganoderma lucidum sample to be detected into a sample solution by using a solvent;
(2) preparing a positive control substance into a positive control substance solution by using a solvent;
(3) constructing a 2D or 3D culture model of the tumor cells, diluting the sample solution and the positive reference substance solution into different concentrations by using a culture solution, and incubating the sample solution and the positive reference substance solution together with the tumor cells;
(4) respectively sucking the solutions incubated in the step (3), adding a fresh culture solution, adding a cell activity indicator, reacting for 1-5 hours, detecting the absorbance or fluorescence intensity of the reaction solution by using an enzyme-linked immunosorbent assay, calculating the inhibition rate of the ganoderma sample solution and the positive control solution on the proliferation of tumor cells, calculating corresponding IC50, and evaluating the quality of the ganoderma by using the inhibition rate of cell proliferation; the method for calculating the cell proliferation inhibition rate comprises the following steps:
cell proliferation inhibition rate (%) ([ 1- (OD)Sample group or positive control group-ODBlank group/ODBlank control group-ODBlank group)]×100％。
2. The method of claim 1, wherein the method is as follows:
(1) preparing a ganoderma lucidum sample to be detected into a sample solution by using a solvent;
(2) preparing a positive control substance into a positive control substance solution by using a solvent;
(3) constructing a 2D or 3D culture model of the tumor cells, diluting the sample solution and the positive reference substance solution into different concentrations by using a culture solution, and respectively incubating with the tumor cells;
(4) constructing a 3D culture model of tumor cells, mixing a sample solution diluted by a culture solution with a reference solution, and incubating the obtained mixed solution with the tumor cells;
(5) respectively sucking the solutions incubated in the steps (3) and (4), adding a fresh culture solution, adding a cell viability indicator, reacting for 1-5 hours, detecting the absorbance or fluorescence intensity of the reaction solution by using an enzyme-linked immunosorbent assay, calculating the inhibition rate of the ganoderma sample solution, the positive control solution and the mixed solution on the proliferation of tumor cells, calculating the corresponding IC50 or proliferation inhibition rate, and evaluating the quality of the ganoderma by using the cell proliferation inhibition rate or growth inhibition rate; the method for calculating the cell proliferation inhibition rate comprises the following steps:
cell proliferation inhibition rate (%) ([ 1- (OD)Sample group or positive control group-ODBlank group/ODBlank control group-ODBlank group)]×100％
Percent (%) increase inhibition ratioMixed solutionInhibition RatePositive control solution。
3. The method of claim 1 or 2, wherein the tumor cell is one of: a liver cancer cell, a breast cancer cell, a colon cancer cell, a lung cancer cell, a stomach cancer cell, or a pancreatic cancer cell; the positive control is one of the following: fluorouracil, cisplatin, irinotecan, or docetaxel; the concentration of the ganoderma lucidum sample in the mixed solution in the step (4) is 0.001-10 mg/mL.
4. The method according to claim 1 or 2, wherein the cell viability indicator is one of: acridine orange/ethidium bromide, DAPI, Hoechst, MTT, XTT, SRB, MTS, CCK-8, CTG, or Calcein/PI.
5. The method according to claim 4, wherein the cell viability indicator is CCK-8, the reaction time is 3.5 hours, and the cell proliferation inhibition (%) is [1- (OD)Sample (I)Group or Positive control group-ODCulture medium/ODBlank control group-ODCulture medium)]×100％。
6. The method according to claim 4, wherein the cell viability indicator is MTT, the reaction time is 4 hours, and the cell proliferation inhibition ratio (%) [1- (OD)Sample group or positive control group-ODDimethyl sulfoxide/ODBlank control group-ODDimethyl sulfoxide)]×100％。
7. The method according to claim 1 or 2, characterized in that said 2D cultured tumor cells are obtained by: after the tumor cells are digested by pancreatin, the tumor cells are diluted by a culture medium and resuspended, the cells are uniformly mixed to obtain cell suspension, and the cell suspension is uniformly dispersed in a micropore plate in the same volume to obtain a tumor cell 2D culture model.
8. The method according to claim 1 or 2, characterized in that the 3D cultured tumor cells are obtained by: digesting the tumor cells by pancreatin, diluting the tumor cells by using a culture medium, resuspending the tumor cells, uniformly mixing to obtain a cell suspension, dissolving a matrix material, uniformly dispersing the matrix material in a microporous plate in an equal volume manner, and after the matrix material is gelatinized, uniformly dispersing the cell suspension in the microporous plate in an equal volume manner to obtain the tumor cell 3D culture model.
9. The method according to claim 1 or 2, wherein the Ganoderma lucidum sample to be tested in step (1) is obtained by the following method: adding Ganoderma into solvent, extracting by reflux extraction, ultrasonic extraction, Soxhlet extraction, leaching or pressure countercurrent extraction to obtain sample solution, and drying by nitrogen blowing, evaporating, concentrating under reduced pressure, centrifuging, or lyophilizing to obtain sample to be tested; the solvent is one of the following: water, ethanol, 5-95% ethanol solution, methanol, 5-95% methanol solution, ethyl acetate and trichloromethane.
10. The method according to claim 1 or 2, wherein the ganoderma lucidum sample in step (1) or (2) comprises ganoderma lucidum medicinal materials, ganoderma lucidum decoction pieces, ganoderma lucidum extracts, compound traditional Chinese medicine products containing ganoderma lucidum, wall-broken ganoderma lucidum spore powder extracts, compound traditional Chinese medicine products containing wall-broken ganoderma lucidum spore powder, wall-removed ganoderma lucidum spore powder extracts, compound traditional Chinese medicine products containing wall-removed ganoderma lucidum spore powder; the Ganoderma lucidum includes Ganoderma lucidum (Ganoderma lucidum) and Ganoderma sinense (Ganoderma sinense).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010668871.6A CN111826416A (en) | 2020-07-13 | 2020-07-13 | Ganoderma lucidum quality evaluation method based on in-vitro tumor cell culture model |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010668871.6A CN111826416A (en) | 2020-07-13 | 2020-07-13 | Ganoderma lucidum quality evaluation method based on in-vitro tumor cell culture model |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111826416A true CN111826416A (en) | 2020-10-27 |
Family
ID=72901376
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010668871.6A Pending CN111826416A (en) | 2020-07-13 | 2020-07-13 | Ganoderma lucidum quality evaluation method based on in-vitro tumor cell culture model |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111826416A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115572754A (en) * | 2022-10-09 | 2023-01-06 | 上海市农业科学院 | Method for evaluating activity of edible and medicinal fungus liquid strain |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104666368A (en) * | 2014-10-17 | 2015-06-03 | 成都大学 | Ganoderma lucidum total triterpene purified product with high anthropogenic tumor cell activity and purification method of ganoderma lucidum total triterpene purified product |
| CN106924301A (en) * | 2017-04-14 | 2017-07-07 | 北京大学 | The application of Ganodenna Lucidum P.E and ganodenic acid monomer in autosomal dominant polycystic kidney disease is treated |
| CN109758486A (en) * | 2019-03-25 | 2019-05-17 | 广州白云山汉方现代药业有限公司 | Ganodenna Lucidum P.E is preparing the application in artitumor multi-medicine-resistant medicine |
-
2020
- 2020-07-13 CN CN202010668871.6A patent/CN111826416A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104666368A (en) * | 2014-10-17 | 2015-06-03 | 成都大学 | Ganoderma lucidum total triterpene purified product with high anthropogenic tumor cell activity and purification method of ganoderma lucidum total triterpene purified product |
| CN106924301A (en) * | 2017-04-14 | 2017-07-07 | 北京大学 | The application of Ganodenna Lucidum P.E and ganodenic acid monomer in autosomal dominant polycystic kidney disease is treated |
| CN109758486A (en) * | 2019-03-25 | 2019-05-17 | 广州白云山汉方现代药业有限公司 | Ganodenna Lucidum P.E is preparing the application in artitumor multi-medicine-resistant medicine |
Non-Patent Citations (3)
| Title |
|---|
| PAN H T等: "Autophagic flux disruption contributes to Ganoderma lucidum polysaccharide-induced apoptosis in human colorectal cancer cells via MAPK/ERK activation", CELL DEATH AND DISEASE * |
| 王杰等: "构建三阴性乳腺癌的三维体外培养模型用于抗肿瘤药物的活性评价", 安徽医科大学学报 * |
| 王樟根: "赤芝抗乳腺癌细胞增殖活性成分及其构效关系研究", 中国优秀硕士学位论文电子期刊医药卫生科技辑 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115572754A (en) * | 2022-10-09 | 2023-01-06 | 上海市农业科学院 | Method for evaluating activity of edible and medicinal fungus liquid strain |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lu et al. | Ganoderma lucidum extracts inhibit growth and induce actin polymerization in bladder cancer cells in vitro | |
| Zhang et al. | A sensitive and rapid radiolabelling method for the in vivo pharmacokinetic study of lentinan | |
| KR20110023804A (en) | Use of lanostane and poria extract in treating diabetes | |
| CN101869633B (en) | Enteric-coated medicament combination containing epigallocatechin gallate | |
| CN105708801A (en) | Preparation method and application of 20 (R)-ginsenoside Rg3/soya bean lecithin/cholesterol/folic acid lipidosome medicine | |
| Wu et al. | Role of particle aggregates in herbal medicine decoction showing they are not useless: considering Coptis chinensis decoction as an example | |
| Tong et al. | An acetylated mannan isolated from Aloe vera induce colorectal cancer cells apoptosis via mitochondrial pathway | |
| CN111826416A (en) | Ganoderma lucidum quality evaluation method based on in-vitro tumor cell culture model | |
| CN101301455A (en) | Chinese medicine compound turmeric rhizome solid dispersion for treating hyperlipemia | |
| CN109674742A (en) | A kind of curcumin hydrogel matrix slow releasing composition, preparation method and its application in anti-cancer field | |
| CN100518768C (en) | Cassia twig tuckahoe effervescence tablet and testing method | |
| CN111388602B (en) | Lucid ganoderma and dendrobium officinale suspension and preparation method thereof | |
| CN105193867B (en) | A kind of ganoderma lucidum spore oil and preparation method thereof rich in ergosterol | |
| CN110478379B (en) | Selaginella chinensis total biflavone precursor liposome and preparation method thereof | |
| CN108371653B (en) | A kind of nano-micelle and its application | |
| CN110623928A (en) | Preparation method of ergosterol and gefitinib combined liposome | |
| US20010018076A1 (en) | Fagopyrum cymosum (Trev.) meisn composition, method to prepare and analyze the same and uses thereof | |
| CN102319420B (en) | The application of turtle peptide in pharmacy | |
| Arib et al. | A pegylated flavin adenine dinucleotide PEG complex to boost immunogenic and therapeutic effects in a liver cancer model | |
| CN108310226A (en) | It is a kind of to have effects that prevent the composition and the preparation method and application thereof of diabetes | |
| CN103432090B (en) | Cyclovirobuxine D sublingual tablet as well as preparation method and application thereof | |
| CN111773185A (en) | Hyaluronic acid modified bufalin-loaded nano liposome as well as preparation method and application thereof | |
| CN110967430A (en) | Method for measuring dissolution curve of coenzyme Q10 capsule | |
| CN110507617A (en) | Dimethylcurcumin solid dispersions, Dimethylcurcumin solid dispersion tablet and preparation method thereof | |
| Ilić et al. | Bioactivity Profiling of Daedaleopsis confragosa (Bolton) J. Schröt. 1888: Implications for Its Possible Application in Enhancing Women’s Reproductive Health |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |