Phellinus igniarius and lucid ganoderma composition with anti-tumor effect as well as extraction method and application of phellinus igniarius and lucid ganoderma composition

Abstract

The invention discloses a traditional Chinese medicine composition with an anti-tumor effect and an extraction method and application thereof, and belongs to the technical field of traditional Chinese medicine compositions. The traditional Chinese medicine composition is composed of ganoderma lucidum spore powder and phellinus igniarius, ethyl acetate extract obtained through ethanol ultrasonic-assisted extraction and fractional extraction has a remarkable anti-tumor effect, and compared with a traditional Chinese medicine composition, a single traditional Chinese medicine extract and the ethyl acetate extract, the traditional Chinese medicine composition has a remarkable synergistic effect and is used for preparing the anti-tumor medicine. According to the present invention, the ganoderma lucidum spore powder or the phellinus igniarius ethanol extract is adopted as the raw material, auxiliary materials are added to prepare the ganoderma lucidum spore powder or the phellinus igniarius ethanol extract into tablets, granules, capsules, powder or injection for use, and compared with the single ganoderma lucidum spore powder or phellinus igniarius ethanol extract, the traditional Chinese medicine composition and the ethyl acetate part have characteristics of antitumor synergistic effect, significantly enhanced antitumor effect, wide antitumor potential and wide clinical application value.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a traditional Chinese medicine composition with anti-tumor efficacy and an extraction method and application thereof. Background Art

[0002] The incidence and mortality of tumors have remained high, posing a serious threat to human health and life expectancy. Therefore, the prevention, treatment and research of malignant tumors have become a research field of great concern to medical workers around the world.

[0003] Traditional Chinese medicine can improve the body's immunity and play an important role in prolonging the patient's survival and improving the quality of life due to its multi-target, multi-link and multi-effect characteristics in treating cancer. Clinical applications have confirmed that traditional Chinese medicine has definite efficacy and unique advantages in assisting the treatment of tumors. Current studies have shown that many natural plant extracts have good anti-tumor activity, and many natural products or their structurally modified compounds have entered the scope of clinical research. The discovery and in-depth research of anti-tumor traditional Chinese medicine will provide new drug options for the clinical application of traditional Chinese medicine anti-tumor, and contribute to improving the effect of tumor treatment and promoting human health.

[0004] Ganoderma lucidum is the dried fruiting body of Ganoderma lucidum (Leyss.ex Fr.) Karst. or Ganoderma sinense Zhao, Xu et Zhang, a fungus of the Polyporaceae family. It is sweet in taste, neutral in nature, and enters the heart, lung, liver, and kidney meridians. It has the effects of replenishing qi and calming the mind, relieving cough and relieving asthma. It is mainly used to treat restlessness, insomnia, palpitations, cough and asthma with excessive sputum, and asthenia. Ganoderma lucidum spore powder is the extremely tiny oval reproductive cells, i.e., spores of Ganoderma lucidum, ejected from the gills of Ganoderma lucidum during the growth and maturity period. Ganoderma lucidum spore powder has many genetic materials of Ganoderma lucidum, and its main components include polysaccharides, triterpenes, proteins, amino acids, nucleosides, etc. It has pharmacological effects such as immunomodulation, anti-oxidation, anti-tumor, anti-aging, hypoglycemic, and liver protection.

[0005] Phellinus igniarius (L.ex Fr.) Quel. [Fomesigniarius (L.) Fr.; Boletus igniarius L.; Polyporus igniarius Fr.] of the Polyporaceae family. It tastes slightly bitter, is cold in nature, and enters the liver and kidney meridians. It has the effects of promoting blood circulation, stopping bleeding, transforming fluid, and stopping diarrhea. It is often used for metrorrhagia, hematuria, rectal prolapse and bleeding, leucorrhea, amenorrhea, accumulation of lumps, drinking habit, and diarrhea caused by spleen deficiency. Its chemical components mainly include polysaccharides, flavonoids, triterpenes, and polyphenols, and have pharmacological effects such as anti-inflammatory, hypoglycemic, antioxidant, liver protection, and anti-tumor.

[0006] Although there are many anti-tumor drugs on the market, most of them have serious side effects, easy to develop drug resistance, and limited therapeutic effects. Although single-ingredient natural products have certain anti-tumor activity, they often have a single mechanism of action and cannot fully cope with the complexity of tumors. Although Ganoderma lucidum spore powder and Coriolus versicolor each have anti-tumor and other pharmacological effects, in-depth research on the specific mechanism, dosage ratio and clinical effect of their combined use is still insufficient.

[0007] Chinese patent (CN107519327A) discloses a Chinese medicinal composition of Phellinus igniarius and its preparation and use in the preparation of drugs for treating gastric cancer, liver cancer, colon cancer, etc. The Chinese medicinal composition includes Phellinus igniarius, Ganoderma lucidum and Tripterygium wilfordii. The extract of the composition has a significant improvement effect on cyclophosphamide-induced immune loss mice and has a significant inhibitory effect on gastric cancer, liver cancer, and colon cancer strains. However, in actual applications, the composition has a high administration concentration, a low tumor inhibition rate, and the anti-tumor effect needs to be further improved. Summary of the invention

[0008] In view of the technical problems that the existing Chinese medicinal compositions of Phellinus igniarius have complex compositions, high proportions of Ganoderma lucidum and high costs, and high dosage concentrations and low tumor inhibition rates in practical applications, the present invention aims to provide an anti-tumor Chinese medicinal composition and an extraction method and application thereof, which can be used as a new type of anti-tumor drug.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] The invention provides an anti-tumor Chinese medicinal composition, which consists of 1 to 3 parts of ganoderma lucidum spore powder and 1 to 3 parts of phellinus linteus by weight.

[0011] The present invention provides a method for extracting an anti-tumor Chinese medicine composition, comprising:

[0012] (1) weighing Ganoderma lucidum spore powder and Phellinus linteus, mixing them, adding ethanol, heating and ultrasonic-assisted extraction, filtering, concentrating and drying under reduced pressure to obtain an ethanol extract of the traditional Chinese medicine composition;

[0013] (2) resuspending the ethanol extract of the Chinese medicine composition in step (1) in distilled water, performing graded extraction, collecting and concentrating the extract to obtain an extract concentrate of the Chinese medicine composition.

[0014] The amount of ethanol added is 20 to 30 times the mass of the raw material, and the volume fraction of ethanol is 70%.

[0015] The mass ratio of the ganoderma lucidum spore powder to mulberry linterus is 1-3:1-3.

[0016] The conditions for the heating ultrasonic assisted extraction are: the heating temperature is 36° C. to 38° C., and the ultrasonic extraction time is 1 h to 2 h.

[0017] The distilled water is added for re-suspending, and the amount of distilled water added is 40 to 60 times the mass of the ethanol extract of the traditional Chinese medicine composition.

[0018] The graded extraction solvent is any one of petroleum ether, chloroform, ethyl acetate and n-butanol, the added amount of the extraction solvent is 1:1 in volume ratio with distilled water, and the number of extractions is 2 to 4 times.

[0019] Furthermore, the extraction solvent is ethyl acetate.

[0020] The anti-tumor Chinese medicine composition is used in the preparation of anti-tumor drugs, wherein the anti-tumor drugs are drugs for treating colon cancer, liver cancer, breast cancer, cervical cancer and lung cancer.

[0021] The anti-tumor drug may further include one or more pharmaceutically acceptable excipients.

[0022] Furthermore, the pharmaceutically acceptable excipient is selected from at least one of pharmaceutically acceptable solvents, solubilizers, cosolvents, adhesives, emulsifiers, colorants, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, preservatives, flavoring agents, suspending agents, coating materials, fragrances, anti-adhesives, pH regulators, integrators, penetration enhancers, buffers, plasticizers, surfactants, humectants, thickeners, inclusion agents, absorbents, release retardants, diluents, flocculants and deflocculating agents, filter aids, polymer skeleton materials and film-forming materials.

[0023] The anti-tumor drug is in the form of capsules, gels, creams, mixtures, tablets, powders, pills, granules, wet compresses, solutions, syrups, suppositories, decoctions, aerosols, ointments, plasters, injections, sprays, tinctures, liniments, pastes or lotions.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] The invention provides a novel Chinese medicine composition with anti-tumor efficacy, which is composed of ganoderma lucidum spore powder and mulberry linterus. Compared with the single use of ganoderma lucidum spore powder or mulberry linterus ethanol extract, the Chinese medicine composition has a synergistic anti-tumor effect and enhances the anti-tumor effect. The Chinese medicine composition of the invention provides a new drug formula for treating tumors, and also provides a new idea for the research and development of innovative drugs, and has high scientific research value and good application prospects.

[0026] The extraction method of the anti-tumor Chinese medicine composition provided by the present invention efficiently extracts components with anti-tumor activity from Ganoderma lucidum spore powder and Phellinus igniarius through ethanol heating ultrasound-assisted extraction and subsequent water resuspension, graded extraction and other steps, can more comprehensively retain the effective ingredients in the medicinal materials, while removing impurities, improving the purity and biological activity of the extract, and is simple to operate and suitable for industrial-scale production.

[0027] Furthermore, different types of active ingredients can be selectively extracted by fractional extraction with different polar solvents. The purity and activity of the extract can be further improved by multiple extractions. Non-polar solvents can extract fat-soluble components in the composition, medium-polar solvents can extract components that are slightly more polar than non-polar solvents but still belong to non-polar to weak polarity, and strong polar solvents can extract polysaccharides, glycosides, certain amino acids and other components with greater polarity in the composition.

[0028] Furthermore, ethyl acetate can extract active ingredients of medium polarity such as flavonoids, phenolic acids, and certain alkaloids. Ethyl acetate extract plays an important role in improving the body's immunity and regulating physiological functions.

[0029] The application provided by the present invention, the Chinese medicine composition and ethyl acetate extraction extract provided by the present invention have significant synergistic effect compared with single Ganoderma lucidum spore powder or Phellinus igniarius ethanol extract, both show synergistic inhibition effect on HCT116 and COLO205, and have the strongest cell inhibition effect; compared with single Chinese medicine extract, the Chinese medicine composition and ethyl acetate extraction extract of the present invention have significant inhibition effect on the growth of nude mouse HCT116 transplanted tumor model and C57BL / 6 mouse Lewis lung cancer transplanted tumor model, when the administration concentration is 800mg / kg, the inhibition rate of the two transplanted tumors reaches 66.54%, 73.15% and 53.33%, 68.93% respectively, which is 20% to 40% higher than that of single extract, showing very good anti-tumor synergistic effect, and no toxic side effects on experimental animals, can be used to prepare drugs for treating various tumors (such as colon cancer, liver cancer, breast cancer, cervical cancer and lung cancer), and has wide anti-tumor potential and clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The graph shows the inhibitory effect of the Chinese medicine composition on the proliferation of human colon cancer HCT116 and COLO205 cells, wherein A is the inhibition rate of the Chinese medicine composition on the proliferation of human colon cancer HCT116 cells, and B is the inhibition rate of the Chinese medicine composition on the proliferation of human colon cancer COLO205 cells. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0032] The present invention is described in further detail below:

[0033] Example 1

[0034] An anti-tumor Chinese medicine composition, wherein the extraction method of the composition comprises:

[0035] 1) Extraction of Chinese herbal medicine composition

[0036] Weigh 5 g of Ganoderma lucidum spore powder and 5 g of Phellinus igniarius, add 70% ethanol with a volume fraction of 25 times the weight of the medicinal materials, perform ultrasonic extraction for 1 hour at 37° C., filter, concentrate the ethanol extract under reduced pressure, and vacuum dry to obtain a dry powder of the Chinese medicine composition, i.e., the ethanol extract of the Chinese medicine composition;

[0037] 2) adding water to the ethanol extract of the traditional Chinese medicine composition to resuspend it, wherein the volume of water is 50 times the mass of the ethanol extract of the traditional Chinese medicine composition; extracting with petroleum ether, chloroform, ethyl acetate and n-butanol with the same volume as water for 3 times respectively, combining and collecting the ethyl acetate extract, recovering the solvent in the ethyl acetate extract under reduced pressure, and concentrating to obtain the extract of the anti-tumor traditional Chinese medicine composition.

[0038] Example 2

[0039] An anti-tumor Chinese medicine composition, wherein the extraction method of the composition comprises:

[0040] Weigh 4 g of Ganoderma lucidum spore powder and 6 g of Phellinus igniarius, add 70% ethanol with a volume fraction of 25 times the weight of the medicinal materials, perform ultrasonic extraction for 1 hour at a temperature of 37° C., filter, concentrate the ethanol extract under reduced pressure, and vacuum dry to obtain a dry powder of the Chinese medicine composition extract, i.e., the ethanol extract of the Chinese medicine composition;

[0041] 2) adding water to the ethanol extract of the traditional Chinese medicine composition to resuspend it, wherein the volume of water is 50 times the mass of the ethanol extract of the traditional Chinese medicine composition; extracting with petroleum ether, chloroform, ethyl acetate and n-butanol with the same volume as water for 3 times respectively, combining and collecting the ethyl acetate extract, recovering the solvent in the ethyl acetate extract under reduced pressure, and concentrating to obtain the extract of the anti-tumor traditional Chinese medicine composition.

[0042] Example 3

[0043] This embodiment provides an anti-tumor Chinese medicine composition, and the extraction method of the composition comprises:

[0044] 1) Weigh 6 g of Ganoderma lucidum spore powder and 4 g of Phellinus igniarius, add 70% ethanol with a volume fraction of 25 times the weight of the medicinal materials, perform ultrasonic extraction for 1 h at 37° C., filter, concentrate the ethanol extract under reduced pressure, and vacuum dry to obtain a dry powder of the Chinese medicine composition extract, i.e., the ethanol extract of the Chinese medicine composition;

[0045] 2) adding water to the ethanol extract of the traditional Chinese medicine composition to resuspend it, wherein the volume of water is 50 times the mass of the ethanol extract of the traditional Chinese medicine composition; extracting with petroleum ether, chloroform, ethyl acetate and n-butanol with the same volume as water for 3 times respectively, combining and collecting the ethyl acetate extract, recovering the solvent in the ethyl acetate extract under reduced pressure, and concentrating to obtain the extract of the anti-tumor traditional Chinese medicine composition.

[0046] Example 4

[0047] The anti-tumor effect of the Chinese medicine composition and its extract provided by the present invention is illustrated by experiments below.

[0048] 1. Screening of anti-tumor active extracts from Ganoderma lucidum spore powder and Phellinus igniarius

[0049] (1) Instruments and Materials

[0050] SW-CJ-1F clean bench (Suzhou Antai Air Technology Co., Ltd.); Ti-U inverted microscope (Nikon); MAC 15AC carbon dioxide constant temperature incubator (SANYO); 80-2 desktop electric centrifuge (Shanghai Analytical Instrument Factory); GH water-proof constant temperature incubator (Beijing Kewei Yongxing Instrument Co., Ltd.); pipette (Eppendorf); UPT-IV-5T ultrapure water preparation machine (Chengdu Ultrapure Technology Co., Ltd.); SB-5200 ultrasonic cleaning machine (Ningbo Xinzhi Biotechnology Co., Ltd.); BP211D electronic balance (Shanghai Guangzheng Medical Instrument Co., Ltd.); cell counting plate (Shanghai Qiujing Biochemical Reagent Co., Ltd.); culture plate (Costar).

[0051] The human colon cancer cell lines HCT-116 and COLO205 used in the present invention were purchased from Air Force Medical University, the human cloned colon adenocarcinoma cell line Caco-2 was purchased from Jiangsu Qi's Biotechnology Co., Ltd., the human liver cancer cell lines Bel-7404 and SMMC-7721 were purchased from Shanghai Jikai Biochemical Technology Co., Ltd., the human liver cancer cell line MHCC-97L was donated by the First Affiliated Hospital of Xi'an Jiaotong University, the human breast cancer cell line MCF-7 was purchased from Shanghai Jikai Biochemical Technology Co., Ltd., the human breast cancer cell line SK-BR-3 was purchased from the National Experimental Cell Resource Sharing Platform (Beijing Headquarters), the human breast cancer cell line MDA-MB-231 was purchased from Shanghai Jikai Biochemical Technology Co., Ltd., and the human cervical cancer cell line HeLa, the human cervical epidermal cancer cell line MS751, the human large cell lung cancer cell line NCI-H460, and the human non-small cell lung cancer cell line NCI-H1299 were all purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences.

[0052] (2) Experimental methods

[0053] Ganoderma lucidum spore powder and Phellinus igniarius medicinal material powder are weighed separately or by mass, and are respectively loaded into a stoppered conical flask, and then water or ethanol with a volume fraction of 70% is added, the conical flask is fixed in an ultrasonic cleaning machine, the power is turned on, ultrasonic extraction is performed by heating, filtering, and the extract is concentrated under reduced pressure and vacuum dried to obtain 6 kinds of extracts of Ganoderma lucidum spore powder and Phellinus igniarius. The extraction process parameters are the same as those in Example 1. Among the numbers 1-6, 1 is an extract obtained by extracting Ganoderma lucidum spore powder with water; No. 2 is an extract obtained by extracting Ganoderma lucidum spore powder with a volume fraction of 70% ethanol; No. 3 is an extract obtained by extracting Phellinus igniarius with water; No. 4 is an extract obtained by extracting Phellinus igniarius with a volume fraction of 70% ethanol; No. 5 is an extract obtained by extracting Ganoderma lucidum spore powder and Phellinus igniarius with water; No. 6 is an extract obtained by extracting Ganoderma lucidum spore powder and Phellinus igniarius with a volume fraction of 70% ethanol; and screening is performed using tumor cells.

[0054] (3) Experimental results

[0055] On the basis of Example 1, two solvents, water and 70% ethanol by volume, were used for extraction, and six extracts obtained by ultrasonic-assisted extraction of Ganoderma lucidum spore powder and Phellinus linteus were subjected to MTT experiments. The results are shown in Table 1.

[0056] Table 1: Concentrations corresponding to 90%-100% inhibition rates of Ganoderma lucidum spore powder and Phellinus igniarius extracts on tumor cell growth (mg / mL)

[0057]

[0058]

[0059] From the data in Table 1, it can be seen that the ethanol extracts of Ganoderma lucidum spore powder and Phellinus igniarius (2, 4, 6) have a significant inhibitory effect on the growth of tumor cells. The extract obtained by extracting the Chinese medicine composition with 70% ethanol by volume has anti-tumor active ingredients, and the anti-tumor activity is significantly enhanced compared with a single Chinese medicine extract; among them, the extract No. 6 obtained by extracting the Chinese medicine composition with 70% ethanol by volume has a minimum inhibitory concentration of 0.4 mg / mL with a 90% to 100% inhibition rate on the growth of all tumor cell lines, indicating that it has a significant inhibitory effect on human colon cancer, liver cancer, breast cancer, cervical cancer and lung cancer cells, and the minimum inhibitory concentration is significantly reduced, with the strongest anti-tumor activity.

[0060] Therefore, the extraction process of Ganoderma lucidum spore powder and Phellinus linteus in the traditional Chinese medicine composition of the present invention is determined to select 70% ethanol by volume, the addition amount is 25 times the mass of the traditional Chinese medicine composition, ultrasonic extraction is performed for 1 hour, and the temperature is 37°C.

[0061] 2. Ganoderma lucidum spore powder and phellinus linteus cell viability test and combined action index analysis

[0062] (1) Instruments and Materials

[0063] SW-CJ-1F clean bench (Suzhou Antai Air Technology Co., Ltd.); Ti-U inverted microscope (Nikon); MAC 15AC carbon dioxide constant temperature incubator (SANYO); 80-2 desktop electric centrifuge (Shanghai Analytical Instrument Factory); GH water-proof constant temperature incubator (Beijing Kewei Yongxing Instrument Co., Ltd.); pipette (Eppendorf); UPT-IV-5T ultrapure water preparation machine (Chengdu Ultrapure Technology Co., Ltd.); BP211D electronic balance (Shanghai Guangzheng Medical Instrument Co., Ltd.); cell counting chamber (Shanghai Qiujing Biochemical Reagent Co., Ltd.); culture plate (Costar).

[0064] The human colon cancer cells HCT116 and COLO205 used in the experiment were purchased from Air Force Medical University, cultured in a culture medium containing 10% fetal bovine serum (containing 100 U / ml penicillin and streptomycin) in a 37°C, 5% CO2 incubator, and passaged every 2 to 3 days.

[0065] (2) Experimental methods

[0066] Weigh 10 g of Ganoderma lucidum spore powder, add ethanol, perform heating and ultrasonic extraction, filter, concentrate the ethanol extract under reduced pressure, and then vacuum dry to obtain a Ganoderma lucidum spore powder extract for stand-alone use.

[0067] Weigh 10 g of Phellinus igniarius medicinal material powder, add ethanol, heat and ultrasonically extract, filter, reduce pressure and concentrate the ethanol extract, and then vacuum dry to obtain Phellinus igniarius extract dry powder for stand-alone use.

[0068] The effect of the test drug on the proliferation of human colon cancer cells HCT116 and COLO205 was detected by the MTT experiment. HCT116 and COLO205 cells in the exponential growth phase were digested with trypsin, blown into a single cell suspension, counted, and inoculated into a 96-well culture plate. Cultured in an incubator for 24 hours, after the cells adhered to the wall, different concentrations of test drugs were added, and the culture was terminated after 48 hours of action, 20μMTT (5mg / mL) was added to each well, and incubated at 37℃ in the dark for 4 hours. Then the liquid in each well was discarded, 150μL DMSO was added, and the crystals were shaken for 15 minutes to fully dissolve, and the cell optical density value was measured at a wavelength of 490nm on an ELISA reader.

[0069] The above-mentioned MTT method was used to detect the cell inhibition effect of the combination of Ganoderma lucidum spore powder and Phellinus igniarius, and the Chou-Talalay method was used to determine whether the combination of the two drugs had a synergistic effect. The calculation method of the drug action relationship was:

[0070] Combination index (CI) = D A / DX A +D B / DX B +D A ×D B / DX A ×DX B , where DX A DX B represents the concentration of drug A and drug B when used alone to produce the inhibitory effect X, D A , D B It represents the corresponding drug concentration when the combined use of drug A and drug B produces the inhibitory effect X. The CI value calculated according to the formula reflects the combined action mode of drug A and drug B, where CI>1 means that drugs A and B are antagonistic, CI=1 means that drugs A and B are additive, and CI<1 means that drugs A and drug B are synergistic.

[0071] The experimental data were expressed as mean ± standard error (Mean ± SEM) and analyzed using Prism 8.0 statistical software. P < 0.05 indicated a significant difference.

[0072] The MTT method was used to investigate the inhibitory effects of Ganoderma lucidum spore powder and Phellinus igniarius alone and in combination on tumor cells, and the drug combination index CI was calculated. The inhibitory effect of the Chinese medicine combination on HCT116 and COLO205 cells is shown in the attached figure. Figure 1 As shown, it was found through experiments that when the ethanol extract of Ganoderma lucidum spore powder and the ethanol extract of Phellinus igniarius were used in combination, they could show a synergistic inhibitory effect on HCT116 and COLO205 at all concentrations. The experimental results are shown in Table 2.

[0073] Table 2: IC values ​​of Ganoderma lucidum spore powder combined with Phellinus igniarius against HCT116 and COLO205 50 (mg / mL)

[0074]

[0075] Note: GLEE represents Ganoderma lucidum spore powder ethanol extract, SVEE represents Phellinus igniarius ethanol extract, GSEE represents Chinese medicine composition (Ganoderma lucidum spore powder and Phellinus igniarius ethanol extract); ▲: CI <1, a combination showing a synergistic effect.

[0076] Attached Figure 1 The results show that the attached Figure 1 In the figure, A is the inhibition rate of the Chinese medicine composition on the proliferation of human colon cancer HCT116 cells, and B is the inhibition rate of the Chinese medicine composition on the proliferation of human colon cancer COLO205 cells. The IC values ​​of the Chinese medicine composition on HCT116 and COLO205 cells were calculated from the data. 50 The values ​​were 0.06mg / mL and 0.11mg / mL respectively.

[0077] It can be seen from the data in Table 2 that the CI values ​​of all combinations (2:1 combination, 1:2 combination, 1:1 combination) are less than 1, which clearly shows that Ganoderma lucidum spore powder and Coriolus linteus show synergistic effects at different concentration ratios, which means that the effect of the two drugs when used in combination is greater than the sum of the effects of each of them used alone, which is of great significance for improving therapeutic effects, reducing drug dosage and reducing side effects.

[0078] For HCT116 cells, regardless of the concentration ratio, the IC 50 The values ​​are lower than the IC of GLEE or SVEE alone. 50 value, especially when Ganoderma lucidum spore powder and Phellinus linteus were used in a 1:1 ratio, its IC 50 The value was the lowest, showing the strongest inhibitory effect; a similar trend was observed for the COLO205 cell line, and the IC 50 The values ​​were significantly lower than those when used alone, and the 1:1 combination also showed a lower IC 50 The IC values ​​of different combinations and Chinese medicine compositions were compared. 50 The IC values ​​of the Chinese herbal medicine composition were compared and analyzed. 50 The value is the lowest, indicating that the Chinese medicine composition of the present invention has a stronger inhibitory effect on human colon cancer cells than the single use of Ganoderma lucidum spore powder or Phellinus igniarius ethanol extract, further proving the significant synergistic effect of the combined use of Ganoderma lucidum spore powder and Phellinus igniarius. Through scientific matching, better therapeutic effects can be achieved.

[0079] 3. Experiment on nude mouse HCT116 transplanted tumor model using traditional Chinese medicine composition

[0080] (1) Instruments and Materials

[0081] Ti-U inverted microscope (Nikon); 80-2 tabletop electric centrifuge (Shanghai Analytical Instrument Factory); pipette (Eppendorf); UPT-IV-5T ultrapure water preparation machine (Chengdu Ultrapure Technology Co., Ltd.); BP211D electronic balance (Shanghai Guangzheng Medical Instrument Co., Ltd.); cell counting plate (Shanghai Qiujing Biochemical Reagent Co., Ltd.); disposable syringe (Jiangxi Jinshan Medical Instrument Co., Ltd.); vernier caliper (Wuxi Xigong Measuring Tool Co., Ltd.); surgical scissors (Shanghai Medical Instrument Co., Ltd.); ultra-low temperature refrigerator (Qingdao Haier Group); cell factory culture system (NUNC).

[0082] Human colon cancer cell line HCT-116 was purchased from Air Force Medical University.

[0083] Healthy BALB / c male nude mice, 18-22 g, were provided by the Experimental Animal Center of Xi'an Jiaotong University. They were given free access to food and water and were cultured in an SPF animal room at 22-24°C, 50%-70%, and a 12h-12h light-dark cycle.

[0084] (2) Experimental methods

[0085] The inhibitory effect of the Chinese medicine composition on the nude mouse HCT116 transplanted tumor model was detected using the nude mouse HCT116 transplanted tumor model.

[0086] Prepared HCT116 cells at 1x10 7 The cells were resuspended in sterile saline at a density of 100 μg / mL, and 200 μL of the cell suspension was injected subcutaneously into the right armpit of each nude mouse.

[0087] After inoculation, the nude mice were closely observed for tumor formation and the size of the transplanted tumors was recorded with a vernier caliper. When the average tumor volume reached 100 mm 3 The nude mice were then randomly divided into groups of 6 in each group, and the drugs were administered according to the designed concentration of the median lethal dose for 21 days. The mice were killed 21 days later, the tumors were removed and weighed, and the inhibition rate of the Chinese medicine composition on the growth of HCT116 transplanted tumors in nude mice was calculated.

[0088] The calculation formula of tumor inhibition rate is as follows:

[0089]

[0090] The experimental data were expressed as mean ± standard error (Mean ± SEM) and analyzed using Prism 8.0 statistical software. P < 0.05 indicated a significant difference. Specific test data are shown in Table 3.

[0091] Table 3: Animal test results of Chinese medicine composition

[0092]

[0093]

[0094] Note: GLEE represents ethanol extract of Ganoderma lucidum spore powder, SVEE represents ethanol extract of Phellinus igniarius, and GSEE represents Chinese medicine composition (Ganoderma lucidum spore powder and ethanol extract of Phellinus igniarius).

[0095] (3) Experimental results

[0096] From the data in Table 3, it can be seen that when the concentration of Ganoderma lucidum spore powder alone was 800 mg / kg, the inhibition rate of nude mouse HCT116 transplanted tumor was 22.60%, indicating that Ganoderma lucidum spore powder alone had a certain anti-tumor effect, but the effect was relatively limited; when the concentration of Phellinus igniarius alone was 800 mg / kg, the inhibition rate of Phellinus igniarius on transplanted tumors reached 44.63%, significantly higher than that of Ganoderma lucidum spore powder, showing stronger anti-tumor activity; when Ganoderma lucidum spore powder and Phellinus igniarius were mixed in equal proportions to form a Chinese medicine composition, and used at a total dosage concentration of 800 mg / kg, the inhibition rate of transplanted tumors reached 64.31%, which was much higher than the inhibition rate when any drug was used alone. This result strongly proves the synergistic effect of Ganoderma lucidum spore powder and Phellinus igniarius in anti-tumor treatment, that is, combined use can significantly improve the therapeutic effect.

[0097] Animal weight changes: The weight changes of nude mice in each medication group before and after medication were not significant. Except for the Chinese medicine combination group with a slightly lower weight growth rate (0.36%), the other groups maintained a relatively stable weight gain, which indicates that the drugs did not cause obvious adverse effects on the physiological state of nude mice during the experiment.

[0098] Toxic side effects evaluation: Although the weight growth rate of the Chinese medicine composition group was the lowest, there was no significant weight loss, and there was no significant difference compared with other groups. Therefore, it is believed that the Chinese medicine composition of the present invention did not produce obvious toxic side effects on experimental animals at the administered concentration.

[0099] In summary, the Chinese medicine composition (GSEE) showed significant anti-tumor effect in the nude mouse HCT116 transplanted tumor model, and its inhibition rate was much higher than that of using Ganoderma lucidum spore powder or Phellinus igniarius alone; the composition had no obvious adverse effects on the physiological state of the experimental animals and did not show obvious toxic side effects. These results provide a strong experimental basis for further research and application of Chinese medicine compositions in anti-tumor treatment.

[0100] 4. Isolation of anti-tumor active fractions from Chinese herbal medicine compositions

[0101] (1) Instruments and Materials

[0102] SW-CJ-1F clean bench (Suzhou Antai Air Technology Co., Ltd.); Ti-U inverted microscope (Nikon); MAC 15AC carbon dioxide constant temperature incubator (SANYO); 80-2 desktop electric centrifuge (Shanghai Analytical Instrument Factory); GH water-proof constant temperature incubator (Beijing Kewei Yongxing Instrument Co., Ltd.); pipette (Eppendorf); UPT-IV-5T ultrapure water preparation machine (Chengdu Ultrapure Technology Co., Ltd.); SB-5200 ultrasonic cleaning machine (Ningbo Xinzhi Biotechnology Co., Ltd.); BP211D electronic balance (Shanghai Guangzheng Medical Instrument Co., Ltd.); cell counting plate (Shanghai Qiujing Biochemical Reagent Co., Ltd.); culture plate (Costar).

[0103] The human colon cancer cells HCT116 and COLO205 used in the experiment were purchased from Air Force Medical University, cultured in a culture medium containing 10% fetal bovine serum (containing 100 U / ml penicillin and streptomycin) in a 37°C, 5% CO2 incubator, and passaged every 2 to 3 days.

[0104] (2) Experimental methods

[0105] Add water to the extract powder of the composition to resuspend, transfer it into a separatory funnel, and then extract it with petroleum ether, chloroform, ethyl acetate and n-butanol in sequence, collect the extracts of the corresponding parts, and spin dry to obtain solids of 5 parts of the Chinese medicine composition. Human colon cancer cells HCT116 and COLO205 were used for screening. Five solvents: petroleum ether, chloroform, ethyl acetate, n-butanol and water were used to extract the 5 parts of the Chinese medicine composition by graded extraction to perform MTT experiments. Among the numbers 1-5, No. 1 is extracted with petroleum ether, No. 2 is extracted with chloroform, No. 3 is extracted with ethyl acetate, No. 4 is extracted with n-butanol, and No. 5 is extracted with water. The results are shown in Table 4.

[0106] Table 4: Inhibition rate of colon cancer cell growth by various parts of the Chinese medicine composition (%)

[0107]

[0108] From the data in Table 4, it can be seen that No. 3 is the ethyl acetate extract of the traditional Chinese medicine composition, which exhibits extremely high growth inhibition rates on both HCT116 and COLO205 colon cancer cell lines, which are 98.88% and 96.10%, respectively, indicating that the ethyl acetate extract is the main anti-tumor active part in the traditional Chinese medicine composition.

[0109] Fractional extraction is a commonly used method for separating chemical components of traditional Chinese medicine, which uses different solvents to separate different components in traditional Chinese medicine based on their solubility differences. In this experiment, the polarity of petroleum ether, chloroform, ethyl acetate and n-butanol gradually increased, and they were able to extract the components with gradually increasing polarity in the traditional Chinese medicine composition in turn. Ethyl acetate has moderate polarity and can extract many biologically active medium-polar compounds. In this experiment, petroleum ether, chloroform, n-butanol and water extracts also showed a certain inhibitory effect on the growth of colon cancer cells, but the activity of these extracts was significantly lower than that of ethyl acetate extracts. The ethyl acetate extract showed significant anti-tumor activity, indicating that the extract is rich in compounds with anti-tumor effects.

[0110] Through this experiment, it was found that the part with the strongest anti-tumor activity in the Chinese medicine composition of the present invention is the ethyl acetate part, and its extraction process was optimized. This provides an important experimental basis and reference for further separation and purification of anti-tumor active ingredients and development of new anti-tumor drugs.

[0111] 5. Ethyl acetate nude mouse HCT116 transplant tumor model experiment

[0112] (1) Instruments and Materials

[0113] Ti-U inverted microscope (Nikon); 80-2 tabletop electric centrifuge (Shanghai Analytical Instrument Factory); pipette (Eppendorf); UPT-IV-5T ultrapure water preparation machine (Chengdu Ultrapure Technology Co., Ltd.); BP211D electronic balance (Shanghai Guangzheng Medical Instrument Co., Ltd.); cell counting plate (Shanghai Qiujing Biochemical Reagent Co., Ltd.); disposable syringe (Jiangxi Jinshan Medical Instrument Co., Ltd.); vernier caliper (Wuxi Xigong Measuring Tool Co., Ltd.); surgical scissors (Shanghai Medical Instrument Co., Ltd.); ultra-low temperature refrigerator (Qingdao Haier Group); cell factory culture system (NUNC).

[0114] Human colon cancer cell line HCT-116 was purchased from Air Force Medical University.

[0115] Healthy BALB / c male nude mice, 18-22 g, were provided by the Experimental Animal Center of Xi'an Jiaotong University. They were given free access to food and water and were cultured in an SPF animal room at 22-24°C, 50%-70%, and a 12h-12h light-dark cycle.

[0116] (2) Experimental methods

[0117] The inhibitory effect of the ethyl acetate part on the nude mouse HCT116 transplanted tumor model was detected using the nude mouse HCT116 transplanted tumor model.

[0118] Prepared HCT116 cells at 1x10 7 The cells were resuspended in sterile saline at a density of 100 μg / mL, and 200 μL of the cell suspension was injected subcutaneously into the right armpit of each nude mouse.

[0119] After inoculation, the nude mice were closely observed for tumor formation and the size of the transplanted tumors was recorded with a vernier caliper. When the average tumor volume reached 100 mm 3 The nude mice were then randomly divided into groups of 6 in each group, and the drugs were administered according to the concentration gradient designed according to the median lethal dose for 21 days. The mice were killed 21 days later, the tumors were removed and weighed, and the inhibition rate of the ethyl acetate site on the growth of nude mouse HCT116 transplanted tumors was calculated.

[0120] The calculation formula of tumor inhibition rate is as follows:

[0121]

[0122] The experimental data were expressed as mean ± standard error (Mean ± SEM) and analyzed using Prism 8.0 statistical software. P < 0.05 indicated a significant difference. Specific data are shown in Table 5.

[0123] Table 5: Animal test results of ethyl acetate

[0124]

[0125] Note: GSEAE stands for ethyl acetate moiety.

[0126] As can be seen from the data in Table 5, the tumor inhibition effect of the ethyl acetate site showed a significant dose dependence, and the tumor inhibition rate gradually increased with the increase of the administration concentration. When the ethyl acetate site administration concentration was 200 mg / kg, the inhibition rate of the nude mouse HCT116 transplanted tumor model was 18.50%; when the ethyl acetate site administration concentration was 400 mg / kg, the inhibition rate of the nude mouse HCT116 transplanted tumor model was 44.02%; and when the ethyl acetate site administration concentration was 800 mg / kg, the inhibition rate could reach 73.15%, indicating that the ethyl acetate site at this concentration has extremely strong anti-tumor activity.

[0127] Toxic side effects: During the administration process, there was no significant difference in the average body weight of all the administration groups compared with the negative control group, indicating that the ethyl acetate part effectively inhibited tumor growth without causing obvious negative effects on the physiological state of the experimental animals.

[0128] In summary, the ethyl acetate fraction showed significant antitumor activity in the nude mouse HCT116 transplant tumor model, and its tumor inhibition effect was dose-dependent. At the same time, this fraction had no obvious toxic side effects on experimental animals, indicating that it has good safety and application prospects. Therefore, the ethyl acetate fraction is expected to become one of the important candidate ingredients for the development of new antitumor drugs.

[0129] 6. Experiment on Lewis lung cancer transplanted tumor model in C57BL / 6 mice with Chinese medicine composition and ethyl acetate

[0130] (1) Instruments and Materials

[0131] Ti-U inverted microscope (Nikon); 80-2 tabletop electric centrifuge (Shanghai Analytical Instrument Factory); pipette (Eppendorf); UPT-IV-5T ultrapure water preparation machine (Chengdu Ultrapure Technology Co., Ltd.); BP211D electronic balance (Shanghai Guangzheng Medical Instrument Co., Ltd.); cell counting plate (Shanghai Qiujing Biochemical Reagent Co., Ltd.); disposable syringe (Jiangxi Jinshan Medical Instrument Co., Ltd.); vernier caliper (Wuxi Xigong Measuring Tool Co., Ltd.); surgical scissors (Shanghai Medical Instrument Co., Ltd.); ultra-low temperature refrigerator (Qingdao Haier Group); cell factory culture system (NUNC).

[0132] Mouse Lewis lung cancer cell line was purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences.

[0133] Healthy C57BL / 6 male mice, 18-22 g, were provided by the Experimental Animal Center of Xi'an Jiaotong University. They were given free access to food and water and were cultured in a clean animal room at 22-24°C, 50%-70%, and a 12h-12h light-dark cycle.

[0134] (2) Experimental methods

[0135] The inhibitory effect of the Chinese medicine composition and the ethyl acetate fraction on the Lewis lung cancer transplanted tumor model in C57BL / 6 mice was detected by using the Lewis lung cancer transplanted tumor model in C57BL / 6 mice.

[0136] Lewis lung cancer cells were prepared at 1x10 7 The cells were resuspended in sterile saline at a density of 100 μg / mL, and 200 μL of the cell suspension was injected subcutaneously into the right armpit of each C57BL / 6 mouse.

[0137] After inoculation, the tumor formation of mice was closely observed and the size of transplanted tumors was recorded with a vernier caliper. When the average tumor volume reached 100 mm 3The mice were then randomly divided into groups of 6 in each group and administered according to the designed concentration of the median lethal dose for 10 days. The mice were killed 10 days later, the tumors were removed and weighed, and the inhibition rate of the Chinese medicine composition and the ethyl acetate fraction on the growth of Lewis lung cancer transplanted tumors in C57BL / 6 mice was calculated.

[0138] The calculation formula of tumor inhibition rate is as follows:

[0139]

[0140] The experimental data were expressed as mean ± standard error (Mean ± SEM) and analyzed using Prism 8.0 statistical software. P < 0.05 indicated a significant difference. Specific data are shown in Table 6.

[0141] Table 6: Animal test results of Chinese medicine composition and ethyl acetate part

[0142]

[0143] As shown in Table 6, both the Chinese medicine composition and its ethyl acetate fraction significantly inhibited the growth of Lewis lung cancer transplanted tumors in C57BL / 6 mice at a concentration of 800 mg / kg. When the concentration of Ganoderma lucidum spore powder alone was 800 mg / kg, the inhibition rate of Lewis lung cancer transplanted tumor model in C57BL / 6 mice was 26.69%; when the concentration of Phellinus igniarius alone was 800 mg / kg, the inhibition rate of Lewis lung cancer transplanted tumor model in C57BL / 6 mice was 40.00%; when the concentration of the Chinese medicine composition was 800 mg / kg, the inhibition rate was 53.33%, which was close to the tumor inhibition rate of the positive control drug gefitinib; and when the concentration of the ethyl acetate fraction was 800 mg / kg, the inhibition rate could reach 68.93%, which was close to the tumor inhibition rate of the positive control drug cisplatin, indicating that it has strong anti-tumor activity.

[0144] Synergistic effect: The inhibition rate of the Chinese medicine composition (GLEE) at the same administration concentration was 53.33%, which was higher than the inhibition rates of Ganoderma lucidum spore powder (GLEE) and Phellinus igniarius (SVEE) when administered alone (26.69% and 40.00%, respectively), indicating that the multiple components in the Chinese medicine composition may have a synergistic effect and jointly enhance the anti-tumor effect. The inhibition rate of the ethyl acetate fraction (GSEAE) at the same administration concentration (68.93%) was higher than that of the Chinese medicine composition (53.33%), indicating that the ethyl acetate fraction is one of the main contributors to the anti-tumor activity in the Chinese medicine composition, which further emphasizes the potential of the ethyl acetate fraction in the development of new anti-tumor drugs.

[0145] Safety assessment: Compared with the positive control drug cisplatin, the weight of mice in the group receiving the Chinese medicine composition and ethyl acetate did not decrease significantly. While the Chinese medicine composition and ethyl acetate effectively inhibited tumor growth, they did not have a significant negative impact on the physiological state of mice, indicating that they have better safety.

[0146] In summary, the Chinese medicine composition of the present invention and its ethyl acetate fraction showed significant antitumor activity in the Lewis lung cancer transplanted tumor model of C57BL / 6 mice, and the inhibitory effect of the ethyl acetate fraction was more prominent. At the same time, they are highly safe for experimental animals and do not show obvious toxic and side effects. Therefore, the Chinese medicine composition and its ethyl acetate fraction are expected to become important candidate ingredients for the development of new antitumor drugs.

[0147] When used specifically, the Chinese medicine composition and the ethyl acetate fraction of the present invention can be used as medicinal ingredients, and auxiliary materials can be added to prepare them into tablets, granules, capsules, powders or injections.

[0148] The present invention finds that the Chinese medicinal composition of Ganoderma lucidum spore powder and Phellinus igniarius and the ethyl acetate fraction thereof have good effects in treating cancers such as colon cancer, liver cancer, breast cancer, cervical cancer, lung cancer, etc. The Chinese medicinal composition and the ethyl acetate fraction provided by the present invention can be used to prepare drugs for treating cancers such as colon cancer, liver cancer, breast cancer, cervical cancer, lung cancer, etc., and this finding has not been reported so far. In addition, the present invention has conducted a large number of experiments, aiming to maximize the therapeutic effect through the combination of drugs, and the large amount of experimental data in the present invention provides an experimental basis for preparing a formula preparation of Ganoderma lucidum spore powder and Phellinus igniarius that inhibits the growth of colon cancer.

[0149] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. An anti-tumor Chinese medicine composition, characterized in that: The traditional Chinese medicine composition consists of 1 to 3 parts of ganoderma lucidum spore powder and 1 to 3 parts of mulberry linterus by weight.

2. The method for extracting the anti-tumor Chinese medicine composition according to claim 1, characterized in that: include: (1) weighing Ganoderma lucidum spore powder and Phellinus linteus, mixing them, adding ethanol, heating and ultrasonic-assisted extraction, filtering, concentrating and drying under reduced pressure to obtain an ethanol extract of the traditional Chinese medicine composition; (2) resuspending the ethanol extract of the Chinese medicine composition in step (1) in distilled water, performing fractional extraction, and concentrating the extract to obtain an extract concentrate of the Chinese medicine composition.

3. The method for extracting the anti-tumor Chinese medicine composition according to claim 2, characterized in that: The amount of ethanol added is 20 to 30 times the mass of the raw material, and the volume fraction of ethanol is 70%.

4. The method for extracting the anti-tumor Chinese medicine composition according to claim 2, characterized in that: The mass ratio of the ganoderma lucidum spore powder to mulberry linterus is 1-3:1-3.

5. The method for extracting the anti-tumor Chinese medicine composition according to claim 2, characterized in that: The conditions for the heating ultrasonic assisted extraction are: the heating temperature is 36° C. to 38° C., and the ultrasonic extraction time is 1 h to 2 h.

6. The method for extracting the anti-tumor Chinese medicine composition according to claim 2, characterized in that: The distilled water is added for re-suspending, and the amount of distilled water added is 40 to 60 times the mass of the ethanol extract of the traditional Chinese medicine composition.

7. The method for extracting the anti-tumor Chinese medicine composition according to claim 2, characterized in that: The graded extraction solvent is any one of petroleum ether, chloroform, ethyl acetate and n-butanol, the added amount of the extraction solvent is 1:1 in volume ratio with distilled water, and the number of extractions is 2 to 4 times.

8. Use of the anti-tumor Chinese medicine composition according to claim 1 in the preparation of anti-tumor drugs, characterized in that: The anti-tumor drug is a drug for treating colon cancer, liver cancer, breast cancer, cervical cancer and lung cancer.

9. The use of the anti-tumor Chinese medicine composition according to claim 8 in the preparation of anti-tumor drugs, characterized in that: The anti-tumor drug may further include one or more pharmaceutically acceptable excipients.

10. Use of the anti-tumor Chinese medicine composition according to claim 8 in the preparation of anti-tumor drugs, characterized in that: The anti-tumor drug is in the form of capsules, gels, creams, mixtures, tablets, powders, pills, granules, wet compresses, solutions, syrups, suppositories, decoctions, aerosols, ointments, plasters, injections, sprays, tinctures, liniments, pastes or lotions.

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