Preparation method and application of spanishneedles herb anti-tumor active extract
By preparing and purifying the anti-tumor active extract of the Ghost Cymbidium, the problems of poor selectivity and toxic side effects of existing anti-tumor drugs were solved, and by inhibiting the transformation of macrophages into M2 macrophages, tumor growth was significantly inhibited, achieving safer and more effective anti-tumor effects.
Patent Information
- Application Number
- CN202311421666.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-10-30
- Publication Date
- 2025-05-02
AI Technical Summary
Existing anti-tumor drugs have problems such as poor selectivity, great toxic and side effects, limited use of patients and prone to drug resistance. Tumor-related macrophages (TAMs), especially M2 macrophages, play an important role in tumor progression, and the prior art is difficult to effectively inhibit their generation or transformation.
The anti-tumor active extract of Germine Cyclops was prepared by methanol extraction, petroleum ether, ethyl acetate extraction, silica gel column chromatography, MCI column chromatography and Sephadex LH-20 gel column chromatography, and it was found that the extract could inhibit the transformation of macrophages to M2 macrophages.
The anti-tumor active extract of the genus cerevisiae has cytotoxic activity on a variety of tumor cells and can significantly inhibit the growth of transplanted tumors in mice. Its anti-tumor effect is significantly better than that of 5-fluorouracil alone, and has the potential for anti-tumor immunomodulation.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of medicines, and particularly relates to a preparation method of a Bidens pilosa L. anti-tumor active extract and application thereof. Background Art
[0003] At present, the sources of anti-tumor drugs mainly include metabolites of plants, animals, microorganisms, minerals and marine organisms, as well as their artificial synthesis and artificial modification products. According to their different action characteristics, the anti-tumor drugs currently used in clinical practice mainly include: tumor cytotoxic anti-tumor drugs, molecular targeted anti-tumor drugs and tumor immunotherapy drugs, which have different clinical uses. Among them, traditional tumor cytotoxic drugs are prone to cause serious adverse reactions due to poor selectivity. Although molecular targeted drugs have certain selectivity and reduced toxic side effects, they also have disadvantages such as limited use by patients and easy development of drug resistance. Tumor immunotherapy is called the fourth tumor treatment method after surgery, chemotherapy and radiotherapy. It can activate specific immune cells and directly target cancer cells. It has high efficacy and safety. It is currently a hot spot in global tumor treatment research. Therefore, tumor immunotherapy drugs are also a hot spot in the field of anti-tumor drug research and development.
[0004] Tumor-associated macrophages (TAMs) are macrophages infiltrating in tumor tissues. They are the most numerous immune cells in the tumor microenvironment, accounting for more than half of the total number of tumor interstitial immune cells. They play a very important role in the occurrence and development, invasion and metastasis, immune escape, and angiogenesis and lymphangiogenesis of malignant tumors. They are important target cells for tumor immunotherapy and anti-tumor metastasis therapy. During the occurrence and development of tumors, mononuclear macrophages in peripheral blood are recruited into tumor tissues and become tumor-associated macrophages. Macrophages in the tumor microenvironment have strong plasticity. Under the stimulation of a variety of different cytokines, inflammatory factors and chemokines in the tumor microenvironment, they can develop into two phenotypes, namely M1 (classical activation type) and M2 (alternative activation type) macrophages. Tumor-associated macrophages of different activation phenotypes play completely different roles. After being activated, M1 TAMs secrete high levels of TNF-α, IL-6, IL-12, and IL-23, etc., and induce the production of iNOS to generate NO and reactive oxygen species. This type of TAMs also has an antigen-presenting function, which initiates a Th1 immune response and kills tumor cells, thereby inhibiting the occurrence of tumors. After being activated, M2 TAMs can produce a large number of anti-inflammatory factors, arginase 1 (ARG1), and indoleamine 2,3-dioxygenase (IDO). At the same time, they also highly express mannose receptors CD204 / 206, scavenger receptors CD163, and cytokines L-10, TGF-β, etc., inducing Th2 immune responses, clearing residues, promoting the formation of blood vessels and lymphatic vessels, participating in tissue remodeling and repair, causing tumor cell proliferation, invasion, and metastasis, and ultimately promoting tumor progression. Therefore, inhibiting the generation of M2 macrophages in the tumor microenvironment or promoting their transformation into M1 macrophages has become one of the strategies for anti-tumor immunotherapy.
[0005] In the field of anticancer drugs, Chinese herbal medicine has played an important role. Natural products from plants play an important role and dominate the current anti-tumor drug market. For example, the anti-tumor drugs used in clinical practice, such as paclitaxel, camptothecin, vinblastine and podophyllotoxin, are all derived from traditional Chinese herbal medicine. Therefore, the anti-tumor effect of Chinese herbal medicine is increasingly valued by people. Bidens Pilosa L. is an annual herbaceous plant of the genus Bidens in the family Asteraceae. It is a commonly used folk medicine with the effects of removing dampness and wind, dispersing blood stasis and reducing swelling. It is mainly used to treat abdominal pain, diarrhea, gastroenteritis, acute appendicitis, sore throat, sprains and other symptoms. Modern pharmacological and clinical studies have shown that Bidens Pilosa has the effects of sterilization, lowering blood pressure, anti-inflammatory and analgesic, protecting the liver, protecting brain damage, treating gout, protecting myocardial ischemia, and lowering blood sugar.
[0006] The present invention aims to provide a safe and effective Bidens pilosa L. extract with anti-tumor activity, so as to provide more choices for drugs or functional foods for preventing and treating tumors. Summary of the invention
[0007] In view of the problems existing in the prior art, the present invention provides a preparation method of Bidens pilosa L. extract and application thereof.
[0008] The present invention is achieved through the following technical solutions:
[0009] In one aspect, the present invention provides a method for preparing an extract of Bidens pilosa L. with anti-tumor activity, comprising the following steps:
[0010] (1) adding a methanol solvent to the Bidens pilosa plant raw material to obtain an extract, and concentrating the extract under reduced pressure to obtain an extract;
[0011] (2) adding water to the extract obtained in step 1 to disperse it to a relative density of 1.05 to 1.20 (measured value at room temperature) to obtain an aqueous dispersion, adding petroleum ether for extraction, collecting the aqueous phase, and then extracting with ethyl acetate, collecting the ethyl acetate portion, and concentrating under reduced pressure to obtain an ethyl acetate extract;
[0012] (3) separating and purifying the ethyl acetate extract obtained in step 2 by silica gel column chromatography to obtain column chromatography sample I;
[0013] (4) separating and purifying the column chromatography sample I obtained in step 3 by MCI column chromatography to obtain a column chromatography sample II;
[0014] (5) The column chromatography sample II obtained in step 4 is separated and purified by Sephadex LH-20 gel column chromatography to obtain the Bidens pilosa L. anti-tumor active extract.
[0015] As a preferred embodiment of the present invention, the Bidens pilosa plant raw material in step 1 of the present invention is the above-ground part of Bidens pilosa, which is any one of its leaves, stems and flowers, or a combination of at least two of them.
[0016] Preferably, the mass-to-volume ratio of the Bidens pilosa plant raw material to the solvent in the extraction in step 1 is 1:3-10, expressed in g / ml. The mass-to-volume ratio of the raw material to the solvent refers to the ratio of the mass of the raw material to the volume of the solvent. For example, 3-8 ml of solvent is used for extraction per gram of raw material.
[0017] Preferably, the extraction time in step 1 is 1-2 days and the number of times is 2-4 times.
[0018] Preferably, the concentration of the methanol solvent in step 1 is 85%-100%.
[0019] Preferably, in step 1, the reduced pressure concentration is concentrated until there is no organic solvent smell.
[0020] Preferably, in step 2, in the petroleum ether extraction, the volume ratio of the aqueous dispersion to the petroleum ether is 1:1 to 3, more preferably 1:1.
[0021] Preferably, in step 2, in the ethyl acetate extraction, the volume ratio of the aqueous phase to ethyl acetate is 1:1 to 3, more preferably 1:1.
[0022] Preferably, in step 3, the silica gel column filler is silica gel with a particle size of 60-100 mesh, the mass ratio of the ethyl acetate extract to the silica gel filler is 1:15-20, and the height-to-diameter ratio of the silica gel column is 3-4:1.
[0023] Preferably, the silica gel column chromatography separation and purification elution method in step 3 is: first elute with petroleum ether for 5 to 7 column volumes to remove the small polar part, and then elute with elution solvents with a dichloromethane-methanol volume ratio of 1:0, 50:1, 25:1, 10:1, 1:1, and 0:1 in sequence, with each gradient elution being 7 to 10 column volumes, collecting the part with a dichloromethane-methanol volume ratio of 10:1, and concentrating and drying under reduced pressure to obtain the column chromatography sample I.
[0024] Preferably, in the MCI column chromatography in step 4, the mass ratio of the column chromatography sample I to the MCI filler is 1:13-17; the height-to-diameter ratio of the chromatography column is 9-10:1, and the elution method is: the elution flow rate is 20-30 mL / min, 9-11 column volumes are eluted with 50% methanol-water solvent, all the eluent is collected, and the column chromatography sample II is obtained by concentrating and drying under reduced pressure.
[0025] Preferably, in the Sephadex LH-20 gel column chromatography in step 5, the mass ratio of the column chromatography sample II to the gel filler is 1:25-100, the height-to-diameter ratio of the gel column is 50-100:1, the elution solvent is methanol, the elution flow rate is 3-5 seconds / drop, the eluate from 220 minutes to 450 minutes is collected, and the Bidens pilosa anti-tumor active extract is obtained by concentrating and drying under reduced pressure.
[0026] The temperature of the reduced pressure concentration in the above steps of the present invention is 50-65°C, such as 50°C, 55°C, 60°C or 65°C, but is not limited to the listed values, and other values not listed within the numerical range are also applicable; the vacuum degree of the reduced pressure concentration is -0.08 to -0.1Mpa, such as -0.08Mpa, -0.085Mpa, -0.09Mpa or -0.095Mpa, but is not limited to the listed values, and other values not listed within the numerical range are also applicable.
[0027] On the other hand, the present invention provides the use of the Bidens pilosa L. anti-tumor active extract prepared by the above method in the preparation of anti-tumor drugs.
[0028] Preferably, the tumor is liver cancer, breast cancer, colon cancer and lung cancer.
[0029] Preferably, the dosage form of the drug includes any one of tablets, capsules, solutions, aerosols, sprays, ointments or films.
[0030] Preferably, the drug further comprises a pharmaceutically acceptable excipient.
[0031] Preferably, the auxiliary materials include any one of diluents, flavoring agents, adhesives or fillers, or a combination of at least two of them. The combination of at least two of them can be, for example, a combination of an adhesive and a diluent, a combination of an adhesive and a flavoring agent, a combination of an adhesive and a filler, etc. Any other combination can be selected and will not be described in detail here.
[0032] On the other hand, the present invention provides the use of the Bidens pilosa L. anti-tumor active extract prepared by the above method in the preparation of tumor immunotherapy drugs.
[0033] On the other hand, the present invention provides the use of the Bidens pilosa anti-tumor active extract prepared by the above method and 5-fluorouracil in the preparation of anti-tumor drugs.
[0034] The present invention unexpectedly discovered that the combination of the Bidens pilosa anti-tumor active extract and 5-fluorouracil can significantly inhibit the growth of mouse transplanted tumors, and its anti-tumor effect is significantly better than that of 5-fluorouracil alone.
[0035] Finally, the present invention provides a pharmaceutical composition, which comprises the Bidens pilosa L. anti-tumor active extract prepared by the above method and 5-fluorouracil, as well as a pharmaceutical carrier suitable for preparing anti-tumor drugs.
[0036] Beneficial effects of the present invention:
[0037] The present invention uses Bidens pilosa as a research object, and prepares a Bidens pilosa anti-tumor active extract through the steps of methanol extraction, petroleum ether, ethyl acetate extraction, silica gel column chromatography, MCI column chromatography and Sephadex LH-20 gel column chromatography separation and purification. The preparation method has good controllability and reproducibility, less sample loss, low cost, convenient operation, and the solvent can be repeatedly recycled and reused, and is suitable for industrial production. And through pharmacological experiments, it is found that the Bidens pilosa anti-tumor active extract has good cytotoxic activity on tumor cells such as human liver cancer cells HepG2, human breast cancer cells MDA-MB-231, human colon cancer cells DLD-1, SW480 and HCT116, and human lung cancer cells A549, and can be used to prepare anti-tumor drugs. It is also unexpectedly found that the Bidens pilosa anti-tumor active extract can inhibit the transformation of macrophages into M2 macrophages with a cancer-promoting effect, has the potential for anti-tumor immune regulation, and can be used to prepare tumor immunotherapy drugs. It was also found that the combination of the Bidens pilosa anti-tumor active extract and 5-fluorouracil can significantly inhibit the growth of mouse transplanted tumors, and its anti-tumor effect is significantly better than that of 5-fluorouracil alone. BRIEF DESCRIPTION OF THE DRAWINGS
[0038] Figure 1 Effects of Bidens pilosa L. extract with antitumor activity on IL-4 and IL-13-induced macrophage differentiation.
[0039] Figure 2 Antitumor active extract of Bidens pilosa L. inhibits the growth of colon cancer cell transplanted tumors in mice.
[0040] Figure 3 Tumor growth curves of mice in different drug groups.
[0041] Figure 4 Tumor weights of mice in different drug groups after administration. Specific embodiments
[0042] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.
[0043] The process, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art, and the present invention has no particular restrictions. The experimental methods without specifying specific conditions in the embodiments are usually carried out under conventional conditions or under conditions recommended by the manufacturer.
[0044] Unless otherwise specified, all technical terms and scientific terms used in this specification have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. However, in case of any conflict, the present specification including the definitions shall prevail.
[0045] The Bidens pilosa sample was collected from Liangwang Mountain, Kunming City, Yunnan Province in June 2020, and was identified as the whole herb of Bidens pilosa L. by Yunnan Zhifen Biotechnology Co., Ltd. (Zhang Jun).
[0046] Human liver cancer cells HepG2, human breast cancer cells MDA-MB-231, human lung cancer cells A549, and human colon cancer cells SW480 were from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, and human colon cancer cells DLD-1 and HCT116 were from Guangzhou Saiku Biotechnology Co., Ltd.
[0047] Mouse macrophage Raw264.7 was obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences.
[0048] Balb / c male mice were purchased from Henan Sikebes Biotechnology Co., Ltd.
[0049] Preparation Example 1 Preparation of Bidens pilosa L. anti-tumor active extract
[0050] (1) Take 800 g of Bidens pilosa, add 8 L of 85% methanol for cold soaking extraction, the extraction time is 1 day, the extraction is performed 4 times, the extracts are combined, and the solvent is recovered by vacuum concentration to obtain 183 g of extract.
[0051] (2) The extract obtained in step (1) was dispersed in water to a relative density of 1.09 (measured at 27° C.) to obtain an aqueous dispersion, and 1 volume of petroleum ether was added for extraction and defatting. The aqueous phase was collected and extracted with 3 volumes of ethyl acetate for 1 time. The ethyl acetate portions were combined and concentrated under reduced pressure to obtain an ethyl acetate extract (16 g);
[0052] (3) The ethyl acetate extract obtained in step (2) was separated and purified by silica gel column chromatography (the mass ratio of ethyl acetate extract to silica gel filler was 1:15, the model was 60-100 mesh, and the column height-to-diameter ratio was 3:1), first eluted with petroleum ether for 5 column volumes to remove the small polar part, and then eluted with elution solvents with a dichloromethane-methanol volume ratio of 1:0, 50:1, 25:1, 10:1, 1:1, and 0:1, each gradient elution was 10 column volumes, and the part with a dichloromethane-methanol volume ratio of 10:1 was collected, and the solvent was recovered by vacuum concentration to obtain 5.4 g of column chromatography sample I;
[0053] (4) The column chromatography sample I obtained in step 3 was separated and purified by MCI column chromatography (the mass ratio of column chromatography sample I to MCI was 1:17, model MCI GEL CHP20 / P120, column height-to-diameter ratio was 9:1), the elution method was: elution flow rate was 20-30 mL / min, elution was performed with 50% methanol-water solvent for 9-11 column volumes, all the eluate was collected, concentrated and dried under reduced pressure, and 3.46 g of the column chromatography sample II was obtained.
[0054] (5) The column chromatography sample II obtained in step 4 was separated and purified by Sephadex LH-20 gel column chromatography (the mass ratio of sample II to gel filler was 1:100, and the height-to-diameter ratio of the gel column was 100:1), eluted with methanol solvent at a flow rate of 3-5 seconds / drop, and the liquid was collected for 220 to 450 minutes. The liquid was concentrated and dried under reduced pressure to obtain 2.97 g of the Bidens pilosa L. anti-tumor active extract.
[0055] Preparation Example 2 Preparation of Bidens pilosa L. Antitumor Active Extract
[0056] (1) Take 800 g of Bidens pilosa, add 5 L of 90% methanol for cold soaking extraction, extract for 2 days, extract 3 times, combine the extracts, concentrate under reduced pressure to recover the solvent, and obtain 187 g of extract.
[0057] (2) The extract obtained in step (1) was dispersed in water to a relative density of 1.10 (measured at 27° C.) to obtain an aqueous dispersion, 3 volumes of petroleum ether were added for extraction and defatting, the aqueous phase was collected, and then extracted with 1 volume of ethyl acetate for 3 times, the ethyl acetate portions were combined, and concentrated under reduced pressure to obtain an ethyl acetate extract (16.3 g);
[0058] (3) The ethyl acetate extract obtained in step (2) was separated and purified by silica gel column chromatography (the mass ratio of ethyl acetate extract to silica gel filler was 1:13, the model was 60-100 mesh, and the column height-to-diameter ratio was 4:1), firstly eluted with petroleum ether for 7 column volumes to remove the small polar part, and then eluted with elution solvents with a dichloromethane-methanol volume ratio of 1:0, 50:1, 25:1, 10:1, 1:1, and 0:1, each gradient elution was 10 column volumes, and the part with a dichloromethane-methanol volume ratio of 10:1 was collected, and the solvent was recovered by vacuum concentration to obtain 5.47 g of column chromatography sample I;
[0059] (4) The column chromatography sample I obtained in step 3 was separated and purified by MCI column chromatography (the mass ratio of column chromatography sample I to MCI was 1:15, model MCI GEL CHP20 / P120, column height-to-diameter ratio was 10:1), the elution method was: elution flow rate was 20-30 mL / min, elution was performed with 50% methanol-water solvent for 11 column volumes, all the eluents were collected, concentrated and dried under reduced pressure, and 3.56 g of the column chromatography sample II was obtained.
[0060] (5) The column chromatography sample II obtained in step 4 was separated and purified by Sephadex LH-20 gel column chromatography (the mass ratio of sample II to gel filler was 1:75, and the height-to-diameter ratio of the gel column was 75:1), eluted with methanol solvent at a flow rate of 3-5 seconds / drop, and the liquid was collected for 220 to 450 minutes. The liquid was concentrated and dried under reduced pressure to obtain 3.03 g of the Bidens pilosa L. anti-tumor active extract.
[0061] Preparation Example 3 Preparation of Bidens pilosa L. Antitumor Active Extract
[0062] (1) Take 800 g of Bidens pilosa, add 2.4 L of 100% methanol for cold soaking extraction, extract for 2 days, extract 4 times, combine the extracts, concentrate under reduced pressure to recover the solvent, and obtain 181 g of extract.
[0063] (2) The extract obtained in step (1) was dispersed in water to a relative density of 1.08 (measured at 28° C.) to obtain an aqueous dispersion, and 2 volumes of petroleum ether were added for extraction and defatting. The aqueous phase was collected and extracted with 2 volumes of ethyl acetate for 2 times. The ethyl acetate portions were combined and concentrated under reduced pressure to obtain an ethyl acetate extract (15.5 g);
[0064] (3) The ethyl acetate extract obtained in step (2) was separated and purified by silica gel column chromatography (the mass ratio of ethyl acetate extract to silica gel filler was 1:15, the model was 60-100 mesh, and the column height-to-diameter ratio was 4:1), firstly eluted with petroleum ether for 7 column volumes to remove the small polar part, and then eluted with elution solvents with a dichloromethane-methanol volume ratio of 1:0, 50:1, 25:1, 10:1, 1:1, and 0:1, each gradient elution was 10 column volumes, and the part with a dichloromethane-methanol volume ratio of 10:1 was collected, and the solvent was recovered by vacuum concentration to obtain 5.40 g of column chromatography sample I;
[0065] (4) The column chromatography sample I obtained in step 3 was separated and purified by MCI column chromatography (the mass ratio of column chromatography sample I to MCI was 1:15, model MCI GEL CHP20 / P120, column height-to-diameter ratio was 10:1), the elution method was: elution flow rate was 20-30 mL / min, elution was performed with 50% methanol-water solvent for 11 column volumes, all the eluents were collected, concentrated and dried under reduced pressure, and 3.53 g of the column chromatography sample II was obtained.
[0066] (5) The column chromatography sample II obtained in step 4 was separated and purified by Sephadex LH-20 gel column chromatography (the mass ratio of sample II to gel filler was 1:75, and the height-to-diameter ratio of the gel column was 75:1), eluted with methanol solvent at a flow rate of 3-5 seconds / drop, and the liquid was collected for 220 to 450 minutes. The liquid was concentrated and dried under reduced pressure to obtain 2.96 g of the Bidens pilosa L. anti-tumor active extract.
[0067] Comparative Preparation Example 1
[0068] Steps 1-3 are the same as in Example 1. The sample obtained in step 3 is directly separated and purified on a Sephadex LH-20 gel column to obtain comparative sample 1.
[0069] Comparative Preparation Example 2
[0070] Steps 1-4 are the same as in Example 1, except that in step 5 the gel filler is replaced with Sephadex G-120 to obtain comparative sample 2
[0071] Experimental Example 1 Tumor Cytotoxic Activity Detection
[0072] Experimental principle: After the drug and cells are co-cultured, the survival rate of tumor cells is detected. Sulforhodamine B (SRB) is a water-soluble protein dye. The sulfonic acid anion in its molecule combines with the basic amino acids of intracellular proteins in a weakly acidic environment. The SRB in the cells is dissolved with an alkaline solution and its absorbance value is detected. The protein content in the cells can be known from the SRB content, which represents the cell survival rate.
[0073] Experimental samples: methanol total extract of Bidens pilosa (BPT1), petroleum ether extraction fraction of the total extract (BPE1) and ethyl acetate extraction fraction (BPE2), samples prepared in Preparation Examples 1-3 and Comparative Preparation Examples 1-2, respectively prepared into samples with a concentration of 50 mg / mL using dimethyl sulfoxide (DMSO) solvent; the positive control was camptothecin, which was prepared into a sample with a concentration of 10 mmol / L using DMSO solvent.
[0074] Experimental method: Human liver cancer cell HepG2, human breast cancer cell MDA-MB-231, human colon cancer cell DLD-1, SW480 and HCT116, and human lung cancer cell A549 were prepared into single cell suspension in culture medium containing 10% fetal bovine serum. 3 Cells were inoculated into 96-well plates. After the cells adhered to the wall, different concentrations of the sample solution were added. After 48 hours of incubation, the cells were fixed with pre-cooled trichloroacetic acid solution. Then, the living cells were stained with SRB solution. Finally, Tris solution was added to dissolve SRB and the absorbance was detected at 520nm. The results were recorded, and a curve was drawn with concentration as the horizontal axis and cell survival rate as the vertical axis. The IC value of the compound was calculated using the Reed and Muench method. 50 value.
[0075] The experimental results are shown in Table 1: The results show that compared with the Bidens pilosa methanol total extract (BPT1), the petroleum ether extraction fraction of the total extract (BPE1) and the ethyl acetate extraction fraction (BPE2), the Bidens pilosa anti-tumor active extract of the present invention significantly enhances the tumor cell toxicity of six types of tumor cells.
[0076] Table 1 Cytotoxic effects of different extracts from Bidens pilosa L. on tumor cells.
[0077]
[0078] Experimental Example 2 Detection of the immunoregulatory activity of tumor-associated macrophages
[0079] Experimental principle: RT-PCR is used to detect the expression of tumor-associated macrophage biomarkers induced by cytokines in samples, so as to examine the regulatory effect of samples on the generation of different types of tumor-associated macrophages.
[0080] Experimental sample: The anti-tumor active extract of Bidens pilosa (BPA) prepared in Example 1 was dissolved in DMSO solvent to prepare a sample solution with a concentration of 50 mg / ml.
[0081] Experimental method: Mouse macrophage Raw264.7 cells were prepared into single cell suspension in culture medium containing 10% fetal bovine serum, with 5×10 4 Cells were inoculated into 12-well plates. After the cells adhered to the wall, the sample solutions with concentrations of 50, 10, and 2 μg / mL were added. After 1 hour, 20 ng / mL of IL-4 and IL-13 were added respectively and cultured for 72 hours. Then the supernatant was discarded, 350 μL of Trizol was added to each well and placed on ice to lyse the cells. The cell lysate was transferred to a centrifuge tube, 100 μL of chloroform was added to each tube, and the mixture was placed on ice for 10 minutes and then centrifuged at 4°C, 12000 rpm for 10 minutes. 200 μL of the upper aqueous phase was aspirated into another centrifuge tube, 200 μL of isopropanol was added, and the mixture was gently mixed and placed on ice for 10 minutes to precipitate RNA. After centrifugation at 4°C, 12000 rpm for 10 minutes, the supernatant was discarded, 350 μL of 75% ethanol containing DEPC was added, and the mixture was thoroughly washed with RNA to remove isopropanol. After centrifugation at 4°C and 12,000 rpm for 10 min, discard the supernatant, open the lid to fully evaporate the residual ethanol, dissolve the RNA precipitate at the bottom of the centrifuge tube with sterile water containing DEPC, and measure the RNA concentration with an ultra-micro spectrophotometer. The extracted RNA was reverse transcribed using the ⅡqRT SuperMix for QPCRT kit to obtain cDNA. The reverse transcribed cDNA was analyzed by quantitative PCR using ChamQ Universal SYBR qPCR Master Mix and 2 -ΔΔCT The differential expression of target genes was calculated.
[0082] The test results are shown in Figure 1:The results showed that compared with the blank group without cytokine stimulation, IL-4 and IL-13 could significantly induce Raw264.7 macrophages to differentiate into M2 macrophages, and the mRNA expression level of Arg-1, a marker of M2 macrophages, was significantly increased (P<0.001), while the mRNA expression level of iNOS, a marker of M1 macrophages, did not change significantly under this condition (P>0.05). The anti-tumor active extract of Bidens pilosa BPA at concentrations of 50, 10 and 2 μg / ml could significantly reduce the mRNA level of Arg-1, a marker of M2 macrophages, induced by IL-4 and IL-13, but did not significantly affect the mRNA level of iNOS, a marker of M1 macrophages, indicating that the anti-tumor active extract of Bidens pilosa BPA could inhibit the transformation of macrophages into M2 macrophages with pro-cancer effects, and had the potential for anti-tumor immunomodulation.
[0083] Experimental Example 3 In vivo anti-tumor effect detection
[0084] Experimental principle: The mouse colon cancer CT26 tumor cells cultured in vitro are transplanted subcutaneously into mice to form tumors. After the tumor grows to a certain volume, the corresponding drug treatment is given. The volume of the tumor is measured every day, and the tumor growth curve is drawn to evaluate the in vivo anti-tumor potential of the sample.
[0085] Experimental drugs: 5-fluorouracil, Bidens pilosa methanol total extract (BPT), Bidens pilosa anti-tumor active extract (BPA) from Preparation Example 1.
[0086] Experimental method: After 6-8 weeks of adaptive feeding, the right armpit hair of Balb / c male mice was shaved and CT26 cells were prepared in serum-free medium at 8.5×10 6 / mL of single cell suspension was injected subcutaneously into the armpit of each mouse at a volume of 0.1mL, and the tumor volume was measured with a vernier caliper every day. When the tumor volume grew to about 50-80mm 3 The animals were divided into model control group, positive control group (5-fluorouracil, 5-FU), administration group 1 (BPT), administration group 2 (BPA) and administration group 3 (BPA+5-FU), with 6 animals in each group and an average volume of approximately 70 mm 3, with an average body weight of about 24g. On the day of grouping, except for the positive control group, the other groups began to be medicated. Group 1 was orally administered with 400mg / kg of BPT, Group 2 was orally administered with 200mg / kg of BPA, Group 3 was orally administered with 100mg / kg of BPA, and the model control group was orally gavaged with an equal volume of 0.5% sodium carboxymethyl cellulose solution once a day. On the 4th day after grouping, the positive control group and Group 3 began to intraperitoneally inject 30mg / kg of 5-FU into the mice, once every 3 days, for a total of 12 days. The tumor volume of the mice was measured with a vernier caliper every day, and the tumor growth curve was drawn.
[0087] The test results are shown in Figure 2-4 : The results showed that compared with the model group, 400 mg / kg of BPT could not significantly inhibit the growth of mouse transplanted tumors, 200 mg / kg of BPA could significantly inhibit the growth of mouse transplanted tumors, and 100 mg / kg of BPA combined with 5-FU could significantly inhibit the growth of mouse transplanted tumors (P < 0.001). Its anti-tumor effect was significantly better than that of the 5-FU group alone, indicating that the combination of the two has a synergistic effect.
[0088] The applicant declares that the above are only a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.
Claims
1. A method for preparing an extract with anti-tumor activity from Bidens pilosa L., the method comprising the following steps: (1) adding a methanol solvent to the Bidens pilosa plant raw material to obtain an extract, and concentrating the extract under reduced pressure to obtain an extract; (2) adding water to the extract obtained in step 1 to disperse it to a relative density of 1.05 to 1.20 to obtain an aqueous dispersion, adding petroleum ether for extraction, collecting the aqueous phase, and then extracting with ethyl acetate, collecting the ethyl acetate portion, and concentrating under reduced pressure to obtain an ethyl acetate extract; (3) separating and purifying the ethyl acetate extract obtained in step 2 by silica gel column chromatography to obtain column chromatography sample I; (4) separating and purifying the column chromatography sample I obtained in step 3 by MCI column chromatography to obtain a column chromatography sample II; (5) The column chromatography sample II obtained in step 4 is separated and purified by Sephadex LH-20 gel column chromatography to obtain the Bidens pilosa L. anti-tumor active extract.
2. The preparation method according to claim 1, characterized in that: In step 1, the mass volume ratio of Bidens pilosa plant raw material to solvent is 1:3-10, in g / ml; the extraction time is 1-2 days, and the number of times is 2-4 times; the concentration of methanol solvent is 85%-100%.
3. The preparation method according to claim 1, characterized in that: In the petroleum ether extraction in step 2, the volume ratio of the aqueous dispersion to the petroleum ether is 1:1-3; in the ethyl acetate extraction, the volume ratio of the aqueous phase to the ethyl acetate is 1:1-3.
4. The preparation method according to claim 1, characterized in that: The silica gel column filler in step 3 is silica gel with a particle size of 60-100 mesh, the mass ratio of the ethyl acetate extract to the silica gel filler is 1:15-20; the height-to-diameter ratio of the silica gel column is 3-4:1; Preferably, the silica gel column chromatography separation and purification elution method in step 3 is: first elute with petroleum ether for 5 to 7 column volumes to remove the small polar part, and then elute with elution solvents with a dichloromethane-methanol volume ratio of 1:0, 50:1, 25:1, 10:1, 1:1, and 0:1 in sequence, with each gradient elution being 7 to 10 column volumes, collecting the part with a dichloromethane-methanol volume ratio of 10:1, and concentrating and drying under reduced pressure to obtain the column chromatography sample I.
5. The preparation method according to claim 1, characterized in that: The mass ratio of the column chromatography sample I to the MCI filler in the MCI column chromatography in step 4 is 1:13-17; the height-to-diameter ratio of the chromatography column is 9-10:1; the elution method is: the elution flow rate is 20-30 mL / min, 9-11 column volumes are eluted with 50% methanol-water solvent, all the eluents are collected, and the column chromatography sample II is obtained by concentrating and drying under reduced pressure.
6. The preparation method according to claim 1, characterized in that: In the Sephadex LH-20 gel column chromatography described in step 5, the mass ratio of the column chromatography sample II to the gel filler is 1:25-100, the height-to-diameter ratio of the gel column is 50-100:1, the elution solvent is methanol, the elution flow rate is 3-5 seconds / drop, and the eluate from 220 minutes to 450 minutes is collected, concentrated and dried under reduced pressure to obtain the Bidens pilosa anti-tumor active extract.
7. Use of the Bidens pilosa L. anti-tumor active extract prepared by the preparation method according to any one of claims 1 to 6 in the preparation of anti-tumor drugs; Preferably, the tumor is liver cancer, breast cancer, colon cancer and lung cancer.
8. Use of the Bidens pilosa L. anti-tumor active extract prepared by the preparation method according to any one of claims 1 to 6 in the preparation of tumor immunotherapy drugs.
9. Use of the Bidens pilosa L. antitumor active extract prepared by the preparation method according to any one of claims 1 to 6 and 5-fluorouracil in the preparation of antitumor drugs.
10. A pharmaceutical composition comprising the Bidens pilosa L. extract with anti-tumor activity obtained by the preparation method according to any one of claims 1 to 6 and 5-fluorouracil, and a pharmaceutical carrier suitable for preparing anti-tumor drugs.