CN114366800B - Traditional Chinese medicine for treating colorectal cancer and preparation method thereof - Google Patents
Traditional Chinese medicine for treating colorectal cancer and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a traditional Chinese medicine for treating colorectal cancer and a preparation method thereof, wherein the traditional Chinese medicine is prepared from the following raw material medicines in parts by weight: 6-10 parts of fructus psoraleae, 10-15 parts of radix rehmanniae, 3-9 parts of baked ginger, 6-18 parts of pawpaw, 3-6 parts of fructus amomi, 10-25 parts of poria with hostwood, 10-14 parts of hairyvein agrimony, 10-30 parts of coix seed, 3-6 parts of ginseng, 5-10 parts of rhizoma sparganii, 6-12 parts of curcuma zedoary, 2-5 parts of coptis chinensis and 5-9 parts of radix sophorae flavescentis; the invention has the function of treating colorectal cancer, and is particularly suitable for patients with 'impairment of yin and yang and cancer toxin inside' after colorectal cancer surgery, radiotherapy and chemotherapy; natural plants are adopted, no obvious toxic or side effect is found in the research, the safety is high, and the raw materials are easy to obtain; the product can be made into pharmaceutical tablets, capsules or granules and can be widely used.
Description
Technical Field
The invention relates to a traditional Chinese medicine preparation and a preparation method thereof, in particular to a traditional Chinese medicine preparation which is prepared by taking Chinese herbal medicines as raw materials and is used for treating colorectal cancer, and a preparation method and application thereof.
Background
Global statistical data in 2020 shows that colorectal cancer becomes a high-incidence malignant tumor with the 3 rd morbidity position in males, the 2 nd morbidity position in females and the 3 rd mortality position in females; over 180 million patients are diagnosed with colorectal cancer each year, about 90 million patients die from colorectal cancer; by 2030, there are about 220 million new cases of colorectal cancer and 110 million death cases each year worldwide, and the burden of the disease increases by about 60%. The Chinese cancer pedigree is in the transition stage from developing countries to developed countries, the colorectal cancer belongs to western lifestyle-related cancer, in recent years, the incidence rate of the Chinese colorectal cancer is increased, the incidence rate of the Chinese colorectal cancer is in the 3 rd position, the incidence rate of the Chinese colorectal cancer is in the trend of increasing year by year, and the burden of the colorectal cancer is more rapidly increased. Therefore, the colorectal cancer becomes a high-incidence malignant tumor which seriously threatens the life health of the whole human, the morbidity and the mortality of the colorectal cancer are reduced, and the survival rate and the survival quality of colorectal cancer patients are improved, thus becoming a difficult problem to be solved urgently.
The traditional Chinese medicine is used for treating colorectal cancer, and the curative effect is undoubted after thousands of years of clinical practice, so that the traditional Chinese medicine is an important component of comprehensive treatment of malignant tumors; the traditional Chinese medicine is used for treating colorectal cancer, is guided by the theory of traditional Chinese medicine, adopts a treatment method based on syndrome differentiation, is widely researched and applied in global anti-colorectal cancer through a traditional Chinese medicine compound, has the characteristics of low cost, multiple targets, multiple components, less toxic and side effects and the like, and plays an important role in enhancing the immunity of an organism, controlling the size of a tumor, prolonging the life cycle of a tumor patient, resisting tumor metastasis and the like; throughout the world, traditional Chinese medicine has proven to be an effective method for treating colorectal cancer in clinical practice.
Disclosure of Invention
The invention provides a traditional Chinese medicine for treating colorectal cancer with remarkable curative effect and small side effect, in particular to a traditional Chinese medicine for treating patients with 'yin-yang dual impairment and cancer toxicity internal bias' after colorectal cancer surgery, radiotherapy and chemotherapy and a preparation method thereof, and solves the technical problems of large toxic and side effect, high cost, difficulty in preventing and treating the recurrence and metastasis of colorectal cancer and the like of the existing western medicines.
The invention adopts the following technical scheme: a traditional Chinese medicine for treating colorectal cancer is prepared from the following raw materials in parts by weight: 6-10 parts of fructus psoraleae, 10-15 parts of radix rehmanniae, 3-9 parts of baked ginger, 6-18 parts of pawpaw, 3-6 parts of fructus amomi, 10-25 parts of poria with hostwood, 10-14 parts of hairyvein agrimony, 10-30 parts of coix seed, 3-6 parts of ginseng, 5-10 parts of rhizoma sparganii, 6-12 parts of curcuma zedoary, 2-5 parts of coptis chinensis and 5-9 parts of radix sophorae flavescentis.
The traditional Chinese medicine for treating colorectal cancer is prepared from the following raw material medicines in parts by weight: 8 parts of fructus psoraleae, 12 parts of radix rehmanniae, 6 parts of baked ginger, 9 parts of pawpaw, 4 parts of fructus amomi, 15 parts of poria with hostwood, 12 parts of hairyvein agrimony, 20 parts of coix seed, 5 parts of ginseng, 7 parts of rhizoma sparganii, 9 parts of curcuma zedoary, 4 parts of coptis chinensis and 7 parts of radix sophorae flavescentis.
In the traditional Chinese medicine for treating colorectal cancer, the coix seed is fried coix seed, and the zedoary is vinegar-processed zedoary.
The preparation method of the traditional Chinese medicine for treating colorectal cancer comprises the following steps: extracting volatile oil from the thirteen raw materials, namely the baked ginger, the rhizoma sparganii and the curcuma zedoary by using a steam distillation method, preserving the distilled aqueous solution in another container, adding water into the decoction dregs and the rest of ten raw materials, such as the bone fat, decocting twice, adding water with the weight of 10 times of the total medicinal materials for the first time, adding water with the weight of 8 times of the total medicinal materials for the second time, combining the decoction and filtering for 1.5-2 hours each time, combining the filtrate with the aqueous solution, concentrating the filtrate under reduced pressure to obtain an extract with the crude drug quantity of 2g/mL, adding auxiliary materials, mixing the mixture uniformly, preparing the mixture into granules, drying the granules, spraying the volatile oil, mixing the volatile oil uniformly, and preparing the solid preparation.
The solid preparation of the traditional Chinese medicine for treating colorectal cancer is one of tablets, capsules or granules.
The auxiliary material of the traditional Chinese medicine for treating colorectal cancer is one of dextrin or sucrose.
The application of the traditional Chinese medicine for treating colorectal cancer in a medicine for treating colorectal cancer.
The traditional Chinese medicine for treating colorectal cancer is applied to the medicine for treating colorectal cancer, and the traditional Chinese medicine syndrome of colorectal cancer is the syndrome of 'yin and yang deficiency and cancer toxin internal stagnation' after colorectal cancer operation, radiotherapy and chemotherapy.
Has the advantages that: the theory of healthy qi and pathogenic factors in traditional Chinese medicine considers that malignant tumor is closely related to healthy qi and deficiency, and ancient people cloud: "strong person has no accumulation, but weak person has it"; the book of Wai Zheng Yi Bing Lu (compilation of medical records for external diseases) cloud: "rock" in the case of positive deficiency; the book of medical records is recorded: the formation of accumulation, the deficiency of healthy qi and the retention of pathogenic qi; the theory of yin and yang in traditional Chinese medicine considers that malignant tumors are the products of yin-yang disharmony, and the theory of pathogenesis records that the accumulation is caused by disharmony of yin and yang, weakness of viscera, wind evil and qi struggling in viscera. The professor Zhongying of national physicians and university brings forward the pathogenesis theory of cancer virus according to more than 60 years of clinical experience, considers that cancer virus is a special pathological factor generated in the body in the process of malignant tumor, and the pathological factors such as wind, fire (heat), phlegm, stasis, dampness, cold and the like mutually beat and merge into a whole body, so that the cancer virus is gradually grown, and the cancer virus is the key for the occurrence, development, transfer and recurrence of the malignant tumor. Colorectal cancer belongs to the categories of 'anorectal hemorrhoid', 'visceral toxin' and 'accumulation' in the traditional Chinese medicine, and is a local manifestation of systemic diseases.
On the basis of the traditional Chinese medicine yin-yang theory, the theory of healthy qi and pathogenic factors and the cancer toxin theory, the applicant combines the clinical practice of many years of tumors, proposes that malignant tumor patients mostly show the syndrome of 'yin-yang deficiency and cancer toxin internal stagnation', proposes a 'Wen Zi detoxification method' to treat malignant tumors in a differentiation mode, obtains good effects frequently, and forms an effective prescription aiming at the colorectal cancer patients with the syndrome of yin-yang deficiency and cancer toxin internal stagnation in the clinical accumulation process of many years of tumors, namely the Wen Zi detoxification formula (hereinafter referred to as WJR). The prescription takes the psoralea fruit as the monarch drug for tonifying the kidney and warming yang and takes the rehmannia root as the monarch drug for tonifying the kidney and nourishing yin; the traditional Chinese medicine composition takes 4 traditional Chinese medicines of baked ginger, villous amomum fruit, indian bread with hostwood and pawpaw as ministerial medicines, has the effects of warming the middle-jiao and strengthening the middle-jiao, strengthening the spleen and stomach, soothing the liver and soothing the nerves, combines the hairyvein agrimony, not only can tonify qi and blood, but also can remove heat toxin, also can astringe and stop bleeding, and the coix seed has the effects of eliminating dampness and phlegm and strengthening the middle-jiao, and takes 6 traditional Chinese medicines as ministerial medicines; ginseng radix has effects of invigorating qi, rhizoma Sparganii and Curcumae rhizoma have effects of promoting blood circulation, removing blood stasis, softening hard masses, and breaking knots, and are used as adjuvant drugs; kuh-seng and coptis root, rhizoma coptidis, radix sophorae flavescentis and coptis root, rhizoma coptidis are used as guiding drugs for clearing heat and drying dampness. The whole formula takes psoralea fruit and traditional Chinese medicine classic famous formula Qiongyu paste (4 kinds of original formula dried rehmannia root, ginseng, tuckahoe and white honey) as basic prescription drugs, a small amount of ginseng for keeping the vital essence of five internal organs and a small prescription for eliminating pathogenic factors of relieving heat toxin, damp toxin, phlegm toxin and stasis toxin are added, the malade formula consists of 13 traditional Chinese medicines, and the malade formula plays roles of Wen Zi in the same use, regulating yin and yang and strengthening body resistance and eliminating pathogenic factors aiming at patients with 'yin and yang deficiency and cancer toxin internal stagnation' after operation, radiotherapy and chemotherapy.
The prescription is different from other anti-colorectal cancer traditional Chinese medicine compound and is mainly characterized in that the traditional Chinese medicine anti-colorectal cancer compound mostly takes qi-tonifying and blood-nourishing medicines such as ginseng or codonopsis pilosula, astragalus and bighead atractylodes rhizome as main body-strengthening and anti-cancer medicines, and takes heat-clearing and detoxifying medicines such as sculellaria barbata, oldenlandia diffusa, sargentgloryvine stem and herba patriniae as main body-strengthening and anti-cancer medicines and blood-activating and stasis-removing medicines such as salvia miltiorrhiza, cortex moutan, angelica and leech as main body-eliminating and anti-cancer medicines. Wen Zi formula for removing toxic substance is guided by warm-nourishing and detoxicating method, and uses warm-tonifying kidney yang medicine and nourishing kidney yin medicine as main body resistance strengthening and anticancer medicine, uses spleen invigorating, stomach strengthening, tranquilizing, liver soothing, and dampness removing as auxiliary anticancer medicine, and uses small amount of qi invigorating, blood circulation promoting, blood stasis removing, and heat clearing and detoxicating medicine as adjuvant anticancer medicine. The traditional Chinese medicine composition is an important treatment method integrating the conventional methods, namely Wen Zi detoxification, in addition to the conventional heat-clearing and detoxifying method, the blood circulation promoting and stasis removing method, the qi and blood nourishing method, the spleen and kidney tonifying method and other conventional traditional Chinese medicine treatment methods in clinical traditional Chinese medicine treatment, and is condensed into a Wen Zi detoxification method which is good in clinical treatment effect, less in toxic and side effects and suitable for long-term administration, so that the traditional Chinese medicine prevention and treatment means are enhanced, and the curative effect of the traditional Chinese medicine on the colorectal cancer is improved.
Drawings
FIG. 1 is a schematic staining diagram of the anti-tumor effect H & E in vivo of the present invention; a is a negative control group (NC), B is a low-dose WJR group (LD), C is a medium-dose WJR group (MD), D is a high-dose WJR group (HD), and E is a positive control group (PC).
FIG. 2 is a schematic staining diagram of Hoechst 33258, an in vitro anti-tumor effect of the present invention; a is a negative control group (NC), B is a low-dose WJR group (LD), C is a medium-dose WJR group (MD), and D is a high-dose WJR group (HD).
Detailed Description
Example 1: taking 6g of fructus psoraleae, 15g of radix rehmanniae, 3g of baked ginger, 18g of pawpaw, 3g of fructus amomi, 25g of poria with hostwood, 10g of hairyvein agrimony, 30g of coix seed, 3g of ginseng, 10g of rhizoma sparganii, 6g of rhizoma zedoariae, 5g of coptis chinensis and 5g of radix sophorae flavescentis, extracting volatile oil from the baked ginger, the rhizoma sparganii and the rhizoma zedoariae by a steam distillation method, preserving the distilled water solution in another container, adding water into the residue and the rest ten medicines such as bone fat and decocting the residue twice, adding water with the weight of 10 times of the total medicines for the first time, adding water with the weight of 8 times of the total medicines for the second time, 1.5 hours each time, combining the decoction, filtering, combining the filtrate and the water solution, concentrating the filtrate under reduced pressure to obtain an extract with the crude drug amount of 2g/mL, adding starch, mixing the mixture, preparing the granules, drying, spraying the volatile oil, mixing the mixture uniformly, tabletting and preparing the tablets.
Example 2: taking 10g of fructus psoraleae, 10g of radix rehmanniae, 9g of baked ginger, 6g of pawpaw, 6g of fructus amomi, 10g of poria with hostwood, 14g of hairyvein agrimony, 10g of coix seed, 6g of ginseng, 5g of rhizoma sparganii, 12g of rhizoma zedoariae, 2g of coptis chinensis and 9g of radix sophorae flavescentis, extracting volatile oil from the baked ginger, the rhizoma sparganii and the rhizoma zedoariae by a steam distillation method, preserving distilled water solution in another container, adding water into the decoction dregs and other ten kinds of bone fat for decoction twice, adding water with the weight of 10 times of the total medicinal materials for the first time, adding water with the weight of 8 times of the total medicinal materials for the second time, 1.5 hours each time, combining decoctions, filtering, combining the filtrate with the water solution, concentrating the filtrate under reduced pressure to obtain an extract with the raw medicine amount of 2g/mL, adding auxiliary materials, mixing, preparing granules, drying, spraying the volatile oil, mixing uniformly, and encapsulating to prepare the capsules.
Example 3: taking 8g of fructus psoraleae, 12g of radix rehmanniae, 6g of baked ginger, 9g of pawpaw, 4g of fructus amomi, 15g of poria with hostwood, 12g of hairyvein agrimony, 20g of coix seed, 5g of ginseng, 7g of rhizoma sparganii, 9g of curcuma zedoary, 4g of coptis chinensis and 7g of radix sophorae flavescentis, extracting volatile oil from the baked ginger, the rhizoma sparganii and the curcuma zedoary by a steam distillation method, storing the distilled aqueous solution in another container, adding water into the residues and the rest of ten medicines such as bone fat and the like, decocting the residues and the rest of the medicines twice, adding water with the weight of 10 times of the total medicinal materials for the first time, adding water with the weight of 8 times of the total medicinal materials for the second time, mixing 2 hours each time, decocting the decoction, filtering, combining the filtrate with the aqueous solution, concentrating the filtrate into extract with the crude drug amount of 2g/mL under reduced pressure, adding auxiliary materials, mixing, preparing the granules, drying, spraying the volatile oil, and mixing the volatile oil to prepare granules.
Example 4: in vivo antitumor Effect
Experimental equipment: inverted fluorescence microscope Nikon Eclipse Ti-SR (Nikon, japan); JB-P5 Paraffin embedding machine (Wuhan Junjie electronics, inc.); RM2016 paraffin slicer (shanghai come instrument limited); non-toxic environmental protection hematoxylin-eosin (H-E) dye liquor (Nanjing Jiansheng science and technology Co., ltd., batch number: 20190404)
Experimental animals: BALB/C male mice, 60, 4-6 weeks old, body mass (20. + -.2) g, were purchased from Shanghai Si Laike laboratory animals, inc.
The experimental process comprises the following steps:
1. preparation of a medicament
A Wen Zi detoxification formulation (hereinafter abbreviated as WJR) was prepared as described in example 3 above and stored at-20 ℃. The drug is divided into low dose, medium dose and high dose groups according to a conversion formula of equivalent doses of human and mice.
2. Animal feeding and molding
Feeding: these animal experiments were conducted according to guidelines approved by the institutional animal ethics committee of the university of medicine in south kyo. The mice are raised in cages, 5 mice are raised in each cage under the specific pathogen-free condition, and the mice are fed with free water. The room temperature is controlled to be 20-25 ℃, the relative humidity is 65-70%, the illumination/dark illumination cycle period is 12 hours, and the indoor ventilation is good.
Molding: CT26 cells (5X 10) 6 And/or) subcutaneous injection to the ventral side of the mouse to establish a colorectal cancer xenograft model. Mice were evaluated daily for tumor formation, tumor diameter>Tumor formation was considered to be 3 mm.
Administration and grouping: when the tumor diameter reached 5-6mm, the mice were randomized into 5 groups (10 per group): control (NC, equal volume saline), WJR low dose group (WJRL), WJR medium dose group (WJRM), WJR high dose group (WJRH), and oxaliplatin group (PC). Mice were given the corresponding dose of drug by gavage once a day for 10 consecutive days, and the control group was given an equivalent dose of physiological saline. The positive control group is given oxaliplatin intraperitoneal injection of 8mg/kg for 1 time/3 days. The administration was continued for 14d. Mice were sacrificed within 24h after the last dose and tumors were collected for further analysis.
In vivo anti-tumor Effect of WJR
Within 14d of the administration, the mice were weighed 1 time every 2-3d, and the long diameter (a) and short neck (b) of the transplanted tumor were measured and recorded with a vernier caliper by the formula V = (a × b) 2 ) The tumor volume was calculated as/2. Within 24h of the last administration, mice were sacrificed by cervical molars, and tumor tissues of the blank control group, the WJR low dose group, the WJR medium dose group, the WJR high dose group and the positive control group were taken out by a surgical method, respectively. Changes in different groups of tumor tissues were visualized by HE staining.
(1) Tumor tissue was fixed with formalin fixation.
(2) Paraffin embedding and slicing: dewaxing and hydrating by a conventional method, soaking the slices in xylene for 5min, replacing the xylene, and soaking for 5min; soaking in anhydrous ethanol for 5min; soaking in 95% ethanol for 5min; soaking in 85% ethanol for 5min; soaking in 70% ethanol for 5min, and soaking in PBS for 3min × 3 times.
The specific experimental steps are as follows:
(1) immersing into a reagent-nuclear dye solution in the kit, dyeing in a dye vat for 3-5min, and washing for about 30-60s;
(2) immersing the sample in a reagent bicolor solution I in the kit for about 20s, and washing the sample with water for about 30-60s;
(3) immersing the test sample in the reagent trichromatic liquid II in the kit for about 40 seconds, and washing the test sample with water for about 30 to 60 seconds;
(4) placing in reagent IV dye solution in kit for dyeing for 2min, washing with reagent V hyperchromic solution to remove excess dye solution, washing twice, and blotting mounting with filter paper for microscopic examination;
(3) The change of pathological forms of tumor cells is observed under a light microscope by professionals, and according to the pathological degree, the tumor tissues can be marked in a semi-quantitative way, which is shown in a table 1:
TABLE 1 semi-quantitative markers for different tumor tissues
The experimental results are as follows:
we studied the in vivo anti-tumor efficacy of Wen Zi detoxification prescription (WJR) using a colorectal cancer-bearing mouse model, see fig. 1 and table 2, the model group (NC) tumors grow rapidly. The WJR low dose group (LD) and medium dose group (MD) suppressed tumor progression to some extent compared to the NC group. The High Dose (HD) and oxaliplatin (PC) groups showed the strongest inhibition of tumor progression. The tumor volumes of the HD group and the PC group are far smaller than those of the NC group, and the HD group and the PC group have no significant difference. At the same time, we also performed TGI scoring during the treatment period. We found TGI significant for the LD and MD groups. But TGI was more pronounced in the HD and PC groups, consistent with the results for tumor volume. Pathological observation shows that the tumor tissue structures of the HD group and the PC group have more obvious extensive damage, serious necrosis and irregular widening of intercellular spaces; however, tumor tissue in the LD and MD groups was moderately damaged. Taken together, these results indicate that WJR can inhibit tumor progression in colorectal cancer in vivo, and that high doses have a stronger inhibitory effect.
TABLE 2 comparison of tumor volumes in different groups of mice
Note: NC stands for blank control group, LD is WJR low dose group, MD is WJR medium dose group, HD is WJR high dose group, PC is positive control group, WJR is Wen Zi detoxification formula. * Represents that P is less than 0.05 compared with NC group; # represents P < 0.05 compared to LD; & represents P < 0.05 compared to group MD.
Example 5: in vitro anti-tumor effect
Experimental equipment:
a spectrophotometer (impelen, germany); pipettor (Eppendorf, germany Co., ltd.)) (ii) a Horizontal electrophoresis tank DYCP-31DN (BIO RAD, USA); enzyme-linked immunosorbent assay (PerkinElmer company, USA)
Experimental cells: DLD-1 human colorectal cancer cell strain is purchased from Shanghai cell bank of Chinese academy of sciences; SD rats (6-8 weeks, 220-250 g) were purchased from Shanghai Si Laike laboratory animals, inc.
The experimental process comprises the following steps:
1. preparation of serum containing medicine
24 adult SD rats (6 to 8 weeks, 220 to 250 g) were divided into WJR group (n = 12) and control group (n = 12). Rats were fasted for 12 hours. According to the preparation method of the serum containing the medicine, the medicine is administrated by intragastric administration for 2 times/d and is continuously administrated for 3d. Collecting blood from retinal vein within 1-2 h after the last administration, centrifuging at 3000rpm for 15min, and centrifuging to separate serum. Putting at 56 deg.C for 30min, extinguishing complement in serum, filtering with 0.22 μm filter membrane, and storing the processed serum containing medicine at-20 deg.C. The control group was given the same volume of physiological saline and a non-drug-containing blank serum was prepared as described above.
2. Cell culture
(1) Cell recovery: taking out the frozen cells, placing the cells in a water bath kettle at 37 ℃, slightly shaking and heating the cells, and completely thawing the cells. Centrifuging at 1000rpm for 5min, removing supernatant, adding 10% fetal calf serum-containing completely cultured and blown cells, mixing, inoculating the resuspended cells into culture dish, placing at 37 deg.C, and 5% CO 2 Overnight, and 2d replaced fresh medium.
(2) Cell passage: when the cells are fused to about 80%, sucking out the original culture medium, adding a proper amount of PBS, cleaning for 3 times, adding 1ml of pancreatin, placing in an incubator at 37 ℃ for digestion for 2min, and adding complete culture solution to stop digestion. Centrifuging at 1000rpm for 5min, discarding supernatant, adding 5ml complete culture medium, repeatedly blowing with a pipette to obtain uniform cell suspension, respectively inoculating into fresh culture bottles, and adding appropriate amount of complete culture medium. At 37 ℃ C, 5% CO 2 Overnight, and 2d replaced fresh medium.
3.MTT
(1) DLD-1 of colorectal cancer cells in logarithmic growth phase at 5 x 10 3 Perwell in 96-well plates and placed at 37 ℃ with 5% CO 2 The culture chamber of (2) was cultured overnight to attach the cells to a 96-well plate.
(2) Cells were divided into 4 groups: control group (blank serum), WJR group (XJR medicated serum). The cells are added into the corresponding culture medium according to the above groups respectively. At 37 ℃ C, 5% CO 2 The culture boxes of (2) are respectively cultured for 12h, 24h, 36h and 48h.
(3) After treatment, 50. Mu.l of MTT was added to each well and incubated for a further 4h to fully reduce MTT.
(4) Then, 150. Mu.L of DMSO was added to each well and the plate was shaken.
(5) The absorbance (OD) of each well was measured at a wavelength of 570nm with a microplate reader. Cell activity (%) = (average OD value of administration group/average OD value of control group) × 100%.
Hoechst 33258 staining
Placing a cell slide with the diameter of 18mm in a 6-well plate, taking DLD-1 cells in a logarithmic growth phase, adjusting the concentration, inoculating 4 multiplied by 105 cells in each well on the cell slide, culturing for 24h, and then administering according to 4 concentrations (0, 40, 80, 160 mu g.mL < -1 >); culturing for 48h, sucking clear supernatant, adding 4% paraformaldehyde 1.0 ml/hole, placing on a shaking table, oscillating at low speed for 10min for fixation, adding Hoechst 33258 staining solution 1 ml/hole, placing on the shaking table, oscillating at low speed for 10min, sucking clear, washing with PBS for 2 times, dripping anti-fluorescence quenching solution, taking out cell slide, placing on a sheet, and observing apoptosis by using a fluorescence microscope.
5. Apoptosis assay
DLD-1 cells were collected in logarithmic growth phase, adjusted for concentration and placed at 4X 10/well 5 Inoculating into six-well plate, culturing for 24 hr, and adding medicated serum; the culture was continued for 48h, washed twice with PBS, centrifuged at 2000rpm for 5min in a centrifuge and the cells were collected in 1.5ml centrifuge tubes. Gently suspending cells by using a Binding Buffer 500 mu L/tube, evenly mixing 5 mu Lannexin V-FITC, then adding 5 mu L Propidium Iodide (PI), gently mixing, reacting for 5-15min in a dark place at room temperature, and placing the mixture into a flow cytometer for detection in groups.
Western blot detection of Bcl-2 and BAX protein expression in different groups
And (3) inoculating DLD-1 cells in the logarithmic growth phase to a six-well plate, culturing for 24h, culturing for 48h according to the administration of serum containing medicine, scraping the cells, adding RIPA lysate to extract total protein, and determining the protein concentration by using a BCA test box to determine that the protein loading amount is 20 mu g. Sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis followed by wet transfer to PVDF membrane, 5% skim milk blocking for 1h, TBST washing, addition of primary antibody Bax (1. The ratio of the target protein to the internal reference optical density is the relative expression quantity of the target protein.
The experimental results are as follows:
MTT assay showed that WJR significantly inhibited the proliferative capacity of cells in a dose-dependent manner compared to NC group (table 3). Hoechst 33258 staining showed that a dose-dependent increase in the proportion of apoptotic cells was found following treatment with WJR (see figure 2). The apoptosis rate was determined by flow cytometry, and the percentage of apoptotic cells after treatment with WJR was significantly increased compared to the NC group, and the inhibitory effect of large doses of WJR was the best (table 4). Furthermore, expression of pro-apoptotic Bax protein increased in a concentration-dependent manner following XJR treatment, with a concomitant decrease in expression of anti-apoptotic Bcl-2 protein (table 5). Taken together, these results indicate that WJR has significant anti-tumor effects in vitro.
TABLE 3 MTT results for different treatment groups
Note: NC stands for blank control group, LD is WJR low dose group, MD is WJR medium dose group, HD is WJR high dose group, WJR is Wen Zi antidote. * Represents that P is less than 0.05 compared with NC group; # represents P < 0.05 compared to LD; & represents P < 0.05 compared to group MD.
TABLE 4 apoptotic results of different treatment groups
Note: NC stands for blank control group, LD is WJR low dose group, MD is WJR medium dose group, HD is WJR high dose group, and WJR is Wen Zi detoxification formula. * Represents that P is less than 0.05 compared with NC group; # represents P < 0.05 compared to LD; & represents P < 0.05 compared to group MD.
TABLE 5 WB results for different treatment groups
NC stands for blank control group, LD is WJR low dose group, MD is WJR medium dose group, HD is WJR high dose group, WJR is Wen Zi antidote. * Represents that P is less than 0.05 compared with NC group; # represents P < 0.05 compared to LD; & represents P < 0.05 compared to group MD.
The above examples are merely illustrative for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. The scientific and technical terms used in this patent have the same meaning as understood by one of ordinary skill in the art.
Claims (7)
1. A traditional Chinese medicine for treating colorectal cancer is characterized in that: the traditional Chinese medicine composition is prepared from the following raw material medicines in parts by weight: 6-10 parts of fructus psoraleae, 10-15 parts of radix rehmanniae, 3-9 parts of baked ginger, 6-18 parts of pawpaw, 3-6 parts of fructus amomi, 10-25 parts of poria with hostwood, 10-14 parts of hairyvein agrimony, 10-30 parts of coix seed, 3-6 parts of ginseng, 5-10 parts of rhizoma sparganii, 6-12 parts of curcuma zedoary, 2-5 parts of coptis chinensis and 5-9 parts of radix sophorae flavescentis, and the traditional Chinese medicine syndrome of colorectal cancer is the syndrome of 'yin-yang dual impairment and cancer toxin internal stagnation' after colorectal cancer surgery, radiotherapy and chemotherapy.
2. The traditional Chinese medicine for treating colorectal cancer according to claim 1, wherein: the traditional Chinese medicine composition is prepared from the following raw material medicines in parts by weight: 8 parts of fructus psoraleae, 12 parts of radix rehmanniae, 6 parts of baked ginger, 9 parts of pawpaw, 4 parts of fructus amomi, 15 parts of poria with hostwood, 12 parts of hairyvein agrimony, 20 parts of coix seed, 5 parts of ginseng, 7 parts of rhizoma sparganii, 9 parts of curcuma zedoary, 4 parts of coptis chinensis and 7 parts of radix sophorae flavescentis.
3. The traditional Chinese medicine for treating colorectal cancer according to claim 1, wherein: the Coicis semen is parched Coicis semen, and Curcumae rhizoma is vinegar Curcumae rhizoma.
4. The traditional Chinese medicine for treating colorectal cancer according to claim 1, wherein the traditional Chinese medicine comprises the following components: the preparation method of the traditional Chinese medicine comprises the following steps: extracting volatile oil from the thirteen raw materials, namely the baked ginger, the rhizoma sparganii and the curcuma zedoary by using a steam distillation method, preserving the distilled water solution in another container, adding water into the decoction dregs and the rest of ten raw materials, such as the rest of the bone fat, decocting twice, adding water with the weight of 10 times of the total medicinal materials for the first time, adding water with the weight of 8 times of the total medicinal materials for the second time, 1.5-2 hours each time, merging decoction, filtering, merging filtrate with the water solution, concentrating under reduced pressure to obtain an extract with the crude drug quantity of 2g/mL, adding auxiliary materials, uniformly mixing, granulating, drying, spraying the volatile oil, uniformly mixing, and preparing into a solid preparation.
5. The traditional Chinese medicine for treating colorectal cancer according to claim 4, wherein: the solid preparation is one of tablet, capsule or granule.
6. The traditional Chinese medicine for treating colorectal cancer according to claim 4, wherein: the auxiliary material is one of dextrin or sucrose.
7. The use of the Chinese medicament for the treatment of colorectal cancer according to claim 1 in the preparation of a medicament for the treatment of colorectal cancer.
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