A special medical food formula for colorectal cancer patients and a preparation method and application thereof

Abstract

This invention relates to the field of special medical foods, and particularly to a special medical food formula for colorectal cancer patients, its preparation method, and its application. The raw materials, in parts by weight, include: 20-30 parts Codonopsis pilosula, 20-30 parts Rehmannia glutinosa (processed), 20-30 parts Astragalus membranaceus, 10-20 parts Coix lacryma-jobi (raw), 10-20 parts Atractylodes macrocephala, 10-20 parts Poria cocos, 10-20 parts Angelica sinensis, 10-20 parts Paeonia lactiflora, 10-20 parts Prunus persica, 3-6 jujubes, 3-15 parts Glycyrrhiza uralensis, 6-15 parts phycocyanin, and 6-15 parts citric acid. This formula can have anti-tumor effects, reduce the toxic side effects of chemotherapy, enhance the reticuloendothelial cell system, promote the enhancement of specific and non-specific immune functions in the body, thereby reducing tumor volume and inhibiting tumor proliferation and metastasis. The drug is encapsulated in Dunaliella salina exosomes and targeted modified to ensure accurate delivery to the lesion site, increasing bioavailability and reducing the drawbacks of a small therapeutic window and high toxicity.

Description

Technical Field

[0001] This invention relates to the field of special medical foods, and in particular to a special medical food formulation for colorectal cancer patients, its preparation method, and its application. Background Technology

[0002] Epidemiological data shows that with the increasing intake of high-fat and high-animal-protein foods and the decreasing intake of dietary fiber, the incidence of colorectal cancer is rising year by year. Globally, colorectal cancer is the third most common malignant tumor and the second leading cause of death among malignant tumors. Surgery, chemotherapy, and radiotherapy are currently the standard medical treatments. However, some patients are diagnosed at an advanced stage or with metastasis, missing the optimal surgical window. Postoperative recurrence rates are high, and complications such as infection and weakened immune function are common. Biotherapy is an emerging treatment method, but it is currently only effective for certain specific tumors and is easily affected by individual genotype differences. Traditional drug formulations also have drawbacks such as low bioavailability, significant toxic side effects, difficulty in crossing biological barriers, and easy clearance by the reticuloendothelial system and liver.

[0003] Traditional Chinese medicine (TCM) has rich clinical experience in treating colorectal cancer, playing an important role in enhancing immune function, improving patients' quality of life, increasing survival rates, and mitigating adverse reactions after chemotherapy. TCM combined with radiotherapy and chemotherapy shows good efficacy in treating colorectal cancer. TCM treatment can participate in the entire treatment process for colorectal cancer, with particularly prominent effects in improving patients' quality of life and prolonging survival during the maintenance therapy phase. Special medical purpose foods (STPS) are specially processed and formulated foods designed to meet the special nutritional or dietary needs of individuals with restricted food intake, digestive and absorption disorders, metabolic disorders, or specific disease states. These include complete nutritional formula foods, specific complete nutritional formula foods, and incomplete nutritional formula foods. These products must be consumed alone or in combination with other foods under the guidance of a doctor or clinical nutritionist. Existing STPS foods have limited functions and low nutritional content, are generally in powder form, and can easily lead to nutritional imbalances when consumed. Furthermore, the powdered form can clump and even spoil after prolonged storage. In terms of nutrition and efficacy, special medical foods have abundant medicinal and edible resources, such as Poria cocos, ginseng, and yam. Nutritionally, they contain rich protein, various essential amino acids, polysaccharides, and trace elements. Pharmacologically, their main components have anti-tumor, immune-enhancing, and antioxidant effects, which are closely related to the occurrence and development of tumors. Summary of the Invention

[0004] The purpose of this invention is to provide a special medical food formulation for colorectal cancer patients, its preparation method, and its application, so as to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a special medical food formula for colorectal cancer patients, the special medical food formula comprising the following ingredients in the indicated weight proportions: Codonopsis pilosula 20-30 parts, Rehmannia glutinosa 20-30 parts, Astragalus membranaceus 20-30 parts, Coix lacryma-jobi 10-20 parts, Atractylodes macrocephala 10-20 parts, Poria cocos 10-20 parts, Angelica sinensis 10-20 parts, Paeonia lactiflora 10-20 parts, Prunus persica 10-20 parts, Jujube 3-6 pieces, Glycyrrhiza uralensis 3-15 parts, Phycocyanin 6-15 parts, Citric acid 6-15 parts.

[0006] The formula for a special medical food for colorectal cancer patients is based on the following preferred weight ratio of ingredients: Codonopsis pilosula 20 parts, Rehmannia glutinosa 20 parts, Astragalus membranaceus 20 parts, Coix lacryma-jobi 10 parts, Atractylodes macrocephala 10 parts, Poria cocos 10 parts, Angelica sinensis 10 parts, Paeonia lactiflora 10 parts, Prunus persica 10 parts, Jujube 3 pieces, Glycyrrhiza uralensis 3 parts, Phycocyanin 6 parts, Citric acid 6 parts.

[0007] The formula for a special medical food for colorectal cancer patients is based on the following preferred weight proportions of ingredients: Codonopsis pilosula 30 parts, Rehmannia glutinosa 30 parts, Astragalus membranaceus 30 parts, Coix lacryma-jobi 20 parts, Atractylodes macrocephala 20 parts, Poria cocos 20 parts, Angelica sinensis 20 parts, Paeonia lactiflora 20 parts, Prunus persica 20 parts, Jujube 6 pieces, Glycyrrhiza uralensis 15 parts, Phycocyanin 15 parts, Citric acid 15 parts.

[0008] The formula for a special medical food for colorectal cancer patients is based on the following preferred weight proportions of ingredients: Codonopsis pilosula 25 parts, Rehmannia glutinosa 25 parts, Astragalus membranaceus 25 parts, Coix lacryma-jobi 15 parts, Atractylodes macrocephala 15 parts, Poria cocos 15 parts, Angelica sinensis 15 parts, Paeonia lactiflora 15 parts, Prunus persica 15 parts, Jujube 5 pieces, Glycyrrhiza uralensis 9 parts, Phycocyanin 9 parts, Citric acid 9 parts.

[0009] The efficacy of the raw materials in the above-mentioned traditional Chinese medicine compound is as follows:

[0010] Codonopsis pilosula: Rich in ginsenosides, it can effectively enhance the function of the reticuloendothelial system in cancer patients, improve the autonomic nerve function of patients, reduce the bone marrow suppression effect of chemotherapy drugs, and thus enhance the appetite of patients and increase the body's immune function.

[0011] Rehmannia glutinosa (processed): It has the effects of replenishing essence and marrow, nourishing blood and yin. Rehmannia glutinosa polysaccharides are the main anti-tumor substances in it. They can not only improve the activity of the reticuloendothelial system and enhance the phagocytic effect of mononuclear macrophages, but also promote the activation of T cells in the body, thereby improving immune function and enhancing anti-tumor effects.

[0012] Astragalus: It is warm in nature and sweet in taste. It enters the spleen and lung meridians. It has the functions of tonifying qi and raising yang, promoting diuresis and reducing swelling. It has good biological activity and good immunomodulatory effects. It can effectively increase the ability of cancer patients to induce interferon and promote the production of complement and antibodies, thereby achieving anti-cancer effects.

[0013] Raw Job's tears: It is sweet and bland in taste, and enters the spleen, stomach and lung meridians. It has the effects of promoting diuresis and clearing heat, strengthening the spleen and nourishing the lungs.

[0014] Atractylodes macrocephala: It is warm in nature and sweet in taste. It enters the spleen and stomach meridians and has the functions of invigorating qi and strengthening the spleen, drying dampness and promoting diuresis. It has the effects of anti-oxidation, scavenging oxygen free radicals and activating the phagocytic function of macrophages. Atractylodes macrocephala lactone I, II and atractylone are the main anti-tumor substances in Atractylodes macrocephala. They can effectively inhibit the activity of endogenous antioxidant enzymes, thereby inducing cell apoptosis.

[0015] Poria cocos: It is neutral in nature and sweet in taste. It enters the heart, lung and spleen meridians. It has the effects of strengthening the spleen and calming the mind, and promoting diuresis and dampness removal. Among them, Poria cocos polysaccharide can effectively enhance the phagocytic and recognition effects of macrophages, inhibit the DNA replication and RNA transcription of tumor cells, thereby inhibiting the proliferation of tumor cells.

[0016] Angelica sinensis: It is warm in nature and sweet in taste. It enters the liver, heart and spleen meridians. It has the effects of promoting blood circulation and regulating qi. Angelica sinensis extract and Angelica sinensis polysaccharide can enhance the specific and non-specific immune functions in the body, and at the same time, can restore the immune function under the state of suppression.

[0017] White peony root: Nourishes blood and softens the liver, relieves spasms and pain. It is a blood-nourishing medicine that can accelerate the blood flow velocity of microarteries and microveins, improve circulation volume, dilate microvessels, increase the diameter of microvessels, and reduce the viscosity of whole blood, serum and plasma to inhibit platelet aggregation.

[0018] Peach kernel: Enhances the effect of promoting blood circulation and removing blood stasis.

[0019] Jujubes: They have the effects of replenishing qi and blood, calming the mind, and regulating the spleen and stomach.

[0020] Licorice: It is warm in nature and sweet in taste. It enters the heart, lung, stomach and spleen meridians. It has the effects of tonifying the spleen and replenishing qi, clearing heat and detoxifying. Glycyrrhizic acid and glycyrrhetinic acid have the effect of inhibiting the carcinogenesis of normal cells.

[0021] Phycocyanin: It has anti-tumor effects, promotes drug absorption, and enhances the body's immunity.

[0022] Citric acid: Improves the taste of medicine.

[0023] Of the selected ingredients, Codonopsis pilosula invigorates the spleen and stomach, promotes the production of body fluids, and nourishes the blood and yin; Rehmannia glutinosa nourishes the blood and yin, replenishes essence and marrow; Astragalus membranaceus assists Codonopsis pilosula in invigorating the spleen and stomach. Codonopsis pilosula, Rehmannia glutinosa, and Astragalus membranaceus together serve as the principal herbs, exerting their effects of invigorating the spleen and nourishing the blood, strengthening the spleen and kidneys, and supporting the body's vital energy. Atractylodes macrocephala and Poria cocos invigorate the spleen and eliminate dampness; Coix lacryma-jobi removes numbness, drains pus, detoxifies, and disperses nodules. These three herbs together serve as the assistant herbs, exerting their effects of invigorating the spleen and eliminating dampness. Angelica sinensis, Paeonia lactiflora, and Prunus persica nourish the blood and promote blood circulation to help dissipate accumulations. These three herbs together serve as the adjuvant herbs. Jujube harmonizes the spleen and stomach to promote the production of qi and blood; Glycyrrhiza uralensis invigorates the spleen and harmonizes the middle jiao. These two herbs serve as the guiding herbs, harmonizing the effects of the other herbs and balancing the functions of the spleen and kidneys. Additionally, the addition of phycocyanin helps enhance the anti-tumor effect and also aids in drug absorption; finally, citric acid is added to improve the taste.

[0024] A method for preparing a special medical food formula for colorectal cancer patients includes the following steps:

[0025] Step S1: Weigh the raw materials according to the ratio and place them in a clean 1000mL beaker. Add an appropriate amount of ultrapure water and soak for 30-60 minutes. Then decoct for 1-1.5 hours. Filter with 3-8 layers of gauze. Put the dregs back into ultrapure water and decoct again. After filtering the liquid, centrifuge the two filtered liquids. Take the supernatant, mix and concentrate. Transfer the concentrated liquid to an evaporating dish, seal with plastic wrap and poke several small holes in the plastic wrap. Then freeze dry to obtain the Chinese herbal extract powder.

[0026] Step S2: After thoroughly mixing the drug powder obtained in step S1, weigh 0.3-0.6g and dissolve it in 1mL of DMSO solution to prepare a stock solution with a concentration of 300-600mg / mL. Take the stock solution, add 99mL of DMEM medium to dilute it, and filter it through a 0.22μm microporous membrane to prepare a stock solution with a concentration of 3-6mg / mL.

[0027] Step S3: After rapidly thawing the Dunaliella salina cells in the cryopreservation tubes in a 37°C water bath, slowly add them to PKS liquid medium using a pipette. Incubate overnight in an artificial light simulator at 26°C with a light-to-dark ratio of 14:10. The next day, determine the Dunaliella salina cell concentration using a visible spectrophotometer. Inoculate 50 μL of the Dunaliella salina cell suspension evenly onto PKS solid medium and continue culturing in the artificial light simulator until monoclonal algal colonies are formed. Pick these colonies onto solid medium and inoculate them into 5 mL of liquid medium for 3 days to amplify the cell concentration to 1 × 10⁻⁶ cells / mL. 6 After passing through multiple passages at a rate of / mL, the second to fourth generations of Dunaliella salina are typically selected for exosome extraction.

[0028] Step S4: Using the Dunaliella salina cell culture medium from Step S3 as a sample, centrifuge at low speed 300-600xg for 5-10 min to separate the cells; retain the supernatant, centrifuge at 2000-3000xg for 10-20 min to remove dead cells, and continue to increase the centrifugation force to 10000-15000xg for 20-30 min to remove cell debris. The processed supernatant is ready for ultracentrifugation at 100,000xg for 60-70 min to obtain crude exosome precipitate (containing a small amount of impurities). Resuspend the exosome precipitate with PBS and centrifuge again at 100,000xg for 70 min to obtain pure exosomes.

[0029] Step S5: Treat cRGDfk in HEPES solution at 50-60°C for 15 min to form micelles. Then, sonicate the micelles at an amplitude of 8-10 μm for 10-20 seconds to reduce the size of the micelles and promote their separation from EXO. Then, mix the exosome solution obtained in step S4 with the above micelle solution at 40°C for 2-3 h, immediately cool to 4°C, and purify the above mixed solution by centrifugation at 80,000-100,000 x g for 50-70 min to obtain cRGD-EXO solution.

[0030] Step S6: Using the average fluorescence intensity taken up by cells as an indicator, the mass ratio of EXO and cRGDfk obtained in step S5 was set to 5:1, 1:1, 1:5, 1:10, and 1:15. Single-factor optimization was performed using flow cytometry, with HCT-116 at 5 × 10⁻⁶ cells / cells. 5 HCT-116 cells were seeded at a density of 1 / mL in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h. After cell attachment, the culture medium was discarded, and different mass ratios of PKH67-labeled cRGD-EXO and EXO were added. The uptake of HCT-116 cells by different proportions of cRGD-EXO and EXO was quantitatively evaluated using fluorescence intensity.

[0031] Step S7: At room temperature, mix the cRGD-EXO obtained in step S6 with the drug evenly, and incubate on a shaker for 60-90 min at a speed of 200-300 r / min. In order to select the optimal ratio of cRGD-EXO to drug, the mass ratio of cRGD-EXO to drug was set to 10:1, 5:1, 1:1, 1:5, and 1:10, with drug encapsulation efficiency and drug loading as evaluation criteria, and single-factor investigation was conducted. Finally, the cRGD-EXO loaded with traditional Chinese medicine was purified by ultracentrifugation, that is, the purified cRGD-EXO / traditional Chinese medicine was obtained, and the drug content in cRGD-EXO / traditional Chinese medicine was detected by high performance liquid chromatography.

[0032] Preferably, in step S1, the ratio of ultrapure water to drug raw material is 4 to 13:1, the freezing condition is freezing in a refrigerator at (-20) to (-80)℃ for 6 to 8 hours, and the drying condition is freezing in a freeze dryer at a low temperature (-56℃) to (-60℃) under a vacuum of 10Pa until the drug concentrate is dried.

[0033] Compared with the prior art, the beneficial effects of the present invention are:

[0034] 1. Based on traditional Chinese medicine theory and the pathogenesis of colorectal cancer, this invention uses a method of syndrome differentiation and treatment, and systemic conditioning to screen several Chinese medicinal herbs. The components and weights were determined according to the principle of "principal, assistant, adjuvant, and guide" in traditional Chinese medicine formulation. This special medical food formula and its active ingredients have functions such as anti-tumor, anti-inflammatory, immune regulation, spleen-strengthening and qi-tonifying, essence-replenishing and marrow-tonifying, heat-clearing and detoxifying, and blood-activating and stasis-removing. Finally, citric acid and phycocyanin are added to improve the taste and promote drug absorption, enhancing the anti-tumor and health-preserving effects. Compared with other Chinese medicine formulas, this formula has advantages such as stable efficacy, food-medicine homology, fewer drug side effects, and a wide range of drug sources.

[0035] 2. This invention uses Dunaliella salina exosomes for encapsulation, which greatly increases the lipid solubility of the drug, making it easier to cross biological barriers. At the same time, it exerts the "high-penetration and strong retention effect" of nanotechnology, reducing the clearance function of the reticuloendothelial cell system and the liver, and improving the bioavailability of the drug.

[0036] 3. This invention involves targeted modification of exosomes, which allows the drug to reach the target site to the greatest extent possible, overcoming problems such as narrow therapeutic window and large toxic side effects, improving the bioavailability and targeting of the drug, and greatly improving the quality of life of patients.

[0037] 4. The special medical food formulation provided by this invention can be made into different dosage forms according to clinical needs, making it convenient for patients to carry and take.

[0038] 5. The special medical food formula for the prevention and treatment of colorectal cancer provided by this invention has a reasonable ratio, stable efficacy, is easy to absorb, has a stable preparation process, and is easy to mass-produce. Attached Figure Description

[0039] Figure 1 The diagram shows the effect of the embodiments of the present invention on cell proliferation, indicating that as the drug concentration increases, the cell survival rate gradually decreases, and the growth of most cells is inhibited.

[0040] Figure 2 This is a flow cytometry image of HCT-116 cells induced by the drug of this invention;

[0041] Figure 3This is a diagram showing the in vivo targeting and efficacy of the drug of this invention. The cGRD-modified Dunaliella salina exosomes have good targeting properties in vivo and a good anti-tumor effect.

[0042] Figure 4 The figure shows the effect of different drug concentrations on apoptosis in mouse tumor tissue. As the drug concentration increases, the apoptosis rate gradually increases, indicating that the drug inhibits the growth of tumor cells through apoptosis. Detailed Implementation

[0043] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] Example 1

[0045] Step 1: Take 20-30 parts of Codonopsis pilosula, 20-30 parts of Rehmannia glutinosa, 20-30 parts of Astragalus membranaceus, 10-20 parts of Coix lacryma-jobi, 10-20 parts of Atractylodes macrocephala, 10-20 parts of Poria cocos, 10-20 parts of Angelica sinensis, 10-20 parts of Paeonia lactiflora, 10-20 parts of Prunus persica, 3-6 jujubes, 3-15 parts of Glycyrrhiza uralensis, 6-15 parts of phycocyanin, and 6-15 parts of citric acid, and set aside.

[0046] Step 2: Place the above-mentioned Chinese herbal medicine formula in a clean 1000mL beaker, add an appropriate amount of ultrapure water (M drug: V ultrapure water = 1:6) and soak for 40 minutes. Bring to a boil over high heat on an electric stove, then simmer over low heat for 1.5 hours. Filter the decoction through three layers of gauze, and then add the dregs back into ultrapure water for simmering. After filtering the decoction, centrifuge the two filtered herbs, collect the supernatant, mix well and concentrate. Transfer the concentrated decoction to an evaporating dish, seal it with plastic wrap and poke several small holes in the plastic wrap, then freeze it in a -40℃ refrigerator. After 8 hours, transfer it to a freeze dryer and freeze it at a low temperature of -56℃ under a vacuum of 10Pa until the concentrated decoction is dried, preferably into powder form.

[0047] Step 3: After thoroughly mixing the drug powder obtained in Step 1, weigh 0.3g and dissolve it in 1mL of DMSO solution to prepare a stock solution with a concentration of 300mg / mL. Take the stock solution, add 99mL of DMEM medium to dilute it, and filter it through a 0.22μm micropore to prepare a 3mg / mL stock solution.

[0048] Step 4: After rapidly thawing the Dunaliella salina cells in the cryopreservation tubes in a 37°C water bath, slowly add them to PKS liquid medium using a pipette. Incubate overnight in an artificial light simulator at 26°C with a light-to-dark ratio of 14:10. The next day, determine the Dunaliella salina cell concentration using a visible spectrophotometer. Inoculate 50 μL of the Dunaliella salina cell suspension evenly onto PKS solid medium and continue culturing in the artificial light simulator until monoclonal algal colonies are formed. Pick these colonies onto solid medium and inoculate them into 5 mL of liquid medium for 3 days to amplify the cell concentration to 1 × 10⁻⁶ cells / mL. 6 After passing through multiple passages at a rate of / mL, the second to fourth generations of Dunaliella salina are typically selected for exosome extraction.

[0049] Step 5: Using the Dunaliella salina cell culture medium from Step 3 as a sample, centrifuge at low speed (400xg) for 8 min to separate the cells; retain the supernatant, centrifuge at 2000xg for 10 min to remove dead cells. Continue centrifugation of the remaining supernatant at 10000xg for 25 min to remove cell debris. The processed supernatant is ready for ultracentrifugation. Centrifuge at 100,000xg for 60 min to obtain a crude exosome precipitate (containing a small amount of impurities). Resuspend the exosome precipitate in PBS and centrifuge again at 100,000xg for 70 min to obtain purified exosomes.

[0050] Step 6: Treat cRGDfk in HEPES at 50°C for 15 min to form micelles. Then, sonicate the micelles at an amplitude of 8 μm for 10 seconds to reduce their size and promote their separation from EXO. Then, mix the exosome solution obtained in Step 4 with the above micelle solution at 40°C for 3 h, immediately cool to 4°C, and purify the above mixed solution by centrifugation at 8000 x g for 70 min to obtain the cRGD-EXO solution.

[0051] Step 7: Using the average fluorescence intensity taken up by cells as an indicator, the mass ratio of EXO to cRGDfk obtained in Step 5 was set to 5:1, 1:1, 1:5, 1:10, and 1:15, and single-factor optimization was performed using flow cytometry. HCT-116 cells were cultured at 5 × 10⁻⁶ cells / year. 5 HCT-116 cells were seeded at a density of 1 / mL in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h. After cell attachment, the culture medium was discarded, and different mass ratios of PKH67-labeled cRGD-EXO and EXO were added. The uptake of HCT-116 cells by different proportions of cRGD-EXO and EXO was quantitatively evaluated using fluorescence intensity.

[0052] Step 8: At room temperature, mix the cRGD-EXO obtained in Step 6 with the drug evenly, and incubate on a shaker for 70 min at a speed of 200 r / min. In order to select the optimal ratio of cRGD-EXO to drug, the mass ratio of cRGD-EXO to drug was set to 10:1, 5:1, 1:1, 1:5, and 1:10, with drug encapsulation efficiency and drug loading as evaluation criteria, and single-factor investigation was conducted. Finally, the cRGD-EXO loaded with traditional Chinese medicine was purified by ultracentrifugation, that is, the purified cRGD-EXO / traditional Chinese medicine was obtained, and the content of traditional Chinese medicine in cRGD-EXO / drug was detected by high performance liquid chromatography.

[0053] Step 9: The release behavior of the traditional Chinese medicine in cRGD-EXO / TCM was determined by dialysis. Dialysis bags were cut into 6cm segments, boiled in distilled water for 30 minutes, rinsed with distilled water, and stored at 4℃, ensuring the bags remained submerged in the solution. A 2cm segment of cRGD-EXO / TCM was transferred to the dialysis bag, and both ends were tied tightly. The dialysis bag was placed in 50mL PBS containing 0.1% Tween 80 at pH 7.4 and pH 5.5, and then placed in a constant temperature water bath shaker (37℃, 100r / min). 5mL samples were taken at 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours for testing. Simultaneously, the same volume and temperature of fresh release medium were added to concentrate the sample medium. The release rate of the TCM was calculated using HPLC. Data were processed and analyzed using SPSS statistical software, and graphs were plotted using Graphpad. ANOVA was used for comparisons between groups, and data are expressed as mean ± standard deviation (MEAN SD). A p-value < 0.05 is considered to indicate a significant difference.

[0054] Step 10: Add 200 μL of cRGD-EXO / traditional Chinese medicine solution to 800 μL of distilled water, physiological saline, PBS buffer solution and DMEM cell culture medium respectively, shake well, centrifuge at 50000xg for 20 min, and observe its stability in various physiological solutions.

[0055] Example 2

[0056] Step 1: Take 20 parts of Codonopsis pilosula, 20 parts of Rehmannia glutinosa, 20 parts of Astragalus membranaceus, 10 parts of Coix lacryma-jobi, 10 parts of Atractylodes macrocephala, 10 parts of Poria cocos, 10 parts of Angelica sinensis, 10 parts of Paeonia lactiflora, 10 parts of Prunus persica, 3 jujubes, 3 parts of Glycyrrhiza uralensis, 6 parts of phycocyanin, and 6 parts of citric acid, and set aside.

[0057] Step 2: Place the above-mentioned Chinese herbal medicine formula in a clean 1000mL beaker, add an appropriate amount of ultrapure water (M drug: V ultrapure water = 1:4) and soak for 30 minutes. Bring to a boil over high heat on an electric stove, then simmer over low heat for 1 hour. Filter the decoction through three layers of gauze, and then add the dregs back into ultrapure water to simmer again. After filtering the decoction, centrifuge the two filtered herbs, collect the supernatant, mix well and concentrate. Transfer the concentrated decoction to an evaporating dish, seal it with plastic wrap and poke several small holes in the plastic wrap, then freeze it in a -20℃ refrigerator. After 8 hours, transfer it to a freeze dryer and freeze it at a low temperature of -56℃ under a vacuum of 10Pa until the concentrated decoction is dried, preferably into powder form.

[0058] Step 3: After thoroughly mixing the drug powder obtained in Step 1, weigh 0.3g and dissolve it in 1mL of DMSO solution to prepare a stock solution with a concentration of 300mg / mL. Take the stock solution, add 99mL of DMEM medium to dilute it, and filter it through a 0.22μm micropore to prepare a 3mg / mL stock solution.

[0059] Step 4: After rapidly thawing the Dunaliella salina cells in the cryopreservation tubes in a 37°C water bath, slowly add them to PKS liquid medium using a pipette. Incubate overnight in an artificial light simulator at 26°C with a light-to-dark ratio of 14:10. The next day, determine the Dunaliella salina cell concentration using a visible spectrophotometer. Inoculate 50 μL of the Dunaliella salina cell suspension evenly onto PKS solid medium and continue culturing in the artificial light simulator until monoclonal algal colonies are formed. Pick these colonies onto solid medium and inoculate them into 5 mL of liquid medium for 3 days to amplify the cell concentration to 1 × 10⁻⁶ cells / mL. 6 After passing through multiple passages at a rate of / mL, the second to fourth generations of Dunaliella salina are typically selected for exosome extraction.

[0060] Step 5: Using the Dunaliella salina cell culture medium from Step 3 as a sample, centrifuge at low speed (300xg) for 10 min to separate the cells; retain the supernatant, centrifuge at 2000xg for 15 min to remove dead cells. Continue centrifugation of the remaining supernatant at 10000xg for 20 min to remove cell debris. The processed supernatant is ready for ultracentrifugation. Centrifuge at 100,000xg for 60 min to obtain a crude exosome precipitate (containing a small amount of impurities). Resuspend the exosome precipitate in PBS and centrifuge again at 100,000xg for 70 min to obtain pure exosomes.

[0061] Step 6: Treat cRGDfk in HEPES solution at 50℃ for 20 min to form micelles. Then, sonicate the micelles at an amplitude of 8 μm for 10 seconds to reduce the size of the micelles and promote their separation from EXO. Then, mix the exosome solution obtained in Step 4 with the above micelle solution at 40℃ for 2 h, immediately cool to 4℃, and purify the above mixed solution by centrifugation at 9000 x g for 60 min to obtain cRGD-EXO solution.

[0062] Step 7: Using the average fluorescence intensity taken up by cells as an indicator, the mass ratio of EXO to cRGDfk obtained in Step 5 was set to 5:1, 1:1, 1:5, 1:10, and 1:15, and single-factor optimization was performed using flow cytometry. HCT-116 cells were cultured at 5 × 10⁻⁶ cells / year. 5 HCT-116 cells were seeded at a density of 1 / mL in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h. After cell attachment, the culture medium was discarded, and different mass ratios of PKH67-labeled cRGD-EXO and EXO were added. The uptake of HCT-116 cells by different proportions of cRGD-EXO and EXO was quantitatively evaluated using fluorescence intensity.

[0063] Step 8: At room temperature, the cRGD-EXO obtained in Step 6 was loaded with drug using electroporation at a voltage of 600V and a capacitance of 500μF. To select the optimal cRGD-EXO to drug ratio, the drug encapsulation efficiency and drug loading were used as evaluation criteria. The mass ratio of cRGD-EXO to drug was set to 10:1, 5:1, 1:1, 1:5, and 1:10 for single-factor analysis. Finally, the cRGD-EXO loaded with traditional Chinese medicine was purified by ultracentrifugation, thus obtaining purified cRGD-EXO / traditional Chinese medicine. The content of traditional Chinese medicine in cRGD-EXO / drug was detected by high performance liquid chromatography.

[0064] Step 9: The release behavior of the traditional Chinese medicine in cRGD-EXO / TCM was determined by dialysis. Dialysis bags were cut into 6cm segments, boiled in distilled water for 20 minutes, rinsed with distilled water, and stored at 4℃, ensuring the bags remained submerged in the solution. A 2cm segment of cRGD-EXO / TCM was transferred to the dialysis bag, and both ends were tied tightly. The dialysis bag was placed in 50mL PBS containing 0.1% Tween 80 at pH 7.4 and pH 5.5, and then placed in a constant temperature water bath shaker (37℃, 100r / min). 5mL samples were taken at 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours for testing. Simultaneously, the same volume and temperature of fresh release medium were added to concentrate the sample medium. The release rate of the TCM was calculated using HPLC. Data were processed and analyzed using SPSS statistical software, and graphs were plotted using Graphpad. ANOVA was used for comparisons between groups, and data are expressed as mean ± standard deviation (MEAN SD). A p-value < 0.05 is considered to indicate a significant difference.

[0065] Step 10: Add 200 μL of cRGD-EXO / traditional Chinese medicine solution to 800 μL of distilled water, physiological saline, PBS buffer solution and DMEM cell culture medium respectively, shake well and centrifuge at 50000xg for 20 min, and observe its stability in various physiological solutions.

[0066] Example 3

[0067] Take 30 parts of Codonopsis pilosula, 30 parts of Rehmannia glutinosa, 30 parts of Astragalus membranaceus, 20 parts of Coix lacryma-jobi, 20 parts of Atractylodes macrocephala, 20 parts of Poria cocos, 20 parts of Angelica sinensis, 20 parts of Paeonia lactiflora, 20 parts of Prunus persica, 6 jujubes, 15 parts of Glycyrrhiza uralensis, 15 parts of phycocyanin, and 15 parts of citric acid, and set aside.

[0068] Step 2: Place the above-mentioned Chinese herbal medicine formula in a clean 1000mL beaker, add an appropriate amount of ultrapure water (M drug: V ultrapure water = 1:4~13) and soak for 60 minutes. Bring to a boil over high heat on an electric stove, then simmer over low heat for 2 hours. After filtering the decoction through 8 layers of gauze, put the dregs back into ultrapure water and simmer again. After filtering the decoction, centrifuge the two filtered herbs, take the supernatant, mix and concentrate. Transfer the concentrated decoction to an evaporating dish, seal it with plastic wrap and poke several small holes in the plastic wrap, then freeze it in a -80℃ freezer. After 8 hours, transfer it to a freeze dryer and freeze it at a low temperature of -60℃ under a vacuum of 10Pa until the concentrated decoction is dried, preferably into powder form.

[0069] Step 3: After thoroughly mixing the drug powder obtained in Step 1, weigh 0.6g and dissolve it in 1mL of DMSO solution to prepare a stock solution with a concentration of 600mg / mL. Take the stock solution, add 99mg / mL of DMEM medium to dilute it, and filter it through a 0.22μm micropore to prepare a 6mg / mL stock solution.

[0070] Step 4: After rapidly thawing the Dunaliella salina cells in the cryopreservation tubes in a 37°C water bath, slowly add them to PKS liquid medium using a pipette. Incubate overnight in an artificial light simulator at 26°C with a light-to-dark ratio of 14:10. The next day, determine the Dunaliella salina cell concentration using a visible spectrophotometer. Inoculate 50 μL of the Dunaliella salina cell suspension evenly onto PKS solid medium and continue culturing in the artificial light simulator until monoclonal algal colonies are formed. Pick these colonies onto solid medium and inoculate them into 5 mL of liquid medium for 3 days to amplify the cell concentration to 1 × 10⁻⁶ cells / mL. 6 After passing through multiple passages at a rate of / mL, the second to fourth generations of Dunaliella salina are typically selected for exosome extraction.

[0071] Step 5: Using the Dunaliella salina cell culture medium from Step 3 as a sample, centrifuge at low speed (600xg) for 5 min to separate the cells; retain the supernatant, centrifuge at 3000xg for 10 min to remove dead cells. Continue centrifugation of the remaining supernatant at 15000xg for 20 min to remove cell debris. The processed supernatant is ready for ultracentrifugation. Centrifuge at 100,000xg for 70 min to obtain a crude exosome precipitate (containing a small amount of impurities). Resuspend the exosome precipitate in PBS and centrifuge again at 100,000xg for 70 min to obtain pure exosomes.

[0072] Step 6: Treat cRGDfk in HEPES solution at 60°C for 15 min to form micelles. Then, sonicate the micelles at an amplitude of 10 μm for 20 seconds to reduce the size of the micelles and promote their separation from EXO. Then, mix the exosome solution obtained in Step 4 with the above micelle solution at 40°C for 3 h, immediately cool to 4°C, and purify the above mixed solution by centrifugation at 100,000 x g for 70 min to obtain cRGD-EXO solution.

[0073] Step 7: Using the average fluorescence intensity taken up by cells as an indicator, the mass ratio of EXO to cRGDfk obtained in Step 5 was set to 5:1, 1:1, 1:5, 1:10, and 1:15, and single-factor optimization was performed using flow cytometry. HCT-116 cells were cultured at 5 × 10⁻⁶ cells / year. 5 HCT-116 cells were seeded at a density of 1 / mL in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h. After cell attachment, the culture medium was discarded, and different mass ratios of PKH67-labeled cRGD-EXO and EXO were added. The uptake of HCT-116 cells by different proportions of cRGD-EXO and EXO was quantitatively evaluated using fluorescence intensity.

[0074] Step 8: At room temperature, mix the cRGD-EXO obtained in Step 6 with the drug evenly, and incubate on a shaker for 90 min at a speed of 200 r / min. In order to select the optimal ratio of cRGD-EXO to drug, the mass ratio of cRGD-EXO to drug was set to 10:1, 5:1, 1:1, 1:5, and 1:10 for single-factor investigation, with drug encapsulation efficiency and drug loading as evaluation criteria. Finally, the cRGD-EXO loaded with traditional Chinese medicine was purified by ultracentrifugation, that is, the purified cRGD-EXO / traditional Chinese medicine was obtained, and the content of traditional Chinese medicine in cRGD-EXO / drug was detected by high performance liquid chromatography.

[0075] Step 9: The release behavior of the traditional Chinese medicine in cRGD-EXO / TCM was determined by dialysis. Dialysis bags were cut into 6cm segments, boiled in distilled water for 20 minutes, rinsed with distilled water, and stored at 4℃, ensuring the bags remained submerged in the solution. A 2cm segment of cRGD-EXO / TCM was transferred to the dialysis bag, and both ends were tied tightly. The dialysis bag was placed in 50mL PBS containing 0.1% Tween 80 at pH 7.4 and pH 5.5, and then placed in a constant temperature water bath shaker (37℃, 150r / min). 5mL samples were taken at 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours for testing, with the same volume and temperature of fresh release medium added simultaneously. The sampled medium was concentrated, and the release rate of the TCM was calculated using HPLC. Data were processed and analyzed using SPSS statistical analysis software, plotted using Graphpad, and ANOVA was used for intergroup comparisons. Data are expressed as mean ± standard deviation (MS / SD). A p-value < 0.05 is considered to indicate a significant difference.

[0076] Step 10: Add 200 μL of cRGD-EXO / traditional Chinese medicine solution to 800 μL of distilled water, physiological saline, PBS buffer solution and DMEM cell culture medium respectively, shake well and centrifuge at 50000xg for 20 min, and observe its stability in various physiological solutions.

[0077] Example 4

[0078] Step 1: Take 25 parts of Codonopsis pilosula, 25 parts of Rehmannia glutinosa, 25 parts of Astragalus membranaceus, 15 parts of Coix lacryma-jobi, 15 parts of Atractylodes macrocephala, 15 parts of Poria cocos, 15 parts of Angelica sinensis, 15 parts of Paeonia lactiflora, 15 parts of Prunus persica, 5 jujubes, 9 parts of Glycyrrhiza uralensis, 9 parts of phycocyanin, and 9 parts of citric acid, and set aside.

[0079] Step 2: Place the medicine in a clean 1000mL beaker, add an appropriate amount of ultrapure water (M drug: V ultrapure water = 1:6) and soak for 40 minutes. Bring to a boil over high heat on an electric stove, then simmer over low heat for 1.5 hours. Filter the liquid through 6 layers of gauze, then add the dregs back into ultrapure water and simmer again. After filtering the liquid, centrifuge the two filtered liquids, collect the supernatant, mix well and concentrate. Transfer the concentrated liquid to an evaporating dish, seal it with plastic wrap and poke several small holes in the plastic wrap, then freeze it in a -60℃ freezer. After 7 hours, transfer it to a freeze dryer and freeze it at a low temperature of -58℃ under a vacuum of 10Pa until the concentrated liquid is dried, preferably into powder.

[0080] Step 3: After thoroughly mixing the drug powder obtained in Step 1, weigh 0.5g and dissolve it in 1mL of DMSO solution to prepare a stock solution with a concentration of 500mg / mL. Take the stock solution, add 99mg / mL of DMEM medium to dilute it, and filter it through a 0.22μm micropore to prepare a 5mg / mL stock solution.

[0081] Step 4: After rapidly thawing the Dunaliella salina cells in the cryopreservation tubes in a 37°C water bath, slowly add them to PKS liquid medium using a pipette. Incubate overnight in an artificial light simulator at 26°C with a light-to-dark ratio of 14:10. The next day, determine the Dunaliella salina cell concentration using a visible spectrophotometer. Inoculate 50 μL of the Dunaliella salina cell suspension evenly onto PKS solid medium and continue culturing in the artificial light simulator until monoclonal algal colonies are formed. Pick these colonies onto solid medium and inoculate them into 5 mL of liquid medium for 3 days to amplify the cell concentration to 1 × 10⁻⁶ cells / mL. 6 After passing through multiple passages at a rate of / mL, the second to fourth generations of Dunaliella salina are typically selected for exosome extraction.

[0082] Step 5: Using the Dunaliella salina cell culture medium from Step 3 as a sample, centrifuge at low speed (500xg) for 8 min to separate the cells; retain the supernatant, centrifuge at 1500xg for 15 min to remove dead cells. Continue centrifugation of the remaining supernatant at 13000xg for 25 min to remove cell debris. The processed supernatant is ready for ultracentrifugation. Centrifuge at 100,000xg for 70 min to obtain a crude exosome precipitate (containing a small amount of impurities). Resuspend the exosome precipitate in PBS and centrifuge again at 100,000xg for 70 min to obtain pure exosomes.

[0083] Step 6: Treat cRGDfk in HEPES at 55°C for 15 min to form micelles. Then, sonicate the micelles at an amplitude of 9 μm for 15 seconds to reduce their size and promote their separation from EXO. Then, mix the exosome solution obtained in Step 4 with the above micelle solution at 40°C for 2.5 h, immediately cool to 4°C, and purify the above mixed solution by centrifugation at 9000 x g for 70 min to obtain the cRGD-EXO solution.

[0084] Step 7: Using the average fluorescence intensity taken up by cells as an indicator, the mass ratio of EXO to cRGDfk obtained in Step 5 was set to 5:1, 1:1, 1:5, 1:10, and 1:15, and single-factor optimization was performed using flow cytometry. HCT-116 cells were cultured at 5 × 10⁻⁶ cells / year. 5 HCT-116 cells were seeded at a density of 1 / mL in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h. After cell attachment, the culture medium was discarded, and different mass ratios of PKH67-labeled cRGD-EXO and EXO were added. The uptake of HCT-116 cells by different proportions of cRGD-EXO and EXO was quantitatively evaluated using fluorescence intensity.

[0085] Step 8: At room temperature, mix the cRGD-EXO obtained in Step 6 with the drug evenly, and incubate on a shaker for 80 min at a speed of 250 r / min. In order to select the optimal ratio of cRGD-EXO to drug, the drug encapsulation rate and drug loading were used as evaluation criteria. The mass ratio of cRGD-EXO to drug was set to 10:1, 5:1, 1:1, 1:5, and 1:10 for single-factor investigation. Finally, the cRGD-EXO loaded with traditional Chinese medicine was purified by ultracentrifugation, that is, the purified cRGD-EXO / traditional Chinese medicine was obtained. The content of traditional Chinese medicine in cRGD-EXO / drug was detected by high performance liquid chromatography.

[0086] Step 9: The release behavior of the traditional Chinese medicine in cRGD-EXO / TCM was determined by dialysis. Dialysis bags were cut into 6cm segments, boiled in distilled water for 20 minutes, rinsed with distilled water, and stored at 4℃, ensuring the bags remained submerged in the solution. A 2cm segment of cRGD-EXO / TCM was transferred to the dialysis bag, and both ends were tied tightly. The dialysis bag was placed in 50mL PBS containing 0.1% Tween 80 at pH 7.4 and pH 5.5, and then placed in a constant temperature water bath shaker (37℃, 120r / min). 5mL samples were taken at 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours for testing. Simultaneously, the same volume and temperature of fresh release medium were added to concentrate the sampled medium. The release rate of the TCM was calculated using HPLC. Data were processed and analyzed using SPSS statistical analysis software, plotted using Graphpad, and ANOVA was used for intergroup comparisons. Data are expressed as mean ± standard deviation (MS / SD). A p-value < 0.05 is considered to indicate a significant difference.

[0087] Step 10: Add 200 μL of cRGD-EXO / traditional Chinese medicine solution to 800 μL of distilled water, physiological saline, PBS buffer solution and DMEM cell culture medium respectively, shake well and centrifuge at 50000xg for 20 min, and observe its stability in various physiological solutions.

[0088] Since the Chinese medicine compositions obtained in each embodiment have basically the same efficacy in preventing and treating colorectal cancer, the Chinese medicine preparations prepared in Examples 1 to 4 are randomly selected for efficacy evaluation.

[0089] Effect Example

[0090] I. Basic Experimental Research

[0091] To demonstrate that the formulation of this special medical food has a more significant therapeutic effect compared to other traditional Chinese medicine compounds, a cytotoxicity experiment was used to evaluate its efficacy.

[0092] (a) Cytotoxicity test

[0093] Remove the cryovials from the liquid nitrogen container and thaw them rapidly in a 37°C water bath within 1 minute. Transfer the cells to a culture dish using a pipette, add 10 mL of complete culture medium, and culture until the cell density reaches 80%–90%. Then, passage and seed the cells into plates at a density of 3 × 10⁻⁶ cells / mL. 4 Seeds were seeded into 96-well plates, and the drug was added the next day. Six different concentration groups were set up, with three replicates for each group. After culturing for 48 or 72 hours, 20 μL of MTT was added, and after incubation for 4 hours, the culture medium was aspirated, and 150 μL of LDMSO was added. After thorough shaking, the absorbance at 450 nm was measured.

[0094] (II) In vitro experiments:

[0095] 1. Cell proliferation experiment

[0096] Re-plate the cells, adding 100 μL of the drug at concentrations of 100, 50, 25, 12.5, 6.25, and 3.125 mg / mL to each well. The control group only received culture medium. Each group had 6 replicates. 100 μL of DMEM was added to the wells around the perimeter of the culture plate as blank wells. After culturing for 24 h, the culture medium was discarded, and 10% volume of CCK-8 was added to each well. The cells were incubated in the dark for 1 h. The absorbance at 450 nm was measured using a microplate reader. The cell viability was calculated as [AS-AB] ÷ [AC-AB] × 100%.

[0097] (AS: Experimental group, absorbance of wells with drug and CCK-8 solution; AB: Blank group, absorbance of wells with culture medium but without drug and CCK-8 solution; AC: Control group, absorbance of wells with CCK-8 solution but without drug), results are as follows. Figure 1 As shown in the figure, the cell survival rate gradually decreases with increasing drug concentration, and most cells are inhibited from growing.

[0098] 2. Apoptosis experiment

[0099] When HCT-116 cells reached 80% confluence, they were digested with trypsin and counted at a concentration of 3 × 10⁻⁶. 4 Inoculate cells into 12-well plates at 37°C and 5% CO2 for 24 hours. Aspirate the supernatant, wash three times with PBS, and add 1 mL of drug at concentrations of 100, 50, 25, 12.5, 6.25, and 3.125 mg / mL respectively. Continue culturing for another 24 hours. Collect the old culture medium, wash once with PBS, digest cells with trypsin without EDTA, centrifuge the old culture medium and cell suspension together to obtain a pellet, wash once with 1 mL of pre-cooled PBS, resuspend cells in binding buffer, and adjust the cell concentration to 1 × 10⁻⁶ cells / well. 6 ~1×10 7 Add 5 μL Annexin V-FITC and PI to 100 μL of cell suspension, mix well, and incubate in the dark for 15 min. No washing is required. Add 385 μL of binding buffer to each tube, mix well, and perform flow cytometry analysis as soon as possible. Results are as follows: Figure 2 As shown, the apoptosis rate gradually increases with increasing drug concentration.

[0100] 3. Cell cycle experiment

[0101] When HCT-116 cells reached 80% confluence, they were digested with trypsin and counted at a concentration of 3 × 10⁻⁶. 4The cells were seeded into 12-well plates and cultured at 37°C with 5% CO2 for 24 hours. The supernatant was aspirated, and the cells were washed three times with PBS. 1 mL of the drug at concentrations of 100, 50, 25, 12.5, 6.25, and 3.125 mg / mL were added, respectively. The blank control group received only culture medium. The cells were cultured for another 24 hours, centrifuged, and collected. 1 mL of PBS was added to wash the cells, and the cells were resuspended in pre-cooled 75% ethanol. The cells were gently pipetted to mix and fixed overnight at 4°C. After centrifugation and removal of the supernatant, each sample was washed with 1 mL of PBS. Then, 0.5 mL of propidium iodide staining solution was added to each tube, and the cells were slowly and thoroughly resuspended. The cells were incubated at 37°C in the dark for 30 minutes before being analyzed.

[0102] 4. Cell migration experiment

[0103] HCT-116 cells were starved in serum-free medium for 12 hours until they reached 80% confluence. After washing three times with PBS, the cells were digested with trypsin. After stopping digestion, the cells were centrifuged, the supernatant was removed, and the cells were washed twice with PBS. The cells were then resuspended in serum-free medium and the density was adjusted to 1×10⁶ cells / cm². 4 Cells were seeded in the upper chamber at a depth of 8 μm. 1 mL of drug at concentrations of 100, 50, 25, 12.5, 6.25, and 3.125 mg / mL were added, respectively. The blank control group received only culture medium. 800 μL of DMEM containing 10% FBS was added to the lower chamber as a chemokine. Cells were cultured at 37°C and 5% CO2 for 24 h. Cells on the surface of the upper chamber were wiped with sterile cotton swabs. Cells that migrated to the lower chamber were fixed with 4% paraformaldehyde for 15 min, then stained with 0.1% crystal violet for 10 min, and washed three times with PBS. Fields of view were randomly selected for observation.

[0104] Note: Compared with the blank control group, *P<0.05

[0105] Results: The data show that cell migration rate decreases with increasing drug concentration in a concentration-dependent manner.

[0106] 5. Cell invasion assay

[0107] The Matrigel was diluted 1:8 with serum-free medium. 50 μL of the diluted solution was evenly applied to the upper surface of the Transwell chamber and dried at 37°C for 2 h to allow the Matrigel to solidify. The procedure was then the same as that for cell migration experiments.

[0108] (III) In vivo experiments

[0109] 1. Establishment and gross observation of animal models

[0110] A tumor xenograft model was established using tumor-bearing mice. Under aseptic conditions, intraperitoneal injections were administered at a dose of 100 μL per mouse, once every three days for a total of four times. During the treatment period, tumor size and mouse weight were recorded every three days using calipers, and tumor growth was photographed. Blood samples were collected 24 hours after injection to detect blood biochemical indicators (ALT, AST, BUN, CRE, WBC). Mice were sacrificed 30 days after the first administration, tumors were removed and photographed, and organs such as the heart, liver, spleen, lungs, and kidneys were removed to evaluate biocompatibility.

[0111] Note: Compared with the blank model group, *P<0.05, **P<0.01

[0112] Results: Drugs encapsulated in exosomes and targeted were more effective at inhibiting tumor growth in mice.

[0113] 2. Near-infrared imaging for observing drug targeting in vivo

[0114] A final concentration of 5 μm DIR near-infrared fluorescent dye was prepared. Appropriate amounts of DIR dye were added to 0.5 mg / mL of single Dunaliella salina exosomes (FreeDExo) and Dunaliella salina exosomes modified with cRGD peptides (cRGD-DExo), respectively. The mixtures were incubated at 37°C in the dark for 30 min, followed by centrifugation at 100,000 x g for 60 min to remove free DIR dye. The precipitates were washed and resuspended to obtain DIR-labeled Free-DExo and cRGD-EXO. Near-infrared small animal in vivo imaging was used to verify the tumor-targeting effect of engineered exosomes in mice. When the tumor volume in mice was approximately 500 m³... 3 At that time, 100 μL of DIR-labeled Free-DExo and cRGD-EXO were injected into nude mice via the tail vein. After 24 hours, images were taken using a small animal in vivo imaging system. The results are as follows: Figure 3 As shown, cGRD-modified Dunaliella salina exosomes exhibit good targeting in vivo and have a good anti-tumor effect.

[0115] 3. Biocompatibility evaluation

[0116] (1) Dewax the paraffin sections of heart, liver, spleen, lung, kidney and tumor tissue in each group to water according to the following procedure: put them in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 15 min, anhydrous ethanol II for 5 min, 75% alcohol for 5 min, wash with distilled water, put the sections in hematoxylin staining solution for 3-8 min, wash with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, then use 0.6% ammonia water to return to blue, rinse with running water, then put the sections in 85% and 95% graded alcohol for 5 min each for dehydration, stain with eosin for 1-3 min, then put the sections in anhydrous ethanol I for 15 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, xylene II for 5 min, mount with neutral resin, and examine under an optical microscope.

[0117] (2) Blood samples were taken from different groups of mice, and the levels of biochemical indicators ALT, AST, BUN, CRE and WBC in the blood were measured to evaluate the biosafety of the novel vector.

[0118]

[0119] Note: *P<0.05 compared with the model control group.

[0120] Results: The results show that the cGRD-modified Dunaliella salina exosomes, this novel drug carrier, are non-toxic to humans.

[0121] 4. Tunel staining of tumor tissue

[0122] Dewax each group of tumor tissue paraffin sections to water using the above procedure. Add an appropriate amount of proteinase K to cover the tissue and incubate at room temperature for 15 minutes. Place the slides on a destaining shaker and wash with PBS for 5 minutes each time (3 times). After the sections are slightly dry, add membrane-breaking working solution to cover the tissue and incubate at room temperature for 10 minutes. Place the slides on a destaining shaker and wash with PBS for 5 minutes each time (3 times). According to the number of sections and tissue size, use the Tunel kit, mixing TdT and DUTP reagents at a 1:9 volume ratio, cover the tissue, and incubate in a humidified chamber at 37°C for 2 hours. Counterstain cell nuclei with DAPI and incubate at room temperature in the dark for 10 minutes. Place the slides on a destaining shaker and wash with PBS for 5 minutes each time (3 times). After slightly drying the sections, mount them with anti-fluorescence quenching mounting medium and examine under a fluorescence microscope. The results are as follows: Figure 4 As shown, the apoptosis rate gradually increases with increasing drug concentration, suggesting that the drug may inhibit tumor cell growth through apoptosis.

[0123] II. Evaluation of Clinical Efficacy

[0124] Digestive system: Symptoms such as abdominal pain, diarrhea, constipation, bloating, nausea, vomiting, and anorexia are reduced or even disappear, and the patient has a good appetite.

[0125] Hematologic system: The patient's hemoglobin levels returned to normal, anemia symptoms improved, complexion became rosy, and nail beds became shiny.

[0126] Systemic symptoms: The patient gains weight, is in good mental condition, edema or ascites disappears, and body temperature returns to normal.

[0127] The principle of this invention: In ancient Chinese medicine texts, colorectal cancer is similar to diseases such as "intestinal masses," "visceral toxins," "hematochezia," and "abdominal masses." It is often caused by congenital deficiency, improper diet, invasion of pathogenic toxins, or spleen damage due to worry, leading to an imbalance of Yin and Yang in the body. This includes both deficiency (including spleen deficiency, kidney deficiency, and insufficient Qi and blood) and excess (including phlegm-dampness, damp-heat, fire-toxin, and blood stasis). Spleen deficiency, kidney deficiency, and insufficient Qi and blood are the root causes of the disease. The entire pathogenesis is a mixture of deficiency and excess, manifesting as a state of extreme weakness. Traditional Chinese medicine believes that the spleen and stomach are the foundation of acquired constitution. The spleen transforms and transports the essence of food and water, which is the material basis for maintaining strong Qi and expelling pathogenic factors. The kidneys govern bones and produce marrow. Bone marrow, nourished by kidney essence and the essence of food and water, is the foundation for the generation of Qi. Therefore, this formula uses Chinese herbs that tonify Qi and benefit the spleen, strengthen the spleen and stomach, invigorate blood and remove blood stasis, replenish essence and marrow, nourish Qi and blood, and improve the body's immunity. Dunaliella salina is a eukaryotic photosynthetic autotrophic organism, suitable for large-scale industrial cultivation. Its cultivation cost is low, and it is non-toxic and harmless; the extracted exosomes are also non-toxic. Integrins are a class of transmembrane glycoproteins, heterodimers formed by an α-subunit and a β-subunit linked by a non-covalent bond. This family contains 24 members. Integrin αvβ3 is the most studied member of the integrin family. It is expressed at low levels or not at all in dormant endothelial cells and other normal tissues, but its expression is abnormally high in various tumor cells and tumor neovascularization endothelial cells, such as in breast cancer and colon cancer. cGRD cyclic peptides are polypeptides composed of arginine, glycine, aspartic acid, and other amino acids, and are ligands for integrin αvβ3. We use cGRD to modify Dunaliella salina exosomes, enabling them to specifically bind to integrin αvβ3, thereby precisely targeting the colon cancer site, increasing drug bioavailability, and reducing side effects.

[0128] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A formula for a special medical food for colorectal cancer patients, characterized in that, The formula of this special medical food contains the following ingredients in the indicated weight proportions: Codonopsis pilosula 20-30 parts, Rehmannia glutinosa 20-30 parts, Astragalus membranaceus 20-30 parts, Coix lacryma-jobi 10-20 parts, Atractylodes macrocephala 10-20 parts, Poria cocos 10-20 parts, Angelica sinensis 10-20 parts, Paeonia lactiflora 10-20 parts, Prunus persica 10-20 parts, Jujube 3-6 pieces, Glycyrrhiza uralensis 3-15 parts, Phycocyanin 6-15 parts, Citric acid 6-15 parts.

2. The formula for a special medical food for colorectal cancer patients according to claim 1, characterized in that, The ingredients contain the following proportions by weight: 20 parts Codonopsis pilosula, 20 parts Rehmannia glutinosa, 20 parts Astragalus membranaceus, 10 parts Coix lacryma-jobi, 10 parts Atractylodes macrocephala, 10 parts Poria cocos, 10 parts Angelica sinensis, 10 parts Paeonia lactiflora, 10 parts Prunus persica, 3 jujubes, 3 parts Glycyrrhiza uralensis, 6 parts Phycocyanin, and 6 parts Citric acid.

3. The formula for a special medical food for colorectal cancer patients according to claim 1, characterized in that, The ingredients contain the following proportions by weight: Codonopsis pilosula 30 parts, Rehmannia glutinosa 30 parts, Astragalus membranaceus 30 parts, Coix lacryma-jobi 20 parts, Atractylodes macrocephala 20 parts, Poria cocos 20 parts, Angelica sinensis 20 parts, Paeonia lactiflora 20 parts, Prunus persica 20 parts, Jujube 6 pieces, Glycyrrhiza uralensis 15 parts, Phycocyanin 15 parts, Citric acid 15 parts.

4. The formula for a special medical food for colorectal cancer patients according to claim 1, characterized in that, The ingredients contain the following proportions by weight: Codonopsis pilosula 25 parts, Rehmannia glutinosa 25 parts, Astragalus membranaceus 25 parts, Coix lacryma-jobi 15 parts, Atractylodes macrocephala 15 parts, Poria cocos 15 parts, Angelica sinensis 15 parts, Paeonia lactiflora 15 parts, Prunus persica 15 parts, Jujube 5 pieces, Glycyrrhiza uralensis 9 parts, Phycocyanin 9 parts, Citric acid 9 parts.

5. A method for preparing a special medical food formula for colorectal cancer patients, as described in any one of claims 1-4, characterized in that... Includes the following steps: Step S1: Weigh the raw materials according to the ratio and place them in a clean 1000mL beaker. Add an appropriate amount of ultrapure water and soak for 30-60 minutes. Then decoct for 1-1.5 hours. Filter with 3-8 layers of gauze. Put the dregs back into ultrapure water and decoct again. After filtering the liquid, centrifuge the two filtered liquids. Take the supernatant, mix and concentrate. Transfer the concentrated liquid to an evaporating dish, seal with plastic wrap and poke several small holes in the plastic wrap. Then freeze dry to obtain the Chinese herbal extract powder. Step S2: After thoroughly mixing the drug powder obtained in step S1, weigh 0.3-0.6g and dissolve it in 1mL of DMSO solution to prepare a stock solution with a concentration of 300-600mg / mL. Take the stock solution, add 99mL of DMEM medium to dilute it, and filter it through a 0.22μm microporous membrane to prepare a stock solution with a concentration of 3-6mg / mL. Step S3: After rapidly thawing the Dunaliella salina cells in the cryopreservation tubes in a 37°C water bath, slowly add them to PKS liquid medium using a pipette. Incubate overnight in an artificial light simulator at 26°C with a light-to-dark ratio of 14:

10. The next day, determine the Dunaliella salina cell concentration using a visible spectrophotometer. Inoculate 50 μL of the Dunaliella salina cell suspension evenly onto PKS solid medium and continue culturing in the artificial light simulator until monoclonal algal colonies are formed. Pick these colonies onto solid medium and inoculate them into 5 mL of liquid medium for 3 days to amplify the cell concentration to 1 × 10⁻⁶ cells / mL. 6 After passing through multiple passages at a rate of / mL, the second to fourth generations of Dunaliella salina are typically selected for exosome extraction. Step S4: Using the Dunaliella salina cell culture medium from Step S3 as a sample, centrifuge at low speed 300-600xg for 5-10 min to separate the cells; retain the supernatant, centrifuge at 2000-3000xg for 10-20 min to remove dead cells, and continue to increase the centrifugation force to 10000-15000xg for 20-30 min to remove cell debris. The processed supernatant is ready for ultracentrifugation at 100,000xg for 60-70 min to obtain crude exosome precipitate (containing a small amount of impurities). Resuspend the exosome precipitate with PBS and centrifuge again at 100,000xg for 70 min to obtain pure exosomes. Step S5: Treat cRGDfk in HEPES solution at 50-60°C for 15 min to form micelles. Then, sonicate the micelles at an amplitude of 8-10 μm for 10-20 seconds to reduce the size of the micelles and promote their separation from EXO. Then, mix the exosome solution obtained in step S4 with the above micelle solution at 40°C for 2-3 h, immediately cool to 4°C, and purify the above mixed solution by centrifugation at 80,000-100,000 x g for 50-70 min to obtain cRGD-EXO solution. Step S6: Using the average fluorescence intensity taken up by cells as an indicator, the mass ratio of EXO and cRGDfk obtained in step S5 was set to 5:1, 1:1, 1:5, 1:10, and 1:

15. Single-factor optimization was performed using flow cytometry, with HCT-116 at 5 × 10⁻⁶ cells / cells. 5 HCT-116 cells were seeded at a density of 1 / mL in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h. After cell attachment, the culture medium was discarded, and different mass ratios of PKH67-labeled cRGD-EXO and EXO were added. The uptake of HCT-116 cells by different proportions of cRGD-EXO and EXO was quantitatively evaluated using fluorescence intensity. Step S7: At room temperature, mix the cRGD-EXO obtained in step S6 with the drug evenly, and incubate on a shaker for 60-90 min at a speed of 200-300 r / min. In order to select the optimal ratio of cRGD-EXO to drug, the mass ratio of cRGD-EXO to drug was set to 10:1, 5:1, 1:1, 1:5, and 1:10, with drug encapsulation efficiency and drug loading as evaluation criteria, and single-factor investigation was conducted. Finally, the cRGD-EXO loaded with traditional Chinese medicine was purified by ultracentrifugation, that is, the purified cRGD-EXO / traditional Chinese medicine was obtained, and the drug content in cRGD-EXO / traditional Chinese medicine was detected by high performance liquid chromatography.

6. The method for preparing a special medical food formula for colorectal cancer patients according to claim 5, characterized in that, In step S1, the ratio of ultrapure water to drug raw materials is 4 to 13:1, the freezing conditions are (-20) to (-80)℃ freezer freezing, and the freezing time is 6 to 8 hours, and the drying conditions are freeze-drying in a freeze dryer at a low temperature (-56℃) to (-60℃) vacuum 10Pa until the drug concentrate is dried.

7. The use of a special medical food formulation as described in any one of claims 1 to 4 or a drug-loaded composition prepared by the method described in any one of claims 5 to 6 in the preparation of a food or medicine for the prevention and / or adjuvant treatment of colorectal cancer.

8. The use of the drug-loaded composition according to claim 7 in the preparation of food or medicine for the prevention and / or adjuvant treatment of colorectal cancer, characterized in that, The dosage form of the food or drug is granules, capsules, oral liquid, tablets, decoction, injection, or enema.

9. A targeted carrier for delivering therapeutic active ingredients for colorectal cancer, characterized in that, The carrier is a Dunaliella salina exosome modified with cRGD polypeptide.

Images