Preparation method of purple corn anthocyanin with efficacy of inhibiting hela cells and Caco-2 cells of colon cancer

Abstract

The invention relates to the technical field of natural product extraction, and discloses a preparation method of purple corn anthocyanin with the efficacy of inhibiting hela cells and Caco-2 cells of colon cancer, and the preparation method comprises the following steps: crushing purple corn cobs to prepare an acid solution; the method comprises the following steps: weighing purple corn cob powder, adding an acid solution, mixing, extracting, and drying an extracting solution to obtain a purple corn anthocyanin crude extract; and carrying out graded alcohol precipitation by using 20%, 40%, 60%, 80% and 100% ethanol solutions, and collecting 20%-40% and 80%-100% ethanol precipitation parts to obtain a target separated extract. According to the purple corn anthocyanin prepared by the preparation method disclosed by the invention, polar components with the highest activity are directionally enriched through an ethanol fractional precipitation process, and the components have a remarkable inhibition effect on the activity of in-vitro cells hela cells and Caco-2 cells of colon cancer and have a remarkable effect of inhibiting the colon cancer.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction technology, and more specifically, to a method for preparing purple corn anthocyanins that have the effect of inhibiting colon cancer HeLa cells and Caco-2 cells. Background Technology

[0002] Purple corn (Zea mays L.), as a distinctive grain resource, is rich in anthocyanins, which have become a hot topic in recent years for research on natural active ingredients. Anthocyanins are a class of water-soluble pigments widely found in plants, belonging to the flavonoid family. Numerous studies have shown that anthocyanins possess a variety of biological activities, including but not limited to powerful antioxidant and free radical scavenging abilities, as well as anti-mutagenic, anti-tumor, anti-cardiovascular disease, and cholesterol absorption-inhibiting effects, showing particular potential application value in inhibiting colon cancer.

[0003] Currently, the main extraction methods for anthocyanins from purple corn include solvent extraction, ultrasonic-assisted extraction, and microwave-assisted extraction. However, existing extraction processes primarily focus on increasing the total extraction rate of anthocyanins, with poor enrichment of specific anthocyanin monomers with colorectal cancer-inhibiting activity (such as cyanidin-3-O-glucoside and pelargonidin-3-O-glucoside). The final product is a crude extract containing all soluble components, without targeted separation and enrichment of the biologically active components. Other studies have attempted purification using macroporous resins, but their aim is either to obtain brightly colored pigments or simply to increase the purity of total anthocyanins, without being associated with specific colorectal cancer-inhibiting activity superior to that of crude extracts. Therefore, existing preparation methods have a significant drawback: the obtained product is a mixture with uncertain composition, and its colorectal cancer-inhibiting efficacy is accidental, failing to guarantee batch-to-batch stability and efficacy. Furthermore, they fail to provide a reliable process for specifically enriching and effectively inhibiting colorectal cancer components. Furthermore, most studies remain at the level of total extract activity, lacking in-depth analysis of the synergistic effects of different polar components and monomeric components, as well as the structure-activity relationship between them and specific colorectal cancer inhibitory activities. This results in a low proportion of highly active ingredients in the prepared anthocyanin products, limiting their colorectal cancer inhibitory effects and making it difficult to meet the application needs of the pharmaceutical, health product, and other fields.

[0004] Therefore, developing a method for preparing purple corn anthocyanins that can effectively enrich highly active components that inhibit colon cancer, and overcoming the technical defects of insufficient enrichment of active ingredients in existing processes, is of great practical significance. Summary of the Invention

[0005] In view of this, the present invention proposes a method for preparing purple corn anthocyanins that can effectively enrich highly active components that inhibit colon cancer, aiming to solve the problems in the current technology.

[0006] On one hand, this invention proposes a method for preparing purple corn anthocyanins with inhibitory effects on colon cancer HeLa cells and Caco-2 cells, characterized by comprising the following steps: (1) Take purple corn cobs, crush them into purple corn cob powder of 20~500 mesh, and prepare an acid solution with pH=2~5.5. The acid solution is one or two of hydrochloric acid, citric acid, malic acid, tartaric acid, acetic acid, phosphoric acid, taurine, fumaric acid and lactic acid. (2) The purple corn cob powder is mixed with an acid solution and then subjected to ultrasonic extraction. After ultrasonic extraction, static extraction is performed. After extraction, the mixture is filtered or centrifuged to obtain the extract. (3) The extract is dried to obtain crude extract of purple corn anthocyanins; (4) The crude extract of purple corn anthocyanin was subjected to fractional ethanol precipitation with 20%, 40%, 60%, 80% and 100% ethanol solutions in sequence, and the ethanol precipitates of 20%~40% and 80%~100% were collected to obtain purple corn anthocyanin with anti-colon cancer HeLa cells and Caco-2 cells.

[0007] More preferably, the ethanol precipitation portion collected in step (4) includes a first component obtained by precipitation from a 20% to 40% ethanol solution and a second component obtained by precipitation from an 80% to 100% ethanol solution; the first component and the second component are used separately or mixed in any proportion.

[0008] More preferably, the ratio of purple corn cob powder to acid solution in step (2) is 1:6-1:50.

[0009] More preferably, the ultrasonic extraction time in step (2) is 30~180 min and the extraction temperature is 20~90℃.

[0010] More preferably, the static extraction time in step (2) is 30~360 min and the extraction temperature is 20~90℃.

[0011] More preferably, the drying method in step (3) is selected from one of hot air drying, vacuum drying, and freeze drying.

[0012] On the other hand, this application also provides an application of purple corn anthocyanin, which is prepared according to the above-mentioned preparation method, characterized in that it includes the application of purple corn anthocyanin in the preparation of food and medicine that inhibit colon cancer HeLa cells and Caco-2 cells.

[0013] Compared with existing technologies, the present invention provides a method for preparing purple corn anthocyanins that inhibits the activity of colon cancer HeLa cells and Caco-2 cells, which has the following significant beneficial effects: 1. It achieved targeted enrichment of active ingredients that inhibit colon cancer, with clear targets and significantly enhanced activity. Existing technologies produce crude extracts of purple corn anthocyanins with complex compositions and unclear active ingredients, resulting in limited and unstable inhibitory effects against colon cancer. This invention creatively employs an ethanol fractionation precipitation process to successfully separate and enrich polarly specific components from the crude extract. These enriched components exhibit significantly superior inhibitory activity against colon cancer compared to the unseparated crude extract (whole powder) and other polar fractions. This represents a leap from "crude extraction" to "refined extraction," resolving the problems of unclear active ingredients and poor efficacy in existing technologies. 2. The process is stable and reliable, easy to scale up, and suitable for industrial production. The ultrasonic-assisted extraction and ethanol fractionation precipitation methods used in this invention are both mature and easily controllable unit operations in the field, requiring conventional equipment and controllable costs. The entire process is clear, highly reproducible, and does not involve complex or harsh reaction conditions, which is very conducive to the transformation from laboratory to large-scale production. This lays a solid industrial foundation for the development of high-value-added purple corn anthocyanins for inhibiting colon cancer in foods, drugs, or health products.

[0014] By optimizing the extraction agent system, the stability and extraction efficiency of the active ingredients were significantly improved. Compared to conventional extraction systems using single inorganic acids or simple organic acids, this invention creatively screens and optimizes the composition of the acid solution. Specifically, it employs a composite acidic extractant containing specific organic acids. This system not only provides a suitable pH environment, but its specific components also interact beneficially with anthocyanin molecules from purple corn, more effectively stabilizing the active structure of anthocyanins during extraction and reducing their degradation, thus ensuring the high activity of the final product from the source. This optimized extractant, synergistically with the subsequent fractional precipitation process, constitutes the key to obtaining highly inhibitory active components.

[0015] In summary, this invention has successfully prepared a purple corn anthocyanin product with significantly enhanced activity against colon cancer, clearly defined components, and controllable quality through a specific separation and enrichment process. This effectively overcomes the shortcomings of existing technologies and has outstanding substantive features and significant progress. Attached Figure Description

[0016] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings: Figure 1 A flowchart illustrating a method for preparing purple corn anthocyanins that inhibits colon cancer HeLa cells and Caco-2 cells, as provided in this embodiment of the invention; Figure 2 This is a schematic diagram illustrating the effect of 5-fluorouracil on HeLa cell activity according to an embodiment of the present invention. Figure 3 This is a schematic diagram illustrating the effect of 5-fluorouracil on the activity of Caco-2 cells according to an embodiment of the present invention. Figure 4 A schematic diagram showing the effect of sample 1 (20-40% fraction) provided in this embodiment of the invention on the activity of HeLa cells; Figure 5 A schematic diagram showing the effect of sample 1 (20%~40% of the sample) provided in this embodiment of the invention on the activity of Caco-2 cells; Figure 6 A schematic diagram illustrating the effect of sample 2 (40%~60% of the sample) provided in this embodiment of the invention on the activity of HeLa cells; Figure 7 A schematic diagram illustrating the effect of sample 2 (40%~60% of the sample) provided in this embodiment of the invention on the activity of Caco-2 cells; Figure 8 A schematic diagram illustrating the effect of sample 3 (60%~80% of the sample) provided in this embodiment of the invention on the activity of HeLa cells; Figure 9 A schematic diagram illustrating the effect of sample 3 (60%~80% of the sample) provided in this embodiment of the invention on the activity of Caco-2 cells; Figure 10 A schematic diagram showing the effect of sample 4 (80%~100% of the sample) provided in this embodiment of the invention on the activity of HeLa cells; Figure 11 This is a schematic diagram showing the effect of sample 4 (80%~100% of the sample) provided in the embodiment of the present invention on the activity of Caco-2 cells. Figure 12 A schematic diagram showing the effect of sample 5 (whole powder) on HeLa cell activity in an embodiment of the present invention; Figure 13 This is a schematic diagram showing the effect of sample 5 (whole powder) provided in the embodiment of the present invention on the activity of Caco-2 cells. Detailed Implementation

[0017] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present disclosure and to fully convey the scope of the disclosure to those skilled in the art. It should be noted that, unless otherwise specified, embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0018] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0021] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0022] This invention proposes a method for preparing purple corn anthocyanins with inhibitory effects on HeLa and Caco-2 colon cancer cells, characterized by comprising the following steps: (1) Take purple corn cobs, crush them into purple corn cob powder of 20~500 mesh, and prepare an acid solution with pH=2~5.5. The acid solution is one or two of hydrochloric acid, citric acid, malic acid, tartaric acid, acetic acid, phosphoric acid, taurine, fumaric acid and lactic acid. (2) The purple corn cob powder is mixed with an acid solution and then subjected to ultrasonic extraction. After ultrasonic extraction, static extraction is performed. After extraction, the mixture is filtered or centrifuged to obtain the extract. (3) The extract is dried to obtain crude extract of purple corn anthocyanins; (4) The crude extract of purple corn anthocyanin was subjected to fractional ethanol precipitation with 20%, 40%, 60%, 80% and 100% ethanol solutions in sequence, and the ethanol precipitates of 20%~40% and 80%~100% were collected to obtain purple corn anthocyanin with the effect of inhibiting colon cancer HeLa cells and Caco-2 cells.

[0023] Step (1) Take purple corn cobs, crush them into 20-500 mesh purple corn cob powder, and prepare an acid solution with pH=2-5.5. The acid solution is one or two of hydrochloric acid, citric acid, malic acid, tartaric acid, acetic acid, phosphoric acid, taurine, fumaric acid, and lactic acid. The present invention firstly involves pulverizing and cooling a certain amount of purple corn cobs dried to constant weight, and then transferring the resulting purple corn cob powder into a plastic bag for storage in the dark; weighing an acid solid and placing it in a beaker, measuring pure water, while continuously stirring with a glass rod, and correcting the pH value with a pH meter, and transferring the resulting acid solution with a pH of 2 to 5.5 into a wide-mouth bottle for storage.

[0024] Understandably, physically disrupting the cell structure of purple corn cobs increases the contact area between the raw material and the extraction solvent. Heat is generated during the pulverization process; if not cooled promptly, the high temperature can damage the chemical structure of anthocyanins, leading to their inactivation. The chemical structure of anthocyanins changes with the pH of the environment. Under acidic conditions, anthocyanins exist in a stable form; however, under neutral or alkaline conditions, their structure alters, their color fades, and they rapidly decompose and become ineffective. Creating an acidic environment helps maintain their chemical stability and biological activity, preventing damage during extraction.

[0025] Step (2) Mix the purple corn cob powder with an acid solution, and then perform ultrasonic extraction. After ultrasonic extraction, perform static extraction again. After extraction, filter or centrifuge to obtain the extract. In this invention, the preferred material-to-liquid ratio of the purple corn cob powder to the acid solution is 1:6-1:50, the preferred ultrasonic extraction time is 30-180 min, and the preferred extraction temperature is 20-90℃; the preferred static extraction time is 30-360 min, and the preferred extraction temperature is 20-90℃.

[0026] Understandably, the extraction employs a synergistic "ultrasonic-static" extraction principle. The ultrasonic cavitation effect instantaneously disrupts cell walls, prompting rapid release of contents; subsequent static extraction utilizes concentration gradient-driven diffusion to ensure full dissolution of active ingredients. The feed-to-liquid ratio and temperature range jointly optimize mass transfer efficiency and extraction kinetics. Centrifugation, as a key solid-liquid separation method, efficiently retains solids, ensuring a clear extract and laying the foundation for subsequent concentration and enrichment steps.

[0027] Step (3) Dry the extract obtained in step (2) to obtain crude extract of purple corn anthocyanins; In this invention, the drying method is selected from one of hot air drying, vacuum drying, and freeze drying.

[0028] Understandably, drying can quickly remove a large amount of solvent from the extract, allow ice crystals to sublimate directly, and completely remove residual moisture, resulting in a stable, easy-to-store, and fully active crude extract of purple corn anthocyanins in solid powder form, providing standardized raw materials for subsequent ethanol fractionation and precipitation.

[0029] Step (4) The crude extract of purple corn anthocyanins is subjected to fractional ethanol precipitation with 20%, 40%, 60%, 80%, and 100% ethanol solutions in sequence, and the ethanol precipitates of 20%~40% and 80%~100% are collected to obtain purple corn anthocyanins that have the effect of inhibiting colon cancer HeLa cells and Caco-2 cells.

[0030] In this invention, the collection of ethanol precipitate in step (4) includes a first component obtained by precipitation from a 20% to 40% ethanol solution and a second component obtained by precipitation from an 80% to 100% ethanol solution; the first component and the second component are used separately or mixed in any proportion.

[0031] Understandably, this invention, through the innovative use of an ethanol fractionation precipitation process, successfully separates and enriches polarly specific components from the crude extract. The enriched components exhibit significantly superior inhibitory activity against colon cancer compared to the unseparated crude extract (whole powder) and other polar fractions. This represents a leap from "crude extraction" to "refined extraction," resolving the problems of unclear active ingredients and poor efficacy in existing technologies.

[0032] The present invention also provides an application of purple corn anthocyanin, which is prepared according to the above preparation method, characterized in that it includes the application of purple corn anthocyanin in the preparation of food and medicine that inhibit colon cancer HeLa cells and Caco-2 cells.

[0033] It is understood that the process flow of this invention is clear, highly reproducible, and does not involve complex or harsh reaction conditions, which is highly conducive to the transformation from laboratory to large-scale production. Compared with conventional single inorganic acid or simple organic acid extraction systems, this invention has creatively screened and optimized the composition of the acid solution. In particular, the use of a composite acidic extractant containing specific organic acids, in synergy with the subsequent fractional precipitation process, constitutes the key to obtaining highly inhibitory active components, laying a solid industrial foundation for the development of high-value-added drugs, foods, or health products that inhibit colon cancer using purple corn anthocyanins.

[0034] Example 1 I. Experimental Procedure 1. Crude extraction of anthocyanins from purple corn (1) A certain amount of purple corn cobs dried to constant weight were pulverized and cooled in a high-speed pulverizer, and the resulting ultrafine powder of purple corn cobs was transferred to a plastic bag for storage in the dark. 0.420g of citric acid solid was weighed and placed in a beaker, and 1000ml of ultrapure water was measured. At the same time, the mixture was stirred continuously with a glass beaker, and the pH value was corrected with a pH meter. The resulting citric acid solution with pH=4 was transferred to a wide-mouth bottle for storage.

[0035] (3) Accurately weigh 1.0000g of purple corn cob ultrafine powder into a 50ml centrifuge tube, and use citric acid solution with pH=4 as the extraction solvent to mix the purple corn cob ultrafine powder thoroughly. The purple corn anthocyanin was extracted by ultrasonic-assisted citric acid-water solvent extraction. First, the purple corn cob sample solution was placed in a high-speed centrifuge and centrifuged at 8000r / min for 10min. After being transferred to an ultrasonic cleaner and sonicated for the required time, it was statically extracted in a 40℃ constant temperature water bath. Then, it was vacuum filtered, rotary evaporated, and placed in an ultra-low temperature storage box at -80℃ for at least 24h before being freeze-dried in a freeze dryer.

[0036] (4) The purple corn anthocyanin extract obtained from the crude extraction was used as the main separation sample. It was vacuum concentrated, the reagent was evaporated, and then it was sequentially extracted by fractional alcohol precipitation with different concentrations of ethanol (20%, 40%, 60%, 80%, 100%) to obtain separation extracts of different polarities (sample 1 (20%~40% fraction), sample 2 (40~60% fraction), sample 3 (60~80% fraction), sample 4 (80%~100% fraction), sample 5 (whole powder)). The separation samples were vacuum concentrated, freeze-dried, and the reagent was evaporated for later use in in vitro cell activity screening studies.

[0037] 2. Screening of in vitro cell activity of purple corn anthocyanins against colon cancer Extracts with different polarities were used in an in vitro growth inhibition experiment on colon cancer cells, with the MTT assay used as the activity screening method. The specific experimental steps are as follows: (1) Take Caco-2 and HeLa cells that are in the logarithmic growth phase and in good growth condition, and use 5×10 3 Cells / well were seeded into 96-well cell culture plates and incubated overnight at 37°C in a 5% CO2 incubator.

[0038] (2) The cells were treated according to the following grouping: Grouping: Group A: HeLa cells Group B: HeLa cells + 1 mg / mL medication Group C: HeLa cells + 5 mg / mL drug Group D: HeLa cells + 10 mg / mL medication Group E: HeLa cells + 15 mg / mL drug Group F: HeLa cells + 20 mg / mL drug Group G: Caco-2 cells Group H: Caco-2 cells + 1 mg / mL Group J: Caco-2 cells + 5 mg / mL drug Group K: Caco-2 cells + 10 mg / mL drug Group L: Caco-2 cells + 15 mg / mL drug Group M: Caco-2 cells + 20 mg / mL drug Treatment time: Five anthocyanin extracts and 5-fluorouracil were treated for 24 hours according to their respective group concentrations.

[0039] After the required cell culture time, add 10 μl MTT to each well and incubate at 37°C for 1 h; aspirate the culture medium, add 150 μl DMSO and shake for 10 min; measure the absorbance of each well with an ELISA reader to determine the optimal isolation site for anti-colon cancer activity.

[0040] II. Experimental Results (I) Screening and component identification of the anti-colon cancer cell activity of purple corn anthocyanins 1. Screening study on the in vitro cell activity of purple corn anthocyanins against colon cancer (1) Effects of 5-fluorouracil on the in vitro cell viability of colon cancer cells The effect of 2-fluorouracil on the activity of HeLa cells was determined, and the results were obtained. Figure 2 The effect of 5-fluorouracil on the activity of Caco-2 cells was determined, and the results were obtained. Figure 3 .from Figure 2 , Figure 3 It can be seen that the 5-fluorouracil control group has a significant inhibitory effect on both HeLa cells and Caco-2 cells in vitro. With the increase of 5-fluorouracil concentration, the cell activity of HeLa cells and Caco-2 cells decreased rapidly, and the proliferation rate of 20 mg / mL 5-fluorouracil was only 8.90%.

[0041] (2) Effect of Sample 1 (20-40% of the sample) on the in vitro viability of colon cancer cells The effect of sample 1 (20%–40% of the sample) on the viability of HeLa cells was determined, and the results were obtained. Figure 4 The effect of sample 1 (20-40% of the sample) on the viability of Caco-2 cells was determined, and the results were obtained. Figure 5 .from Figure 4, Figure 5 It can be seen that Sample 1 (20%~40% fraction) has a certain inhibitory effect on both HeLa cells and Caco-2 cells in vitro. As the concentration of Sample 1 (20%~40% fraction) increases, the cell activity of HeLa cells and Caco-2 cells gradually decreases. The proliferation rate of HeLa cells with 20 mg / mL Sample 1 (20%~40% fraction) added is 69.14%, and the proliferation rate of Caco-2 cells with 20 mg / mL Sample 1 (20%~40% fraction) added is 65.21%.

[0042] (3) Effect of Sample 2 (40%~60% of the sample) on the in vitro viability of colon cancer cells The effect of sample 2 (40%–60% of the sample) on the viability of HeLa cells was determined, and the results were obtained. Figure 6 The effect of sample 2 (40%–60% of the sample) on the viability of Caco-2 cells was determined, and the results were obtained. Figure 7 ;from Figure 6 , Figure 7 It can be seen that, compared with sample 1, sample 2 (40%~60% fraction) had a smaller inhibitory effect on HeLa cells and Caco-2 cells in vitro. As the concentration of sample 2 (40%~60% fraction) increased, the cell activity of HeLa cells and Caco-2 cells decreased slowly. The proliferation rate of HeLa cells with 20 mg / mL sample 2 (40%~60% fraction) was 89.88%, and the proliferation rate of Caco-2 cells with 20 mg / mL sample 2 (40%~60% fraction) was 87.10%.

[0043] (4) Effect of sample 3 (60%~80% of the sample) on the in vitro viability of colon cancer cells. The effect of sample 3 (60%–80% of the sample) on the viability of HeLa cells was determined, and the results were obtained. Figure 8 The effect of sample 3 (60%–80% of the sample) on the viability of Caco-2 cells was determined, and the results were obtained. Figure 9 .from Figure 8 , Figure 9 It can be seen that sample 3 (60%~80% fraction) has a slightly better inhibitory effect on HeLa cells and Caco-2 cells in vitro than sample 2, but it is still worse than sample 1. As the concentration of sample 3 (60%~80% fraction) increases, the cell activity of HeLa cells and Caco-2 cells decreases slowly. The proliferation rate of HeLa cells with 20 mg / mL sample 3 (60%~80% fraction) added is 75.19%, and the proliferation rate of Caco-2 cells with 20 mg / mL sample 3 (60%~80% fraction) added is 77.33%.

[0044] (5) Effect of sample 4 (80%~100% of the sample) on the in vitro viability of colon cancer cells. The effect of sample 4 (80%–100% of the sample) on the viability of HeLa cells was determined, and the results were obtained. Figure 10 The effect of sample 4 (80%–100% concentration) on the viability of Caco-2 cells was determined, and the results were obtained. Figure 11 .from Figure 10 , Figure 11 It can be seen that sample 4 (80%–100% fraction) has a significant inhibitory effect on both HeLa cells and Caco-2 cells in vitro. With increasing concentration of sample 4 (80%–100% fraction), the cell activity of both HeLa and Caco-2 cells rapidly decreased. The proliferation rate of HeLa cells with 20 mg / mL sample 4 (80%–100% fraction) was 41.46%, and the proliferation rate of Caco-2 cells with 20 mg / mL sample 4 (80%–100% fraction) was 62.80%. Among the four fractions, sample 4 showed the best inhibitory effect.

[0045] (6) Effect of Sample 5 (whole powder) on the in vitro cell viability of colon cancer cells The effect of sample 5 (whole powder) on HeLa cell viability was determined, and the results were obtained. Figure 12 The effect of sample 5 (whole powder) on the activity of Caco-2 cells was determined, and the results were obtained. Figure 13 .from Figure 12 , Figure 13 It can be seen that Sample 5 (whole powder) also has a significant inhibitory effect on HeLa cells and Caco-2 cells in vitro. With the increase of the concentration of Sample 5 (whole powder), the cell activity of HeLa cells and Caco-2 cells decreased rapidly. The proliferation rate of HeLa cells with 20 mg / mL Sample 5 (whole powder) was 55.40%, and the proliferation rate of Caco-2 cells with 20 mg / mL Sample 5 (whole powder) was 64.20%. The inhibitory effect of the whole powder was second only to that of Sample 4.

[0046] Based on the above results, it can be seen that the 5-fluorouracil control group had a significant inhibitory effect on both HeLa cells and Caco-2 cells in vitro for colon cancer. The inhibitory effects of each sample group on HeLa cells and Caco-2 cells in vitro for colon cancer were as follows: Sample 4 (80%~100% fraction) > Sample 5 (whole powder) > Sample 1 (20%~40% fraction) > Sample 3 (60%~80% fraction) > Sample 2 (40%~60% fraction). Among them, the cell proliferation rates of Sample 1, Sample 4, and Sample 5 were all less than 70%, indicating that (20%~40%), (80%~100%), and whole powder had better anti-colon cancer effects, and Sample 4 (80%~100%) had the best anti-colon cancer effect, which was better than that of whole powder.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing purple corn anthocyanins with inhibitory effects on colon cancer HeLa cells and Caco-2 cells, characterized in that, Includes the following steps: (1) Take purple corn cobs, crush them into purple corn cob powder of 20~500 mesh, and prepare an acid solution with pH=2~5.

5. The acid solution is one or two of hydrochloric acid, citric acid, malic acid, tartaric acid, acetic acid, phosphoric acid, taurine, fumaric acid and lactic acid. (2) The purple corn cob powder is mixed with an acid solution and then subjected to ultrasonic extraction. After ultrasonic extraction, static extraction is performed. After extraction, the mixture is filtered or centrifuged to obtain the extract. (3) The extract is dried to obtain crude extract of purple corn anthocyanins; (4) The crude extract of purple corn anthocyanin was subjected to fractional ethanol precipitation with 20%, 40%, 60%, 80% and 100% ethanol solutions in sequence, and the 20%~40% and 80%~100% ethanol precipitates were collected to obtain purple corn anthocyanin with the effect of inhibiting colon cancer HeLa cells and Caco-2 cells.

2. The preparation method according to claim 1, characterized in that, The collection of ethanol precipitate in step (4) includes a first component obtained by precipitation from a 20% to 40% ethanol solution and a second component obtained by precipitation from an 80% to 100% ethanol solution; the first component and the second component are used separately or mixed in any proportion.

3. The preparation method according to claim 1, characterized in that, The ratio of purple corn cob powder to acid solution in step (2) is 1:6-1:

50.

4. The preparation method according to claim 1, characterized in that, The ultrasonic extraction time in step (2) is 30~180 min, and the extraction temperature is 20~90℃.

5. The preparation method according to claim 1, characterized in that, The static extraction time in step (2) is 30~360 min, and the extraction temperature is 20~90℃.

6. The preparation method according to claim 1, characterized in that, The drying method described in step (3) is selected from one of hot air drying, vacuum drying, and freeze drying.

7. An application of purple corn anthocyanin, wherein the purple corn anthocyanin is prepared by the preparation method according to any one of claims 1-6, characterized in that, include: Application of purple corn anthocyanins in the preparation of foods and drugs that inhibit colon cancer HeLa cells and Caco-2 cells.

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