阅读说明：本技术 一种猴头菇抗人结肠癌肿瘤细胞（hct-8）提取物的制备方法 (Preparation method of hericium erinaceus anti-human colon cancer tumor cell (HCT-8) extract ) 是由 陈晓明 丁舒 陈颖 宋兆伟 崔瀚元 张越 李淑芳 陈龙 张晓� 张峻 张志军 于 2021-09-06 设计创作，主要内容包括：本发明提供一种猴头菇抗人结肠癌肿瘤细胞(HCT-8)提取物的制备方法,采用超临界CO-(2)萃取装置萃取得到猴头菇提取物,其制备成本相对较低,所制备的提取物对人结肠癌肿瘤细胞(HCT-8)增殖抑制活性强,且工序简单,无溶剂残留,食用安全,既可以开发保健功能食品,也可以开发药品,因此本发明具有很好的推广价值和市场潜力。同时,本发明也提供了以开发猴头菇精深加工新方向,丰富了猴头菇高值化加工技术,为建立和完善其他食用菌多级联产的加工技术体系奠定基础。本发明普遍适用于猴头菇。(The invention provides a preparation method of a hericium erinaceus anti-human colon cancer tumor cell (HCT-8) extract, which adopts supercritical CO 2 The hericium erinaceus extract is obtained by extraction of the extraction device, the preparation cost is relatively low, the prepared extract has strong proliferation inhibition activity on human colon cancer tumor cells (HCT-8), the process is simple, no solvent residue is generated, the hericium erinaceus extract is safe to eat, and health-care functional food and medicines can be developed. Meanwhile, the invention also provides a new direction for developing the fine and deep processing of the hericium erinaceus, enriches the high-valued processing technology of the hericium erinaceus and aims at establishing and perfecting the processing technology body of the multi-stage co-production of other edible fungiLays a foundation. The invention is generally applicable to hericium erinaceus.)

1. A preparation method of a hericium erinaceus HCT-8 extract for resisting human colon cancer tumor cells is characterized by comprising the following steps:

1) firstly, crushing a dried hericium erinaceus product;

2) putting the hericium erinaceus powder crushed in the step 1) into an extraction kettle of a supercritical CO2 extraction device, setting supercritical CO2 extraction condition parameters, and obtaining a hericium erinaceus supercritical CO2 extract after the extraction is finished, wherein the supercritical extraction condition parameters are as follows: the extraction pressure is 20-40Mpa, the extraction temperature is 25-45 ℃, the separation kettle is 3-8 Mpa, the entrainer absolute ethyl alcohol is 4-10 mL g-1, and the extraction time is 0.5-2.5 h;

3) vacuum concentrating the hericium erinaceus supercritical CO2 extract obtained in the step 2), and after all absolute ethyl alcohol is recovered, finishing concentration to obtain the concentrated hericium erinaceus extract.

4) Filtering the concentrated hericium erinaceus extract obtained in the step 3) by a cellulose acetate CA membrane, collecting filtrate, and preparing the hericium erinaceus HCT-8 extract for resisting human colon cancer tumor cells through the steps.

2. The method for preparing the hericium erinaceus HCT-8 extract as claimed in claim 1, wherein the extract comprises the following steps: in the step 2), the concentration temperature is 60-70 ℃, and the vacuum degree is 0.5-0.6 MPa.

3. The method for preparing the hericium erinaceus HCT-8 extract as claimed in claim 1, wherein the extract comprises the following steps: in the step 3), the aperture of the cellulose acetate CA membrane is 0.2-0.8 μm.

4. The method for preparing the hericium erinaceus HCT-8 extract as claimed in claim 1, wherein the extract comprises the following steps: the number of the crushed meshes in the step 1) is 20-120 meshes.

Technical Field

The invention provides a preparation method of a hericium erinaceus anti-human colon cancer tumor cell (HCT-8) extract, belonging to the technical field of agricultural product processing.

Background

Hericium erinaceus (Hericium erinaceus) belongs to large edible fungi and medicinal fungi of Hericium of Hydnaceae of Basidiomycotina, Basidiomycetes, Polyporales, and Basidiomycetes, and is commonly called Hericium erinaceus, Hedgehog fungus, Hypsizygus marmoreus, Hericium erinaceus, cauliflower fungi, and Tricholoma giganteum. China is an important production area of the hericium erinaceus, and various provinces in northeast and Henan, Hebei, Tibet, Shanxi, Sichuan and the like have production. The hericium erinaceus is rich in nutrition, contains rich protein, fat, cellulose, polysaccharide and 16 amino acids, wherein 8 amino acids which are necessary for human bodies are famous medical and edible dual-purpose bacteria, are tender and delicious, are delicacies on banquets, are often combined with bear palms, sea cucumbers and shark fins as four famous dishes by people, and have the saying of 'mountain delicacies hericium erinaceus and sea-flavor cubilose'. The hericium erinaceus has unique medicinal value, is considered by traditional Chinese medicine to be neutral in nature, sweet in taste, beneficial to five internal organs, capable of promoting digestion, nourishing and treating neurasthenia. Researches show that the traditional Chinese medicine composition has the curative effects of resisting ulcer, resisting inflammation, resisting aging, protecting liver, reducing blood sugar, reducing blood fat, reducing blood pressure and the like, and has wide development and application prospects.

At present, related documents report the application of hericium erinaceus polysaccharide in preventing and treating colon cancer, documents introduce tea containing hericium erinaceus for conditioning colorectal cancer, report that hericium erinaceus extract has an improvement effect on Dextran Sodium Sulfate (DSS) -induced colitis in mice, and study on inhibition of helicobacter pylori by hericium erinaceus fermentation liquor is related. The related literature reports that the neurotoxicity of rat pheochromocytoma PC12 cells extracted from fruiting bodies and mycelia of hericium erinaceus has a protective effect.

In the aspect of extraction research of hericium erinaceus, documents disclose a hericium erinaceus in-vitro efficient antioxidant component, and an extraction method and application thereof. Related documents introduce a method for preparing a hericium erinaceus fruiting body extract, an effervescent tablet containing the extract and an application of the hericium erinaceus fruiting body extract, and the effervescent tablet has the effects of resisting oxidation, reducing blood sugar, enhancing immunity, resisting aging, maintaining gastrointestinal tract health and the like. There are documents that hericium erinaceus/shiitake mushroom beta-glucan is extracted by adopting a compound enzyme method to assist hot water leaching, and the extraction process conditions are optimized through orthogonal experiments. There are documents that a novel fungal chitin is extracted from hericium erinaceus residues by adopting a mild chemical sequential extraction method, and the influence of deproteinization conditions (NaOH concentration, reaction temperature and reaction time) on chitin yield, purity, molecular weight and acetylation degree is studied. But the extraction process used is not a supercritical CO2 extraction process.

In summary, the basic research of hericium erinaceus at present mainly focuses on the aspects of pharmacology and health care efficacy, and the functional activity of hericium erinaceus has a large development space, but no relevant report that the extract prepared by the method disclosed by the invention has anti-colon cancer activity exists at present. Therefore, the research on the hericium erinaceus for enriching the anti-colon cancer active substances is necessary by combining the early research foundation of high-valued fine and deep processing of the edible fungi, so that a new direction for fine and deep processing of the hericium erinaceus is developed, high-valued processing technologies of the hericium erinaceus are enriched, and a foundation is laid for establishing and perfecting a processing technology system for multi-stage co-production of other edible fungi.

Disclosure of Invention

The invention aims to provide a preparation method of a hericium erinaceus anti-human colon cancer tumor cell (HCT-8) extract, which comprises the following steps:

1) firstly, crushing a dried hericium erinaceus into 20-120 meshes;

2) putting the hericium erinaceus powder crushed in the step 1) into supercritical CO₂In an extraction kettle of the extraction device, supercritical CO is set₂Extracting condition parameters, and obtaining the hericium erinaceus supercritical CO after extraction is finished₂And (3) extracting. Wherein, the supercritical extraction condition parameters are as follows: the extraction pressure is 20-40Mpa, the extraction temperature is 40-50 ℃, the pressure of a separation kettle I is 7-9Mpa, the temperature is 30-40 ℃, the pressure of a separation kettle II is 3-5Mpa, the temperature is 20-30 ℃, and the entrainer absolute ethyl alcohol is 4-10 mL/g^-1The extraction time is 0.5-2.5 h;

3) subjecting the hericium erinaceus obtained in the step 2) to supercritical CO₂And (3) concentrating the extract in vacuum, and after all the absolute ethyl alcohol is recovered, finishing the concentration to obtain the concentrated hericium erinaceus extract. Wherein the concentration temperature is 60-70 ℃, and the vacuum degree is 0.5-0.6 MPa.

4) Filtering the concentrated hericium erinaceus extract obtained in the step 3) through a 0.2-0.8-micron Cellulose Acetate (CA) membrane, and collecting filtrate.

Through the steps, the hericium erinaceus anti-human colon cancer tumor cell (HCT-8) extract is prepared.

The invention provides a preparation method of a hericium erinaceus anti-human colon cancer tumor cell (HCT-8) extract, which is invented in the process of solving the key technical link in a hericium erinaceus multistage co-production system according to the physiological growth characteristics of the hericium erinaceus and combining with the professional bases of agricultural product processing raw material science, natural product research, biochemistry and the like. The extract prepared by the invention has extremely strong inhibiting effect on the in vitro growth of human colon cancer tumor cells (HCT-8). Research shows that the hericium erinaceus extract prepared by the method of the invention and the screened IC₅₀The value (half inhibitory concentration) is 0.17-0.21 μ g/ml, and the inhibition rate on HCT-8 cell proliferation reaches 70% or more, wherein the extract obtained under the optimal extraction condition is used for treating HCT-8 cells with fine particles at the final concentration of 5 μ g/mlThe cell proliferation inhibition rate reaches 71.67 percent, and the rescreened IC₅₀The value (semi-inhibitory concentration) is 0.17 mug/ml, the inhibition rate of positive drug 5-fluorouracil with the same concentration is 74.06%, and the IC of the rescreening is₅₀The value (half inhibitory concentration) was 1.59. mu.g/ml, and the antitumor activity against colon cancer of the extract was very significant.

As can be seen from FIGS. 2 and 3, the fruiting body powder of Hericium erinaceus extracted by supercritical CO2 has significant changes in appearance and microstructure. The edible fungus mycelium has extremely firm cell walls, and under the pressure state of 20-40MPa, obvious structural change appears when the cell walls are pressurized and decompressed. Further investigation was needed as to whether the cell wall of the submerged mycelium in supercritical CO2 had a change in permeability. The supercritical CO2 extraction method is helpful for improving the breaking degree of the cell wall of the fruiting body of the hericium erinaceus and is more beneficial to further processing and absorption and utilization by human bodies. Separating and vacuum concentrating the supercritical extract of the hericium erinaceus, volatilizing the solvent, and performing primary GC-MS analysis and detection, wherein the result shows that: the Hericium erinaceum extract contains palmitic acid, ethyl linoleate, linoleic acid, etc.

The preparation method of the hericium erinaceus extract for resisting the human colon cancer tumor cells (HCT-8) provided by the invention is relatively low in preparation cost, the prepared extract is high in activity for resisting the human colon cancer tumor cells (HCT-8), the process is simple, no solvent residue is generated, the extract is safe to eat, and health-care functional food and medicines can be developed. Meanwhile, the invention also provides a new direction for developing the fine and deep processing of the hericium erinaceus, enriches the high-valued processing technology of the hericium erinaceus, and lays a foundation for establishing and perfecting a processing technology system for multi-stage co-production of other edible fungi.

Drawings

FIG. 1: the hericium erinaceus extract (1 st sampling of separation kettle I and II, 2 nd sampling of separation kettle I and II in sequence from left to right in the figure).

FIG. 2: comparing the appearance of the fruiting body powder of Hericium erinaceus before and after extraction (a. before and b. after extraction).

FIG. 3: comparing the microstructure of the extracted Hericium erinaceus fruiting body powder before and after extraction (a. before extracting Hericium erinaceus fruiting body powder; b. after extracting Hericium erinaceus fruiting body powder).

FIG. 4: and (4) detecting the GC-MS of the supercritical CO2 extract of the hericium erinaceus.

FIG. 5: effect of hericium erinaceus extract on mRNA expression.

FIG. 6: effect of hericium erinaceus extract on mouse body weight.

FIG. 7: effect of hericium erinaceus extract on tumor volume in mice.

Detailed Description

The processing method of the present invention will be described below with reference to specific examples, but the present invention is not limited thereto. The reagents and materials and equipment are commercially available, unless otherwise specified.

Example 1

A preparation method of Hericium erinaceus (Hericium erinaceus) anti-human colon cancer tumor cell (HCT-8) extract comprises the following steps:

1) firstly, crushing the dried hericium erinaceus into 20 meshes;

2) putting the hericium erinaceus powder crushed in the step 1) into supercritical CO₂In an extraction kettle of the extraction device, supercritical CO is set₂Extracting condition parameters, and obtaining the hericium erinaceus supercritical CO after extraction is finished₂The extract, wherein the supercritical extraction condition parameters are as follows: the extraction pressure is 40Mpa, the extraction temperature is 50 ℃, the pressure of the separation kettle I is 9Mpa, the temperature is 40 ℃, the pressure of the separation kettle II is 5Mpa, the temperature is 30 ℃, the entrainer absolute ethyl alcohol is 4mL g^-1The extraction time is 2.5 h;

3) subjecting the hericium erinaceus obtained in the step 2) to supercritical CO₂And (3) concentrating the extract in vacuum, and after all the absolute ethyl alcohol is recovered, finishing the concentration to obtain the concentrated hericium erinaceus extract. Wherein the concentration temperature is 70 ℃, and the vacuum degree is 0.50 MPa;

4) filtering the concentrated Hericium erinaceus extract obtained in the step 3) through a 0.8-micron Cellulose Acetate (CA) membrane, and collecting the filtrate.

Through the steps, the hericium erinaceus anti-human colon cancer tumor cell (HCT-8) extract is prepared.

Example 2.

A preparation method of Hericium erinaceus (Hericium erinaceus) anti-human colon cancer tumor cell (HCT-8) extract comprises the following steps:

1) firstly, crushing the dried hericium erinaceus into 60 meshes;

2) putting the hericium erinaceus powder crushed in the step 1) into supercritical CO₂In an extraction kettle of the extraction device, supercritical CO is set₂Extracting condition parameters, and obtaining the hericium erinaceus supercritical CO after extraction is finished₂The extract, wherein the supercritical extraction condition parameters are as follows: the extraction pressure is 30Mpa, the extraction temperature is 45 ℃, the pressure of the separation kettle I is 8Mpa, the temperature is 35 ℃, the pressure of the separation kettle II is 4Mpa, the temperature is 25 ℃, the entrainer absolute ethyl alcohol is 9mL g^-1The extraction time is 2.0 h;

3) subjecting the hericium erinaceus obtained in the step 2) to supercritical CO₂And (3) concentrating the extract in vacuum, and after all the absolute ethyl alcohol is recovered, finishing the concentration to obtain the concentrated hericium erinaceus extract. Wherein the concentration temperature is 60 ℃, and the vacuum degree is 0.6 MPa;

4) filtering the concentrated Hericium erinaceus extract obtained in the step 3) through a 0.2-micron Cellulose Acetate (CA) membrane, and collecting the filtrate.

Through the steps, the hericium erinaceus anti-human colon cancer tumor cell (HCT-8) extract is prepared.

Example 3.

A preparation method of Hericium erinaceus (Hericium erinaceus) anti-human colon cancer tumor cell (HCT-8) extract comprises the following steps:

1) firstly, crushing the dried hericium erinaceus into 120 meshes;

2) putting the hericium erinaceus powder crushed in the step 1) into supercritical CO₂In an extraction kettle of the extraction device, supercritical CO is set₂Extracting condition parameters, and obtaining the hericium erinaceus supercritical CO after extraction is finished₂The extract, wherein the supercritical extraction condition parameters are as follows: the extraction pressure is 20Mpa, the extraction temperature is 40 ℃, the pressure of the separation kettle I is 7Mpa, the temperature is 30 ℃, the pressure of the separation kettle II is 3Mpa, the temperature is 20 ℃, the entrainer absolute ethyl alcohol is 10mL g^-1The extraction time is 0.5 h;

3) mixing the product obtained in the step 2)Hericium erinaceus supercritical CO₂And (3) concentrating the extract in vacuum, and after all the absolute ethyl alcohol is recovered, finishing the concentration to obtain the concentrated hericium erinaceus extract. Wherein the concentration temperature is 60 ℃, and the vacuum degree is 0.5 MPa;

4) filtering the concentrated Hericium erinaceus extract obtained in the step 3) through a 0.5-micron Cellulose Acetate (CA) membrane, and collecting the filtrate.

Through the steps, the hericium erinaceus anti-human colon cancer tumor cell (HCT-8) extract is prepared.

Comparative example 1.

The hericium erinaceus was replaced with hericium erinaceus mycelia, and the remaining steps were the same as in example 1.

Comparative example 2.

And (3) replacing the steps 2) to 3) of the embodiment 1 with adding 10 times of deionized water into the hericium erinaceus powder, heating, extracting for 30min, cooling, filtering, concentrating the filtrate, adding 10 times of anhydrous ethanol to dissolve, and concentrating under reduced pressure.

Comparative example 3.

Replacing hericium erinaceus with pleurotus eryngii, and the other steps are the same as in example 1.

Comparative example 4.

The hericium erinaceus was replaced with agaricus bisporus, and the other steps were the same as in example 1.

experiment-HCT-8 tumor cell growth inhibition in vitro experiment

Cells in logarithmic growth phase are selected, trypsinized and inoculated in 96-well culture plate by using 10% fetal calf serum RPMI1640 culture solution, the cell density of each well is 4000/100 mu l, and the cells are cultured for 24h at 37 ℃ and 5% CO 2. The medium was changed to 190. mu.l fresh complete medium before the experiment, 10. mu.l of the sample (final concentration monomeric compound 5. mu.g/ml; mixture 50. mu.g/ml) at the primary screening concentration was added to the experimental group, the medium containing the same volume of solvent was changed to the control group, the culture was incubated at 37 ℃ with 5% CO2 for 3 days, and the medium was discarded. Cells were fixed on plates by gently adding 100. mu.l of 10% TCA (1640 serum-free medium) pre-cooled at 4 ℃. Standing for 5min, and then moving to 4 ℃ for 1 h. The fixative was decanted, washed 5 times with distilled water to remove TCA, and air dried (at least 1 hour). Add 0.4% SRB solution 80 μ l into each hole, dye for 30min at room temperature, discard the dye solution, wash 6 times with 1% TCA (deionized water) to remove the unbound SRB, and air dry. Add 150. mu.l 10mM unbuffered Tris lye (pH10.5) for solubilization and go on a micro shaker for 5 min. The microplate reader M5 detector measures the OD510nm value. If the primary screening result shows that the HCT-8 cell proliferation inhibition rate of the sample exceeds 50%, the sample is rescreened, and the IC50 value is obtained.

Tumor cell growth inhibition (%) was 100% (OD control-OD experiment)/(OD control-OD blank)%

A Hericium erinaceus anti-human colon cancer tumor cell (HCT-8) extract biological activity screening result is shown in Table 1:

TABLE 1 results of HCT-8 tumor cell growth inhibition in vitro experiment (SRB method)

The extract prepared by the invention has extremely strong inhibiting effect on the in vitro growth of human colon cancer tumor cells (HCT-8). Research shows that the hericium erinaceus extract prepared by the method of the invention and the screened IC₅₀The value (half inhibition concentration) is 0.17-0.21 mug/ml, the proliferation inhibition rate on HCT-8 cells reaches more than 70%, wherein the proliferation inhibition rate on HCT-8 cells of the extract obtained under the optimal extraction condition reaches 71.67% when the final concentration is 5 mug/ml, and the screened IC is rescreened₅₀The value (semi-inhibitory concentration) is 0.17 mug/ml, the inhibition rate of positive drug 5-fluorouracil with the same concentration is 74.06%, and the IC of the rescreening is₅₀The value (half inhibitory concentration) was 1.59. mu.g/ml, and the antitumor activity against colon cancer of the extract was very significant. Compared with the comparative example, the hericium erinaceus extract obtained by supercritical CO2 extraction in the invention has unexpected effects compared with the traditional extraction method and the extraction method using other mushrooms or hericium erinaceus mycelium as raw materials.

Experiment two apoptosis rate determination experiment

Taking H in logarithmic growth phaseCT-8 tumor cells, adjusting cell density 5 x 10⁴And (2) inoculating the cells/ml into a 96-well cell culture plate, transferring the cells into an incubator for 24 hours to allow the cells to adhere to the wall, removing the supernatant, treating cell sap with different groups of equal amount of extracts for 48 hours, washing the cell sap with PBS, adding 10 mu l of Annexin V and 5 mu l of Propidium Iodide (PI), reacting for 15 minutes in a dark place, and quantitatively detecting by adopting a flow cytometer to calculate the apoptosis rate. And extracting total RNA of each group of cells from the treated cells according to the kit instruction, synthesizing cDNA, and carrying out qRT-PCR analysis, wherein the blank group is not treated by adding an extract.

TABLE 2 determination of the apoptosis Rate of HCT-8 tumor cells

The promotion of apoptosis is one of the important ways to exert the anti-tumor bioactivity, and the experiment of the tumor cell apoptosis rate in table 2 proves that the hericium erinaceus extract can obviously improve the apoptosis proportion of HCT-8 tumor cells. From the results of fig. 5, it can be seen that the hericium erinaceus extract can significantly reduce the expression level of mRNA compared to the blank group.

Experiment on influence of Hericium erinaceus extract on mouse transplantation tumor model

Randomly dividing one week-old mice into four groups, each group containing ten mice, making each group have uniform quality, selecting HCT-8 tumor cells in logarithmic growth phase, and adjusting cell density to 5 x 10⁴And each cell per mL is injected into 0.5mL of cell suspension subcutaneously at the back of a mouse, nodules appear subcutaneously after one week, which indicates that a tumor model is successfully established, the mouse is placed indoors, proper illumination, temperature and humidity are controlled, water is freely taken, a blank group is subjected to intraabdominal injection with 10g/kg of physiological saline every day, a fluorouracil group is subjected to intraperitoneal injection with 10mg/kg of drugs every day, and an experimental group is subjected to intraabdominal injection with 10g/kg of hericium erinaceus extracts prepared in example 1 and example 2 respectively. Groups of mice were weighed weekly for 5 consecutive weeks, tumor volume and final tumor mass. Tumor volume is tumor lengthDiameter perpendicular to long diameter²。

As can be seen from figures 6-7, the weights of mice in the blank group and the fluorouracil group are lower than those of mice in the example group, the hericium erinaceus extract on the surface can reduce the adverse effect of fluorouracil, the tumor volumes of the examples and the fluorouracil group are obviously reduced, the tumor mass is obviously reduced, the hericium erinaceus extract on the surface can effectively inhibit the growth of tumor cells, and the weight loss caused by fluorouracil drugs is reduced.