Preparation method and application of Coprinus comatus polysaccharide extracted by subcritical water

Abstract

The present invention belongs to the technical field of polysaccharide extraction, and discloses a preparation method and application of Coprinus comatus polysaccharide extracted by subcritical water. The preparation method comprises drying Coprinus comatus at 50°C, crushing and passing through a 50-mesh sieve, and storing in a cool and dry place; placing in a subcritical water extraction device and extracting three times; cooling the extracted extract and centrifuging it at 3000r·min‑1 for 10min. The supernatant obtained by centrifugation is added with ethanol, stirred and allowed to stand for 24h, and then centrifuged at 3000r·min‑1 for 10min after standing, and the precipitate is freeze-dried to obtain a Coprinus comatus polysaccharide extract. The preparation method provided by the present invention is simple to operate, has a high extraction rate, greatly saves extraction time, and the Coprinus comatus polysaccharide extract extracted by the present invention has a good ability to inhibit the proliferation of HCT‑116 and A431 cells. It can be applied in anti-tumor activity.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide extraction, and relates to a preparation method and application of Coprinus comatus polysaccharide extracted with subcritical water. Background Art

[0002] Coprinus comatus (OF Müll.) Pers. belongs to the Coprinus family and the genus Coprinus. It is also known as chicken leg mushroom and chicken leg mushroom. It is mainly distributed in Heilongjiang, Jilin, Liaoning, Shanxi, Hebei, Qinghai and Gansu, and is now cultivated on a large scale. Coprinus comatus is an edible fungus with high nutritional value and health benefits. It is known as the "new star among fungi." Coprinus comatus is sweet and mild in nature, and has the effects of invigorating the stomach, refreshing the mind, digesting food, and enhancing appetite. Modern research shows that the fruiting body of Coprinus comatus contains proteins, polysaccharides, vitamins and trace elements, which can prevent arteriosclerosis, hyperlipidemia, heart disease and diabetes. Polysaccharides, as one of the main active ingredients of Coprinus comatus, have pharmacological activities such as lowering blood sugar, anti-tumor, regulating immunity and anti-oxidation, and are of great development value.

[0003] Reported methods for extracting polysaccharides from Coprinus comatus include hot water extraction, enzymatic hydrolysis, and microwave-assisted extraction. However, these extraction methods have disadvantages such as low extraction yield or long extraction time. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention provides a method for preparing and applying Coprinus comatus polysaccharides extracted through subcritical water extraction. The preparation method provided by the present invention is simple to operate, has a high extraction rate, and significantly reduces extraction time. Furthermore, the Coprinus comatus polysaccharide extract extracted by the present invention has a good ability to inhibit the proliferation of HCT-116 and A431 cells and can be applied for anti-tumor activity.

[0005] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0006] A method for preparing Coprinus comatus polysaccharide by subcritical water extraction specifically comprises the following steps:

[0007] (1) Dry the Coprinus comatus at 50°C, crush it, pass it through a 50-mesh sieve, and store it in a cool, dry place.

[0008] (2) placing the powder of Coprinus comatus prepared in step (1) in a subcritical water extraction device and extracting three times;

[0009] (3) Cool the extract after extraction in step (2) and heat it at 3000 r·min -1 Centrifuge for 10 minutes.

[0010] (4) Concentration step (4) Add ethanol (final ethanol concentration is 80vt%) to the supernatant obtained by centrifugation, stir well and let it stand for 24h.

[0011] (5) After standing still, the -1 The mixture was centrifuged at 400°C for 10 min and the precipitate was freeze-dried to obtain the polysaccharide extract of Coprinus comatus.

[0012] Furthermore, the extraction conditions of step (2) are: extraction temperature of 100-200°C, extraction time of 10-35 min, liquid-to-solid ratio of 5:1-30:1 mL·g -1 .

[0013] Furthermore, in step (2), the amount of Coprinus comatus powder is preferably 5 g.

[0014] Furthermore, the extraction conditions of step (2) are preferably: extraction temperature of 150°C, extraction time of 27 min, liquid-to-solid ratio of 24:1 mL·g -1 .

[0015] The present invention also claims to protect the use of the Coprinus comatus polysaccharide extract prepared by the above preparation method in inhibiting the proliferation of HCT-116 and A431 cells.

[0016] The beneficial effects of the present invention compared with the prior art are:

[0017] In the preparation method provided by the present invention, the polysaccharide extraction rate of Coprinus comatus polysaccharide under the optimal extraction conditions (temperature 150°C, time 27 min, liquid-to-solid ratio 24:1 mL·g-1) is 10.74%. Anti-tumor activity studies have shown that the Coprinus comatus polysaccharide extract has a good ability to inhibit the proliferation of HCT-116 and A431 cells, IC 50 The values ​​were 262.3 μg / mL and 528.7 μg / mL respectively. The invention provides a technical reference for the scientific development and utilization of Coprinus comatus. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a graph showing the effect of different extraction temperatures on the polysaccharide extraction rate in Examples 1 to 6.

[0019] Figure 2 This is a graph showing the effect of different extraction times on the polysaccharide extraction rate in Examples 7 and 12.

[0020] Figure 3 This is a graph showing the effect of different liquid-to-solid ratios on polysaccharide extraction rates in Example 13 and Example 18.

[0021] Figure 4 This is a glucose standard curve.

[0022] Figure 5This is a graph showing the anti-tumor activity of the Coprinus comatus polysaccharide extract obtained by the preparation method of Example 19. DETAILED DESCRIPTION

[0023] The present invention is described in detail below by specific examples, but the scope of protection of the present invention is not limited. Unless otherwise specified, the experimental methods adopted in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained from commercial channels.

[0024] The Coprinus comatus in the embodiment was collected from the northern foot of Qilian Mountain in Wuwei City, Gansu Province in August 2022, dried at 50°C, crushed and passed through a 50-mesh sieve, and stored in a cool and dry place for later use.

[0025] The instruments used in the examples are: HT-500FC subcritical water extraction equipment (Shanghai Huotong Experimental Instrument Co., Ltd.), SMF01 mill (Zhejiang Supor Co., Ltd.), RE-5298A rotary evaporator (Shanghai Yarong Biochemical Instrument Factory), BSA224S electronic balance (Sartorius, Germany), UV-8000A double beam UV-visible spectrophotometer (Suzhou Jiangdong Precision Instrument Co., Ltd.), FreeZone 2.5L desktop freeze dryer (Labconco, USA), 5810R desktop centrifuge (Eppendorf, Germany), Synergy TM H4 full-function microplate reader (BioTek, USA), MCO-17AICCO2 cell culture incubator (Sanyo, Japan).

[0026] The reagents used in the examples were MTT, glucose control and erlotinib control (Shanghai Yuanye Biotechnology Co., Ltd.), DMEM culture medium, fetal bovine serum (FBS), penicillin-streptomycin double antibody (Hyclone Company, USA), concentrated sulfuric acid and ethanol (Tianjin Komeiou Chemical Reagent Co., Ltd.).

[0027] In Examples 1 to 19, the phenol-sulfuric acid method was used to determine the polysaccharide content of Coprinus comatus, and the regression equation y = 0.0608x-0.0253 (where x is the glucose concentration and y is the absorbance value) was obtained. The glucose standard curve is shown in FIG. Figure 4 As shown in Figure 2, within the range of 2.5-15.0 μg / mL, there is a good linear relationship (R 2 =0.9995).

[0028] In Examples 1 to 19, the concentration of polysaccharide was calculated by measuring the absorbance of aqueous solution of Coprinus comatus polysaccharide extract. The calculation formula (1) for the extraction rate of Coprinus comatus polysaccharide is as follows:

[0029]

[0030] Where: Y is the extraction rate of Coprinus comatus polysaccharide (%); x is the concentration of Coprinus comatus polysaccharide (μg·mL -1 ); v is the volume of the aqueous solution of the Coprinus comatus polysaccharide extract (mL); m is the dry mass of the Coprinus comatus medicinal material (g).

[0031] Example 1

[0032] 5.0 g of Coprinus comatus powder was weighed and placed in a subcritical water extraction device. The extraction temperature was 100 °C, the time was 20 min, and the liquid-to-solid ratio was 20:1 mL·g. -1 The extraction was carried out under the following conditions, and the number of extractions was fixed at 3 times. After the extraction was completed, the extract was cooled and heated at 3 000 r·min -1 Then, the supernatant was concentrated and ethanol (final concentration was 80%) was added, stirred and allowed to stand for 24 hours, and then centrifuged at 3000 r·min. -1 The mixture was centrifuged at 400°C for 10 min and the precipitate was freeze-dried to obtain the polysaccharide extract of Coprinus comatus.

[0033] Example 2

[0034] The extraction temperature was 120° C., and the other steps were the same as in Example 1.

[0035] Example 3

[0036] The extraction temperature was 150° C., and the other steps were the same as in Example 1.

[0037] Example 4

[0038] The extraction temperature was 160° C., and the other steps were the same as in Example 1.

[0039] Example 5

[0040] The extraction temperature was 180° C., and the rest was the same as in Example 1.

[0041] Example 6

[0042] The extraction temperature was 200° C., and the rest was the same as in Example 1.

[0043] Example 7

[0044] 5.0 g of Coprinus comatus powder was weighed and placed in a subcritical water extraction device. The extraction temperature was 160 °C, the extraction time was 10 min, and the liquid-to-solid ratio was 20:1 mL·g -1 The extraction was carried out under the following conditions, and the number of extractions was fixed at 3 times. After the extraction was completed, the extract was cooled and heated at 3 000 r·min -1 Then, the supernatant was concentrated and ethanol (final concentration was 80%) was added, stirred and allowed to stand for 24 hours, and then centrifuged at 3000 r·min. -1The mixture was centrifuged at 400°C for 10 min and the precipitate was freeze-dried to obtain the polysaccharide extract of Coprinus comatus.

[0045] Example 8

[0046] The extraction time was 15 min, and the rest was the same as in Example 7.

[0047] Example 9

[0048] The extraction time was 20 min, and the rest was the same as in Example 7.

[0049] Example 10

[0050] The extraction time was 24 min, and the rest was the same as in Example 7.

[0051] Example 11

[0052] The extraction time was 30 min, and the rest was the same as in Example 7.

[0053] Example 12

[0054] The extraction time was 35 min, and the rest was the same as in Example 7.

[0055] Example 13

[0056] 5.0 g of Coprinus comatus powder was weighed and placed in a subcritical water extraction device. The extraction temperature was 160 °C, the extraction time was 20 min, and the liquid-to-solid ratio was 5:1 mL·g -1 The extraction was carried out under the following conditions, and the number of extractions was fixed at 3 times. After the extraction was completed, the extract was cooled and heated at 3 000 r·min -1 Then, the supernatant was concentrated and ethanol (final concentration was 80%) was added, stirred and allowed to stand for 24 hours, and then centrifuged at 3000 r·min. -1 The mixture was centrifuged at 400°C for 10 min and the precipitate was freeze-dried to obtain the polysaccharide extract of Coprinus comatus.

[0057] Example 14

[0058] The liquid-to-solid ratio is 10:1 mL·g -1 The rest is the same as Example 13.

[0059] Example 15

[0060] The liquid-to-solid ratio is 15:1 mL·g -1 The rest is the same as Example 13.

[0061] Example 16

[0062] The liquid-to-solid ratio is 20:1 mL·g -1 The rest is the same as Example 13.

[0063] Example 17

[0064] The liquid-to-solid ratio is 27:1 mL·g -1 The rest is the same as Example 13.

[0065] Example 18

[0066] Liquid-to-solid ratio: 30:1 mL·g -1 The rest is the same as Example 13.

[0067] Example 19

[0068] 5.0 g of Coprinus comatus powder was weighed and placed in a subcritical water extraction device. The extraction temperature was 150 °C, the extraction time was 27 min, and the liquid-to-solid ratio was 24:1 mL·g -1 The extraction was carried out under the following conditions, and the number of extractions was fixed at 3 times. After the extraction was completed, the extract was cooled and heated at 3 000 r·min -1 Then, the supernatant was concentrated and ethanol (final concentration was 80%) was added, stirred and allowed to stand for 24 hours, and then centrifuged at 3000 r·min. -1 The mixture was centrifuged at room temperature for 10 minutes, and the precipitate was freeze-dried to obtain the polysaccharide extract of Coprinus comatus. The polysaccharide extraction rate was 10.74%.

[0069] like Figure 1-Figure 3 As shown in the results, when the subcritical water temperature increased from 100°C to 150°C, the extraction rate of Coprinus comatus polysaccharides increased from 7.04% to 9.79%. However, when the subcritical water temperature was further increased to 200°C, the extraction rate of Coprinus comatus polysaccharides decreased to 5.34%. This may be because, within the range of 100-150°C, increasing the water temperature reduces surface tension, increasing the mass transfer rate of polysaccharides from the solid phase to the liquid phase, thereby improving the extraction rate of polysaccharides. However, when the temperature exceeded 150°C, the higher the temperature and the lower the polarity of the water, the less suitable it was for dissolving polysaccharides. At the same time, other less polar components in Coprinus comatus competed with the polysaccharides for dissolution, resulting in a decrease in the extraction rate of polysaccharides. When the extraction time increased from 10 min to 27 min, the extraction rate of Coprinus comatus polysaccharides increased from 6.05% to 9.35%. However, when the extraction time was further extended to 35 min, the extraction rate of Coprinus comatus polysaccharides decreased to 7.44%. This may be because the amount of polysaccharide dissolved in the first 25 min continued to increase, but when the extraction time reached 27 min, the polysaccharide concentration reached solid-liquid equilibrium, and some polysaccharides were degraded due to prolonged heating, resulting in a decrease in the polysaccharide extraction rate. -1 Increased to 15:1 mL·g -1 The extraction rate of Coprinus comatus polysaccharide increased from 7.75% to 9.68% when the liquid-to-solid ratio was further increased to 30:1 mL·g -1The extraction rate of polysaccharides from Coprinus comatus decreased to 7.45% when the liquid-to-solid ratio was increased within a certain range. The reason for this phenomenon may be that when the liquid-to-solid ratio increases within a certain range, the concentration of polysaccharides inside and outside the Coprinus comatus cell tissue increases, which can promote the dissolution of polysaccharides from the Coprinus comatus cell tissue, thereby improving the polysaccharide extraction rate.

[0070] The MTT method was used to determine the proliferation inhibitory effect of the Coprinus comatus polysaccharide extract extracted by the preparation method in Example 19 on the human colon cancer cell line HCT-116 and the human skin squamous cell carcinoma cell line A431. The activity of the Coprinus comatus polysaccharide extract in inhibiting the proliferation of HCT-116 and A431 cells was determined by the method reported by Elmetwally et al. (ELMETWALLY SA, SAIED KF, EISSA IH, et al. Design, synthesis and anticancer evaluation of thieno[2,3-d]pyrimidine derivatives as dual EGFR / HER2 inhibitors and apoptosis inducers[J]. Bioorganic Chemistry, 2019, 88: 102944.). Erlotinib was used as a positive control. GraphPad Prism 9.5 software was used to calculate the half inhibitory concentration (IC50) of the antitumor activity of Coprinus comatus polysaccharide. 50 ).

[0071] Depend on Figure 5 It can be seen that when the concentration of the polysaccharide extract of Coprinus comatus is in the range of 62.5 to 2000 μg·mL -1 When the concentration of β-actin was 0.04, the ability of β-actin to inhibit the proliferation of HCT-116 and A431 cells increased with the increase of the concentration, showing a certain dose-dependent effect. 50 The values ​​were 262.3 μg / mL and 528.7 μg / mL respectively (the IC values ​​of the positive control drug erlotinib for inhibiting the proliferation of HCT-116 and A431 cells were 50 The values ​​were 0.23 μM and 0.14 μM respectively). When the concentration of the polysaccharide extract of Coprinus comatus was 2000 μg·mL -1 The inhibition rates of HCT-116 and A431 cell proliferation were 81.32% and 68.34%, respectively, indicating that Coprinus comatus polysaccharide has certain anti-tumor efficacy. Coprinus comatus polysaccharide has the potential to be developed as a natural anti-tumor drug.

[0072] The above-described embodiments are only preferred embodiments of the present invention, and are not intended to be all feasible embodiments of the present invention. Any obvious modifications made by a person skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A use of subcritical water extracted Coprinus comatus polysaccharide in the preparation of a natural anti-tumor drug for inhibiting the proliferation of HCT-116 and A431 cells, characterized in that: The specific steps include: (1) Dry the Coprinus comatus at 50°C, crush it, pass it through a 50-mesh sieve, and store it in a cool and dry place. (2) placing the powder of Coprinus comatus prepared in step (1) in a subcritical water extraction device and extracting three times; (3) Cool the extract obtained in step (2) and spin it at 3000 r·min -1 Centrifuge for 10 min; (4) Concentration step (3) Add ethanol to the supernatant obtained by centrifugation, stir well and let it stand for 24 hours. (5) After standing still, rotate at 3000 r·min -1 The mixture was centrifuged for 10 min and the precipitate was freeze-dried to obtain Coprinus comatus polysaccharide. The extraction conditions of step (2) are as follows: extraction temperature of 100-200°C, extraction time of 10-35 min, liquid-to-solid ratio of 5:1-30:1 mL·g -1 .

2. The use of a subcritical water-extracted Coprinus comatus polysaccharide according to claim 1 in the preparation of a natural anti-tumor drug for inhibiting the proliferation of HCT-116 and A431 cells, characterized in that: In step (2), the powder of Coprinus comatus is 5 g.

3. Use of the subcritical water-extracted Coprinus comatus polysaccharide according to claim 1 in the preparation of a natural anti-tumor drug for inhibiting the proliferation of HCT-116 and A431 cells, characterized in that: The extraction conditions of step (2) were as follows: extraction temperature of 150 °C, extraction time of 27 min, liquid-to-solid ratio of 24:1 mL·g -1 .

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