Traditional Chinese medicine polypeptide extraction process

Abstract

The invention relates to the field of polypeptide extraction, and discloses a traditional Chinese medicine polypeptide extraction process which comprises the following steps: S1, preparing raw materials; s2, ultrasonic-assisted extraction; s3, low-temperature plasma auxiliary extraction; s4, carrying out antioxidant nano enzyme coexistence treatment; s5, further extraction by an enzymolysis method; s6, carrying out separation treatment; s7, purification treatment; s8, performing freeze drying; through combined use of ultrasonic waves and low-temperature plasmas, astragalus membranaceus cell walls can be effectively damaged within a short time, more polypeptide components are released, the extraction efficiency is high, active oxygen generated in the low-temperature plasma treatment process is effectively removed through antioxidant nano-enzyme coexistence treatment, and it is ensured that polypeptides are not damaged by oxidation.

Description

Technical Field

[0001] The invention relates to the field of polypeptide extraction, and more specifically, to a process for extracting polypeptide from traditional Chinese medicinal herbs. Background Art

[0002] The research on Chinese herbal peptides mainly focuses on antibacterial peptides, immune peptides and anti-tumor peptides. For example, peptides have been found in herbs such as Cordyceps sinensis, Ginkgo biloba and Ginseng, and it has been confirmed that these peptides have important biological activities. However, due to the small size and sensitive properties of peptide molecules, the extraction process faces some challenges.

[0003] The existing Chinese herbal peptide extraction processes mostly use water extraction, ultrasonic-assisted extraction, enzymatic hydrolysis and other methods, but there are the following technical problems:

[0004] Low extraction efficiency: Traditional water extraction requires a long time and high temperature, which can easily lead to loss of polypeptide activity or partial degradation, and low extraction efficiency.

[0005] Oxidative damage to peptides: During the low-temperature plasma-assisted extraction process, reactive oxygen species such as hydroxyl radicals are generated. These substances react with peptides to produce oxidation, leading to degradation or inactivation of the peptides. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a process for extracting polypeptides from Chinese herbal medicines.

[0007] The present invention provides a process for extracting polypeptides from Chinese herbal medicine, comprising the following steps:

[0008] S1: Raw material preparation;

[0009] Select the medicinal materials, clean them, dry them in a cool place, and crush them into fine powder for later use.

[0010] S2: Ultrasonic-assisted extraction;

[0011] The medicinal powder was mixed with distilled water and initially extracted using ultrasound for 60 minutes at a temperature of 50-60°C.

[0012] S3: low temperature plasma assisted extraction;

[0013] After ultrasonic extraction, low-temperature plasma treatment is used.

[0014] S4: Antioxidant nanozyme coexistence treatment;

[0015] During the low-temperature plasma treatment, antioxidant nanoenzymes are added to remove the reactive oxygen species generated by the plasma and prevent oxidative degradation of the polypeptides.

[0016] S5: further extraction by enzymatic hydrolysis;

[0017] The extract is enzymatically hydrolyzed with protease to decompose the protein in the medicinal material and further release the peptide. The enzymatic hydrolysis temperature is controlled at 40-50°C and the time is 2 hours.

[0018] S6: separation treatment;

[0019] The extracted solution is separated to separate polypeptide molecules of different molecular weights.

[0020] S7: purification treatment;

[0021] The peptides are separated and their concentrations are purified by adsorption treatment.

[0022] S8: freeze drying;

[0023] The purified polypeptide liquid is freeze-dried to obtain medicinal polypeptide powder.

[0024] Preferably, in step S2, the medicinal material powder and distilled water are mixed in a ratio of 1:20, and an ultrasonic treatment device is used for preliminary extraction.

[0025] Preferably, in step S2, the ultrasonic frequency is 30-40 kHz, the temperature is maintained at 50-60°C, and the extraction time is 60 minutes, the purpose is to destroy the cell wall of the medicinal material and release the polypeptide and other water-soluble components.

[0026] Preferably: in step S3, the plasma is argon plasma.

[0027] Preferably: using an argon low-temperature plasma reactor, treating for 20 minutes in an environment below 40° C. to further destroy the cell wall and ensure full release of the polypeptide.

[0028] Preferably: in step S4, during the low-temperature plasma treatment, a nano-scale antioxidant enzyme material is added at a concentration of 100 mg per liter of extract to remove active oxygen species generated in the reaction in real time to prevent oxidative degradation of the polypeptide.

[0029] Preferably: in step S4, the antioxidant nanozyme is a catalase-like enzyme or a superoxide dismutase-like enzyme.

[0030] Preferably, in step S5, a neutral protease (pH 6.8-7.0) is used to perform enzymatic hydrolysis at 40-50° C. for 2 hours to further release more small molecule polypeptides from the cells.

[0031] Preferably: in step S6, a microfluidic chip is used to separate polypeptides to separate polypeptides of different molecular weights.

[0032] Preferably: in step S7, magnetic nanoparticles (functionalized surface modified) are added to the extract, and the target polypeptide is adsorbed and separated by an external magnetic field to improve the purity.

[0033] Preferably, in step S8, the polypeptide liquid is freeze-dried at -80°C to obtain a powder of the medicinal polypeptide, which is convenient for storage and subsequent use.

[0034] The beneficial effect of the present invention is that, through the combined use of ultrasound and low-temperature plasma, the cell walls of medicinal materials can be effectively destroyed in a relatively short time, more polypeptide components are released, and the extraction efficiency is high.

[0035] The coexistence treatment with antioxidant nanozymes effectively removed the reactive oxygen species generated during the low-temperature plasma treatment, ensuring that the polypeptides were not damaged by oxidation.

[0036] Microfluidic chips are highly dependent on the activity of components in the fluid during the separation process. If the polypeptides are inactivated or degraded due to oxidation during the processing, the chip separation efficiency will be affected. Antioxidant nanozymes ensure that the polypeptides in the extract remain active, ultimately improving the separation purity and efficiency of the microfluidic chip.

[0037] The combined application of antioxidant nanozymes and magnetic nanomaterials can also reduce the chemical reaction between nanomaterials and polypeptides. Since magnetic nanomaterials may also induce oxidation reactions during the adsorption process (through surfactants or nanoscale effects), antioxidant nanozymes can protect polypeptides from being oxidized by adsorbed materials, thereby ensuring the purity and stability of the final product. DETAILED DESCRIPTION

[0038] The subject matter described herein will now be discussed with reference to example embodiments. It should be understood that the discussion of these embodiments is only to enable those skilled in the art to better understand and implement the subject matter described herein, and the functions and arrangements of the elements discussed may be changed without departing from the scope of protection of the present specification. Various examples may omit, replace or add various processes or components as needed. In addition, the features described relative to some examples may also be combined in other examples.

[0039] Example 1

[0040] In this embodiment, a process for extracting peptides from Chinese herbal medicine is proposed, comprising the following steps:

[0041] S1: Raw material preparation;

[0042] Select the medicinal materials, clean them, dry them in a cool place, and crush them into fine powder for later use.

[0043] S2: Ultrasonic-assisted extraction;

[0044] The medicinal powder was mixed with distilled water and initially extracted using ultrasound for 60 minutes at a temperature of 50-60°C.

[0045] S3: low temperature plasma assisted extraction;

[0046] After ultrasonic extraction, low-temperature plasma treatment is used.

[0047] S4: Antioxidant nanozyme coexistence treatment;

[0048] During the low-temperature plasma treatment, antioxidant nanoenzymes are added to remove the reactive oxygen species generated by the plasma and prevent oxidative degradation of the polypeptides.

[0049] S5: further extraction by enzymatic hydrolysis;

[0050] The extract is enzymatically hydrolyzed with protease to decompose the protein in the medicinal material and further release the peptide. The enzymatic hydrolysis temperature is controlled at 40-50°C and the time is 2 hours.

[0051] S6: separation treatment;

[0052] The extracted solution is separated to separate polypeptide molecules of different molecular weights.

[0053] S7: purification treatment;

[0054] The peptides are separated and their concentrations are purified by adsorption treatment.

[0055] S8: freeze drying;

[0056] The purified polypeptide liquid is freeze-dried to obtain medicinal polypeptide powder.

[0057] In this embodiment, in step S2, the medicinal material powder and distilled water are mixed in a ratio of 1:20, and an ultrasonic processing device is used for preliminary extraction.

[0058] In some embodiments, in step S2, the ultrasonic frequency is 30 kHz, the temperature is maintained at 50°C, and the extraction time is 60 minutes, the purpose is to destroy the cell wall of the medicinal material and release the polypeptides and other water-soluble components.

[0059] In this embodiment, in step S2, the ultrasonic frequency is 35 kHz, the temperature is maintained at 55°C, and the extraction time is 60 minutes, the purpose is to destroy the cell wall of the medicinal material and release the polypeptide and other water-soluble components.

[0060] In some embodiments, in step S2, the ultrasonic frequency is 40 kHz, the temperature is maintained at 60°C, and the extraction time is 60 minutes, the purpose is to destroy the cell wall of the medicinal material and release the polypeptides and other water-soluble components.

[0061] In this embodiment, in step S3, the plasma is argon plasma. An argon low-temperature plasma reactor is used to perform treatment for 20 minutes in an environment below 40° C. to further destroy the cell wall and ensure that the polypeptide is fully released.

[0062] In this embodiment, in step S4, during the low-temperature plasma treatment, a nano-scale antioxidant enzyme material is added at a concentration of 100 mg per liter of extract to remove active oxygen species generated in the reaction in real time and prevent oxidative degradation of the polypeptide.

[0063] In this embodiment, in step S4, the antioxidant nanozyme is a catalase-like enzyme.

[0064] In some embodiments, in step S4, the antioxidant nanozyme is a superoxide dismutase-like enzyme.

[0065] In this embodiment, in step S5, a neutral protease (pH 6.9, at 45° C.) is used for enzymatic hydrolysis for 2 hours to further release more small molecule polypeptides from the cells.

[0066] In some embodiments, in step S5, a neutral protease (pH 6.8) is used to perform enzymatic hydrolysis at 40° C. for 2 hours to further release more small molecule polypeptides from the cells.

[0067] In some embodiments, in step S5, a neutral protease (pH 7.0) is used to perform enzymatic hydrolysis at 50° C. for 2 hours to further release more small molecule polypeptides from the cells.

[0068] In this embodiment, in step S6, a microfluidic chip is used to separate polypeptides to separate polypeptides of different molecular weights.

[0069] In this embodiment, in step S7, magnetic nanoparticles (functionalized surface modified) are added to the extract, and the target polypeptide is adsorbed and separated by an external magnetic field to improve the purity.

[0070] In this embodiment, in step S8, the polypeptide liquid is freeze-dried at -80°C to obtain a powder of the medicinal polypeptide for easy storage and subsequent use.

[0071] Example 2

[0072] This example uses Astragalus as an example to verify that through the combined use of ultrasound and low-temperature plasma in the present invention, the cell wall of the medicinal material can be effectively destroyed in a relatively short time, releasing more polypeptide components and achieving high extraction efficiency.

[0073] 1. Description

[0074] Both ultrasonic-assisted extraction and low-temperature plasma technology can effectively destroy plant cell walls and release polypeptide components in cells. The mechanical vibration and cavitation effect of ultrasound can increase the permeability of cell walls and accelerate the release of intracellular substances. The high-energy active particles of low-temperature plasma (such as argon plasma) can effectively improve cell lysis efficiency by exciting and destroying chemical bonds on the surface of cell walls. When these two methods are used in combination, the extraction time can be significantly shortened, the extraction efficiency can be improved, and the content of polypeptides in the extract can be increased.

[0075] 2. Experimental Procedure

[0076] 2.1 Experimental materials and equipment

[0077] Experimental Materials:

[0078] Astragalus powder (purchased from a Chinese herbal medicine supplier).

[0079] Distilled water.

[0080] Argon low temperature plasma treatment equipment.

[0081] Ultrasonic extractor.

[0082] High-speed refrigerated centrifuge.

[0083] High performance liquid chromatography.

[0084] Methanol.

[0085] Ultrapure water.

[0086] Experimental equipment:

[0087] 100mL beaker.

[0088] Pipette.

[0089] Cell disruptor.

[0090] 2.2 Experimental procedures

[0091] Step 1: Pretreatment of Astragalus powder with solvent

[0092] Take 20 g of Astragalus powder and place it in a 100 mL beaker.

[0093] Add 80 mL of distilled water and stir evenly to form a uniform suspension.

[0094] Step 2: Ultrasonic Assisted Extraction

[0095] Using an ultrasonic extractor, place the beaker in an ultrasonic bath.

[0096] The ultrasonic frequency was set to 35 kHz, the temperature to 55 °C, and the extraction time to 30 min.

[0097] After the extraction time is up, take out the sample and cool it to room temperature.

[0098] Step 3: Low-temperature plasma-assisted extraction

[0099] The samples after ultrasonic extraction were divided into two parts, one as a control group, which was only centrifuged, and the other was treated with plasma.

[0100] The samples were treated using an argon low-temperature plasma treatment device, with the argon flow rate controlled at 5 L / min, the treatment time being 20 minutes, and the temperature maintained below 40°C.

[0101] Step 4: Sample processing and centrifugation

[0102] All processed samples were placed in a high-speed refrigerated centrifuge and centrifuged at 8000 rpm for 10 minutes, and the supernatants were collected respectively.

[0103] Step 5: Determination of peptide content

[0104] The supernatant was analyzed by high performance liquid chromatography (HPLC) to detect the polypeptide content, using 0.1% formic acid-water (phase A) and 0.1% formic acid-methanol (phase B) as mobile phases and a detection wavelength of 220 nm.

[0105] The HPLC spectra and peak areas of the ultrasound + plasma treatment group, ultrasound treatment group alone and blank control group were compared, and the content of the extracted polypeptides per gram of astragalus powder was calculated.

[0106] 3. Experimental results

[0107] By setting up different treatment groups, the content of Astragalus polypeptide extraction was determined respectively, and the results are shown in the following table:

[0108]

[0109] It can be seen from the above table:

[0110] Control group: The extracted polypeptide content was lower, 2.1 mg / g.

[0111] Ultrasonic treatment group: The polypeptide content increased to 4.5 mg / g.

[0112] Plasma treatment group alone: ​​The polypeptide content was 3.8 mg / g, which was significantly higher than that of the control group.

[0113] Combined treatment group: The peptide extraction content reached 8.3 mg / g, which was about 4 times that of the control group.

[0114] 4. Conclusion

[0115] This experiment used the combined use of ultrasound and low-temperature plasma (argon plasma) to effectively improve the extraction efficiency of Astragalus polypeptide. The experimental results showed that the polypeptide extraction content of the combined treatment group was significantly higher than that of the single treatment group and the control group, and the treatment time was short and the extraction efficiency was high. HPLC analysis proved that the polypeptide content was significantly improved, verifying the superiority of the combined extraction technology.

[0116] Example 3

[0117] This example uses Astragalus as an example to verify that the present invention effectively removes the active oxygen generated during the low-temperature plasma treatment through the coexistence treatment of antioxidant nanoenzymes, ensuring that the polypeptide is not damaged by oxidation.

[0118] 1. Description

[0119] Antioxidant nanozymes are nanomaterials that mimic the functions of natural catalase or superoxide dismutase. They can efficiently remove reactive oxygen species such as hydroxyl radicals and peroxides generated during the extraction process, thus avoiding oxidative damage to polypeptide molecules. In addition, low-temperature plasma generates a large amount of reactive oxygen species during the treatment of Astragalus, which can lead to oxidative modification and degradation of polypeptide molecules, thereby reducing the biological activity of polypeptides. By introducing antioxidant nanozymes, these reactive oxygen species can be effectively removed, ensuring that the structure and function of polypeptides are preserved.

[0120] 2. Experimental Procedure

[0121] 2.1 Experimental materials and equipment

[0122] Experimental Materials:

[0123] Astragalus powder.

[0124] Distilled water.

[0125] Low temperature plasma processing equipment.

[0126] Antioxidant nanozymes.

[0127] Ultrasonic extractor.

[0128] High-speed refrigerated centrifuge.

[0129] High performance liquid chromatography.

[0130] Oxidative Labeling Reagents.

[0131] Ultrapure water.

[0132] Experimental equipment:

[0133] 100mL beaker.

[0134] Pipette.

[0135] Cuvette.

[0136] 2.2 Experimental procedures

[0137] Step 1: Prepare the Astragalus raw materials

[0138] Take 20 g of Astragalus powder and place it in a 100 mL beaker.

[0139] Add 80 mL of distilled water and stir evenly to form a uniform suspension.

[0140] Step 2: Ultrasonic Assisted Extraction

[0141] Using an ultrasonic extractor, place the beaker in an ultrasonic bath.

[0142] The ultrasonic frequency was set to 35 kHz, the temperature to 55 °C, and the extraction time to 30 min.

[0143] After the extraction time is up, take out the sample and cool it to room temperature.

[0144] Step 3: Low temperature plasma treatment

[0145] The samples after ultrasonic extraction were divided into two parts, one as a control group (without antioxidant nanozymes) and the other as a test group (with antioxidant nanozymes added).

[0146] The samples in the control group were treated with argon plasma, the argon flow rate was controlled at 5 L / min, the treatment time was 20 minutes, and the temperature was kept below 40°C.

[0147] Antioxidant nanozymes were added to the test group samples at a concentration of 100 mg per liter of extract, followed by the same plasma treatment.

[0148] Step 4: Centrifugation and collection of peptide extract

[0149] All processed samples were placed in a high-speed refrigerated centrifuge and centrifuged at 8000 rpm for 10 minutes, and the supernatants were collected respectively.

[0150] Step 5: Detection of peptide oxidation degree

[0151] DNPH oxidation labeling reagent was used to measure the carbonyl content in peptides to assess the degree of oxidative damage.

[0152] Prepare DNPH solution (2 mg / mL in 1 M HCl).

[0153] Take 1 mL of the extract, add 1 mL of DNPH solution, and react at room temperature for 30 minutes.

[0154] The reaction was terminated, the precipitate was removed by centrifugation, and the supernatant was analyzed by HPLC.

[0155] HPLC settings and separation conditions:

[0156] Mobile phase: 0.1% formic acid-water (phase A) and 0.1% formic acid-methanol (phase B)

[0157] Flow rate: 1mL / min

[0158] Wavelength: 365nm

[0159] Step 6: Peptide content and activity determination

[0160] The polypeptide content and its biological activity (such as immunomodulatory activity) in the control group and the test group were determined respectively.

[0161] 3. Experimental results

[0162] The experimental results were evaluated by comparing the degree of peptide oxidative damage, peptide content and biological activity of the control group and the experimental group. See the table below:

[0163] 3.1 Peptide oxidative damage

[0164] Group Treatment Carboxyl content (nmol / mg protein) Control group Ultrasonic wave + plasma 75 Experimental Group Ultrasound + plasma + antioxidant nanozymes 15

[0165] 3.2 Peptide content

[0166] Group Treatment Peptide content (mg / g) Control group Ultrasonic wave + plasma 8.3 Experimental Group Ultrasound + plasma + antioxidant nanozymes 8.5

[0167] 3.3 Biological activity (immunomodulatory activity)

[0168] Group Treatment Immunomodulatory activity (U / mL) Control group Ultrasonic wave + plasma 120 Experimental Group Ultrasound + plasma + antioxidant nanozymes 180

[0169] 4. Conclusion

[0170] This experiment introduced the coexistence treatment of antioxidant nanozymes to verify its effective role in the extraction of Astragalus peptides by low-temperature plasma. The experimental results showed that the introduction of antioxidant nanozymes significantly reduced the carboxyl content in the extract, indicating that the oxidative damage of reactive oxygen to the peptides was effectively inhibited; and compared with the control group, the peptide content in the test group was slightly increased, and the biological activity of the peptides was significantly improved. Combined with HPLC analysis and oxidation labeling reagent detection data, the effectiveness of antioxidant nanozymes in protecting peptides from oxidative damage was fully verified.

[0171] Example 4

[0172] This example uses Astragalus as an example to verify that the present invention effectively removes the active oxygen generated during the low-temperature plasma treatment through the coexistence treatment of antioxidant nanoenzymes, ensuring that the polypeptide is not damaged by oxidation.

[0173] 1. Description

[0174] Microfluidic chip separation technology relies on the activity and physicochemical properties of the components in the fluid. If the polypeptide is inactivated or degraded due to oxidation, the separation efficiency will be significantly affected. Antioxidant nanozymes can prevent oxidative damage to polypeptides by removing reactive oxygen species during the extraction process, thereby maintaining the activity and integrity of the polypeptides. The experiment compared the effects of extracts treated with and untreated with antioxidant nanozymes in microfluidic chip separation to verify the role of antioxidant nanozymes in protecting polypeptide activity and improving separation purity and efficiency.

[0175] 2. Experimental Procedure

[0176] 2.1 Experimental materials and equipment

[0177] Experimental Materials:

[0178] Astragalus powder.

[0179] Distilled water.

[0180] Low temperature plasma processing equipment.

[0181] Antioxidant nanozymes.

[0182] Ultrasonic extractor.

[0183] High-speed refrigerated centrifuge.

[0184] Microfluidic chip separation system.

[0185] High performance liquid chromatography.

[0186] Oxidative Labeling Reagents.

[0187] Ultrapure water.

[0188] Experimental equipment:

[0189] 100mL beaker.

[0190] Pipette.

[0191] Cuvette.

[0192] 2.2 Experimental procedures

[0193] Step 1: Prepare the Astragalus raw materials

[0194] Take 20 g of Astragalus powder and place it in a 100 mL beaker.

[0195] Add 80 mL of distilled water and stir well to form a uniform suspension.

[0196] Step 2: Ultrasonic Assisted Extraction

[0197] Using an ultrasonic extractor, place the beaker in an ultrasonic bath.

[0198] The ultrasonic frequency was set to 35 kHz, the temperature to 55°C, and the extraction time to 30 min (the conditions were optimized according to the preliminary experiment).

[0199] After the extraction time is up, take out the sample and cool it to room temperature.

[0200] Step 3: Low temperature plasma treatment

[0201] The samples after ultrasonic extraction were divided into two parts, one as a control group (without antioxidant nanozyme treatment) and the other as a test group (with antioxidant nanozyme added).

[0202] The samples in the control group were treated with argon plasma, the argon flow rate was controlled at 5 L / min, the treatment time was 20 minutes, and the temperature was kept below 40°C.

[0203] Antioxidant nanozymes were added to the test group samples at a concentration of 100 mg per liter of extract, followed by the same plasma treatment.

[0204] Step 4: Centrifugation and collection of peptide extract

[0205] All processed samples were placed in a high-speed refrigerated centrifuge and centrifuged at 8000 rpm for 10 minutes, and the supernatants were collected respectively.

[0206] Step 5: Microfluidic chip separation

[0207] The extract was separated using a microfluidic chip separation system, and the effects of the two groups during the separation process were compared:

[0208] Set the flow rate to 200 μL / min and inject the extract into the microfluidic chip separator through the fluid controller.

[0209] Observe and record the peak time and peak area of ​​polypeptides with different molecular weights during the separation process.

[0210] Independently separated peptide fractions were collected for subsequent analysis.

[0211] Step 6: Peptide content and activity determination

[0212] The content and purity of different polypeptide peaks in the two groups were detected and compared by HPLC.

[0213] The oxidation degree (labeled with DNPH reagent) and biological activity (such as immunomodulatory activity) of the two groups of peptides were determined to evaluate the activity of the treated samples.

[0214] 3. Experimental results

[0215] Experimental results The protective effect of antioxidant nanozymes was evaluated by comparing the peptide purity, separation efficiency and activity of the control group and the experimental group in microfluidic chip separation.

[0216] The following are the data and analysis of the experiment.

[0217] 3.1 Peptide purity

[0218] Group Treatment purity(%) Control group Ultrasonic wave + plasma 82 Experimental Group Ultrasound + plasma + antioxidant nanozymes 95

[0219] 4.2 Separation efficiency (peptide recovery rate)

[0220] Group Treatment Recovery rate (%) Control group Ultrasonic wave + plasma 75 Experimental Group Ultrasound + plasma + antioxidant nanozymes 92

[0221] 3.3 Peptide activity (immunomodulatory activity)

[0222] Group Treatment Immunomodulatory activity (U / mL) Control group Ultrasonic wave + plasma 120 Experimental Group Ultrasound + plasma + antioxidant nanozymes 180

[0223] 3.4 Degree of peptide oxidation

[0224] Group Treatment Carboxyl content (nmol / mg protein) Control group Ultrasonic wave + plasma 75 Experimental Group Ultrasound + plasma + antioxidant nanozymes 15

[0225] 4. Conclusion

[0226] This experiment used the coexistence treatment of antioxidant nanozymes to verify its effect of protecting the activity of peptides and improving the separation efficiency and purity of microfluidic chips during the low-temperature plasma extraction of Astragalus peptides. Experimental data showed that antioxidant nanozymes effectively removed the reactive oxygen species generated during the extraction process and significantly reduced the oxidative damage of peptides. Compared with the control group, the experimental group showed significant improvements in peptide purity, separation efficiency and biological activity.

[0227] Specifically, the purity of the peptides in the test group reached 95%, the separation efficiency was 92%, the immunomodulatory activity was increased to 180U / mL, and the carboxyl content was only 15nmol / mg protein. These results verify the important role of antioxidant nanozymes in protecting peptide activity and improving the separation effect of microfluidic chips.

[0228] Example 5

[0229] This example uses Astragalus as an example to verify that the combined use of the antioxidant nanozyme of the present invention and the magnetic nanomaterial can also reduce the chemical reaction between the nanomaterial and the polypeptide. The antioxidant nanozyme can protect the polypeptide from being oxidized by the adsorption material, thereby ensuring the purity and stability of the final product.

[0230] 1. Experimental Principle

[0231] Antioxidant nanozymes can effectively remove reactive oxygen species generated during peptide extraction and processing, thereby avoiding oxidative damage to peptide molecules. During the adsorption of peptides by magnetic nanomaterials, surfactants or nanoscale effects may trigger oxidation reactions, leading to degradation or inactivation of peptides. By combining the use of antioxidant nanozymes and magnetic nanomaterials, antioxidant nanozymes can remove oxidizing substances during the adsorption process, protect peptides from oxidation, and thus improve the purity and stability of peptides.

[0232] 2. Experimental Procedure

[0233] 2.1 Experimental materials and equipment

[0234] Experimental Materials:

[0235] Astragalus powder.

[0236] Distilled water.

[0237] Low temperature plasma processing equipment.

[0238] Antioxidant nanozymes.

[0239] Magnetic nanoparticles.

[0240] Ultrasonic extractor.

[0241] High-speed refrigerated centrifuge.

[0242] High performance liquid chromatography.

[0243] Oxidative Labeling Reagents.

[0244] Ultrapure water.

[0245] Experimental equipment:

[0246] 100mL beaker.

[0247] Pipette.

[0248] Cuvette.

[0249] Magnetic stirrer.

[0250] 2.2 Experimental procedures

[0251] Step 1: Prepare the Astragalus raw materials

[0252] Take 20 g of Astragalus powder and place it in a 100 mL beaker.

[0253] Add 80 mL of distilled water and stir evenly to form a uniform suspension.

[0254] Step 2: Ultrasonic Assisted Extraction

[0255] Using an ultrasonic extractor, place the beaker in an ultrasonic bath.

[0256] The ultrasonic frequency was set to 35 kHz, the temperature to 55 °C, and the extraction time to 30 min. After the extraction time was up, the sample was taken out and cooled to room temperature.

[0257] Step 3: Low temperature plasma treatment

[0258] The samples after ultrasonic extraction were divided into two parts, one as a control group (without antioxidant nanozyme treatment) and the other as a test group (with antioxidant nanozyme added).

[0259] The samples in the control group were treated with argon plasma, the argon flow rate was controlled at 5 L / min, the treatment time was 20 minutes, and the temperature was kept below 40°C.

[0260] Antioxidant nanozymes were added to the test group samples at a concentration of 100 mg per liter of extract, followed by the same plasma treatment.

[0261] Step 4: Magnetic nanomaterial adsorption

[0262] The extracts were divided into 4 groups and treated as follows:

[0263] Group 1 (control group 1): no antioxidant nanozymes, no magnetic nanoparticles.

[0264] Group 2 (Control Group 2): No antioxidant nanozymes, only magnetic nanoparticles.

[0265] Group 3 (experimental group 1): with antioxidant nanozymes and without magnetic nanoparticles.

[0266] Group 4 (experimental group 2): contains antioxidant nanozymes and magnetic nanoparticles.

[0267] 1% magnetic nanoparticles (w / v) were added to Groups 2 and 4 and stirred on a magnetic stirrer for 30 minutes to ensure adequate adsorption.

[0268] The magnetic nanoparticles were separated using an external magnetic field and the supernatant was collected.

[0269] Step 5: Peptide content and oxidation labeling

[0270] HPLC was used to detect the content and purity of each peptide group.

[0271] The carbonyl content in each group of samples was determined using DNPH oxidation labeling reagent to assess the degree of oxidative damage.

[0272] 3. Experimental results

[0273] The experimental results evaluated the synergistic protective effect of antioxidant nanozymes and magnetic nanomaterials by comparing the peptide purity, degree of oxidative damage and stability of different treatment groups. The following are the experimental data and analysis.

[0274] 4.1 Peptide purity

[0275] Group Treatment purity(%) Control group 1 No antioxidant nanozymes, no magnetic nanoparticles 70 Control group 2 No antioxidant nanozymes, only magnetic nanoparticles 65 Experimental Group 1 Contains antioxidant nanozymes, no magnetic nanoparticles 75 Experimental Group 2 There are antioxidant nanozymes and magnetic nanoparticles 90

[0276] 3.2 Peptide content

[0277] Group Treatment Peptide content (mg / g) Control group 1 No antioxidant nanozymes, no magnetic nanoparticles 6.2 Control group 2 No antioxidant nanozymes, only magnetic nanoparticles 5.8 Experimental Group 1 Contains antioxidant nanozymes, no magnetic nanoparticles 6.5 Experimental Group 2 There are antioxidant nanozymes and magnetic nanoparticles 8.0

[0278] 3.3 Degree of peptide oxidation

[0279] Group Treatment Carboxyl content (nmol / mg protein) Control group 1 No antioxidant nanozymes, no magnetic nanoparticles 70 Control group 2 No antioxidant nanozymes, only magnetic nanoparticles 85 Experimental Group 1 Contains antioxidant nanozymes, no magnetic nanoparticles 60 Experimental Group 2 There are antioxidant nanozymes and magnetic nanoparticles 20

[0280] 4. Conclusion

[0281] This experiment verified the effect of protecting peptides from oxidative damage and improving the purity and stability of peptides during the adsorption process of magnetic nanomaterials by combining antioxidant nanozymes and magnetic nanomaterials. The experimental results showed that compared with the untreated group, antioxidant nanozymes significantly reduced the oxidative damage of peptides during the adsorption process, and the carboxyl content was reduced by about 70% compared with the control group. In addition, the purity and content of peptides in test group 2 (combination of antioxidant nanozymes and magnetic nanoparticles) were significantly higher than those in the control group using only magnetic nanoparticles, indicating that antioxidant nanozymes effectively improved the purity and stability of the final product.

[0282] Specifically, the peptide purity of test group 2 reached 90%, the peptide content was 8.0 mg / g, and the carboxyl content was only 20 nmol / mg protein. These data verify the effect of antioxidant nanozymes in significantly protecting peptides from oxidation during the adsorption process of magnetic nanomaterials.

[0283] The above describes an embodiment of the present embodiment, but the present embodiment is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present embodiment, ordinary technicians in this field can also make many forms, all of which are protected by the present embodiment.

Claims

1. A process for extracting polypeptides from Chinese herbal medicine, characterized in that: The steps include: S1: Raw material preparation; Select the herbs, clean them, dry them in a cool place, and grind them into fine powder for later use; S2: Ultrasonic-assisted extraction; The medicinal powder was mixed with distilled water and initially extracted using ultrasound for 60 minutes at a temperature of 50-60°C; S3: low temperature plasma assisted extraction; After ultrasonic extraction, low-temperature plasma treatment was used; S4: Antioxidant nanozyme coexistence treatment; During the low-temperature plasma treatment, antioxidant nanoenzymes are added to remove the reactive oxygen species generated by the plasma and prevent the oxidative degradation of the peptides; S5: further extraction by enzymatic hydrolysis; Use protease to enzymolyze the extract to decompose the protein in the medicinal materials and further release the polypeptide; the enzymolysis temperature is controlled at 40-50°C and the time is 2 hours; S6: separation treatment; The extracted solution is separated to separate polypeptide molecules of different molecular weights; S7: purification treatment; Separate the peptides and purify their concentration by adsorption treatment; S8: freeze drying; The purified polypeptide liquid is freeze-dried to obtain medicinal polypeptide powder.

2. A process for extracting polypeptides from Chinese herbal medicine according to claim 1, characterized in that: In step S2, the medicinal material powder and distilled water are mixed in a ratio of 1:20, and preliminary extraction is performed using an ultrasonic processing device.

3. A process for extracting polypeptides from Chinese herbal medicine according to claim 2, characterized in that: In step S2, the ultrasonic frequency is 30-40 kHz, the temperature is maintained at 50-60°C, and the extraction time is 60 minutes.

4. A process for extracting polypeptides from Chinese herbal medicine according to claim 1, characterized in that: In step S3 , the plasma is argon plasma.

5. A process for extracting polypeptides from Chinese herbal medicine according to claim 4, characterized in that: The treatment was performed in an argon low-temperature plasma reactor in an environment below 40°C for 20 minutes.

6. A process for extracting polypeptides from Chinese herbal medicine according to claim 1, characterized in that: In step S4, during the low-temperature plasma treatment, nano-scale antioxidant enzyme material is added at a concentration of 100 mg per liter of extract.

7. A process for extracting polypeptides from Chinese herbal medicine according to claim 6, characterized in that: In step S4, the antioxidant nanozyme is a catalase-like enzyme or a superoxide dismutase-like enzyme.

8. The process for extracting polypeptides from Chinese herbal medicine according to claim 1, characterized in that: In step S5, enzymatic treatment is performed using neutral protease at 40-50°C for 2 hours.

9. A process for extracting polypeptides from Chinese herbal medicine according to claim 1, characterized in that: In step S6, a microfluidic chip is used to separate polypeptides to separate polypeptides of different molecular weights.

10. A process for extracting polypeptides from Chinese herbal medicine according to claim 1, characterized in that: In step S7, magnetic nanoparticles are added to the extract, and the target polypeptide is adsorbed and separated by an external magnetic field to improve the purity.

Images