Gastrodia elata oligopeptides for relieving neuroinflammation, their preparation methods and applications

Abstract

This invention relates to Gastrodia elata oligopeptides for relieving neuroinflammation, their preparation method, and applications. Belonging to the field of food-medicine homologous active peptides, this invention aims to address the problem of relieving neuroinflammation. The key technical points are: S100. Adding a complex glycosidase to a Gastrodia elata powder solution for the first enzymatic hydrolysis; S200. Adding a complex protease to the solution after the first enzymatic hydrolysis for the second enzymatic hydrolysis, obtaining Gastrodia elata hydrolysate; S300. Performing aqueous two-phase extraction on the Gastrodia elata hydrolysate, and collecting the supernatant from the aqueous two-phase extraction to obtain the Gastrodia elata oligopeptides. This invention prepares Gastrodia elata oligopeptides with high gastrodin content through aqueous two-phase extraction combined with complex enzymatic hydrolysis. These oligopeptides inhibit the transformation of microglia to an anti-inflammatory state, thereby relieving neuroinflammation and protecting neurons. They can be applied in foods or health products that alleviate neurological diseases caused by neuronal damage.

Description

Technical Field

[0001] This invention belongs to the field of food-medicine homologous active peptides, specifically relating to a gastrodia oligopeptide that relieves neuroinflammation, its preparation method, and its application. Background Technology

[0002] Gastrodia elata Bl. is a traditional and precious Chinese medicinal herb, a unique and advantageous resource. Its tuber is used medicinally and possesses functions of calming the liver and relieving wind-related convulsions. It is mainly used to treat rheumatic back pain, dizziness, numbness of limbs, neuralgia, convulsions, and stroke. With the deepening of research on Gastrodia elata, the research focus has shifted from cultivation and breeding to the pharmacological activity analysis and efficacy study of its effective components. The research emphasis is mainly on the sedative, hypnotic, anticonvulsant, anti-inflammatory, neuroprotective, and anti-anxiety / depressant effects of Gastrodia elata, as well as the optimization of its active ingredient extraction process and clinical research on compound Gastrodia elata preparations.

[0003] Currently, research on the main active components and pharmacological effects of Gastrodia elata focuses primarily on phenolic compounds, Parishins, and Gastrodia elata polysaccharides. Further research is needed on other potentially pharmacologically active components, such as small-molecule sugars and proteins comprising approximately 10% of the dry weight of Gastrodia elata. Wang et al. recently isolated a new tripeptide, ethyl-S-(4-hydroxybenzyl)glutathione, from Gastrodia elata and discovered its potential neuroprotective effects. Chinese invention patent CN202210082421.8, "Extraction Process of Gastrodia elata Polypeptides," improves the extraction rate of Gastrodia elata polypeptides through ultrasound-assisted enzymatic hydrolysis, but does not clearly obtain the molecular weight distribution, activity, and content of other functional components of the Gastrodia elata polypeptides. Furthermore, the cyclic ultrasound method involves short intervals between ultrasound sessions and long pause times, making it time-consuming and labor-intensive. Chinese invention patent CN202111307848.5, "Use of Gastrodia elata Polypeptides in the Preparation of Anti-COVID-19 External Protective Products," describes the use of salt extraction followed by enzymatic hydrolysis and 10 kDa membrane ultrafiltration to obtain acidic polypeptide components from Gastrodia elata, finding them to have anti-COVID-19 effects. Chinese invention patent CN202410933731.5, "A Small Peptide from Gastrodia elata and Its Preparation Method and Application," obtains a 15-peptide from Gastrodia elata through conventional polypeptide separation and purification methods such as defatting, ammonium sulfate precipitation, proteolytic hydrolysis, and elution. This peptide exhibits anti-aging and cognitive impairment-alleviating effects, but its application in large-scale preparation makes it difficult to obtain large quantities of this peptide, and functional components such as gastrodin are not effectively utilized. Currently, there are no reports on technologies that combine gastrodin and other active ingredients in Gastrodia elata with gastrodin peptides for beneficial development. Therefore, further research on the preparation methods of gastrodin peptides and expanding their application in the prevention and treatment of nervous system diseases caused by neuroinflammation or neuronal damage are urgent technical problems to be solved in this field. Summary of the Invention

[0004] To address the aforementioned objectives, this invention provides a gastrodia oligopeptide for relieving neuroinflammation and its preparation method. The prepared gastrodia oligopeptide contains certain gastrodin and its derivatives, which synergistically work with the active peptide components to effectively reduce neuroinflammation, inhibit microglial cell activation, and protect neurons. Furthermore, the preparation conditions are mild and easy to operate, making it suitable for large-scale preparation and applicable in fields such as food and biomedicine.

[0005] In a first aspect, according to the method for preparing gastrodin in some embodiments of this application, including

[0006] S100. Add a compound glycosidase to the gastrodia powder solution for the first enzymatic hydrolysis;

[0007] S200. Add a complex protease to the solution after the first enzymatic hydrolysis to perform a second enzymatic hydrolysis to obtain Gastrodia elata hydrolysate;

[0008] S300. Perform aqueous two-phase extraction on the hydrolysate of Gastrodia elata, and take the upper phase of the aqueous two-phase extraction to obtain Gastrodia elata oligopeptides.

[0009] According to the method for preparing gastrodia peptides in some embodiments of this application, the preparation of gastrodia powder solution includes the following steps:

[0010] S110. Add Gastrodia elata powder with a particle size of 20-100μm to 10-15 times the volume of pure water, heat and stir to obtain Gastrodia elata powder solution;

[0011] The heating and stirring process involves a pH range of 9.0–10.0, a heating and stirring temperature of 80–95℃, and a heating and stirring time of 1–2 hours.

[0012] According to the preparation method of Gastrodia peptide in some embodiments of this application, the mass of the complex glycosidase in step S100 is 0.2% to 0.5% of the mass of Gastrodia powder.

[0013] According to the preparation method of Gastrodia peptide in some embodiments of this application, the mass ratio of the complex glycosidase in step S100 is cellulase:amylase:pectinase = 1:(3~5):(1~2);

[0014] The first enzymatic hydrolysis temperature is 37–45℃, the first enzymatic hydrolysis pH is 6.0–7.0, and the first enzymatic hydrolysis time is 1–2 hours.

[0015] According to the method for preparing Gastrodia peptide in some embodiments of this application, the mass of the complex protease in step S200 is 0.3% to 0.5% of the volume of the solution after the first enzymatic hydrolysis.

[0016] According to the preparation method of Gastrodia peptide in some embodiments of this application, the mass ratio of the complex protease in step S200 is alkaline protease: neutral protease: fig protease = (3-5): (1-3): (1-2);

[0017] The second enzymatic hydrolysis temperature is 45–55℃, the second enzymatic hydrolysis pH is 7.0–8.0, and the second enzymatic hydrolysis time is 3–6 h.

[0018] According to the preparation method of Gastrodia peptide in some embodiments of this application, in step S200, the solution after the second enzymatic hydrolysis is dried to obtain Gastrodia hydrolysate;

[0019] According to the preparation method of Gastrodia peptide in some embodiments of this application, in step S200, the residue is removed from the solution after the second enzymatic hydrolysis, and then the solution is dried to obtain Gastrodia hydrolysate.

[0020] According to the preparation method of Gastrodia peptide in some embodiments of this application, in step S200, the residue is removed by centrifugation;

[0021] According to the preparation method of Gastrodia peptide in some embodiments of this application, in step S200, the residue is removed by centrifugation at 8000 r / min.

[0022] According to the preparation method of Gastrodia peptide in some embodiments of this application, the aqueous two-phase system in the aqueous two-phase extraction in step S300 is composed of polyethylene glycol (PEG), dextran (DETRAN) and water;

[0023] The mass percentage of polyethylene glycol in the aqueous two-phase system is 20%–30%, and the mass percentage of dextran in the aqueous two-phase system is 5%–20%.

[0024] According to the preparation method of Gastrodia peptide in some embodiments of this application, the mass-volume ratio of Gastrodia hydrolysate to the total volume of the aqueous two-phase system is 1:(10-30).

[0025] According to the preparation method of Gastrodia elata peptide in some embodiments of this application, the mass-volume ratio of Gastrodia elata hydrolysate to the total volume of the aqueous two-phase system is 1:20.

[0026] According to the preparation method of Gastrodia elata peptide in some embodiments of this application, in step S300, polyethylene glycol in the upper phase of aqueous two-phase extraction is removed and dried to obtain Gastrodia elata oligopeptide.

[0027] According to the preparation method of Gastrodia elata peptide in some embodiments of this application, the drying is one of spray drying, freeze drying or vacuum drying.

[0028] According to the preparation method of Gastrodia peptide in some embodiments of this application, Gastrodia is either red Gastrodia or black Gastrodia.

[0029] In a second aspect, the use of gastrodia peptides prepared by any of the methods described herein in the preparation of drugs for relieving neuroinflammatory diseases, or in the preparation of food or in the preparation of functional foods.

[0030] Beneficial effects:

[0031] The present invention can improve the yield and hydrolysis effect of Gastrodia elata peptides by enzymatic hydrolysis with the complex glycosidase and complex protease, and make the enzymatic hydrolysate contain active peptide components with the activity of relieving neuroinflammatory.

[0032] This invention utilizes a PEG-DETRAN aqueous two-phase extraction system to extract gastrodin and gastrodin peptides from the enzymatic hydrolysate of Gastrodia elata. During the process, it was found that under the conditions of the aqueous two-phase system, gastrodin and the active peptide component (TMP-NF) with neuroinflammatory-relieving effects were extracted into the PEG upper phase of the aqueous two-phase system, achieving good separation from starch and other substances, which constitute 60-70% of the main components of Gastrodia elata. After removing the PEG from the upper phase and drying, high-gastrodin-content Gastrodia elata oligopeptides were obtained. The obtained Gastrodia elata oligopeptides, under the synergistic effect of gastrodin and TMP-NF, exhibited superior neuroprotective effects compared to an equivalent amount of gastrodin or Gastrodia elata extract. The entire preparation process is mild, has a short production cycle, and uses a simple PEG recovery method, making it suitable for large-scale preparation and showing promising application prospects in the prevention and treatment of neurological diseases. Attached Figure Description

[0033] Figure 1 Gastrodin content and protein content.

[0034] Figure 2 Survival and activation rates of microglia, including (a) relative levels of NF-κB protein, (b) relative levels of TNF-α protein, and (c) relative levels of iNOS protein. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments do not constitute a limitation on the scope of protection of the present invention.

[0036] TMH: Tianma Hydrolysate;

[0037] PEG: Polyethylene glycol;

[0038] DETRAN: Glucan;

[0039] LP: Lower Phase Extraction Powder;

[0040] TMP: Gastrodia elata oligopeptide.

[0041] TME: Gastrodia elata extract that has not been hydrolyzed by complex proteases.

[0042] The present invention discloses a method for preparing Gastrodia elata oligopeptides that alleviate neuroinflammation, comprising:

[0043] S1. After cleaning the Gastrodia elata, dry it in the sun and then pulverize it into ultrafine powder with a particle size of 20-100μm. Take an appropriate amount of Gastrodia elata powder, add 10-15 times the amount of pure water, adjust the pH to 9.0-10.0, and heat and stir at 80-95℃ for 1-2 hours.

[0044] Furthermore, the gastrodia elata is either red gastrodia elata or black gastrodia elata.

[0045] Add 0.2-0.5% of a compound glycosidase by weight of Gastrodia elata powder to the liquid in S2.S1. The compound glycosidase includes cellulase:amylase:pectinase = 1:3-5:1-2. Enzymatic hydrolysis is carried out at 37-45℃ and pH 6.0-7.0 for 1-2 hours. The residue is removed by centrifugation at 8000r / min to obtain the first enzymatic hydrolysate.

[0046] S3. Add a complex protease to the first enzymatic hydrolysate. The complex protease includes alkaline protease: neutral protease: fig protease = 3-5: 1-3: 1-2. Enzymatically hydrolyze for 3-6 hours at 45-55℃ and pH 7.0-8.0. Centrifuge at 8000r / min to remove the residue to obtain the second enzymatic hydrolysate. Dry to obtain Gastrodia elata hydrolysate.

[0047] S4. Perform aqueous two-phase extraction on the hydrolysate of Gastrodia elata, wherein the aqueous two-phase system in the aqueous two-phase extraction consists of polyethylene glycol (PEG), dextran and water, wherein the mass percentage of polyethylene glycol in the aqueous two-phase system is 20% to 30%, the mass percentage of dextran in the aqueous two-phase system is 5% to 20%, and the remainder is water.

[0048] Furthermore, the mass-to-volume ratio of the gastrodia hydrolysate to the total volume of the aqueous two-phase system is 1:10 to 30, preferably 1:20.

[0049] S5. Take the upper phase of the aqueous two-phase extraction, remove PEG, and dry to obtain Gastrodia elata oligopeptide.

[0050] Furthermore, the drying process can be selected from one of spray drying, freeze drying, or vacuum drying.

[0051] Example 1. Preparation of Gastrodia elata oligopeptide TMP1, comprising the following steps:

[0052] S1. After cleaning the red Gastrodia elata, dry it in the sun and then pulverize it into ultrafine powder with a particle size of 50μm. Take 100g of Gastrodia elata powder, add 10 times the amount of pure water, adjust the pH to 9.0, and heat and stir at 90℃ for 2 hours.

[0053] Add 0.5% of the mass of Gastrodia elata powder to the liquid in S2.S1. The complex glycosidase includes cellulase:amylase:pectinase = 1:4:2. Enzymatic hydrolysis is carried out at 37℃ and pH 6.5 for 2 hours. The residue is removed by centrifugation at 8000 r / min to obtain the first enzymatic hydrolysate.

[0054] S3. Add a complex protease to the first enzymatic hydrolysate. The complex protease consists of alkaline protease, neutral protease, and fig protease in a ratio of 5:2:2. Enzymatically hydrolyze for 4 hours at 50°C and pH 8.0. Centrifuge at 8000 r / min to remove the residue and obtain the second enzymatic hydrolysate. Dry the hydrolysate to obtain Tianma Hydrolysate (TMH1).

[0055] S4. Take 25g of Gastrodia elata hydrolysate TMH1 into a beaker, then add 100g of polyethylene glycol, 75g of dextran, and 325g of pure water. Stir for 1 minute and then transfer to a separatory funnel and let stand for 24 hours.

[0056] S5. After releasing the lower phase of the aqueous two-phase extraction and drying it, the lower phase powder (LP1) is obtained. After releasing the upper phase of the aqueous two-phase extraction and removing polyethylene glycol by vacuum evaporation, the powder is freeze-dried to obtain Tianma Peptide (TMP1).

[0057] Example 2. Preparation of Gastrodia elata oligopeptide TMP2

[0058] S1. After cleaning the red Gastrodia elata, dry it in the sun and then pulverize it into ultrafine powder with a particle size of 100μm. Take 100g of Gastrodia elata powder, add 10 times the amount of pure water, adjust the pH to 9.0, and heat and stir at 90℃ for 2 hours.

[0059] Add 0.5% of the mass of Gastrodia elata powder to the liquid in S2.S1. The complex glycosidase includes cellulase:amylase:pectinase = 1:3:1. Enzymatic hydrolysis is carried out at 37℃ and pH 7.0 for 1 hour. The residue is removed by centrifugation at 8000 r / min to obtain the first enzymatic hydrolysate.

[0060] S3. Add a complex protease to the first enzymatic hydrolysate. The complex protease includes alkaline protease: neutral protease: fig protease = 4:3:2. Enzymatically hydrolyze for 5 hours at 55°C and pH 7.5. Centrifuge at 8000 r / min to remove residue and obtain the second enzymatic hydrolysate. Dry to obtain Gastrodia elata hydrolysate TMH2.

[0061] S4. Take 25g of Gastrodia elata hydrolysate TMH2 into a beaker, then add 150g of polyethylene glycol, 100g of dextran, and 250g of pure water. Stir for 1 minute, then transfer to a separatory funnel and let stand for 36 hours.

[0062] S5. After releasing the lower phase of the aqueous two-phase extraction and drying it, the lower phase powder LP2 is obtained. After releasing the upper phase of the aqueous two-phase extraction and removing polyethylene glycol by vacuum evaporation, it is freeze-dried to obtain Gastrodia elata oligopeptide TMP2.

[0063] Example 3. Preparation of Gastrodia elata oligopeptide TMP3

[0064] S1. After cleaning and drying the Gastrodia elata, pulverize it into ultrafine powder with a particle size of 50μm. Take 100g of Gastrodia elata powder, add 15 times the amount of pure water, adjust the pH to 9.5, and heat and stir at 80℃ for 2 hours.

[0065] Add 0.3% of the mass of Gastrodia elata powder to the liquid in S2.S1. The complex glycosidase includes cellulase:amylase:pectinase = 1:5:2. Enzymatic hydrolysis is carried out at 45℃ and pH 7.0 for 1 hour. The residue is removed by centrifugation at 8000 r / min to obtain the first enzymatic hydrolysate.

[0066] S3. Add a complex protease to the first enzymatic hydrolysate. The complex protease includes alkaline protease: neutral protease: fig protease = 4:1:1. Enzymatically hydrolyze for 6 hours at 50℃ and pH 8.0. Centrifuge at 8000 r / min to remove residue and obtain the second enzymatic hydrolysate. Dry to obtain Gastrodia elata hydrolysate TMH3.

[0067] S4. Take 25g of Gastrodia elata hydrolysate TMH3 into a beaker, then add 150g of PEG, 75g of dextran and 275g of pure water. Stir for 1 minute and then transfer to a separatory funnel and let stand for 24 hours.

[0068] S5. After releasing the lower phase of the aqueous two-phase extraction and drying, the lower phase powder LP3 is obtained. After releasing the upper phase of the aqueous two-phase extraction and removing PEG by vacuum evaporation, the powder is freeze-dried to obtain Gastrodia elata oligopeptide TMP3.

[0069] Example 4. Preparation of Gastrodia elata oligopeptide TMP4

[0070] The preparation steps of Example 4 are basically the same as those of Example 1, except for steps S4 and S5 in Example 4:

[0071] S4. Take 25g of Gastrodia elata hydrolysate TMH1 into a beaker, then add 50g of polyethylene glycol, 150g of dextran and 300g of pure water. Stir for 1 minute and then transfer to a separatory funnel and let stand for 24 hours.

[0072] S5. After releasing the lower phase of the aqueous two-phase extraction and drying it, the lower phase powder (LP4) is obtained. After releasing the upper phase of the aqueous two-phase extraction and removing polyethylene glycol by vacuum evaporation, the powder is freeze-dried to obtain Tianma Peptide (TMP4).

[0073] The amounts of ingredients in step S4 were adjusted to include Gastrodia elata hydrolysate TMH1 (25g), polyethylene glycol (50g), dextran (150g), and purified water (300g), ultimately yielding the lower phase powder LP4 and Gastrodia elata oligopeptide TMP4.

[0074] In Example 4, the hydrolysate TMH1 from Gastrodia elata is referred to as hydrolysate TMH4 from Gastrodia elata.

[0075] Example 5. Preparation of Gastrodia elata oligopeptide TMP5

[0076] The preparation steps of Example 5 are basically the same as those of Example 1, except that Example 5 does not have the complex glycosidase enzymatic hydrolysis step S2 of Example 1.

[0077] In step S3 of Example 1, a complex protease is directly added to the solution obtained in step S1 to obtain Gastrodia elata hydrolysate TMH5.

[0078] After aqueous two-phase extraction of Gastrodia elata hydrolysate TMH5, the lower phase powder LP5 and Gastrodia elata oligopeptide TMP5 were obtained.

[0079] Example 6. Preparation of Gastrodia elata extract TME

[0080] The preparation steps of Example 6 are the same as those of Example 1, except that Example 5 does not have the complex protease hydrolysis step S3 of Example 1.

[0081] That is, after the glycosidase hydrolysis in step S2 of Example 1, the gastrodia hydrolysate TMH6 is dried to obtain gastrodia hydrolysate TMH6. The gastrodia hydrolysate TMH6 is then subjected to aqueous two-phase extraction to obtain the lower phase powder LP6, which is gastrodia extract TME.

[0082] Experimental Example 1. Gastrodin / Peptide Extraction Rate: The content of gastrodin in Gastrodia elata oligopeptides was determined by RP-HPLC high performance liquid chromatography, and the protein content in Gastrodia elata oligopeptides and Gastrodia elata hydrolysate was determined by BCA method. The results are shown in the table below. Compared with the unextracted gastrodin hydrolysates TMH1, TMH2, and TMH3 obtained by drying the upper phase after extraction according to the aqueous two-phase system described in this invention, the gastrodin and protein contents of TMP1, TMP2, and TMP3 were significantly increased (P < 0.05), indicating a good extraction effect. The gastrodin and protein contents of TMP4 obtained by extraction using an aqueous two-phase system with a different ratio than that described in this invention were significantly lower than those of TMP1, TMP2, and TMP3 (P < 0.05), indicating a significant decrease in extraction effect. The extraction efficiency of gastrodin and gastrodin peptides by TMH5 without glycosidase hydrolysis was significantly lower than that of gastrodin hydrolysates from other examples (P < 0.05), resulting in a decrease in the content of gastrodin and gastrodin peptides in the final TMP5. TME was at the same level as TMP1 in terms of gastrodin extraction effect (P > 0.05), but it could not simultaneously extract the unhydrolyzed protein components. The above results demonstrate that the aqueous two-phase extraction method provided by this invention can enrich and purify gastrodin oligopeptides with high gastrodin content, while the glycosidase hydrolysis step can improve the yield of both gastrodin and gastrodin peptides. It should be noted that TMH1 and TMH4 are essentially prepared using the same steps; the very small difference in gastrodin and protein content between the two is a reasonable deviation due to different experimental measurements.

[0083] Group Gastrodin content (%) Protein content (%) TMP1 7.44±0.51 25.33±2.48 TMP2 7.13±0.63 24.07±3.12 TMP3 6.89±0.39 24.58±1.17 TMP4 3.84±0.85 11.91±2.79 TMP5 3.30±0.66 19.15±3.75 TME 7.16±0.30 8.57±1.49 TMH1 1.58±0.42 7.17±0.94 TMH2 1.65±0.36 7.72±1.14 TMH3 1.50±0.22 6.84±0.71 TMH4 1.63±0.31 7.32±0.78 TMH5 0.57±0.29 4.64±1.35 TMH6 1.71±0.18 7.94±1.08

[0084] Experimental Example 2. Inhibition rate of microglia activation in vitro: BV2 microglia were completely cultured in DMEM containing 10% FBS and passaged at 37°C with 5% CO2. Cells in logarithmic growth phase were adjusted to a density of 2 × 10⁻⁶ cells / cells. 5 100 μL of LPS was seeded into each well of a 96-well cell culture plate and cultured for 3–4 h. The experiment was divided into a control group, LPS group, TMP1 group, TMP2 group, TMP3 group, TMP4 group, TMP5 group, and TME group. LPS was diluted to 1 mg / L with ultrapure water, and TMP / TME in each group was dissolved in 0.01 mol / L PBS buffer (pH 7.4) to a concentration of 50 mg / L. After cell attachment, the supernatant was discarded. 100 μL of DMEM medium was added to each well of the control group, 90 μL of DMEM was added to each well of the LPS group, and 80 μL of DMEM and 10 μL of TMP dilution were added to each well of the TMP / TME groups. After 1 h of culture, 10 μL of LPS was added to each well of the LPS and TMP / TME groups, and the 96-well cell culture plates were placed in a 5% CO2, 37°C cell culture incubator for another 24 h. The cell viability of each group was determined by the MTT assay. Cell viability = (A value of intervention group - A value of blank well) / (A value of control group - A value of blank well) × 100%. The microglia activation rate was calculated by randomly photographing 3-5 fields of view for each group and morphologically observing the percentage of "amoebic" activated cells in each field. Results are as follows: Figure 1 As shown, the survival rate of microglia treated with LPS decreased significantly compared with the control group, while the proportion of activated cells increased. The cell survival rate of cells treated with TMP1, TMP2, and TMP3 was significantly increased (P>0.05) and the activation rate was significantly decreased (P>0.05) compared with the LPS group. Compared with the TME group containing the same level of gastrodin but not subjected to protease hydrolysis, the inhibitory effect on microglia activation was significantly improved (P>0.05), indicating that the upper phase of the aqueous two-phase extraction described in this invention contains active peptide components that inhibit microglia activation.

[0085] Example 3. LPS-induced neuroinflammation mouse model: Mice were acclimatized for 7 days before the experiment began. From day 1 to day 7, except for the control group, all other groups received intraperitoneal injections of LPS (0.25 mg / kg). Starting on day 8, all treatment groups received LPS via gavage once daily for 2 weeks. Mice were divided into 8 groups of 3 mice each, including a blank / model group (normal diet + saline), and experimental groups (TMP1, TMP2, TMP3, TMP4, TMP5, TME, etc., normal diet + 1 g / kg / day of TMP / TME). After 2 weeks, cerebral cortex tissue was collected from mice via cardiac perfusion, tissue proteins were extracted using the Trizol method, and the protein expression of NF-κB, TNF-α, and iNOS in the tissue was measured by immunoblotting. Results are as follows: Figure 2 As shown, compared with the control group, the protein expression levels of NF-κB, iNOS, and TNF-α in the model group were increased, and the differences were statistically significant. Compared with the model group, the protein expression levels of NF-κB, iNOS, and TNF-α in the TMP1-5 group and the TME group were decreased, and the differences were statistically significant. The decrease in the TMP1-3 group was greater than that in the TMP4, TMP5, and TME groups, and the differences were statistically significant. This indicates that the TMP1-3 group has a stronger effect in relieving neuroinflammation.

[0086] As can be seen from the above embodiments, the preparation method of Gastrodia elata peptides described in this invention can obtain Gastrodia elata active peptide components that inhibit microglial cell activation and relieve neuroinflammation. At the same time, extraction through the aqueous two-phase system can simultaneously extract gastrodin and the Gastrodia elata active peptide components into the upper phase of the aqueous two-phase system, obtaining Gastrodia elata oligopeptide products with high gastrodin content, further optimizing the effect of relieving neuroinflammation, and showing good application prospects in functional foods, biomedicine and other fields.

[0087] This invention prepares gastrodin oligopeptides with high gastrodin content through aqueous two-phase extraction combined with complex enzymatic hydrolysis. These oligopeptides inhibit the transformation of microglia into an anti-inflammatory state, thereby alleviating neuroinflammation and protecting neurons. They can be applied to foods or health products that alleviate neurological diseases caused by neuronal damage.

[0088] Finally, it should be noted that the above examples are merely a few specific embodiments of the present invention. All derivatives that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. A method for preparing a Gastrodia elata peptide, characterized by, include S100. A compound glycosidase is added to the gastrodia powder solution for the first enzymatic hydrolysis; wherein, the mass ratio of the compound glycosidase is cellulase:amylase:pectinase = 1:(3~5):(1~2); the first enzymatic hydrolysis temperature is 37~45℃, the first enzymatic hydrolysis pH is 6.0~7.0, and the first enzymatic hydrolysis time is 1~2h; S200. A complex protease is added to the solution after the first enzymatic hydrolysis for a second enzymatic hydrolysis to obtain Gastrodia elata hydrolysate; wherein, the mass ratio of the complex protease is alkaline protease: neutral protease: fig protease = (3~5): (1~3): (1~2); the second enzymatic hydrolysis temperature is 45~55℃, the second enzymatic hydrolysis pH is 7.0~8.0, and the second enzymatic hydrolysis time is 3~6h; S300. Aqueous two-phase extraction is performed on the hydrolysate of Gastrodia elata, and the upper phase of the aqueous two-phase extraction is taken to obtain Gastrodia elata oligopeptides with high gastrodin content; wherein, the aqueous two-phase system in the aqueous two-phase extraction is composed of polyethylene glycol (PEG), dextran and water; the mass percentage of polyethylene glycol in the aqueous two-phase system is 20% to 30%, and the mass percentage of dextran in the aqueous two-phase system is 5% to 20%.

2. The method for preparing gastrodia peptide according to claim 1, characterized in that, The preparation of Gastrodia elata powder solution includes the following steps: S110. Gastrodia elata powder with a particle size of 20~100μm is added to 10~15 times the volume of pure water and heated and stirred to obtain Gastrodia elata powder solution; wherein, the pH of heating and stirring is 9.0~10.0, the heating and stirring temperature is 80~95℃, and the heating and stirring time is 1~2h.

3. The method for preparing gastrodia peptide according to claim 1, characterized in that, In step S100, the mass of the complex glycosidase is 0.2% to 0.5% of the mass of Gastrodia elata powder.

4. The method for preparing gastrodia peptide according to claim 1, characterized in that, In step S200, the mass of the complex protease is 0.3% to 0.5% of the volume of the solution after the first enzymatic hydrolysis.

5. The method for preparing gastrodia peptide according to claim 1, characterized in that, In step S200, the solution after the second enzymatic hydrolysis is dried to obtain Gastrodia elata hydrolysate; wherein, in step S200, the residue is removed from the solution after the second enzymatic hydrolysis, and then the solution is dried to obtain Gastrodia elata hydrolysate; In step S200, the residue is removed by centrifugation; wherein, in step S200, the residue is removed by centrifugation at 8000 r / min.

6. The method for preparing gastrodia peptide according to claim 1, characterized in that, In step S300, the mass-to-volume ratio of the gastrodia hydrolysate to the total volume of the aqueous two-phase system is 1:(10~30).

7. The method for preparing gastrodia peptide according to claim 6, characterized in that, In step S300, the mass-to-volume ratio of the total volume of the gastrodia hydrolysate to the aqueous two-phase system is 1:

20.

8. The method for preparing gastrodia peptide according to claim 1, characterized in that, In step S300, polyethylene glycol is removed from the upper phase of the aqueous two-phase extraction, and the product is dried to obtain Gastrodia elata oligopeptides; wherein, the drying is one of spray drying, freeze drying or vacuum drying; and the Gastrodia elata is one of red Gastrodia elata or black Gastrodia elata.

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