A highly active extract of eucommia ulmoides and its preparation method and use

Abstract

This invention discloses a highly active Cornus officinalis extract, its preparation method, and its uses. The preparation method includes hot water extraction, stepwise enzymatic hydrolysis with a compound enzyme, separation, filtration, and concentration. During enzymatic hydrolysis, a specific compound enzyme A (composed of pectinase, cellulase, and hemicellulase in a mass ratio of 1.5-2.5:1.5-2.5:1) and a compound enzyme B (composed of α-amylase and glucosidase in a mass ratio of 0.8-1.5:1) are used to perform stepwise enzymatic hydrolysis of the Cornus officinalis extract. This not only significantly improves the dissolution rate of the active ingredients in the final extract but also transforms large molecules into more easily absorbed small molecules through enzymatic hydrolysis, thereby simultaneously enhancing the bioavailability and physiological activity of the final product.

Description

Technical Field

[0001] This invention relates to the field of Cornus officinalis processing technology, and in particular to a highly active Cornus officinalis extract, its preparation method, and its uses. Background Technology

[0002] Cornus officinalis was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica). It is slightly warm in nature, sour and astringent in taste, and enters the liver and kidney meridians. It has the effects of tonifying the liver and kidneys, and astringing essence and preventing prolapse. Modern pharmacological studies have confirmed that Cornus officinalis is rich in iridoid glycosides (such as monoglobulin and loganin), polysaccharides, saponins, organic acids, and other active ingredients. In animal models and some clinical studies, it has shown significant hypoglycemic, yin-nourishing and kidney-tonifying, anti-inflammatory, and antioxidant stress-relieving effects, making it a representative drug for protecting the kidneys and lowering blood sugar.

[0003] Currently, the main processing methods for Cornus officinalis are water extraction, ultrasonic extraction, alkaline extraction, and alcohol extraction. These methods have problems such as low dissolution rate of active ingredients, long-term and high-temperature extraction that easily leads to the inactivation of heat-sensitive components, and the resulting extract is prone to precipitation during storage due to its high content of pectin, starch and other macromolecules, resulting in poor stability. Summary of the Invention

[0004] The purpose of this invention is to provide a highly active Cornus officinalis extract and its preparation method, which can improve the dissolution rate of active ingredients in the Cornus officinalis extract and also transform macromolecular substances into more easily absorbed small molecule components through enzymatic hydrolysis, thereby simultaneously enhancing the bioavailability and physiological activity of the final product.

[0005] To achieve the above objectives, the solution of the present invention is: a method for preparing a highly active Cornus officinalis extract, comprising the following steps:

[0006] Pretreatment: Mix the pulp of Cornus officinalis with water at a mass ratio of 1:15-40, and then pulp to obtain Cornus officinalis pulp; Extraction: The slurry is extracted to obtain a water extract of Cornus officinalis, which is then homogenized to obtain a mixed extract of Cornus officinalis. Enzymatic hydrolysis: Add 0.2-0.5% (by weight of water) of compound enzyme A to the Cornus officinalis mixture and hydrolyze at 40-50℃ for 60-100 min; then add 0.2-0.5% (by weight of water) of compound enzyme B and hydrolyze at 40-50℃ for 30-60 min; after hydrolysis, inactivate the enzyme at 85-90℃ for 10-20 min to obtain Cornus officinalis enzymatic hydrolysate; The complex enzyme A is composed of pectinase, cellulase and hemicellulase, with a mass ratio of 1.5~2.5:1.5~2.5:1; the complex enzyme B is composed of α-amylase and glucosidase, with a mass ratio of α-amylase and glucosidase of 0.8~1.5:1. Separation, filtration, and concentration: The enzymatic hydrolysate is subjected to solid-liquid separation to obtain the supernatant, which is then filtered and concentrated to obtain the Cornus officinalis extract.

[0007] Furthermore, in the extraction step, Cornus officinalis is mixed with deionized water and then heated to 85-95℃ for 30-90 minutes to obtain an aqueous extract of Cornus officinalis.

[0008] Furthermore, the pectinase is Nanning Pangbo pectinase, with an enzyme activity of 30,000 U / g; The cellulase mentioned is Nanning Pangbo cellulase, with an enzyme activity of 20,000 U / g; The α-amylase mentioned is Nanning Pangbo α-amylase, with an enzyme activity of 5000 U / g; The hemicellulase mentioned is Nanning Pangbo hemicellulase, with an enzyme activity of 100,000 U / g; The glucosidase is Jiangsu Duoyang Bio-glucosidase, with an enzyme activity of 100,000 U / g.

[0009] Furthermore, in the complex enzyme A, the mass ratio of pectinase, cellulase and hemicellulase is 1.6~2.3:1.6~2.3:1.

[0010] Furthermore, in the complex enzyme B, the mass ratio of α-amylase to glucosidase is 1~1.3:1.

[0011] Furthermore, in the separation and filtration steps, the Cornus officinalis enzymatic hydrolysate is centrifuged at 4500–5500 rpm for 5–10 min, the supernatant is collected, and the supernatant is filtered through a membrane microfiltration filter with a pore size of 0.45–0.50 μm to obtain the Cornus officinalis filtrate.

[0012] Furthermore, in the concentration step, the Cornus officinalis filtrate is concentrated to 25-30% solids at 75-80 °C to obtain Cornus officinalis extract.

[0013] Furthermore, it is prepared using the preparation method described in any one of claims 1 to 7.

[0014] Furthermore, the extract contains ≥7.0 mg / mL mononoside and ≥3.0 mg / mL loganin, and has a DPPH free radical scavenging rate of ≥85% and an ABTS free radical scavenging rate of ≥90%.

[0015] The above-mentioned highly active Cornus officinalis extract is used in the preparation of drugs for tonifying the kidney and liver and / or lowering blood sugar.

[0016] After adopting the above solution, the beneficial effects of the present invention are as follows: This invention employs hot water extraction combined with stepwise enzymatic hydrolysis using compound enzymes to prepare a highly active Cornus officinalis extract.

[0017] First, the Cornus officinalis fruit raw material is pulped and extracted with hot water. Hot water extraction can soften the cell walls, initially release active ingredients, and at the same time inactivate the endogenous enzymes and bacteria in the raw material, providing favorable conditions for subsequent enzymatic hydrolysis reactions.

[0018] Then, the pulp is enzymatically hydrolyzed stepwise using specific complex enzymes A (pectinase, cellulase, and hemicellulase) and B (α-amylase and glucosidase) to specifically degrade plant cell walls, completely breaking down the dissolution barrier of active ingredients and significantly improving the dissolution rate. This results in a high content of active ingredients (monoglycosides, loganin, etc.) in the final Cornus officinalis extract, which also possesses antioxidant activity. It can serve as a high-value raw material for the development of biopharmaceuticals with hypoglycemic, kidney-tonifying, and liver-benefiting effects. Furthermore, enzymatic hydrolysis converts large molecules into more easily absorbed small molecules, thereby simultaneously enhancing the bioavailability and physiological activity of the final extract.

[0019] The process conditions of this invention are mild, better preserving the natural medicinal efficacy of Cornus officinalis. Specifically, although it includes a short high-temperature extraction step (for rapid sterilization, softening of tissues, and initial dissolution of active ingredients), the introduction of a highly efficient multi-enzyme stepwise enzymatic hydrolysis process (conducted under mild conditions of 40-50°C) significantly reduces the time required for high-temperature extraction. The enzymatic hydrolysis process specifically disrupts cell walls at a mild temperature, replacing the long high-temperature treatment required in traditional processes to achieve the same extraction efficiency. This reduces the total time that heat-sensitive active ingredients are exposed to high temperatures, effectively preventing degradation caused by prolonged heat exposure.

[0020] Enzymatic hydrolysis can break down large molecular impurities such as pectin and starch that cause product turbidity and precipitation, significantly improving the clarity and long-term storage stability of the extract and extending its shelf life.

[0021] This invention utilizes simple operations such as extraction, enzymatic hydrolysis, and filtration to obtain the desired Cornus officinalis extract. It requires minimal equipment, is simple and easy to operate, and is suitable for large-scale industrial production. The entire process does not use organic solvents, complies with environmental and food safety standards, and has promising market application prospects. Attached Figure Description

[0022] Figure 1 A comparison chart of DPPH free radical scavenging rates of different Cornus officinalis extracts; Figure 2 A comparative chart showing the ABTS free radical scavenging rates of different Cornus officinalis extracts; Figure 3 A comparison of the effects of different Cornus officinalis extracts on GLP-1 secretion in STC-1 cells; Figure 4 A comparative diagram showing the effects of different Cornus officinalis extracts on ROS in the TM3 cell D-gal model. Figure 5 A comparative diagram showing the effects of different Cornus officinalis extracts on gene expression levels in the TM3 cell D-gal model. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. The technical features designed in different implementations of this application described below can be combined with each other as long as they do not conflict with each other. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0024] In the description of this application, it should be noted that all terms used in this application (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, and should not be construed as limiting this application; it should be further understood that the terms used in this application should be understood to have the same meaning as those in the context of this specification and the relevant field, and should not be understood in an idealized or overly formal sense, except as expressly defined in this application.

[0025] This invention provides a method for preparing a highly active Cornus officinalis extract, comprising the following steps: Step 1: Preprocessing Cornus officinalis pulp is mixed with water at a mass ratio of 1:15-40 and then pulped to obtain Cornus officinalis pulp. Deionized water is preferred for this process.

[0026] Step 2, Extraction: The cornelian cherry pulp was heated to 85-95℃ and extracted for 30-90 minutes to obtain a cornelian cherry aqueous extract. The aqueous extract was then homogenized to obtain a cornelian cherry mixture.

[0027] Step 3, Enzymatic hydrolysis: Step 1: Enzymatic hydrolysis: Add compound enzyme A to the Cornus officinalis mixture and hydrolyze for 60–100 min at 40–50℃; the amount of compound enzyme A added is 0.2–0.5% of the water mass. The complex enzyme A is composed of pectinase, cellulase, and hemicellulase, with a mass ratio of 1.5~2.5 : 1.5~2.5 : 1. Alternatively, the mass ratio of pectinase, cellulase, and hemicellulase in the complex enzyme A can be 1.6~2.3 : 1.6~2.3 : 1, 2 : 2 : 1, etc.

[0028] The second step is enzymatic hydrolysis: Compound enzyme B is added to the above enzymatic hydrolysis system, and the mixture is hydrolyzed at 40–50°C for 30–60 minutes; then, the enzyme is inactivated at 85–90°C for 10–20 minutes to obtain the hydrolysate; the amount of compound enzyme B added is 0.2–0.5% of the water mass. The water mass mentioned in this case refers to the amount of water added when forming the slurry.

[0029] The complex enzyme B is composed of amylase and glucosidase, with the mass ratio of α-amylase to glucosidase being 0.8~1.5:1. In the complex enzyme B, the mass ratio of α-amylase to glucosidase can also be 1~1.3:1, 1:1, etc.

[0030] Step 4: Centrifugation and filtration: The enzymatic hydrolysate was centrifuged at 4500–5500 rpm for 5–10 min, and the supernatant was collected. The supernatant was then filtered through a membrane microfiltration system with a pore size of 0.45–0.50 μm to obtain the Cornus officinalis filtrate.

[0031] Step 5: Concentration: The Cornus officinalis filtrate was concentrated to 25-30% solids at 75-80 °C to obtain Cornus officinalis extract.

[0032] This application provides the following embodiments and comparative examples to verify the effectiveness of the proposed solution: Example 1 (1) Raw material pretreatment: Take 200g of Cornus officinalis pulp and add 3kg of water. The material-to-liquid ratio is 1:15. After pulping, stir for 30 minutes until completely mixed to form Cornus officinalis pulp.

[0033] (2) Extraction: The slurry was heated to 85℃ and extracted for 120 minutes to obtain an aqueous extract. The aqueous extract was then uniformly processed to obtain a mixed extract of Cornus officinalis.

[0034] (3) Enzymatic hydrolysis: Add 15g of compound enzyme A to the cornus officinalis mixture and hydrolyze at 50℃ for 90min; compound enzyme A is a mixture of pectinase, cellulase and hemicellulase in a mass ratio of 2:2:1. Then add 6g of compound enzyme B and hydrolyze at 45℃ for 50min. Compound enzyme B is a mixture of amylase and glucosidase in a mass ratio of 1:1. After hydrolysis, inactivate the enzyme at 85℃ for 15min and allow it to cool naturally to obtain the hydrolysate.

[0035] (4) Centrifugation and filtration: The enzymatic hydrolysate was centrifuged at 5000 rpm for 5 min, and the supernatant was microfiltered through a membrane with a pore size of 0.45 μm to obtain the Cornus officinalis filtrate.

[0036] (5) Concentration: The Cornus officinalis filtrate was concentrated to 25% solids at 80 °C to obtain the Cornus officinalis extract.

[0037] Example 2 The difference between this embodiment and Embodiment 1 is that, in the enzymatic hydrolysis step, the mass ratio of pectinase, cellulase, and hemicellulase in compound enzyme A is 1.8:2:1. The mass ratio of amylase and glucoside in compound enzyme B is 0.8:1. All other operations and processes are the same as in Embodiment 1.

[0038] Example 3 The difference between this embodiment and Embodiment 1 is that, in the enzymatic hydrolysis step, the mass ratio of pectinase, cellulase, and hemicellulase in compound enzyme A is 2:2.5:1. The mass ratio of amylase and glucoside in compound enzyme B is 1.5:1. All other operations and processes are the same as in Embodiment 1.

[0039] Comparative Example 1 (unblended and unenzymatically hydrolyzed) The difference between this comparative example and Example 1 is that in the raw material pretreatment step, 100g of Cornus officinalis pulp was accurately weighed, thoroughly mixed with 1.5kg of water, and then the mixture was extracted at 100℃ for 120min, centrifuged at 5000rpm for 5min, and filtered to obtain the supernatant. No pulping or enzymatic hydrolysis was performed.

[0040] Comparative Example 2 (Extraction without enzymatic hydrolysis) The difference between this comparative example and Example 1 is that only the Cornus officinalis pulp was extracted, centrifuged, filtered and concentrated, without enzymatic hydrolysis. All other operations and processes were the same as in Example 1.

[0041] Comparative Example 3 (Single-step enzymatic hydrolysis) The difference between this comparative example and Example 1 is that in the enzymatic hydrolysis step, only compound enzyme A is added for single-step enzymatic hydrolysis, and compound enzyme B is not added. All other operations and processes are the same as in Example 1.

[0042] Comparative Example 4 (Single-step enzymatic hydrolysis) The difference between this comparative example and Example 1 is that in the enzymatic hydrolysis step, only compound enzyme B is added for single-step enzymatic hydrolysis, and compound enzyme A is not added. All other operations and processes are the same as in Example 1.

[0043] Regarding the raw materials used in the examples and comparative examples: The pectinase used was Nanning Pangbo pectinase, with an enzyme activity of 30,000 U / g.

[0044] The cellulase used was Nanning Pangbo cellulase, with an enzyme activity of 20,000 U / g.

[0045] The amylase used was Nanning Pangbo α-amylase, with an enzyme activity of 5000 U / g.

[0046] The selected hemicellulase was Nanning Pangbo hemicellulase, with an enzyme activity of 100,000 U / g.

[0047] The selected glucosidase was Jiangsu Duoyang Bio-glucosidase, with an enzyme activity of 100,000 U / g.

[0048] The performance of the Cornus officinalis extract prepared in the above embodiments and comparative examples was tested: 1. Determination of the content of active ingredients in Cornus officinalis extract According to the detection methods for mononoside and loganin specified in Part I of the 2020 edition of the Chinese Pharmacopoeia, the contents of mononoside and loganin in Cornus officinalis extracts prepared by different processes were detected, and the results are shown in Table 1.

[0049] Table 1. Content of active ingredients in the examples and comparative examples

[0050] Note: Compared with Comparative Example 1 p <0.05, n=3.

[0051] As shown in Table 1, compared with those without extraction and / or enzymatic hydrolysis (Comparative Examples 1 and 2), the present invention, through extraction and stepwise hydrolysis with a specific complex enzyme (Examples 1 to 3), can promote the dissolution rate of active ingredients in Cornus officinalis and effectively increase the content of mononoside and loganin in the Cornus officinalis extract. Specifically, compared with Comparative Example 1, Example 1 showed a 145% increase in mononoside content and a 160% increase in loganin content.

[0052] As can be seen from Comparative Examples 3-4, although single-step enzymatic hydrolysis of Cornus officinalis extract using compound enzyme A and compound enzyme B can increase the content of mononoside and loganin in the extract, the increasing effect is obviously not as good as the stepwise hydrolysis method used in Examples 1 to 3.

[0053] As can be seen from Examples 2-3, the content of cellulase and pectinase has a significant difference in the extraction efficiency of effective substances from Cornus officinalis. Cellulase effectively degrades the cellulose skeleton in the cell wall, while pectinase hydrolyzes pectin, the adhesive of the intercellular layer. Cellulase can directly destroy the cellulose skeleton of the cell wall, fundamentally dismantling its core supporting structure, thereby creating the most sufficient mass transfer channel for the release of effective substances.

[0054] In summary, this invention utilizes specific compound enzymes A and B to sequentially hydrolyze Cornus officinalis extract. The synergistic use of these two compound enzymes significantly increases the content of active ingredients in the Cornus officinalis extract. Specifically: In the first hydrolysis: pectinase preferentially hydrolyzes pectin in the intercellular layer, loosening intercellular connections and creating a larger contact area for subsequent enzymes; subsequently, cellulase can more fully contact and destroy the cellulose skeleton of the cell wall, completely dismantling the structural barrier; then, hemicellulase completely hydrolyzes hemicellulose.

[0055] In the second hydrolysis, amylase further removes intracellular starch, reduces the viscosity of the extraction system, and promotes the diffusion of released active substances; glucosidase specifically hydrolyzes the glucose groups at the ends of glycosides such as monoglucoside and loganin, increasing their content or releasing more active aglycones.

[0056] 2. Determination of the antioxidant activity of Cornus officinalis extract 20 ml of the Cornus officinalis extract from each of the examples and comparative examples was taken as a sample solution, and its DPPH free radical scavenging rate and ABTS free radical scavenging rate were detected. The results are as follows: Figure 1 and Figure 2 As shown. The DPPH and ABTS free radical scavenging rates of Cornus officinalis extract were determined using the DPPH and ABTS methods for antioxidant determination in GB / T 39100-2020.

[0057] Depend on Figure 1 and Figure 2 It can be seen that the DPPH free radical scavenging rate and ABTS free radical scavenging rate of the Cornus officinalis extracts in Examples 1 to 3 are higher than those in Comparative Examples 1 and 2.

[0058] Furthermore, the DPPH and ABTS free radical scavenging rates of the Cornus officinalis extracts in Examples 1 to 3 were higher than those in Comparative Examples 3 and 4, with the increase in ABTS free radical scavenging rate being more significant. Among them, Example 1 showed the highest DPPH and ABTS free radical scavenging rates.

[0059] The above experimental results show that the stepwise enzymatic hydrolysis method of the compound enzyme used in this invention efficiently and directionally degrades the cell wall and intercellular layer, breaking the physical barrier and allowing the active ingredients in Cornus officinalis to be released more fully and completely into the extract, thereby significantly increasing the concentration of effective ingredients in contact with free radicals.

[0060] From the perspective of comprehensive evaluation indicators of antioxidant activity, this indicates that the Cornus officinalis extract prepared by using a combination of specific enzymes for enzymatic hydrolysis, homogenization and extraction has superior antioxidant activity.

[0061] 3. Effects of Cornus officinalis extract on GLP-1 secretion levels in STC-1 cells Glucagon-like peptide-1 (GLP-1) is a glucose-dependent hypoglycemic hormone secreted by the intestine, especially the terminal ileum. Therefore, the incidence of hypoglycemia is low. In addition, current research has confirmed that GLP-1 also has advantages such as cardiovascular protection, improvement of insulin resistance, weight loss, promotion of pancreatic function recovery, and restoration of physiological insulin secretion.

[0062] This experiment investigated the effects of Cornus officinalis extract prepared by different processes on the GLP-1 secretion level of STC-1 cells using the following methods: STC-1 cell culture: STC-1 cells in good condition were seeded at a density of 90% in 12-well plates and cultured at 37°C in a 5% CO2 incubator for 48 hours.

[0063] GLP-1 secretion assay: After observation of adhesion, the upper culture medium was discarded. The experimental groups were treated with complete culture medium containing the corresponding Cornus officinalis extract for 2 hours. After incubation, the supernatant was collected, centrifuged at 1000g for 20 minutes at 4°C, and transferred to a new 1.5 EP tube. GLP-1 was detected according to the kit (Mouse Glucagon-like Peptide 1 (GLP-1) ELISA Kit, KOBO Biotechnology, catalog number CB10786-Mu).

[0064] Test results as follows Figure 3 As shown: The Cornus officinalis extracts of Examples 1 to 3 promoted the secretion of GLP-1 by STC-1 cells, and the promoting effect was higher than that of Comparative Examples 1 and 2. Furthermore, the Cornus officinalis extracts of Examples 1 to 3 promoted the secretion of GLP-1 by STC-1 cells more than that of Comparative Examples 3 and 4.

[0065] In summary, the enzyme treatment process of this invention not only improves extraction efficiency, but more importantly, through efficient and targeted cell wall disruption, it better releases and preserves specific active ingredients (such as iridoid glycosides and polysaccharides) in Cornus officinalis that can stimulate STC-1 cells to secrete GLP-1. These components may not dissolve sufficiently or undergo structural changes during traditional extraction processes due to the physical barrier of the cell wall. However, the stepwise enzymatic hydrolysis process ensures the integrity and high concentration of the active ingredients, thereby more effectively stimulating relevant intracellular signaling pathways when co-cultured with STC-1 cells, leading to a significant increase in GLP-1 secretion.

[0066] 4. Effects of Cornus officinalis extract on reactive oxygen species (ROS) content and related gene expression in D-gal-induced mouse testicular interstitial (TM3) cells. Leydig cells (LCs) are located between seminiferous tubules and have the function of synthesizing and secreting testosterone, playing an important role in the development of male reproductive organs, spermatogenesis, and maintenance of sexual function. LCs use cholesterol as a raw material and synthesize testosterone through the joint catalysis of various testosterone synthases (StAR, 3β-HSD, CYP17a1).

[0067] D-galactose (D-gal) is a recognized aging inducer and a monosaccharide found in certain foods and produced by the human body. Under normal conditions, it is entirely metabolized by D-galactokinase and galactose-1-phosphate uridine transferase. However, elevated D-gal levels can lead to oxidative stress.

[0068] To further investigate the protective effect of Cornus officinalis extract on TM3 cells, an oxidative stress response model was constructed using different concentrations of D-gal, and then the toxic effects of Cornus officinalis extract on mouse LCs TM3 cells were studied.

[0069] 4.1 Detection of Reactive Oxygen Species (ROS) Content in D-gal-induced TM3 Cells by Cornus officinalis Extract (1) Seeding cells: TM3 cells with good growth were seeded in 96-well plates (black) at a density of 10,000 cells / well and cultured in a 37°C incubator with 5% CO2 for 24 hours.

[0070] (2) Experimental groups: NC control group, D-gal model group, and experimental group.

[0071] (3) After observing the adhesion, the upper culture medium was discarded, and the experimental groups were treated with complete culture medium containing the corresponding Cornus officinalis extract for 24 hours.

[0072] (4) After the intervention, replace the culture medium with 160mM D-gal for another 24h.

[0073] (5) Probe preparation: After the processing time is reached, the probe is loaded in situ and the DCFH-DA stock solution is diluted 1:1000 with serum-free DMEM / F-12 medium according to the instructions to make the final concentration of the fluorescent probe 10μM.

[0074] (6) Probe loading: Discard the supernatant, add 100 μL of diluted DCFH-DA, and incubate at 37°C for 30 min.

[0075] (7) Cell washing: Wash the cells 1-2 times with serum-free DMEM / F-12 medium to fully remove DCFH-DA that has not entered the cells.

[0076] (8) Simultaneously, use an ELISA reader (96-well plate): excitation wavelength 488nm, emission wavelength 525nm, and detect the intensity of fluorescence before and after stimulation.

[0077] The results are as follows Figure 4 As shown, compared with the D-gal model group, the relative content of ROS was reduced in all experimental groups, indicating that Cornus officinalis can alleviate D-gal-induced oxidative stress in TM3 cells. Among them, the antioxidant effects of Examples 1 to 3 were better than those of the comparative examples, especially Example 1, which showed the best effect. This indicates that the composite enzymatic hydrolysis method can better release the effective substances in Cornus officinalis, allowing them to exert their effects more effectively.

[0078] 4.2 Gene Expression Level Detection (1) Cell seeding: TM3 cells with good growth were seeded in 12-well plates at a density of 50,000 cells / well and cultured in a 37°C 5% CO2 incubator for 24 hours.

[0079] (2) Experimental groups: NC control group, D-gal model group, and experimental group.

[0080] (3) After observing the adhesion, the upper culture medium was discarded, and the experimental groups were treated with complete culture medium containing the corresponding Cornus officinalis extract for 24 hours.

[0081] (4) After the intervention, replace the culture medium with 160mM D-gal for another 24h.

[0082] (6) After drug incubation, RNA was extracted, reverse transcribed, and real-time qPCR was performed to detect testosterone synthase genes (StAR, 3β-HSD, CYP17a1). Primers were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd. β-actin gene was used as an internal reference gene, with 3 replicates per group. 2 -ΔΔCt Calculate the relative expression levels of each gene.

[0083] The results are as follows Figure 5 As shown, compared with the control group, the expression levels of StAR, 3β-HSD, and CYP17a1 in the D-gal model group were significantly reduced. Examples 1 to 3 all showed varying degrees of upregulation of the expression of StAR, 3β-HSD, and CYP17a1, with Example 1 showing the most significant upregulation of all three genes. The gene expression levels in Comparative Examples 3 and 4 were higher than those in Comparative Examples 1-2, but still significantly lower than those in Examples 1 to 3.

[0084] Upregulation of StAR indicates that the Cornus officinalis extract enhanced cholesterol transport from the outer mitochondrial membrane to the inner membrane, a first step in testosterone synthesis. This suggests that the embodiment may have ensured the smooth progress of StAR-mediated cholesterol transport by protecting mitochondria. Upregulation of 3β-HSD indicates that the Cornus officinalis extract promoted the conversion of pregnenolone and progesterone to androgens (dehydroepiandrosterone (DHEA) and androstenedione), opening the pathway for androgen production from progesterone. Upregulation of CYP17a1 indicates that the embodiment directly catalyzed the final step of androstenedione conversion to testosterone, a direct step in the production of biologically active testosterone.

[0085] In summary, the proposed solution fundamentally enhances the testosterone synthesis capacity of senescent TM3 cells by systematically reversing the downregulation of genes expressing key enzymes in senescence-related testosterone synthesis.

[0086] It should be noted that: (1) Definition: In this article, “~” is used to represent the range of values, and the range of values ​​represented by this expression includes two endpoint values.

[0087] The term "food" as used in this article is used in a broad sense, including human food and drink.

[0088] In the text, "DPPH" stands for 1,1-diphenyl-2-trinitrophenylhydrazine, also known as 1,1-diphenyl-2-picrylhydrazine (free radical).

[0089] In the text, "ABTS" refers to the free radical 2,2'-adiazon-bis-3-ethylbenzothiazoline-6-sulfonic acid.

[0090] (2) Raw materials used: All enzymes used are commercially available and can be purchased by those skilled in the art.

[0091] In summary, the specific parameters or some commonly used reagents or raw materials in the above embodiments are specific or preferred embodiments under the concept of this application, and not limitations thereof; those skilled in the art can make adaptive adjustments within the concept and protection scope of this application.

[0092] In addition, unless otherwise specified, the raw materials used may be commercially available products in the field or prepared by conventional methods in the field; that is, the reagents and instruments used in this embodiment do not specify the manufacturer or other information, and are all conventional products that can be purchased from the market.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A method for preparing a highly active Cornus officinalis extract, characterized in that, Includes the following steps: Pretreatment: Mix the pulp of Cornus officinalis with water at a mass ratio of 1:15-40, and then pulp to obtain Cornus officinalis pulp; Extraction: The slurry is extracted to obtain a water extract of Cornus officinalis, which is then homogenized to obtain a mixed extract of Cornus officinalis. Enzymatic hydrolysis: Add 0.2-0.5% (by weight of water) of compound enzyme A to the Cornus officinalis mixture and hydrolyze at 40-50℃ for 60-100 min; then add 0.2-0.5% (by weight of water) of compound enzyme B and hydrolyze at 40-50℃ for 30-60 min; after hydrolysis, inactivate the enzyme at 85-90℃ for 10-20 min to obtain Cornus officinalis enzymatic hydrolysate; The complex enzyme A is composed of pectinase, cellulase and hemicellulase, with a mass ratio of 1.5~2.5:1.5~2.5:1; the complex enzyme B is composed of α-amylase and glucosidase, with a mass ratio of α-amylase and glucosidase of 0.8~1.5:

1. Separation, filtration, and concentration: The enzymatic hydrolysate is subjected to solid-liquid separation to obtain the supernatant, which is then filtered and concentrated to obtain the Cornus officinalis extract.

2. The method for preparing the highly active Cornus officinalis extract as described in claim 1, characterized in that: In the extraction step, Cornus officinalis is mixed with deionized water and then heated to 85-95℃ for 30-90 minutes to obtain an aqueous extract of Cornus officinalis.

3. The method for preparing the highly active Cornus officinalis extract as described in claim 1, characterized in that: The pectinase is Nanning Pangbo pectinase, with an enzyme activity of 30,000 U / g. The cellulase mentioned is Nanning Pangbo cellulase, with an enzyme activity of 20,000 U / g; The α-amylase mentioned is Nanning Pangbo α-amylase, with an enzyme activity of 5000 U / g; The hemicellulase mentioned is Nanning Pangbo hemicellulase, with an enzyme activity of 100,000 U / g; The glucosidase is Jiangsu Duoyang Bio-glucosidase, with an enzyme activity of 100,000 U / g.

4. The method for preparing the highly active Cornus officinalis extract as described in claim 1, characterized in that: In the complex enzyme A, the mass ratio of pectinase, cellulase and hemicellulase is 1.6~2.3:1.6~2.3:

1.

5. The method for preparing the highly active Cornus officinalis extract as described in claim 1, characterized in that: In the complex enzyme B, the mass ratio of α-amylase to glucosidase is 1~1.3:

1.

6. The method for preparing the highly active Cornus officinalis extract as described in claim 1, characterized in that: In the separation and filtration steps, the Cornus officinalis enzymatic hydrolysate is centrifuged at 4500-5500 rpm for 5-10 min, the supernatant is collected, and the supernatant is filtered through a membrane microfiltration filter with a pore size of 0.45-0.50 μm to obtain Cornus officinalis filtrate.

7. The method for preparing the highly active Cornus officinalis extract as described in claim 6, characterized in that: In the concentration step, the Cornus officinalis filtrate is concentrated to 25-30% solids at 75-80 °C to obtain Cornus officinalis extract.

8. A highly active Cornus officinalis extract, characterized in that: It is prepared by the preparation method described in any one of claims 1 to 7.

9. The highly active Cornus officinalis extract as described in claim 8, characterized in that: The extract contains ≥7.0 mg / mL mononoside and ≥3.0 mg / mL loganin, and has a DPPH free radical scavenging rate of ≥85% and an ABTS free radical scavenging rate of ≥90%.

10. The use of the highly active Cornus officinalis extract as described in claim 8 in the preparation of a medicament for tonifying the kidney and liver and / or lowering blood sugar.

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