A peony seed meal extract, extraction method and application

Abstract

The present application provides a peony seed meal extract, an extraction method and application, and belongs to the technical field of medicine. The extraction method comprises the following steps: after defatting treatment of peony seed meal, methanol solution is used for extraction to prepare a crude extract; gradient elution is performed on the crude extract, in the gradient elution process, an ethanol aqueous solution with a flow rate of 5-15 mL / min and a volume concentration of 8-12%, 18-22%, 28-32%, 38-42%, 48-52%, 58-62%, 68-72% and 78-82% is used for gradient elution, each concentration elution is performed for 4-6 column volumes, and the elution product of the ethanol aqueous solution with a volume concentration of 48%-72% is collected. The total flavone purity of the peony seed meal extract can reach more than 90%. The extract plays an obvious anti-fibrosis role by inhibiting the activation of hepatic stellate cells, and in vivo experiments show that it can significantly improve the liver function indexes of CCl4-induced liver fibrosis mice, and reduce the pathological damage and collagen deposition of active substances in liver tissue.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to a peony seed meal extract, its extraction method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Liver fibrosis is a key pathological stage in the progression of various chronic liver diseases (such as viral hepatitis, alcoholic liver disease, and non-alcoholic fatty liver disease) to cirrhosis and liver cancer. Its core pathological feature is the continuous activation of hepatic stellate cells (HSCs), leading to excessive deposition of the extracellular matrix (ECM), ultimately resulting in liver structural remodeling and functional loss. Natural products, especially luteolin, kaempferol, and quercetin, possess antioxidant, anti-inflammatory, and anti-tumor biological activities. Due to their multi-target and low-toxicity characteristics, they exhibit unique advantages in the prevention and treatment of chronic liver diseases.

[0004] Peony (Paeonia ostii) is a traditional medicinal plant. Peony seed meal is the main byproduct of peony seed oil extraction, accounting for 60% to 80% of the total raw materials. Currently, it is mostly treated as low-value feed or waste, resulting in low resource utilization and causing environmental pollution and resource waste. There are no existing reports on the application of peony seed meal in anti-liver fibrosis. Summary of the Invention

[0005] In view of the current state of technology, the purpose of this invention is to provide a peony seed meal extract, extraction method and application, to efficiently enrich flavonoid extracts from peony seed meal, and to clarify its application in the preparation of anti-liver fibrosis drugs.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows.

[0007] Firstly, a method for extracting peony seed meal extract, comprising the following steps: Peony seed meal was defatted and extracted with methanol solution to prepare crude extract. The crude extract was then subjected to gradient elution at a flow rate of 5-15 mL / min, sequentially using pure water, and aqueous ethanol solutions with volume concentrations of 8-12%, 18-22%, 28-32%, 38-42%, 48-52%, 58-62%, 68-72%, and 78-82%. Each concentration was used for elution for 4-6 column volumes (4-6 BV). The elution product with a volume concentration of 48-72% ethanol solution was collected to obtain peony seed meal extract.

[0008] Secondly, the peony seed meal extract obtained by the above-mentioned extraction method.

[0009] Thirdly, the application of the above-mentioned peony seed meal extract in the preparation of drugs for the prevention and / or treatment of liver fibrosis.

[0010] Fourthly, a drug comprising the aforementioned peony seed meal extract.

[0011] The beneficial effects of this invention are as follows: This invention establishes a process for enriching flavonoid extracts with anti-hepatic fibrosis activity from peony seed meal, achieving a total flavonoid purity of over 90%. The extract exerts a significant anti-fibrotic effect by inhibiting hepatic stellate cell activation (downregulating α-SMA expression). In vivo experiments show that it can significantly improve liver function indicators (ALT, AST, TBIL, ALB) in CCl4-induced hepatic fibrosis mice, and reduce pathological damage and collagen deposition in liver tissue. Attached Figure Description

[0012] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0013] Figure 1 The enrichment and identification results of the flavonoid extract from peony seed meal in Example 1 are shown. A: Content of various active ingredients in the crude extract (total polysaccharides, terpenes, reducing sugars, total flavonoids, stilbenes); B: Elution efficiency of flavonoids in elution sections with different ethanol concentrations.

[0014] Figure 2 The effects of peony seed meal flavonoid extract on liver function in mice with liver fibrosis in Example 2 are shown. A: Quantitative analysis of serum ALT enzyme activity; B: Quantitative analysis of serum AST enzyme activity; C: Quantitative analysis of serum ALB expression; D: Quantitative analysis of serum TBIL expression.

[0015] Figure 3The effect of peony seed meal flavonoid extract on liver tissue pathological damage in mice with liver fibrosis in Example 2. A: Anatomical diagram of mouse liver (scale bar: 1 cm); B: HE staining of liver tissue (scale bar: 200 μm and 10 μm); C: Sirius red staining of liver tissue (scale bar: 200 μm); D: Immunofluorescence staining of α-SMA protein in liver tissue (scale bar: 200 μm).

[0016] Figure 4 The effect of peony seed meal flavonoid extract on TGF-β1-activated human hepatic stellate cells (LX-2) in Example 3. A: CCK-8 assay to detect the effect of different concentrations of PSMF on LX-2 cell viability; B: Immunofluorescence quantitative assessment of the effect of different concentrations of PSMF on TGF-β1-activated LX-2 cells; C: Immunofluorescence staining pattern of PSMF (4 μM) on TGF-β1-activated LX-2 cells (scale bar: 200 μm); D: RT-qPCR quantitative analysis of α-SMA mRNA expression level (*p<0.05). Detailed Implementation

[0017] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0018] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0019] One or more embodiments of the present invention provide a method for extracting peony seed meal extract, comprising the steps of: Peony seed meal was defatted and extracted with methanol solution to prepare crude extract. The crude extract was then subjected to gradient elution with ethanol aqueous solution at a flow rate of 5-15 mL / min and volume concentrations of 8-12%, 18-22%, 28-32%, 38-42%, 48-52%, 58-62%, 68-72%, and 78-82%, respectively. Each concentration was eluted for 4-6 column volumes (4-6 BV), and the eluent with a volume concentration of 48%-72% ethanol aqueous solution was collected.

[0020] In the above process, a crude extract is first prepared with methanol, and then a peony seed meal extract with a total flavonoid concentration of over 90% is obtained by gradient elution.

[0021] Optionally, the peony seed meal is derived from peony seed meal after pressing or subcritical extraction; it is a product after extracting peony seed oil, which enables the high-value utilization of peony seed meal.

[0022] Optionally, the degreasing process includes: soaking in n-butane to degrease and obtain degreased powder; further removing the oil components from the peony seed meal and removing oily impurities.

[0023] Optionally, after extraction with methanol solution, the extract is spray-dried to obtain the crude extract, which contains flavonoid active ingredients. Specifically, defatting powder is added to a methanol aqueous solution at a temperature of 45-55°C and a concentration of 85-95 vol% for soaking and extraction at a ratio of 1:(7-9) to 1, and the extraction is repeated 2-4 times, with each soaking lasting 1.5-2.5 hours.

[0024] Optionally, the crude extract can be dissolved in 35-45 times its weight of warm water at 35-45°C and then subjected to gradient elution.

[0025] Optionally, adsorption can be performed using macroporous adsorption resins such as D101, HPD-100, HPD-BJQH, HPD-450, or AB-8 before elution.

[0026] Optionally, the elution product can be collected from a 60% ethanol aqueous solution.

[0027] Optionally, the step may also include: evaporating and concentrating the eluent and then freeze-drying it.

[0028] One or more embodiments of the present invention provide a peony seed meal extract obtained by the above-described extraction method for peony seed meal extract.

[0029] Flavonoids are the main components, and the total flavonoid purity of the extract is ≥90%.

[0030] One or more embodiments of the present invention provide the use of the above-mentioned peony seed meal extract in the preparation of drugs for the prevention and / or treatment of liver fibrosis.

[0031] Peony seed meal extract exerts its anti-hepatic fibrosis effect by inhibiting the activation of hepatic stellate cells and reducing extracellular matrix deposition. Specifically, it can reduce serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) levels and increase albumin (ALB) levels in animal models of hepatic fibrosis; inhibit the expression of α-smooth muscle actin (α-SMA) and collagen fiber deposition in liver tissue; and inhibit TGF-β1-induced mRNA transcription and protein expression of α-SMA in hepatic stellate cells.

[0032] One or more embodiments of the present invention provide a medicine comprising the above-described peony seed meal extract.

[0033] Optionally, the drug may include other pharmaceutically acceptable carriers and at least one inactive pharmaceutical ingredient.

[0034] The inactive components of the drug can be pharmaceutically commonly used carriers, excipients, and diluents. Furthermore, according to conventional methods, it can be formulated into oral, topical, suppository, and sterile injectable solutions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and sprays.

[0035] The non-pharmaceutical active ingredients that may be included, such as carriers, excipients, and diluents, are well known in the art, and those skilled in the art can determine that they meet clinical standards.

[0036] The present invention will be further described below with reference to specific embodiments.

[0037] Example 1 A peony seed meal extract, the extraction method comprising: S1. Peony (Paeonia ostii) seeds are shelled, pressed into cakes, and crushed for screw pressing to extract peony seed oil. The remaining peony seed meal is then crushed and degreased using subcritical fluid extraction with food-grade n-butane. The volume ratio of peony seed meal to n-butane is 1:10, the extraction pressure is 0.6 MPa, the subcritical extraction temperature is 40℃, the desolvation temperature is 50℃, and the evaporation system temperature is 60℃. The extraction is performed 4 times, each time for 30 minutes, to obtain degreased powder.

[0038] S2. According to the material-liquid ratio of 1:8, the defatting powder is added to a methanol aqueous solution with a temperature of 50℃ and a concentration of 90% for soaking and extraction. The extraction is carried out 3 times, with each soaking lasting 2 hours. After solid-liquid separation, the extract is obtained. The extract is spray-dried to make fine powder to obtain crude extract.

[0039] S3. Dissolve the crude extract in 20 times its mass of water at 40°C, and then adsorb it using D101 macroporous resin as the adsorbent. After that, pack the macroporous resin containing the crude extract into an elution column and perform gradient elution. During the elution process, use pure water, and ethanol aqueous solutions with volume concentrations of 10%, 20%, 30%, 40%, 50%, 60%, 70%, and 80% sequentially at a flow rate of 10 mL / min. Elute 5 column volumes (5 BV) for each concentration. Collect the elution products of each concentration of ethanol aqueous solution.

[0040] S4. The collected eluted fractions are combined and concentrated by rotary evaporation, followed by freeze drying to obtain peony seed meal flavonoid extract (PSMF).

[0041] The crude extract obtained in S2 and the PSMF obtained in S4 were analyzed for biochemical components using standard colorimetric methods. The results are as follows: Figure 1 As shown, Figure 1 A in the figure shows that the crude extract contains 45.89% (w / w) total polysaccharides, 13.29% (w / w) terpenoids, 4.38% (w / w) reducing sugars, 3.84% (w / w) total flavonoids, and 0.96% (w / w) stilbenes. Figure 1 The B-type flavonoids in the sample were mainly concentrated in the 60% ethanol elution section, with an elution efficiency of over 90%, and the purity of the total flavonoids in the collected PSMF was over 90%.

[0042] Example 2 The following tests were performed using the elution product of the 60% ethanol aqueous solution in Example 1, namely the peony seed meal flavonoid extract (PSMF).

[0043] The evaluation of the therapeutic effect of PSMF on CCl4-induced liver fibrosis in mice included the following steps: S1. Animal Grouping and Model Construction: Male C57BL / 6J mice (7-8 weeks old) were acclimatized for one week and then randomly divided into 4 groups (n=5): blank control group (Control), CCl4 model group (CCl4), low-dose PSMF group (200 mg / kg), and high-dose PSMF group (300 mg / kg). The modeling method was as follows: Except for the blank control group, all other groups of mice were intraperitoneally injected with a 20% CCl4-ol-olive oil mixture (200 mg / kg) twice a week for 6 consecutive weeks to induce liver fibrosis; during this period, the blank control group was injected with an equal volume of physiological saline. The low-dose PSMF group and the high-dose PSMF group were administered the corresponding dose of PSMF daily by gavage starting one week before modeling, and continued synchronously during the modeling process for 7 weeks; during this period, the blank control group and the model group were given an equal volume of physiological saline.

[0044] S2. Serum Biochemical Analysis: After the last administration, whole blood was collected from mice, and serum was separated. 10 μL of serum sample was added to a 96-well plate along with 40 μL of diluent. 100 μL of horseradish peroxidase (HRP)-labeled detection antibody was added to each well, and the plate was incubated at 37°C for 1 h. The wells were washed 5 times, and 50 μL of substrates A and B from the ELISA kit were added. The plate was incubated at 37°C in the dark for 15 min. After terminating the reaction, the optical density (OD) at 450 nm was measured using an Infinite F50 microplate reader, and the concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), and albumin (ALB) in the serum were calculated based on the standard curve. Results are as follows: Figure 2 show, Figure 2 In this context, A represents the quantitative analysis of serum ALT enzyme activity; Figure 2 B in the figure represents the quantitative analysis of serum AST enzyme activity; Figure 2 C in the figure represents the quantitative analysis of serum ALB expression levels; Figure 2 D in the figure represents the quantitative analysis of serum TBIL expression. Compared with the blank control group, the serum AST, ALT and TBIL levels of the model group mice were significantly increased (approximately 4-5 times), and the ALB level was significantly decreased. Compared with the model group, the PSMF treatment group (especially the high-dose group) showed a significant decrease in AST, ALT and TBIL levels, and a significant increase in ALB level. This indicates that after PSMF intervention, the liver function of the liver fibrosis mice was significantly improved, and PSMF can effectively improve the abnormal liver biochemical indicators caused by CCl4.

[0045] S3, Liver tissue pathological staining: such as Figure 3 As shown in Figure A, mouse liver tissue was fixed in 4% paraformaldehyde, dehydrated with graded ethanol, cleared with xylene, and then embedded in paraffin to prepare 4 μm thick sections. After spreading and drying at 60°C, the sections were dewaxed with xylene, stained with hematoxylin and eosin (H&E), and images were recorded using a ZEISS Axiocam 506 optical microscope (Zeiss, Germany) to assess the pathological changes in the liver tissue. The H&E staining results are shown below. Figure 3 B in the diagram shows that the liver tissue structure in the control group was clear; the liver tissue in the model group showed extensive hepatocyte vacuolar degeneration, destruction of liver lobule structure, congestion of hepatic sinusoids, and inflammatory cell infiltration, which are typical pathological features of fibrosis. PSMF intervention (PSMF (Low) and PSMF (High)) significantly alleviated the above pathological changes and improved liver tissue structure. Given that excessive extracellular matrix deposition is a core pathological feature of liver fibrosis, Sirius red staining was further used to detect collagen fiber deposition. Sirius red staining results are shown in Figure 1. Figure 3The results showed that, compared with the normal group (Control), the liver tissue of mice in the CCl4 model group showed significant connective tissue hyperplasia and a large amount of red collagen fiber deposition, while the red collagen fibers in the PSMF treatment groups (PSMF (Low) and PSMF (High)) were significantly reduced, suggesting that PSMF can effectively inhibit CCl4-induced liver fibrosis and collagen deposition.

[0046] S4. Immunofluorescence Staining: To verify the role demonstrated in S3 at the molecular level, liver tissue sections from step S3 were immunofluorescently stained using α-SMA antibody. This included: dewaxing paraffin sections with xylene, hydration with graded ethanol, and antigen retrieval. After cooling, the sections were blocked with 5% BSA at room temperature for 1 h, then incubated overnight at 4°C with rabbit anti-α-SMA primary antibody (1:200 dilution). The next day, after washing three times with PBST, the sections were incubated with Alexa Fluor 488 goat anti-rabbit IgG secondary antibody (1:400 dilution) at room temperature in the dark for 1 h, and the cell nuclei were stained with DAPI. Images were acquired and analyzed using a Cytation 5 imaging system (Biotek, USA). The results are as follows: Figure 3 The results showed that α-SMA protein expression was significantly upregulated in the liver tissue of the model group; α-SMA expression was significantly reduced in the PSMF treatment group, indicating that PSMF may exert its anti-fibrotic effect by inhibiting the activation of hepatic stellate cells.

[0047] In vivo experiments showed that the extract could significantly improve liver function indicators (ALT, AST, TBIL, ALB) in CCl4-induced liver fibrosis mice, reduce pathological damage and collagen deposition in liver tissue, and inhibit the expression of α-smooth muscle actin (α-SMA).

[0048] Example 3 The following tests were performed using the elution product of the 60% ethanol aqueous solution in Example 1, namely the peony seed meal flavonoid extract (PSMF).

[0049] Given that TGF-β1 is a key factor driving hepatic stellate cell activation and fibrosis, the inhibitory effect of peony seed meal flavonoid extract on TGF-β1-induced LX-2 cell activation was evaluated. The methods included: S1. Cell Culture and Treatment: Human liver stellate cell line (LX-2, purchased from the American Type Culture Collection Center (ATCC)) was cultured in DMEM / F12 medium containing 10% fetal bovine serum at 37°C and 5% CO2. The experiment was divided into three groups: control group, TGF-β1 treatment group (10 ng / mL), and PSMF treatment group (TGF-β1 + PSMF).

[0050] S2 and CCK-8 cell viability assays: LX-2 cells obtained in S1 were seeded at 5 × 10³ cells / well in 96-well plates. The TGF-β1 treatment group was treated with 10 ng / mL TGF-β1 for 24 h, and the PSMF treatment group was treated with 10 ng / mL TGF-β1 and different concentrations of PSMF (1, 2, 4 μM) for 24 h. 10 μL of CCK-8 solution was added to each well and incubated for 2 h. Absorbance was measured at 450 nm. Results are as follows: Figure 4 A in the study showed that PSMF inhibited the viability of TGF-β1-induced LX-2 cells in the concentration range of 1–4 μM.

[0051] S3. Immunofluorescence staining: LX-2 cells obtained in S1 were seeded onto a spreader slide and treated with TGF-β1 (10 ng / mL) and PSMF (4 μM) for 24 h. Cells were then washed with PBS, fixed with 4% paraformaldehyde at room temperature for 10 min, permeabilized with 0.5% Triton X-100 for 10 min, and blocked with 3% BSA at room temperature for 1 h. Subsequently, they were incubated overnight at 4°C with rabbit anti-α-SMA primary antibody (1:200 dilution). The next day, after washing three times with PBS, they were incubated with Alexa Fluor 488 goat anti-rabbit IgG secondary antibody (1:400 dilution) at room temperature in the dark for 1 h. After DAPI staining of the nuclei, images were acquired using the Cytation 5 imaging system, and the fluorescence intensity of α-SMA was quantitatively analyzed using its accompanying image analysis software. The results are as follows: Figure 4 B in the figure shows that, compared with the TGF-β1 treatment group alone, the expression of α-SMA protein in LX-2 cells in the PSMF intervention group was significantly downregulated, and 4 μM was the optimal effective inhibitory concentration; after α-SMA immunofluorescence staining, the results are as follows: Figure 4 As shown in C, compared with the TGF-β1 treatment group alone, 4 μM PSMF treatment significantly downregulated the mRNA transcription level and protein expression of α-SMA in LX-2 cells.

[0052] S4. Real-time quantitative PCR (RT-qPCR): Total RNA was extracted from LX-2 cells in each group, reverse transcribed into cDNA, and then α-SMA mRNA expression levels were detected by real-time quantitative PCR using the SYBR Green method. Subsequently, real-time quantitative PCR amplification was performed on an Archimed X instrument using ChamQ UniversalSYBR Green qPCR Master Mix. The PCR reaction program included: 95°C pre-denaturation for 10 min; 95°C denaturation for 15 s, 60°C annealing for 30 s, and 72°C extension for 30 s, for a total of 40 cycles. GAPDH was used as an internal reference gene, and 2... -The relative expression level of the target gene α-SMA (gene name ACTA2) was calculated using the ΔΔCt method. Each sample was tested in triplicate, and the experiment was independently repeated three times. The primer sequences involved are shown in Table 1.

[0053] Table 1 Primer Sequences

[0054] The results are as follows Figure 4 The results showed that, compared with the TGF-β1-only treatment group, the PSMF intervention group had a significantly lower α-SMA mRNA transcription level.

[0055] In vitro experiments further confirmed that the extract can inhibit TGF-β1-induced activation of hepatic stellate cells and α-SMAmRNA transcription.

[0056] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for extracting an extract of Paeonia suffruticosa seed meal, characterized by, Including the following steps: Peony seed meal was defatted and extracted with methanol solution to prepare crude extract. The crude extract was then subjected to gradient elution at a flow rate of 5-15 mL / min, sequentially using pure water, and aqueous ethanol solutions with volume concentrations of 8-12%, 18-22%, 28-32%, 38-42%, 48-52%, 58-62%, 68-72%, and 78-82%. Each concentration was used for elution of 4-6 column volumes. The eluent with the 48-72% aqueous ethanol solution was collected.

2. The extraction method of the Paeonia suffruticosa Andr. seed meal extract according to claim 1, characterized in that, The peony seed meal is derived from peony seed meal after pressing or subcritical extraction. Alternatively, the degreasing treatment method includes: soaking in n-butane to degrease and obtain degreasing powder.

3. The extraction method of the Paeonia suffruticosa Andr. seed meal extract according to claim 1, characterized in that, After extraction with methanol solution, the extract was spray-dried to obtain the crude extract.

4. The extraction method of the Paeonia suffruticosa Andr. seed meal extract according to claim 1, characterized in that, The crude extract was dissolved in 35-45 times its weight of warm water at 35-45℃ and then subjected to gradient elution.

5. The extraction method of the Paeonia suffruticosa Andr. seed meal extract according to claim 1, characterized in that, Collect the elution product in a 60% ethanol aqueous solution.

6. The extraction method of the Paeonia suffruticosa Andr. seed meal extract according to claim 1, characterized in that, Before elution, adsorption was performed using macroporous adsorption resins such as D101, HPD-100, HPD-BJQH, HPD-450, or AB-8.

7. The extraction method of the Paeonia suffruticosa Andr. seed meal extract according to claim 1, characterized in that, It also includes the step of evaporating and concentrating the eluent and then freeze-drying it.

8. A peony seed meal extract obtained by the extraction method of peony seed meal extract according to any one of claims 1-7.

9. The use of the peony seed meal extract as described in claim 8 in the preparation of a drug for the prevention and / or treatment of liver fibrosis.

10. A drug, characterized in that, Includes the peony seed meal extract as described in claim 8.

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