Preparation method and application of fresh peony extract

Abstract

The preparation method comprises the following steps: taking a certain amount of fresh peony leaves, adding pectinase, tannase, cellulase and water, performing crushing and grinding to obtain plant slurry, performing solid-liquid separation, taking supernate, adding activated clay, activated carbon and diatomite at the temperature of 60-100 DEG C, performing solid-liquid separation, performing solid-liquid separation at the temperature of 60-100 DEG C, and performing solid-liquid separation to obtain the peony fresh extract with the alcoholic fatty liver prevention and treatment effect. Keeping the temperature for 10 to 60 minutes; performing solid-liquid separation, and taking supernate; and carrying out ultrafiltration separation, and collecting the permeate to obtain the product. Experiments show that the fresh peony extract has a remarkable protection effect on the alcoholic fatty liver, can be used for preparing medicines and health-care foods for preventing and treating the alcoholic fatty liver, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of natural plant preparation and application, and specifically relates to a preparation method of peony fresh extract and a new medical application thereof. Background Art

[0002] As people's quality of life improves, the prevalence of fatty liver increases year by year. Among them, long-term heavy drinking can cause alcoholic fatty liver. The earliest form of alcoholic fatty liver is fatty degeneration. The liver cells increase in size, pile up on each other and compress the liver sinusoids, causing ischemia and hypoxia of liver cells, making the liver susceptible to inflammatory mediators or other toxic substances, and pathological damage is very likely to occur, which may eventually lead to hepatitis and liver fibrosis. Alcoholic fatty liver is a reversible liver damage stage. If drug intervention is used in time in the early stages of alcoholic liver damage to reduce fatty degeneration, the progression of alcoholic liver disease may be slowed down or stopped. Because the pathogenesis of alcoholic fatty liver is still unclear, there is no specific drug to prevent and treat alcoholic fatty liver. Therefore, it is of great significance to develop drugs that can slow down alcoholic fatty liver.

[0003] Peony is a plant of the dicotyledon class, Paeoniaceae, and genus Paeonia. It is a traditional flower unique to my country and has the reputation of "the king of flowers". As early as the Qin and Han Dynasties, peony was used as a medicinal material, and it has been widely used in the pharmaceutical industry as a pharmaceutical raw material. Pharmacological studies in recent years have shown that peony has good antibacterial, anti-inflammatory, whitening and anti-aging effects, and the total flavonoids, total phenols, paeoniflorin, polyphenols, and polysaccharides in peony have obvious antioxidant effects. For example, Chinese patent application CN118634183A discloses an application of peony flower extract in anti-oxidation, whitening, soothing, anti-aging, antibacterial and anti-inflammatory effects.

[0004] At present, there are no reports or studies on the mitigating effect of peony leaf extract on alcoholic fatty liver. Therefore, studying the protective effect of peony leaf extract on alcoholic fatty liver provides a new scientific basis for drugs to prevent and treat alcoholic fatty liver.

[0005] Based on this, this application was developed. Summary of the invention

[0006] The present invention aims to overcome the defects of the prior art and provide a peony fresh extract. The present invention uses alcoholic fatty liver model animals to confirm that the peony fresh extract has a significant therapeutic effect on alcoholic fatty liver, and can be used to prepare medicines, health products, etc. for preventing and treating alcoholic fatty liver.

[0007] The present invention also provides a preparation method of the peony fresh extract and a new medical application thereof in preventing and treating alcoholic fatty liver.

[0008] To achieve the above object, the present invention adopts the following technical solution: A method for preparing peony fresh extract comprises the following steps: 1) Take a certain amount of fresh peony leaves, add pectinase, tannin, cellulase and water, crush and grind (during grinding, the temperature of the colloid mill should be kept at 35-45℃), obtain plant slurry, separate the solid and liquid, and take the supernatant; 2) Adding activated clay, activated carbon and diatomaceous earth to the supernatant obtained in step 1) at 60-100°C and keeping the temperature for 10-60 minutes; separating the solid and the liquid and taking the supernatant; 3) Separating the supernatant obtained in step 2) with an ultrafiltration membrane, collecting the permeate, and obtaining the peony fresh extract.

[0009] Specifically, in step 1), the added amounts of pectinase, tannase and cellulase can be 0.02-0.1%, 0.2-0.5% and 0.1-0.5% of the mass of fresh peony leaves, respectively.

[0010] Furthermore, in step 1), the amount of water added can be 2-4 times the mass of the fresh peony leaves.

[0011] In the above preparation method, specifically, in step 2), the amount of activated clay added can be 0.5-3% of the mass of the supernatant obtained in step 1). The amount of activated carbon added can be 1-4% of the mass of the supernatant obtained in step 1). The amount of diatomaceous earth added can be 0.5-2% of the mass of the supernatant obtained in step 1).

[0012] Specifically, in step 3), the molecular weight cutoff of the ultrafiltration membrane used in ultrafiltration may be 1000-1500.

[0013] Furthermore, in step 1), the fresh peony leaf tissue can be crushed by a crusher and ground by a colloid mill. The solid-liquid separation in step 1) and step 2) includes but is not limited to the use of plate and frame filter filtration, butterfly centrifuge centrifugation and the like.

[0014] The invention provides a peony fresh extract prepared by the preparation method.

[0015] The present invention also provides the use of the above-mentioned peony fresh extract in the preparation of medicines or health products for preventing and treating alcoholic fatty liver.

[0016] The present invention also provides a medicine or health product comprising the peony fresh extract, which is prepared by compounding the peony fresh extract as an active ingredient with conventional auxiliary materials acceptable in pharmacy or health products.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The core problem of the present invention is to discover and provide a new medical use of peony fresh extract, that is, its use in the preparation of drugs or health products for preventing and treating alcoholic fatty liver. Peony, as a plant with ornamental value, edible value and medicinal value, is highly safe, rich in resources and low in price; the fresh extract is easy to prepare and extract, and has broad market prospects.

[0018] The present invention uses alcoholic fatty liver model animals to confirm that peony fresh extract can significantly reduce mouse liver index, reduce ALT, AST and TG content in mouse serum, reduce MDA level in mouse liver tissue and significantly increase SOD level, while reducing IL-6 and TNF-α levels, thereby having significant protective and therapeutic effects on alcoholic fatty liver. Therefore, the present invention provides the use of peony leaf fresh extract in the preparation of drugs and health foods for preventing and treating alcoholic fatty liver, that is, it can be used to prepare drugs and health foods for preventing and treating alcoholic fatty liver, providing a new idea and approach for the application of peony leaves. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 HPLC chromatograms of gallic acid reference (A) and peony leaf sample (B); Figure 2 These are the pathological morphological observations of liver tissues of mice in each group treated with fresh peony leaf extract. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is further described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0021] In the following experiments, the raw materials used are all common commercial products that can be directly purchased, or can be prepared using conventional techniques in the art. For experimental processes or operations not described in detail, conventional techniques in the art can be used.

[0022] 1. Experimental Materials 1.1 Materials Pectinase (50000u / g) and tanninase (200u / g) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; cellulase (10000u / g) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0023] Activated white clay (decolorization rate ≥ 98%) and activated carbon (200 mesh) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd., and diatomaceous earth was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0024] Peony leaf fresh extract: prepared by the preparation method in 2.1; 53° Beijing Red Star Erguotou, purchased from Beijing Red Star Co., Ltd.; Superoxide dismutase (SOD), malondialdehyde (MDA), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and triglyceride (TG) detection kits were purchased from Nanjing Jiancheng Bioengineering Institute; Mouse interleukin-6 (IL-6) and mouse tumor necrosis factor α (TNF-α) ELISA kits were purchased from Tianjin Anoricon Company.

[0025] 1.2 Animals The SPF-grade BALB / c mice used in the experiment were purchased from Henan Sikebes Biotechnology Co., Ltd.

[0026] 2. Test methods 2.1 Preparation of Peony Leaf Extract 1) Take a certain weight of fresh peony leaf raw material, add water containing a certain amount of pectinase, tannase and cellulase (the added amounts of pectinase, tannase and cellulase relative to the mass of fresh peony leaves are 0.04%, 0.3% and 0.2%, respectively, and the added amount of water is 3 times the mass of fresh peony leaves), use a crusher to crush the fresh peony leaves, grind them in a colloid mill at 40℃ to obtain plant slurry, centrifuge in a butterfly centrifuge (10000r / min), and take the supernatant.

[0027] 2) Add activated clay, activated carbon and diatomaceous earth to the supernatant at 80°C (the amount of activated clay, activated carbon and diatomaceous earth added relative to the mass of the supernatant is 1%, 2% and 1%, respectively), keep warm for 30 minutes, centrifuge in a butterfly centrifuge (10000r / min), take the supernatant and separate it through an ultrafiltration membrane with a molecular weight cutoff of 1000-1500, collect the permeate, and obtain the peony leaf fresh extract.

[0028] The peony leaf fresh extract prepared above contains about 5 mg / ml gallic acid, about 10% polyphenols, and about 5% polysaccharides. Gallic acid is determined by high performance liquid chromatography, and the chromatogram is as follows: Figure 1 Polyphenols and polysaccharides were determined by UV spectrophotometry using gallic acid and anhydrous glucose as reference substances, respectively.

[0029] 2.2 Establishment, grouping and drug administration of alcoholic fatty liver mouse model Fifty BALB / c mice were adaptively fed with control fluid feed for 7 days and then randomly divided into 5 groups according to body weight, with 10 mice in each group, namely blank control group, model group, low-dose group of peony leaf fresh extract (1-fold dilution group), medium-dose group of peony leaf fresh extract (original solution group), and high-dose group of peony leaf fresh extract (1-fold concentration group).

[0030] Due to the limitation of oral gavage volume for mice, the high-dose group of peony leaf fresh extract (1-fold concentrated group) was prepared by reducing the pressure and concentrating the original fresh extract at 50°C. The concentrated solution was equivalent to 2 times the concentration of the original solution and was used for the experiment.

[0031] The method of modeling alcoholic fatty liver was as follows: the model group and each drug-treated group were gavaged with Erguotou at a dose of 0.1 mL / 10 g, once a day, for 10 consecutive weeks. The Erguotou dilution was diluted with pure water, and the concentrations of the dilutions were 30%, 35%, 40%, 45% and 50%, respectively. The Erguotou dilution with a concentration of 30% was given in the first and second weeks of the experiment, and the concentration increased to 35% in the third and fourth weeks, 40% in the fifth and sixth weeks, 45% in the seventh and eighth weeks, and 50% in the ninth and tenth weeks. At the same time as modeling, the blank control group and the model group were gavaged with pure water, and each drug-treated group was given different concentrations of peony leaf fresh extract at a volume of 0.1 mL / 10 g, once a day, for 10 consecutive weeks.

[0032] 2.3 Sampling and determination of serum and liver markers After the last administration, mice were fasted but not watered for 12 h and weighed. After blood was collected from the eyeball, mice were killed by dislocation of the neck, left to stand for 30 min, centrifuged at 4°C and 1500 r / min for 15 min, and serum was collected. Serum ALT, AST, IL-6, and TNF-α were determined according to the instructions of the detection kit.

[0033] Remove the mouse liver, weigh the liver mass, and calculate the liver index, liver index (%) = liver mass (g) / body mass (g) × 100%. Take about 0.3 g of liver tissue, rinse with cold saline, dry with filter paper, add 2.7 mL of saline to make 10% liver tissue homogenate, centrifuge at 4000r / min for 10 min; take the supernatant, and measure the TG, MDA and SOD levels in the liver tissue according to the instructions of the kit.

[0034] 2.4 Observation of liver tissue pathology Fresh liver was cut into small pieces, and the liver tissue was fixed with 10% formalin, embedded in paraffin, and sliced. Then, hematoxylin and eosin (H&E) staining was performed, the slices were sealed with neutral resin, and observed under an optical microscope.

[0035] 2.5 Statistical analysis GraphPad Prism 8.0 software was used for statistical analysis and drawing. Single-factor analysis was used for multiple comparisons among groups, and t-test was used for comparisons between two groups. P The difference was statistically significant when the value was less than 0.05.

[0036] 3 Results 3.1 Effects of Peony Leaf Extract on Mouse Liver Index Compared with the blank control group, the liver index of mice in the model group increased significantly ( P <0. 01). Compared with the model group, the low, medium and high dose groups of peony leaf extract could significantly reduce the liver index of mice ( P <0. 05). The experimental results show that the peony leaf fresh extract of the present invention can effectively improve the liver enlargement of mice caused by alcohol. The results are shown in Table 1.

[0037] Table 1 Effects of fresh peony leaf extract on liver indexes of mice in each group Note: a: Compared with the blank control group, P <0.01; b: compared with the model group, P <0.05; c: Compared with the model group, P <0.01.

[0038] 3.2 Effects of fresh peony leaf extract on the levels of ALT, AST and TG in the serum of mice in each group Compared with the blank control group, the levels of ALT, AST and TG in the serum of mice in the model group increased significantly ( P <0. 01). Compared with the model group, the ALT, AST and TG levels in the serum of mice in the high-dose group, medium-dose group and low-dose group of the fresh extract of peony leaves of the present invention were significantly reduced ( P <0. 05), the results are shown in Table 2.

[0039] Table 2 Effects of fresh peony leaf extract on the levels of ALT, AST and TG in the serum of mice in each group Note: a: Compared with the blank control group, P <0.01; b: compared with the model group, P <0.05; c: Compared with the model group, P <0.01.

[0040] 3.3 Effects of fresh peony leaf extract on MDA and SOD levels in liver tissues of mice in each group Compared with the blank control group, the MDA level in the liver tissue of the model group mice increased significantly ( P <0. 05), SOD level was significantly decreased ( P<0. 05). Compared with the model group, the MDA levels in the liver tissues of mice in the high-dose, medium-dose and low-dose peony leaf extract groups of the present invention were significantly reduced ( P <0. 05), while SOD levels increased significantly ( P <0. 05), the results are shown in Table 3.

[0041] Table 3 Effects of fresh peony leaf extract on MDA and SOD levels in liver tissues of mice in each group Note: a: Compared with the blank control group, P <0.01; b: compared with the model group, P <0.05; c: Compared with the model group, P <0.01.

[0042] 3.4 Effects of Peony Leaf Extract on Hepatic Inflammatory Response in Mice Compared with the blank control group, the levels of IL-6 and TNF-α in the serum of the mice in the model group were significantly increased (P < 0.01). Compared with the model group, the levels of IL-6 and TNF-α in the high-dose group, the medium-dose group and the low-dose group of the fresh extract of peony leaves of the present invention were significantly reduced (P < 0.01). The results are shown in Table 4.

[0043] Table 4 Effects of peony leaf extract on IL-6 and TNF-α levels in liver tissues of mice in each group Note: a: Compared with the blank control group, P <0.01; b: compared with the model group, P <0.01.

[0044] 3.5 Effects of fresh peony leaf extract on the pathological morphology of liver tissues in each group of mice H&E staining of mouse liver tissues showed Figure 2 As shown in the figure, it can be seen that the liver cells in the liver tissue of the blank control group mice are arranged in an orderly and neat manner, and the liver lobule structure is normal. Lipid deposition, disordered arrangement of liver cells, and destroyed liver lobule structure appeared in the liver tissue of the model group mice. The degree of fatty degeneration in the liver tissue of mice in the high, medium and low dose groups of the peony leaf fresh extract of the present invention was significantly reduced compared with the model group, the arrangement of liver cells gradually became normal, and the structure of liver lobule was restored.

[0045] In summary, it can be shown that the peony leaf fresh extract of the present invention can significantly reduce the liver index of mice, reduce the ALT, AST and TG content in mouse serum, reduce the MDA level in mouse liver tissue and significantly increase the SOD level, while reducing the IL-6 and TNF-α levels. Therefore, the peony leaf fresh extract of the present invention can effectively prevent and treat alcoholic fatty liver, and is used to prepare drugs or health products for preventing and treating alcoholic fatty liver.

Claims

1. A method for preparing peony fresh extract, characterized in that: The steps include: 1) Take a certain amount of fresh peony leaves, add pectinase, tannin, cellulase and water, crush and grind to obtain plant slurry, separate the solid and liquid, and take the supernatant; 2) Adding activated clay, activated carbon and diatomaceous earth to the supernatant obtained in step 1) at 60-100°C and keeping the temperature for 10-60 minutes; separating the solid and the liquid and taking the supernatant; 3) The supernatant obtained in step 2) is separated by ultrafiltration, and the permeate is collected to obtain the peony fresh extract.

2. The method for preparing the peony fresh extract according to claim 1, characterized in that: In step 1), the added amounts of pectinase, tannase and cellulase are 0.02-0.1%, 0.2-0.5% and 0.1-0.5% of the mass of fresh peony leaves, respectively.

3. The method for preparing the peony fresh extract according to claim 1, characterized in that: In step 1), the amount of water added is 2-4 times the mass of the fresh peony leaves.

4. The method for preparing the peony fresh extract according to claim 1, characterized in that: In step 2), the amount of activated clay added is 0.5-3% of the mass of the supernatant obtained in step 1).

5. The method for preparing the peony fresh extract according to claim 1, characterized in that: In step 2), the amount of activated carbon added is 1-4% of the mass of the supernatant obtained in step 1).

6. The method for preparing the peony fresh extract according to claim 1, characterized in that: In step 2), the amount of diatomaceous earth added is 0.5-2% of the mass of the supernatant obtained in step 1).

7. The method for preparing the peony fresh extract according to claim 1, characterized in that: In step 3), the ultrafiltration membrane used in ultrafiltration has a molecular weight cutoff of 1000-1500.

8. Peony fresh extract prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the peony fresh extract according to claim 8 in the preparation of medicines or health products for preventing and treating alcoholic fatty liver.

10. A medicine or health product comprising the peony fresh extract according to claim 8, characterized in that: The peony fresh extract is used as an active ingredient and conventional auxiliary materials acceptable in pharmacy or health care products are added to prepare the product.

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