White paeony root essence extract, preparation method and application of white paeony root essence extract in preparation of medicine for treating liver injury

Abstract

The invention belongs to the field of traditional Chinese medicines, and particularly relates to a radix paeoniae alba essence extract, a preparation method and application of the radix paeoniae alba essence extract in preparation of medicines for treating liver injury. The white paeony root essence extract is extracted from the traditional Chinese medicine white paeony root through a method of ethanol reflux extraction and C18 filler medium-pressure preparation, and can be prepared into a clinically common oral preparation. Pharmacological experiments show that the essence extract has the prospect of developing and treating the hepatic fibrosis diseases, and a new choice is provided for treatment of the hepatic fibrosis diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a white peony essence extract, a preparation method and application of the extract in preparing a medicine for treating liver damage. Background Art

[0002] White peony root, Radix Paeoniae Alba., is the dried root of Paeonia lactiflora Pall., a plant of the Ranunculaceae family. It is a perennial herb with enlarged dark brown roots. It tastes sour and bitter and is slightly cold in nature. It enters the liver and spleen meridians. It has the effects of nourishing blood and regulating menstruation, restraining yin and stopping sweating, softening the liver and relieving pain, and calming liver yang. White peony root is often used to treat symptoms such as blood deficiency and sallow complexion, irregular menstruation, spontaneous sweating, night sweats, flank pain, abdominal pain, cramps in the limbs, headache and dizziness. White peony root can nourish liver yin to nourish liver blood, and can restrain liver yin to soften liver body. It is the most in line with the physiological characteristics of the liver and is a classic medicine used by doctors in all dynasties to nourish the liver. In recent years, research on white peony root in liver diseases has received more and more attention.

[0003] Hepatic fibrosis (HF) refers to the process of chronic liver disease development, in which hepatocytes are repeatedly destroyed and then regenerated, and extracellular matrix such as collagen, glycoprotein, proteoglycan, etc. diffusely deposits excessively and distributes abnormally in the liver. It is the pathological repair response of the liver to chronic damage, and is a key step in the development of various chronic liver diseases to cirrhosis and an important link affecting the prognosis of chronic liver diseases. Further development of liver fibrosis can cause structural disorder of the liver, nodular regeneration of hepatocytes, and formation of pseudolobular structure, i.e., cirrhosis. Common diseases that cause liver fibrosis include viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, cholestasis, autoimmune liver disease, etc. At present, magnesium isoglycyrrhizinate, obeticholic acid, and ursodecholic acid are commonly used anti-hepatic fibrosis drugs in clinical practice, but the existing anti-hepatic fibrosis drugs have not achieved the expected effect in treatment, and long-term use is prone to adverse reactions such as increased blood pressure, hypokalemia, skin itching, dizziness, palpitations, and vomiting. Therefore, it is very urgent to find drugs that can effectively treat liver fibrosis in clinical practice.

[0004] Traditional Chinese medicine has a long history, outstanding efficacy, few side effects, and abundant production. With the continuous advancement of medical research, Chinese medicine has gradually become one of the hottest research fields. The present invention prepares the essence extract of the traditional Chinese medicine white peony root and uses it to treat rats and mice with liver fibrosis. It is found that the essence extract of white peony root can effectively alleviate liver damage and reduce liver fibrosis, which provides a new idea for the treatment of liver fibrosis with traditional Chinese medicine. Summary of the invention

[0005] The invention aims to provide a white peony essence extract, which contains paeoniflorin, paeoniflorin, ethyl gallate and 1,2,3,4,6-pentagalloyl glucose in peony and has the effect of oral treatment of liver damage.

[0006] Another object of the present invention is to provide a method for preparing the white peony essence extract, which can effectively concentrate the extracted components into the desired preferred components and remove the remaining low-polarity and high-polarity components.

[0007] The technical solution adopted by the present invention to solve its technical problem is:

[0008] A method for preparing a white peony essence extract, the method comprising the following steps:

[0009] S1, reflux extraction: add 6-10 times the mass volume of 50-90% ethanol solution to the white peony root slices for reflux extraction 1-3 times, each time for 1-3 hours, filter and concentrate the filtrate;

[0010] S2, medium-pressure preparation with C18 filler: The concentrated filtrate obtained in S1 is passed through an ODS medium-pressure preparation column with C18 filler, and eluted with methanol at volume fractions of 20%, 40%, 60%, 80% and 100% at 6-15 retention volumes in sequence; the 40% elution fraction is collected, and the methanol and water are recovered under reduced pressure to obtain the white peony essence extract.

[0011] Furthermore, the detection wavelength in S2 is 254 nm.

[0012] A white peony essence extract obtained by the preparation method of the invention.

[0013] A use of the white peony essence extract of the present invention in preparing a medicine for treating liver damage.

[0014] Preferably, in the preparation of the drug for treating liver injury, the dosage of white peony root extract is 1-10 g / person / day, preferably 5 g / person / day. One person is calculated as 60 kg.

[0015] The beneficial effects of the present invention are:

[0016] The white peony essence extract of the invention is extracted from the traditional Chinese medicine white peony by ethanol reflux extraction and C18 filler medium pressure preparation method, and can be prepared into an oral preparation commonly used in clinic.

[0017] Pharmacological experiments show that the white peony essence extract of the present invention has an oral therapeutic effect on liver damage, and its oral therapeutic effect on liver fibrosis is stronger than that of the white peony methanol extract at a dose of 4 times its dose.

[0018] The white peony essence extract of the invention has the prospect of being developed for treating liver fibrosis diseases, and provides a new option for treating liver fibrosis diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0020] Figure 1 UPLC spectrum and characteristic peak identification of white peony essence extract, wherein A is the fingerprint of white peony methanol extract (MPRA) and white peony essence extract (MP-40), and B is the characteristic peak identification of white peony essence extract MP-40;

[0021] Figure 2 The results of the efficacy test of white peony extract in protecting rats from liver damage. A: Experimental process of mice; B: Survival rate of mice; C: Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) indicators; D: Liver tissue entity diagram; Liver tissue hematoxylin and eosin (H&E) staining, Sirius red staining, Masson staining results; Data were analyzed by one-way ANOVA and expressed as mean ± standard deviation *P<0.05 is statistically significant * Compared with the control group, *P<0.05, **P<0.01, ***P<0.001, # Compared with the model group, #P<0.05, ## P<0.01, ### P<0.001, the same below;

[0022] Figure 3 The results of the detection of marker proteins of white peony extract in protecting rats from liver damage. A: CollagenⅠ, α-SMA and GAPDH protein blot bands in liver tissue; B: α-SMA immunofluorescence image in liver tissue;

[0023] Figure 4 Results of the efficacy test of white peony extract in protecting mice from liver damage. A: Survival rate of rats; B: Survival rate of rats; C: Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) indicators; D: Liver tissue entity diagram; Liver tissue hematoxylin and eosin (H&E) staining, Sirius red staining, and Masson staining results.

[0024] Figure 5 The results of the detection of marker proteins of white peony extract in protecting rats from liver damage. A: α-SMA and GAPDH protein blot bands in liver tissue; B: α-SMA immunofluorescence image in liver tissue;

[0025] Figure 6 The results of the signaling pathway detection of white peony extract in protecting mice from liver damage. A: Protein imprints of NLRP3, α-SMA, Cleaved Caspase-1, 1L-1β and GAPDH in liver tissue; B: Western Blot analysis of the expression of NLRP3, TGF-β, Cleaved Caspase-1, 1L-1β and GAPDH in liver tissue. DETAILED DESCRIPTION

[0026] In order to further illustrate the present invention, the application of the white peony essence extract provided by the present invention in the preparation of a drug for treating liver fibrosis is described in detail below in conjunction with the accompanying drawings and examples, but they should not be understood as limiting the scope of protection of the present invention. The test samples and test processes used in the following examples include the following (if the specific experimental conditions are not indicated in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels).

[0027] The invention provides application of white peony root essence extract in preparing medicine for treating liver fibrosis.

[0028] The liver fibrosis described in the present invention is cholestatic liver fibrosis. In the embodiments, the liver fibrosis model mice and rats induced by bile duct ligation are preferably used as examples for verification to confirm the anti-liver fibrosis activity of the white peony essence extract.

[0029] The therapeutic effect of the drug of the present invention is preferably manifested as at least one of the following:

[0030] (1) reducing the expression of fibrosis characteristic proteins in fibrotic liver tissue; the characteristic proteins preferably include one or more of Collagen I and α-SMA;

[0031] (2) Reduce inflammatory infiltration of liver tissue.

[0032] Example 1

[0033] The preparation method of white peony root extract comprises the following specific steps:

[0034] S1, reflux extraction: select white peony root slices, add 6-10 times the mass volume concentration of 70% ethanol solution, reflux extraction 1-3 times, each time 1-3h, filter and concentrate the filtrate to obtain white peony root methanol extract (MPRA);

[0035] S2, C18 medium-pressure preparation: The concentrated filtrate obtained from S1 was passed through an ODS medium-pressure preparation column with a detection wavelength of 254 nm and C18 filler. Eight retention volumes of methanol with volume fractions of 20%, 40%, 60%, 80% and 100% were used for elution.

[0036] The 40% elution fraction was collected, and methanol and water were recovered under reduced pressure to obtain the white peony root extract (MP-40).

[0037] The UPLC spectrum and characteristic peak identification of the white peony extract obtained are as follows: Figure 1 As shown. The essence contains paeoniflorin, paeoniflorin, ethyl gallate and 1,2,3,4,6-pentagalloyl glucose. The yield of white peony methanol extract (MPRA) is 13.6%, and the yield of white peony essence extract (MP-40) is 3.45%.

[0038] Example 2

[0039] 1.1 Drug preparation

[0040] Accurately weigh the white peony essence extract and the white peony methanol extract, dissolve them in 0.5% CMC-Na solution respectively, prepare the dosing solution (the dosing dose of the white peony essence extract is 0.78 g / kg, and the dosing dose of the white peony methanol extract is 3.1 g / kg), and store them in a refrigerator at 4°C for later use.

[0041] 1.2 Modeling and drug administration

[0042] 1.2.1 Mouse liver fibrosis model

[0043] 22 C57BL / 6J male mice, weighing 17-22g, were randomly divided into 4 groups. After one week of adaptation and feeding, follow-up experiments were conducted. The experimental groups were divided into a blank group (5 mice), a bile duct ligation group (BDL group, 7 mice), a white peony extract group (5 mice), and a white peony methanol extract group (5 mice). The mice were anesthetized with pentobarbital and then sutured after bile duct ligation surgery. The treatment of the blank group was similar to the above, but no bile duct ligation surgery was performed. 24h after the operation, the white peony extract group and the white peony methanol extract group were treated with drugs, and the model group and the blank group were treated with 0.5% CMC-Na for 14 days. The number of mice surviving each day was recorded during this period. The drug administration experiment was administered by a one-time intraperitoneal gavage method. After gavage, the diet and water were normal. After the last dose, all groups of mice were fasted (no water was allowed).

[0044] 1.2.2 Rat liver fibrosis model

[0045] 20 rats were randomly divided into 3 groups, and the follow-up experiments were carried out after one week of adaptation and feeding. The experimental groups were divided into a blank group (5 rats), a bile duct ligation group (BDL group, 8 rats), and a white peony essence extract group (7 rats). The mice were anesthetized with pentobarbital and then sutured after bile duct ligation surgery. The treatment of the blank group was similar to the above, but no bile duct ligation surgery was performed. 24 hours after the operation, the rats in the white peony essence extract group were treated with drugs for 29 days. The model group and the blank group were treated with 0.5% CMC-Na, and the number of surviving rats was recorded every day. The drug administration experiment adopted a one-time intraperitoneal gavage method. After gavage, the diet and water were normal. After the last dose, all groups of mice were fasted (but not water).

[0046] 1.3 Tissue and serum collection

[0047] 24 hours after the last administration, blood was collected from the heart after anesthesia, and the blood of mice / rat was collected in a centrifuge tube, centrifuged at 3000 rpm for 10 min, and the serum was separated and stored in a -80°C refrigerator for later use. C57BL / 6J male mice were dissected, the liver of mice / rat was removed, a part was cut and fixed with 10% formalin, and the rest was stored at -80°C for subsequent related experiments.

[0048] 1.4 Determination of AST and ALT biochemical indicators in animal serum

[0049] The separated serum samples were analyzed by standard techniques of clinical chemistry to determine the content of AST and ALT in mouse / rat serum.

[0050] The results of the efficacy test of white peony root extract in protecting liver damage are shown in Figure 2 and Figure 4 .

[0051] Figure 2 B is the survival rate of mice, and the survival numbers are blank group (5 / 5), bile duct ligation group (5 / 7), white peony essence extract group (5 / 5) and methanol extract group (4 / 5).

[0052] Figure 4 B is the survival ratio of rats in each group, and the survival numbers are blank group (5 / 5), bile duct ligation group (6 / 8) and white peony root extract group (7 / 7).

[0053] The above results show that white peony root extract can significantly improve the survival rate of mice and rats with liver fibrosis induced by bile duct ligation, and the effect of white peony root extract is significantly better than that of methanol extract.

[0054] The results of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice are as follows Figure 2As shown in C, compared with the blank group, the AST and ALT in the serum of mice in the BDL group were significantly increased. Compared with the BDL group, the ALT and AST biochemical indexes in the serum of mice in the white peony essence extract and white peony methanol extract administration groups were significantly reduced, and the white peony essence extract group was significantly lower than the white peony methanol extract group.

[0055] The results of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in rats were as follows: Figure 4 As shown in C, compared with the blank group, the AST and ALT in the serum of the rats in the BDL group were significantly increased. Compared with the BDL group, the AST and ALT biochemical indicators in the serum of the rats in the white peony extract administration group were significantly lower than those in the BDL group.

[0056] In summary, it is confirmed that white peony root extract can significantly alleviate liver damage caused by bile duct ligation (BDL).

[0057] 1.5 Histopathological study of animal liver inflammatory infiltration (H&E, Masson, Sirius red staining)

[0058] Mouse / rat liver tissue samples were fixed with formalin for one week, then taken out and rinsed with running water to remove excess formalin on the tissue surface. A blade was used to cut liver tissue with a volume of about 1 cm3, and it was continuously rinsed with running water for more than 2 hours. All samples were dehydrated using a fully automatic dehydrator. Then, an embedding machine was used to embed the tissue with paraffin. After cooling to room temperature, all samples were sliced ​​with a thickness of about 4 μm, and then sliced ​​with glass slides. The paraffin sections were placed in an oven and melted at 60°C for more than 2 hours. After dewaxing and hydration, H&E, Masson and Sirius red staining were performed. After completion, 1 to 2 drops of neutral gum were dropped on a glass slide, sealed with a cover slip, and placed under an optical microscope to observe the inflammatory infiltration of the liver tissue of mice and rats. Figure 2 C is a solid staining image of liver tissue (including liver tissue hematoxylin and eosin (H&E) staining, Sirius red staining, and Masson staining).

[0059] like Figure 2 D and Figure 4As shown in D, in the H&E result graph, the liver tissues of mice and rats in the BDL group had severe inflammatory infiltration, while the inflammatory cell aggregation degree of the liver tissues of mice in the drug-treated groups (white peony extract group, white peony methanol extract group) and rats in the drug-treated group (white peony extract group) was lighter than that in the BDL group, and the inflammatory infiltration area of ​​the white peony extract group was less than that of the white peony methanol extract group. In the Masson and Sirius Red result graphs, compared with the blank group, the collagen in the liver tissues of mice and rats in the BDL group was severely accumulated. Compared with the BDL group, the collagen-positive area in the drug-treated mice and rats was reduced, and the positive area in the white peony extract group was less than that in the white peony methanol extract group.

[0060] In summary, the white peony essence extract has the activity of alleviating liver inflammation and improving liver fibrosis, and its efficacy is stronger than that of the white peony methanol extract with the same amount of raw medicine (the weight is 4 times that of the white peony essence extract).

[0061] 1.6 Western Blotting to detect the expression of fibrosis proteins in animal liver tissue

[0062] Take fresh liver tissue from each group of mice / rats, accurately weigh about 0.1g of tissue sample, and add 1×PBS buffer containing 1% PMSF, about 5 times the mass volume. After the tissue is cut into pieces, it is homogenized with a tissue homogenizer to obtain lung tissue homogenate. Add RIPA lysis buffer for lysis, place it on ice for 30 minutes, and extract total protein with protein extraction reagent. After centrifugation at 13000rpm for 10 minutes, take the lysate. Then, centrifuge the lysate again with a high-speed centrifuge, collect the supernatant, divide it into portions, and store it in a -20℃ refrigerator for later use. After measuring the BCA protein concentration of the sample, Western Blotting experiment was used to detect protein expression.

[0063] The results of the marker protein test of white peony extract in protecting mice from liver damage are as follows Figure 3 As shown in the figure, the expression levels of CollagenⅠ and α-SMA in the BDL group were significantly higher than those in the blank group. Both the white peony essence extract group and the white peony methanol extract group could down-regulate the expression of fibrosis-characterizing proteins in BDL-induced liver fibrosis mice, and the effect of the white peony essence extract was better than that of the white peony methanol extract with the same amount of crude drug.

[0064] The results of the marker protein test of white peony extract in protecting rats from liver damage are as follows Figure 5 As shown in the figure, the expression level of α-SMA in rats in the BDL group was significantly higher than that in the blank group. The white peony extract group could down-regulate the expression of fibrosis characteristic proteins in BDL-induced liver fibrosis mice.

[0065] The results of the signaling pathway protein test of white peony extract in protecting mice from liver damage are as follows Figure 6As shown in the results, the expression levels of NLRP3, TGF-β, Cleaved Caspase-1, and 1L-1β in the BDL group were significantly higher than those in the blank group. Compared with the BDL group, the expression of NLRP3, TGF-β, Cleaved Caspase-1, and 1L-1β in the white peony extract group was significantly decreased.

[0066] Experiments have shown that white peony root extract can reduce the expression of α-SMA and Collagen I caused by bile duct ligation (BDL) and proteins related to liver fibrosis signaling pathway.

[0067] 1.7 Immunofluorescence detection of the expression of fibrotic proteins in animal liver tissue

[0068] The liver tissue was embedded with embedding medium (OCT). 4 μm sections were sliced ​​on a freezing microtome and stored at -80°C for later use. The liver tissue sections were taken, placed at room temperature for 15 min and fixed with a fixative (methanol: acetone = 1:1) for 10 min. The tablets were then rinsed with PBS, blocked with 5% goat serum for 1 h, and then incubated with the primary antibody at 4°C overnight. The secondary antibody (goat anti-rabbit IgG) was added to the sections and incubated in the dark at room temperature for 1 hour. After washing, the tablets were sealed with an anti-fluorescence quencher containing 4',6-diamidino-2-phenylindole (DAPI) and finally observed under a fluorescence microscope.

[0069] like Figure 3 As shown in the figure, the expression level of α-SMA in the BDL group mice was higher than that in the blank group. Compared with the BDL group, the expression of α-SMA in the white peony essence extract group mice and the white peony methanol extract group mice was reduced, and the white peony essence extract group was significantly less than the methanol extract group.

[0070] like Figure 5 As shown in the figure, compared with the blank group, the expression level of α-SMA in the rats of the BDL group was higher than that of the blank group. Compared with the BDL group, the expression of α-SMA in the rats of the white peony essence extract group was significantly reduced.

[0071] Experiments have shown that both the white peony essence extract group and the white peony methanol extract group can downregulate BDL-induced liver fibrosis, and the white peony essence extract is more effective than the white peony methanol part.

[0072] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part.

[0073] The above is a detailed introduction to a white peony essence extract, a preparation method and an application thereof provided by the present invention. Specific examples are used herein to illustrate the principles and implementation methods of the present invention, and the description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be pointed out that for ordinary technicians in this technical field, without departing from the principle of the present invention, the present invention can also be improved and modified in a number of ways, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a white peony root extract, characterized in that The method comprises the following steps: S1, reflux extraction: add 6-10 times the mass volume of 50-90% ethanol solution to the white peony slices and reflux extract 1-3 times, each time for 1-3h, filter and concentrate the filtrate; S2, medium-pressure preparation with C18 filler: The concentrated filtrate obtained in S1 was passed through an ODS medium-pressure preparation column with C18 filler, and eluted with methanol at volume fractions of 20%, 40%, 60%, 80% and 100% at 6-15 retention volumes in sequence; the 40% elution fraction was collected, and the methanol and water were recovered under reduced pressure to obtain the white peony essence extract.

2. A white peony root extract obtained by the preparation method according to claim 1.

3. The white peony root extract according to claim 2, characterized in that: The white peony root extract contains paeoniflorin, paeoniflorin, ethyl gallate and 1,2,3,4,6-pentagalloyl glucose.

4. Use of the white peony root extract according to claim 2 in preparing a drug for treating liver damage.

5. The use according to claim 4, characterized in that: The dosage of the white peony root extract is 1-10 g / person / day.

6. The use according to claim 4, characterized in that: The dosage of the white peony root extract is 5g / person / day.

7. The use according to claim 4, characterized in that: The medicine contains pharmaceutically acceptable excipients.

8. The use according to claim 4, characterized in that: The medicine is an oral medicine.

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