Application of birch juice separation liquid in improvement of parenchymal hepatic cell injury

Abstract

The invention belongs to the field of medical application of birch juice, and discloses application of a birch juice separating medium to improvement of parenchymal hepatic cell injury, the birch juice separating medium is used for improving parenchymal hepatic cell injury, and the birch juice separating medium is prepared according to the following steps: S1, raw material pretreatment; s2, preliminary filtration; s3, performing ultrafiltration; s4, performing nanofiltration concentration; s5, chromatographic purification; s6, vacuum drying; according to the technical scheme, the birch juice separation liquid is applied to improvement of the parenchymal hepatic cell injury, the parenchymal hepatic cell injury is improved through the birch juice separation liquid, the application field of the birch juice is expanded, and experiments show that the birch juice separation liquid prepared through the technical scheme can remarkably reduce transaminase release of the injured parenchymal hepatic cells, so that the survival rate of the injured parenchymal hepatic cells is increased. The traditional Chinese medicine composition has the advantages that the activity of antioxidant enzymes is improved, the content of lipid peroxidation products is reduced, and the traditional Chinese medicine composition has a good improvement effect on parenchymal hepatic cell injury induced by various factors and provides a new natural active substance source for preventing and treating liver injury.

Description

Technical Field

[0001] This invention relates to the field of birch sap medicinal applications, and more particularly to the application of a birch sap extract in improving liver parenchymal cell damage. Background Technology

[0002] The liver, a vital metabolic and detoxification organ, relies on hepatocytes, the primary cell type responsible for its physiological functions. Damage to hepatocytes directly impacts normal liver function, leading to various liver diseases. Currently, the causes of hepatocyte damage are diverse, including chemical damage (such as from drugs, alcohol, and toxins); pathological damage (such as viral infections and autoimmune diseases); and metabolic damage (such as lipid overload induced by a high-fat diet). Statistics show that global deaths from various liver diseases are rising annually, with hepatocyte damage being a core factor in the progression of most liver diseases.

[0003] Existing methods for improving liver parenchymal cell damage mainly include chemotherapy and natural product intervention. Although chemotherapy may have certain efficacy in the short term, it is often accompanied by significant toxic side effects, and long-term use can easily lead to drug resistance, making it difficult to meet the needs of clinical treatment.

[0004] Birch, a living fossil among woody plants, possesses exceptional vitality and restorative abilities. Its sap, a natural secretion formed when birch trees absorb soil nutrients during early spring snowmelt, has a long history of use in Northeast my country, Russia, and parts of Europe. The Compendium of Materia Medica records that birch bark can be used to treat liver-related diseases such as jaundice, while the Dictionary of Traditional Chinese Medicine and the Chinese Materia Medica also record its expectorant and antitussive medicinal value. Modern research shows that birch sap is rich in minerals, vitamins, and essential amino acids, possessing significant nutritional value. Furthermore, triterpenoids such as betulinol and betulinic acid in birch-related products have been proven to have various biological activities, including anti-inflammatory, antioxidant, and antitumor effects. However, existing research on birch sap largely focuses on its nutritional and drinking value; applications of birch sap and its isolated products in improving liver parenchymal cell damage are not reported, nor are the specific active ingredients and mechanisms of action for its hepatoprotective effects clearly defined. Therefore, this invention is proposed. Summary of the Invention

[0005] The present invention aims to provide an application of birch sap extract in improving liver parenchymal cell damage, in order to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: An application of a birch sap extract in improving liver parenchymal cell damage, characterized in that the birch sap extract is used to improve liver parenchymal cell damage, and the birch sap extract is prepared according to the following steps: S1. Raw material pretreatment: Select fresh birch sap, filter to remove impurities from the birch sap, then add 0.05% to 0.1% (w / v) of vitamin C, stir well, and then put it into a refrigeration device with a storage temperature of 4°C for later use. S2. Preliminary filtration: The birch sap obtained after the pretreatment in step S1 is preliminarily filtered through a microfiltration membrane with a pore size of 0.22-0.45 μm and a filtration pressure of 0.1-0.2 MPa to obtain the permeate. S3. Ultrafiltration: The permeate is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 5000-10000 Da. The ultrafiltration pressure is 0.3-0.5 MPa and the operating temperature is 25-30℃ to obtain the retentate. S4. Nanofiltration Concentration: The retentate is concentrated by passing it through a nanofiltration membrane with a molecular weight cutoff of 100–300 Da at an operating pressure of 1–1.5 MPa and an operating temperature of 30–35 °C to obtain a concentrated solution. S5. Chromatographic purification: Load the concentrated solution onto an AB-8 macroporous adsorption resin chromatography column at a flow rate of 1-2 BV / h; first elute with deionized water at a flow rate of 2-3 BV / h for 2-3 BV, then elute with 30-50% (v / v) ethanol solution at a flow rate of 1-2 BV / h for 3-4 BV, and collect the ethanol eluent; S6. Vacuum drying: Place the ethanol eluent in a vacuum drying oven and dry it to constant weight at a temperature of 40-50℃ and a pressure of -0.08-0.1MPa to obtain birch sap separation powder; dissolve it with sterile physiological saline to prepare birch sap separation solution before use.

[0007] Preferably, the liver parenchymal cell injury is chemical, alcoholic, or immune-mediated liver parenchymal cell injury.

[0008] Preferably, the chemically induced liver parenchymal cell damage is induced by carbon tetrachloride, acetaminophen, or D-galactosamine.

[0009] Preferably, the birch sap used in step S1 is collected from birch trees in spring, from April to May.

[0010] Preferably, the microfiltration membrane, ultrafiltration membrane, and nanofiltration membrane used in steps S2, S3, and S4 are all made of polyethersulfone.

[0011] Preferably, in step S5, the AB-8 macroporous adsorption resin is activated with ethanol and washed with deionized water until neutral before use.

[0012] Preferably, the birch sap extract is used at a concentration of 10–100 μg / mL.

[0013] The beneficial effects of this technical solution compared to existing technologies are as follows: (1) This technical solution applies birch sap separation to improve liver parenchymal cell damage. By improving liver parenchymal cell damage through birch sap separation, the application field of birch sap is expanded. Experiments show that the birch sap separation prepared by this technical solution can significantly reduce the release of transaminase from damaged liver parenchymal cells, increase the activity of antioxidant enzymes, and reduce the content of lipid peroxidation products. It has a good effect on improving liver parenchymal cell damage induced by various factors, and provides a new source of natural active substances for the prevention and treatment of liver injury.

[0014] (2) This technical solution also optimizes the preparation method of birch sap separation liquid. It adopts a three-stage membrane separation process of microfiltration-ultrafiltration-nanofiltration coupled with macroporous resin chromatography to achieve efficient enrichment and purification of active ingredients in birch sap. The purity of polyphenols and polysaccharides in the separation liquid is 50-80 times higher than that of natural birch sap. This solves the problem of low concentration of effective ingredients and insignificant pharmacological effects in natural birch sap, and improves the effect of separation liquid in improving liver parenchymal cell damage. Moreover, the entire preparation process adopts physical separation and gentle drying methods, without the use of toxic and harmful chemical reagents, effectively preserving the structure and biological activity of active ingredients. The product is highly safe and does not produce other side effects when used for medicine. Attached Figure Description

[0015] Figure 1 A schematic flowchart illustrating the preparation method of birch sap separation solution provided by the present invention; Detailed Implementation

[0016] The following is a reference to the accompanying drawings in the instruction manual. Figure 1 The present invention will be further described in detail below with reference to the embodiments: An application of birch sap extract in improving liver parenchymal cell damage, wherein the birch sap extract is used to improve liver parenchymal cell damage, which is chemical, alcoholic, or immune-mediated liver parenchymal cell damage; chemical liver parenchymal cell damage is induced by carbon tetrachloride, acetaminophen, or D-galactosamine. Birch sap extract is prepared according to the following steps: S1. Raw material pretreatment: Select fresh birch sap, which is collected from birch trees in spring (April to May). Filter to remove impurities from the birch sap, then add 0.05% to 0.1% (w / v) of vitamin C, stir well, and store in a refrigerator at 4°C for later use to prevent oxidation of active ingredients in the birch sap. S2. Preliminary filtration: The birch sap obtained after the pretreatment in step S1 is preliminarily filtered through a microfiltration membrane with a pore size of 0.22–0.45 μm and a filtration pressure of 0.1–0.2 MPa to obtain the permeate; removing large molecular impurities such as proteins and colloids. S3. Ultrafiltration: The permeate is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 5000-10000 Da. The ultrafiltration pressure is 0.3-0.5 MPa and the operating temperature is 25-30℃ to obtain a retentate. The retentate is rich in active ingredients such as polysaccharides and polyphenols. S4. Nanofiltration Concentration: The retentate is concentrated by passing it through a nanofiltration membrane with a molecular weight cutoff of 100–300 Da at an operating pressure of 1–1.5 MPa and an operating temperature of 30–35 °C to obtain a concentrated solution. S5. Chromatographic purification: Load the concentrated solution onto an AB-8 macroporous adsorption resin column. Before use, the AB-8 macroporous adsorption resin is activated with ethanol and washed with deionized water until neutral. The flow rate of the concentrated solution is 1-2 BV / h. First, elute with deionized water at a flow rate of 2-3 BV / h for 2-3 BV, then elute with 30-50% (v / v) ethanol solution at a flow rate of 1-2 BV / h for 3-4 BV. Collect the ethanol eluent. S6. Vacuum drying: Place the ethanol eluent in a vacuum drying oven and dry it to constant weight at a temperature of 40-50℃ and a pressure of -0.08-0.1MPa to obtain birch sap separation powder; dissolve it with sterile physiological saline to prepare a birch sap separation solution with a concentration of 10-100μg / mL before use.

[0017] Microfiltration, ultrafiltration, and nanofiltration membranes are all made of polyethersulfone, which gives them good chemical stability and biocompatibility, improving their performance.

[0018] Example 1; Birch sap extract is prepared according to the following steps: S1. Raw material pretreatment: Weigh 10L of fresh birch sap, which is collected from birch trees in spring (April to May). Filter the sap through a 200-mesh nylon mesh to remove impurities. Then add 0.08% (w / v) of vitamin C, stir well, and refrigerate in a refrigerator at 4°C for 1 hour. S2. Preliminary filtration: The birch sap obtained after the pretreatment in step S1 is preliminarily filtered through a microfiltration membrane with a pore size of 0.22 μm and a filtration pressure of 0.15 MPa, yielding 9.2 L of permeate. S3, Ultrafiltration; The permeate is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 8000 Da at an ultrafiltration pressure of 0.4 MPa and an operating temperature of 28 °C, yielding 1.8 L of retentate; S4. Nanofiltration Concentration: The retentate is concentrated by nanofiltration through a nanofiltration membrane with a molecular weight cutoff of 200 Da at an operating pressure of 1.2 MPa and an operating temperature of 32 °C, until it is concentrated to 1 L to obtain the concentrate. S5. Chromatographic purification: Load the concentrated solution onto an AB-8 macroporous adsorption resin column with a column size of φ5cm×50cm and a resin packing volume of 100mL. Before use, the AB-8 macroporous adsorption resin is activated with ethanol and washed with deionized water until neutral. The flow rate for loading the concentrated solution is 1.5BV / h. First, elute 2BV with deionized water at a flow rate of 2.5BV / h, discard the water eluent, and then elute 3BV with 40% (v / v) ethanol solution at a flow rate of 1.5BV / h. Collect the ethanol eluent. S6. Vacuum drying: Place the ethanol eluent in a vacuum drying oven and dry it to constant weight at a temperature of 45℃ and a pressure of -0.09MPa to obtain 2.3g of birch sap separation powder; dissolve it with sterile physiological saline to prepare a birch sap separation solution with a concentration of 50μg / mL before use.

[0019] Experiment Example 1: Experiment on the ameliorative effect of birch sap separation on carbon tetrachloride-induced hepatocyte damage; Experimental cells were selected; the experimental cells were primary rat hepatocytes, which were isolated from SPF-grade male SD rats. Prepare the experimental reagents, including the birch sap separation solution prepared in Example 1 of this invention, carbon tetrachloride, dimethyl sulfoxide, 1640 complete culture medium (containing 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin), ALT detection kit, AST detection kit, GSH-Px (glutathione peroxidase) detection kit, MDA (malondialdehyde) detection kit, and MTT reagent; Prepare experimental equipment, including a carbon dioxide incubator, an ELISA reader, a centrifuge, and an inverted microscope; Experimental steps: Primary hepatocytes were isolated and cultured. Rats were anesthetized and cannulated via the portal vein. The cells were first perfused with calcium-free perfusion fluid, then circulated with type IV collagenase perfusion fluid at 37°C and oxygenated. The liver was then removed, the liver capsule was removed, and a washing solution containing 5% fetal bovine serum was added. The cells were then pipetted into a single-cell suspension, filtered through a 200-mesh nylon filter, centrifuged, and the centrifuged product was collected. The suspension was washed three times. Cells were cultured in 1640 complete culture medium to prepare 1×10⁻⁶ cells. 9 Hepatocyte suspension was seeded into 24-well and 96-well culture plates and incubated at 37°C in a 5% CO2 incubator for 12–16 h. After the cells adhered, subsequent experiments were carried out. The experiment was divided into four groups: a blank control group, a model control group, a high-dose birch sap separation solution group, a medium-dose birch sap separation solution group, and a low-dose birch sap separation solution group; each group had three replicates. The blank control group was not given carbon tetrachloride and birch sap separation solution, and the culture was continued as usual. Model control group: only carbon tetrachloride was added, without birch sap separation solution; the model control group is essentially a carbon tetrachloride-induced liver parenchymal cell injury model; the culture supernatant was aspirated and removed, and culture medium containing carbon tetrachloride (DMSO final concentration 0.1%) was added, and culture was continued for 6 hours to complete the establishment of the carbon tetrachloride-induced liver parenchymal cell injury model and the culture of the model control group. High-dose birch sap extract group (100 μg / mL), medium-dose birch sap extract group (50 μg / mL), and low-dose birch sap extract group (10 μg / mL); birch sap extract of the corresponding concentration was added at the same time as carbon tetrachloride; other culture steps were the same as the model control group, except that birch sap extract of the corresponding concentration was added at the same time as the culture medium containing carbon tetrachloride, and the two groups were cultured together for 6 hours.

[0020] Test data; Cell viability was determined using the MTT assay. After culture, 2020 μL of MTT solution (5 mg / mL) was added to each replicate well in each group, and the cells were cultured for another 4 hours. After culture, the supernatant was discarded, and 150 μL of DMSO was added to each replicate well. The cells were then shaken for 10 minutes. The absorbance (A value) was measured at 490 nm using a microplate reader, and cell viability was calculated. Cell viability (%) = (A value of drug group / A value of blank control group) × 100%. ALT and AST activity were detected using ALT and AST detection kits; culture supernatants from each group were collected and analyzed according to ALT detection methods. Instructions for using the kit and AST detection kit to detect ALT and AST activity in the supernatant; GSH-Px activity and MDA content were detected using GSH-Px and MDA assay kits. The culture supernatant was discarded, and cells were lysed with 0.2% Triton-100 aqueous solution. Cell lysates were collected by centrifugation. GSH-Px activity and MDA content were measured according to the instructions of the GSH-Px and MDA assay kits, respectively. The above operations yielded multiple sets of data. These data were processed, and the experimental data are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 22.0 software. The t-test was used for comparisons between groups, and P < 0.05 was considered statistically significant. The experimental results are shown in the table below. Compared with the blank control group, P < 0.05; compared with the model control group, P < 0.05, which is statistically significant.

[0021] As shown in the table above, the model control group had significantly lower cell viability, significantly increased ALT and AST activities, significantly decreased GSH-Px activities, and significantly increased MDA content. This indicates that the carbon tetrachloride-induced hepatocyte injury model was successfully established. Compared with the model control group, the high-dose, medium-dose, and low-dose birch sap extract groups all significantly improved the viability of damaged hepatocytes, reduced ALT and AST activities, increased GSH-Px activities, and reduced MDA content in a dose-dependent manner. Furthermore, the high-dose birch sap extract group showed effects close to the blank control group, indicating that the birch sap extract prepared in this technical scheme has a significant ameliorative effect on carbon tetrachloride-induced hepatocyte injury.

[0022] Experiment Example 2: Experiment on the ameliorative effect of birch sap separation on alcohol-induced liver parenchymal cell damage; Primary hepatocytes were isolated and cultured; the same procedure as in Experiment 1 was followed. Grouped administration; the experiment was divided into a blank control group, a model control group, and a medium-dose birch sap separation group; each group had 3 replicates; The blank control group was not added with ethanol and birch sap separation solution, and the culture was continued as usual. Model control group: only ethanol was added, without birch sap separation solution; the model control group is essentially an alcohol-induced liver parenchymal cell injury model; the culture supernatant was aspirated and removed, and fresh culture medium containing 100mM ethanol was added, and the culture was continued for 24 hours to complete the establishment of the alcohol-induced liver parenchymal cell injury model and the culture of the model control group. Medium-dose birch sap separation solution group (50 μg / mL): The corresponding concentration of birch sap separation solution was added at the same time as ethanol; other culture steps were the same as the model control group, except that the corresponding concentration of birch sap separation solution was added at the same time as ethanol-containing culture medium, and the two groups were cultured together for 24 h.

[0023] The detection data was obtained in the same manner as in Experiment 1. The experimental results are as follows; Following the same analytical method as in Experimental Example 1, the table above shows that the birch sap extract prepared by this technical solution has a significant preventive and ameliorative effect on alcohol-induced liver parenchymal cell damage.

[0024] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. The application of a birch sap extract in improving liver parenchymal cell damage, characterized in that, The birch sap extract is used to improve liver parenchymal cell damage, and the birch sap extract is prepared according to the following steps: S1. Raw material pretreatment: Select fresh birch sap, filter to remove impurities from the birch sap, then add 0.05% to 0.1% (w / v) of vitamin C, stir well, and then put it into a refrigeration device with a storage temperature of 4°C for later use. S2. Preliminary filtration: The birch sap obtained after the pretreatment in step S1 is preliminarily filtered through a microfiltration membrane with a pore size of 0.22-0.45 μm and a filtration pressure of 0.1-0.2 MPa to obtain the permeate. S3. Ultrafiltration: The permeate is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 5000-10000 Da. The ultrafiltration pressure is 0.3-0.5 MPa and the operating temperature is 25-30℃ to obtain the retentate. S4. Nanofiltration Concentration: The retentate is concentrated by passing it through a nanofiltration membrane with a molecular weight cutoff of 100–300 Da at an operating pressure of 1–1.5 MPa and an operating temperature of 30–35 °C to obtain a concentrated solution. S5. Chromatographic purification: Load the concentrated solution onto an AB-8 macroporous adsorption resin chromatography column at a flow rate of 1-2 BV / h; first elute with deionized water at a flow rate of 2-3 BV / h for 2-3 BV, then elute with 30-50% (v / v) ethanol solution at a flow rate of 1-2 BV / h for 3-4 BV, and collect the ethanol eluent; S6. Vacuum drying: Place the ethanol eluent in a vacuum drying oven and dry it to constant weight at a temperature of 40-50℃ and a pressure of -0.08-0.1MPa to obtain birch sap separation powder; dissolve it with sterile physiological saline to prepare birch sap separation solution before use.

2. The application of a birch sap extract as described in claim 1 in improving liver parenchymal cell damage, characterized in that, The liver parenchymal cell damage is chemical, alcoholic, or immune-mediated.

3. The application of a birch sap extract as described in claim 1 in improving liver parenchymal cell damage, characterized in that, The chemically induced liver parenchymal cell damage was induced by carbon tetrachloride, acetaminophen, or D-galactosamine.

4. The application of a birch sap extract as described in claim 1 in improving liver parenchymal cell damage, characterized in that, The birch sap used in step S1 is collected from birch trees in spring, from April to May.

5. The application of a birch sap extract as described in claim 1 in improving liver parenchymal cell damage, characterized in that, The microfiltration membranes, ultrafiltration membranes, and nanofiltration membranes used in steps S2, S3, and S4 are all made of polyethersulfone.

6. The application of a birch sap extract as described in claim 1 in improving liver parenchymal cell damage, characterized in that, In step S5, the AB-8 macroporous adsorption resin is activated with ethanol and washed with deionized water until neutral before use.

7. The application of a birch sap extract as described in claim 1 in improving liver parenchymal cell damage, characterized in that, The birch sap extract is used at a concentration of 10–100 μg / mL.

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