Application of elsholtzia rugulosa extract in preparation of medicine for preventing and treating acute kidney injury

Abstract

The invention discloses a novel application of elsholtzia rugulosa extract in preparation of a medicine for preventing and treating acute kidney injury, and belongs to the technical field of biology. The medicine is applied to prevention and / or treatment of acute kidney injury, the content of urea nitrogen (BUN) in plasma can be reduced, pathological injury of kidney tissue is improved, and expression of related factors in the kidney tissue is adjusted; the elsholtzia rugulosa extract can effectively reduce the content of BUN in plasma of a cis-platinum (CDDP)-induced acute kidney injury model mouse, effectively improve pathological injury of kidney tissue of the cis-platinum-induced acute kidney injury model mouse and regulate expression of related factors in the kidney tissue of the cis-platinum-induced acute kidney injury model mouse under a relatively low dosage. The raw materials come from natural products and are convenient to collect and high in safety.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to the application of Elsholtzia rugulosa Hemsl extract in the preparation of drugs for preventing and treating acute kidney injury. Background Art

[0002] Acute Kidney Injury (AKI) is a disease in which renal function rapidly declines or disappears within a short period of time due to various reasons, manifested as the accumulation of nitrogenous metabolites in the blood, the increase of end products of nitrogen metabolism (urea and creatinine), and a decrease in urine output. If the patient is not treated in time, it is very likely to lead to chronic kidney disease. Acute kidney injury is characterized by a high incidence, unclear pathogenesis, difficult early diagnosis, lack of effective treatment methods, and poor prognosis, and is a major public health problem recognized globally. Currently, the clinical treatment methods for acute kidney injury usually involve administering potassium-lowering and phosphorus-lowering drugs or hemodialysis. Although hemodialysis can effectively prolong the life of patients, the oxidative stress injury and cytokine storm in the body caused by this treatment method often cause various side effects in patients undergoing long-term treatment, such as loss of appetite, nausea, vomiting, etc., seriously affecting the quality of life and mental health of patients. In order to prevent or treat the process of acute kidney injury, it is urgent to find a safe and effective drug for preventing or treating acute kidney injury from natural products.

[0003] Elsholtzia rugulosa Hemsl, a plant of the genus Elsholtzia in the Lamiaceae family, also known as fragrant perilla herb, fragrant mountain herb, small dam, broom tea, etc., is widely distributed in the southwestern regions of China such as Yunnan, Guizhou, and Sichuan, and is a commonly used traditional Chinese medicine and tea drink among the Yi people in Yunnan. Elsholtzia rugulosa Hemsl contains various substances such as aromatic oils, flavonoid compounds, phenolic substances, steroids, and triterpenoids. In terms of biological activities, it is reported that Elsholtzia rugulosa Hemsl has antioxidant stress activity, antibacterial activity, anti-influenza virus activity, etc., and the extract of Elsholtzia rugulosa Hemsl is safe and has no mutagenic effect. However, there is no literature report on the effect of Elsholtzia rugulosa Hemsl extract on acute kidney injury at home and abroad. Summary of the Invention

[0004] The main purpose of the present invention is to provide a new use of Elsholtzia rugulosa Hemsl extract in the preparation of drugs for preventing and treating acute kidney injury, and it is applied to the preparation of drugs for preventing and treating acute kidney injury caused by cisplatin.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The application of Elsholtzia rugulosa Hemsl extract in the preparation of drugs for preventing and treating acute kidney injury.

[0007] The drugs prepared from the Elsholtzia rugulosa Hemsl extract for preventing and treating acute kidney injury caused by cisplatin include (1)-(4):

[0008] (1) Reduce the content of BUN in the plasma of mice with acute kidney injury model and improve renal function;

[0009] (2) Alleviate the pathological injury of renal tissue in mice with acute kidney injury model;

[0010] (3) The mechanism of action is to regulate the expression of related factors (PTGER4, ADRB2, P-AKT, P-NFκB, COX2) in the renal tissue of mice with acute kidney injury model;

[0011] (4) Reduce the risk of acute kidney injury caused by the chemotherapy drug cisplatin (CDDP).

[0012] The present invention has conducted a series of studies on the preparation of the extract of Elsholtzia rugulosa Hemsl. for preventing and treating acute kidney injury caused by CDDP, and confirmed that the extract of Elsholtzia rugulosa Hemsl. can significantly reduce the content of BUN in the plasma; improve the pathological injury of renal tissue in mice with acute kidney injury model; regulate the expression of related factors in mice with acute kidney injury model. Generally speaking, it effectively improves the renal tissue injury in mice with acute kidney injury model. The extract of Elsholtzia rugulosa Hemsl. provided by the present invention has a significant improvement effect on kidney injury caused by CDDP; and Elsholtzia rugulosa Hemsl. is widely distributed, has high safety, low price, and easily available raw materials; Elsholtzia rugulosa Hemsl. or its extract has good development and application prospects in the preparation of drugs for preventing and treating acute kidney injury caused by CDDP.

[0013] Preferably, the extract of Elsholtzia rugulosa Hemsl. is the methanol extract of Elsholtzia rugulosa Hemsl.; the methanol extract of Elsholtzia rugulosa Hemsl. is the extract obtained by extracting Elsholtzia rugulosa Hemsl. with methanol with a mass concentration of 80%.

[0014] Preferably, the drug contains the methanol extract of Elsholtzia rugulosa Hemsl.

[0015] The extract of Elsholtzia rugulosa Hemsl. is prepared by the following method:

[0016] (1) Take the whole herb of Elsholtzia rugulosa Hemsl., dry it in the sun, powder it, and sieve it to obtain the powder of Elsholtzia rugulosa Hemsl.; add petroleum ether to the powder of Elsholtzia rugulosa Hemsl., and perform ultrasonic extraction for defatting and decolorization to obtain a mixture; centrifuge the obtained mixture, discard the supernatant containing fat, and obtain the residue of Elsholtzia rugulosa Hemsl.;

[0017] (2) Dry the residue of Elsholtzia rugulosa Hemsl. in step (1), add an 80% methanol solution for ultrasonic extraction, and then perform suction filtration to obtain the filter residue and supernatant of Elsholtzia rugulosa Hemsl.;

[0018] (3) Continue to add methanol to the filter residue of Elsholtzia rugulosa Hemsl. in step (2), perform ultrasonic extraction 2-3 times, and suction filter the combined supernatant to obtain a filtrate;

[0019] (4) Rotate and evaporate the filtrate in step (3) to concentrate it, recover methanol until an aqueous phase is obtained; continue to rotary evaporate and concentrate the aqueous phase to obtain a concentrated solution; place the concentrated solution in a refrigerator at -20 °C for pre-freezing, and then perform freeze-drying with a vacuum freeze dryer to obtain the extract of wild basil in 80% methanol solution (lyophilized powder).

[0020] Preferably, the sieving in step (1) is through a sieve of 80 - 100 meshes.

[0021] Preferably, the mass-volume ratio of the wild basil powder to petroleum ether in step (1) is 1 g:10 mL.

[0022] Preferably, the mass-volume ratio of the wild basil residue to 80% methanol in steps (2) and (3) is 1 g:10 mL.

[0023] Preferably, the ultrasonic extraction in steps (2) and (3) is carried out by ultrasonic wave at a power of 70 - 100 w for 25 - 30 min.

[0024] Preferably, the administration dose of the wild basil extract is 200 mg / kg - 600 mg / kg.

[0025] Preferably, the drug contains the wild basil extract; the drug containing the wild basil extract is ingested into the gastrointestinal tract of mammals.

[0026] The new drug for preventing and treating acute kidney injury in the present invention uses the wild basil extract as an active ingredient, and one or more pharmaceutically acceptable excipients can also be added, or it can be compounded with other active ingredients to play a synergistic role in treating acute kidney injury; the drug is an oral preparation, and the oral preparation is a granule, pill, tablet, capsule, powder, etc.

[0027] The beneficial effects of the present invention are as follows:

[0028] 1. The present invention discovers a new medical use for the wild basil extract, proves that the wild basil extract can play a protective role in mice with a cisplatin-induced acute kidney injury model, can be added as an active ingredient to drugs for preventing and treating acute kidney injury, and reduce the occurrence and development degree of acute kidney injury clinically. Therefore, the wild basil extract is expected to be well applied in the preparation of drugs for treating and preventing acute kidney injury.

[0029] 2. Through animal model experiments, the present invention finds that the wild basil extract can effectively reduce the content of BUN in the plasma of model mice at a lower dose; inhibit the shedding of renal tubular epithelial cells and the vacuolar degeneration of renal tubules, and effectively improve the pathological damage caused in the kidney tissue of acute kidney injury model mice.

[0030] 3. The present invention broadens the application field of the wild basil extract and provides new options and ideas for preventing and treating acute kidney injury. Brief Description of the Drawings

[0031] Figure 1 This is the total ion current chromatogram of the negative ion mode of the UPLC-MS / MS principal component analysis of the methanol extract of Elsholtzia rugulosa Hemsl. of the present invention;

[0032] Figure 2 This is the Venn diagram of the component blood entry analysis of the negative ion mode of the UPLC-MS / MS principal component analysis of the methanol extract of Elsholtzia rugulosa Hemsl. of the present invention;

[0033] Figure 3 This is the change of the concentration of BUN in the plasma of mice with acute kidney injury model applied with the extract of Elsholtzia rugulosa Hemsl. of the present invention;

[0034] Figure 4 This is the HE staining diagram of the pathological section of the kidney tissue of mice with acute kidney injury model applied with the extract of Elsholtzia rugulosa Hemsl. of the present invention;

[0035] Figure 5 This is the expression of related factors in the kidney tissue of mice with acute kidney injury model applied with the extract of Elsholtzia rugulosa Hemsl. of the present invention. Detailed Description of the Invention

[0036] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

[0037] Example 1 Preparation of the extract of Elsholtzia rugulosa Hemsl.:

[0038] (1) Extraction of the methanol extract of Elsholtzia rugulosa Hemsl.:

[0039] Dry and powder the whole herb of Elsholtzia rugulosa Hemsl., sieve it through a 80-mesh sieve to obtain the dry powder of the whole herb of Elsholtzia rugulosa Hemsl. Weigh 100 g of the dry powder of the whole herb of Elsholtzia rugulosa Hemsl., add petroleum ether (the mass-volume ratio of the dry powder of the whole herb of Elsholtzia rugulosa Hemsl. to petroleum ether is 1 g:10 mL), ultrasonically extract for 30 min at a power of 70 w, then perform suction filtration, recover the petroleum ether by rotary evaporation, and retain the residue of Elsholtzia rugulosa Hemsl. This step is repeated 3 times; then dry the residue of Elsholtzia rugulosa Hemsl., add 80% methanol (the mass-volume ratio of the residue of Elsholtzia rugulosa Hemsl. to methanol is 1 g:10 mL), ultrasonically extract for 30 min at a power of 100 w, then perform suction filtration, collect the filtrate, continue to add 80% methanol to the obtained residue of Elsholtzia rugulosa Hemsl., ultrasonically extract for 30 min at a power of 100 w, and collect the filtrate; this step is repeated 3 times. Rotate and concentrate the collected filtrate to recover methanol until an aqueous phase is obtained; continue to rotate and concentrate the aqueous phase to obtain a concentrated solution; place the concentrated solution in a -20°C refrigerator for pre-freezing, and then freeze-dry it with a freeze dryer to obtain the freeze-dried powder of the extract of Elsholtzia rugulosa Hemsl.

[0040] (2) Qualitative analysis and blood analysis of the main chemical components of the Elsholtzia rugulosa Hemsl. extract

[0041] UPLC-MS / MS was used for qualitative analysis of the main components of the Elsholtzia rugulosa Hemsl. extract. An Agilent SB-C18 chromatographic column (1.8 μm, 2.1 mm × 100 mm) was used; mobile phase A was ultrapure water (containing 0.1% formic acid), and mobile phase B was acetonitrile (containing 0.1% formic acid); elution program: 0 min, 5% B; 9 min, 95% B; 10 min, 95% B; 11 min, 5% B. The column temperature was 40 °C; the injection volume was 2 μL, and the flow rate was 0.35 mL / min. The analysis results are as Figure 1 shown; in the form of a Venn diagram, the analysis of the components of the Elsholtzia rugulosa Hemsl. extract in blood was performed, and the analysis results are as Figure 2 shown. ERE refers to the methanol extract of Elsholtzia rugulosa Hemsl.; Control refers to the group of mice intragastrically administered with pure water; EREH refers to the group of mice intragastrically administered with the Elsholtzia rugulosa Hemsl. extract.

[0042] It can be seen from Figure 1 that the main compounds contained in the methanol extract of Elsholtzia rugulosa Hemsl. are methyl palmitate, phylloquinone, 3,7-dimethoxyquercetagetin, Isoscoparin I, 1-linoleoylglycerol-2,3-di-O-glucoside, 5β-hydroxycostic acid, gardenia yellow C, (2-carboxyethyl)-L-valine, D-erythrose-4-phosphate, N-acetyl-L-glycine, etc., and also mainly contain other flavonoids, terpenoids and phenolic acids and other compounds.

[0043] It can be seen from Figure 2 that the components of the ethanol extract of Elsholtzia rugulosa Hemsl. contained in the mouse plasma are mainly β-asarone, phosphoethanolamine, 5-acetamidovaleric acid, luteolin-7-O-glucuronide(2→1)-glucuronide, indole-3-lactic acid, N-acetyl-L-glutamic acid, cyclic-3',5'-adenosine monophosphate, D-fructose-6-phosphate, 2-aminohexadecane-1,5,6-triol and 1-naphthol, etc.

[0044] Example 2 Activity evaluation of the Elsholtzia rugulosa Hemsl. extract on the prevention and treatment of acute kidney injury

[0045] 2.1 Establishment of an acute kidney injury mouse model and experimental process

[0046] Using male C57BL / 6J mice (8 weeks old, weighing about 20 g) as the research subjects, a mouse model of acute kidney injury was constructed by inducing kidney injury through intraperitoneal injection of cisplatin (20 mg / kg) for 72 hours. The preventive and therapeutic effects of the 80% methanol extract of Eupatorium adenophorum obtained in Example 1 were evaluated by intragastric administration; after the mice were purchased, they were acclimatized for 7 days in a standard environment and fed with a standard diet. The specific experimental procedures and groupings are as follows:

[0047] Blank control group (Control): Intragastrically administered an equal volume of distilled water for 10 consecutive days;

[0048] Model group (CDDP): Intragastrically administered an equal volume of distilled water for 10 consecutive days, and cisplatin (20 mg / kg) was intraperitoneally injected according to body weight on the 7th day, and the model was established for 72 h;

[0049] Low-dose Eupatorium adenophorum group (CDDP+ERE(200)): Intragastrically administered 200 mg / kg of the 80% methanol extract of Eupatorium adenophorum for 10 consecutive days, and cisplatin (20 mg / kg) was intraperitoneally injected according to body weight on the 7th day, and the model was established for 72 h;

[0050] High-dose Eupatorium adenophorum group (CDDP+ERE(600)): Intragastrically administered 600 mg / kg of the 80% methanol extract of Eupatorium adenophorum for 10 consecutive days, and cisplatin (20 mg / kg) was intraperitoneally injected according to body weight on the 7th day, and the model was established for 72 h;

[0051] Positive control group (CDDP+DXM): Cisplatin (20 mg / kg) was intraperitoneally injected according to body weight on the 7th day, and the model was established for 72 h. Dexamethasone (10 mg / kg) was intragastrically administered for 3 consecutive days starting from the 7th day.

[0052] 2.2 Detection of the effect of the active ingredients of Eupatorium adenophorum on plasma BUN in model mice

[0053] 2.2.1 For each group of mice, blood was collected from the eye socket into an anticoagulant tube, centrifuged at 2000 r / min for 10 min to obtain the supernatant plasma, and the BUN content in the plasma of each group of mice was detected using a BUN kit.

[0054] 2.2.2 Experimental results

[0055] Blood urea nitrogen is the main end product of human protein metabolism, and most of it can be excreted through the kidneys. However, when the renal parenchyma is damaged, the glomerular filtration rate decreases, resulting in an increase in the blood urea nitrogen concentration, such as chronic nephritis, acute nephritis, renal failure, etc. Therefore, the glomerular filtration function can be understood to a certain extent by measuring blood urea nitrogen. In this invention, the effect of the Eupatorium adenophorum extract on acute kidney injury model mice was investigated by detecting the BUN content in the plasma of each group of mice using a BUN kit.

[0056] ByFigure 3 It can be seen that compared with the control group, the BUN concentration in the model group increased significantly. After administration of the high-dose and low-dose groups of Elsholtzia rugulosa Hemsl. extract, it was found that the BUN concentration decreased significantly, and the effect was similar to that of the positive control group, indicating that the active ingredients of Elsholtzia rugulosa Hemsl. can significantly improve renal tissue damage in mice with acute kidney injury model. ***P < 0.001, ****P < 0.0001, with statistical significance.

[0057] 2.3 Detection of the effect of active ingredients on the histopathology of the kidneys of model mice

[0058] 2.3.1 Take the mice in each group, obtain kidney tissue under aseptic conditions, rinse with sterile pre-cooled PBS and then fix to make paraffin sections, and observe the pathological changes of the tissue structure under a microscope after HE staining.

[0059] Preparation method of paraffin sections:

[0060] Sampling: Take fresh kidney tissue and fix it with 10% formalin solution for more than 24 h. Take out the kidney tissue from the fixative, trim the tissue at the target site smoothly with a scalpel in the fume hood, and put the trimmed tissue and the corresponding label into the dehydration box;

[0061] Dehydration and wax infiltration: Put the dehydration box into the dehydrator and dehydrate it successively with gradient alcohol. 75% alcohol for 4 h, 85% alcohol for 2 h, 90% alcohol for 2 h, 95% alcohol for 1 h, absolute ethanol I for 30 min, absolute ethanol II for 30 min, alcohol-benzene for 5 - 10 min, xylene I for 5 - 10 min, xylene II for 5 - 10 min, melted paraffin I at 65℃ for 1 h, melted paraffin II at 65℃ for 1 h, melted paraffin III at 65℃ for 1 h;

[0062] Embedding: Embed the tissue impregnated with wax in the embedding machine. First, put the melted wax into the embedding frame. Before the wax solidifies, take out the tissue from the dehydration box, put it into the embedding frame according to the requirements of the embedding surface and paste the corresponding label, cool it on a -10℃ freezing table. After the wax solidifies, take out the wax block from the embedding frame and trim the wax block;

[0063] Sectioning: Place the trimmed wax block on a paraffin slicer to cut sections with a thickness of 4 μm; float the sections on the warm water at 40℃ of the spreading machine to flatten the tissue, pick up the tissue with a glass slide, bake the slide in an oven at 60℃, take it out after the water is dried and the wax is melted, and store it at room temperature for standby.

[0064] HE staining method is as follows:

[0065] Deparaffinize the paraffin sections to water: Put the sections into xylene I for 20 min - xylene II for 20 min - absolute ethanol I for 5 min - absolute ethanol II for 5 min - 75% alcohol for 5 min in sequence, and wash with tap water;

[0066] Hematoxylin staining: The sections were immersed in hematoxylin staining solution for 3 - 5 minutes, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with bluing solution, and rinsed with running water;

[0067] Eosin staining: The sections were dehydrated in gradient ethanol of 85% and 95% for 5 minutes each in sequence, and then stained in eosin staining solution for 5 minutes.

[0068] Dehydration and mounting: The sections were successively placed in absolute ethanol I for 5 minutes - absolute ethanol II for 5 minutes - absolute ethanol III for 5 minutes - xylene I for 5 minutes - xylene II for 5 minutes for transparency, and then mounted with neutral gum;

[0069] Microscopic examination and image acquisition and analysis.

[0070] 2.3.2 Experimental results

[0071] In this experiment, HE staining was used to observe the renal tissue structure of mice. It can be seen that, compared with the control group, the renal pathological damage in the model group was obvious, the morphology of renal tubular epithelial cells was irregular and exfoliated, the renal tubules were significantly dilated, and vacuolar degeneration occurred. After the high-dose and low-dose groups of Elsholtzia communis extracts were given, it was found that the pathological damage was significantly improved, and the improvement effect was similar to that of the positive control group, indicating that the Elsholtzia communis extract can significantly improve the renal tissue pathological damage in acute kidney injury model mice. Figure 4 It can be seen that, compared with the control group, the renal pathological damage in the model group was obvious, the morphology of renal tubular epithelial cells was irregular and exfoliated, the renal tubules were significantly dilated, and vacuolar degeneration occurred. After the high-dose and low-dose groups of Elsholtzia communis extracts were given, it was found that the pathological damage was significantly improved, and the improvement effect was similar to that of the positive control group, indicating that the Elsholtzia communis extract can significantly improve the renal tissue pathological damage in acute kidney injury model mice.

[0072] 2.4 Detection of the expression of related factors in the renal tissue of model mice by the active ingredient

[0073] 2.4.1 Protein immunoblotting was used to detect the expression of related factors PTGER4, ADRB2, P-AKT, P-NF-κB, and COX2 in the renal tissue of model mice.

[0074] 2.4.2 Experimental results

[0075] The protein encoded by the PTGER4 gene is a member of the G protein-coupled receptor family. This protein is one of the four receptors identified for PGE2, can activate T cell factor signaling, can mediate PGE2-induced EGR1 expression, and regulate the mRNA level and stability of COX2. The ADRB2 receptor is a member of the G protein-coupled transmembrane receptor superfamily, can specifically bind to catecholamines and other endogenous ligands, and is activated by their agonists, generating a series of biological effects.

[0076] It can be seen from Figure 5It can be seen that, compared with the control group, the expressions of ADRB2, PTGER4, P-NF-κB, and COX2 in the model group were significantly increased. After administration of the high-dose and low-dose groups of the wild basil extract, it was found that their expressions were significantly restored. Compared with the control group, the expression of P-AKT in the model group was significantly decreased. After administration of the high-dose and low-dose groups of the wild basil extract, the expression of P-AKT was significantly restored. This indicates that the active components of the wild basil can significantly regulate the expressions of related factors in the renal tissues of mice with acute kidney injury model. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, which is statistically significant.

[0077] In summary, the wild basil extract can significantly reduce the content of BUN in the plasma of mice with acute kidney injury model, improve the pathological damage of the renal tissues of the model mice, and regulate the expressions of related factors in the renal tissues of the model mice. The wild basil extract provided by the present invention has a significant improvement effect on preventing and treating acute kidney injury caused by cisplatin, and the raw materials are from natural products, with high safety, low cost, and are suitable for industrial production and market promotion and application.

[0078] In addition, in the preparation method of the wild basil methanol extract, changing the sieve mesh number of the whole wild basil powder to 90 mesh or 100 mesh, and ultrasonicating for 25 min at 70 w power, or ultrasonicating for 28 min at 80 w power to obtain the wild basil methanol extract also has the above effects.

[0079] The specific embodiments of the invention have been described in detail above, but they are only examples, and the invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modification or substitution of the invention is also within the scope of the invention. Therefore, equivalent transformations, modifications, improvements, etc. made without departing from the spirit and principle of the invention should all be covered within the scope of the invention.

Claims

1. Use of wild basil extract in the preparation of a drug for preventing and treating acute kidney injury caused by cisplatin.

2. The application according to claim 1, characterized in that, The wild basil extract is a methanol extract of wild basil.

3. The application according to claim 1, characterized in that, The drug contains a methanol extract of wild basil.

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