CN115671123A - Application of clematis root saponin in preparation of purine-lowering medicine - Google Patents
Application of clematis root saponin in preparation of purine-lowering medicine Download PDFInfo
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- CN115671123A CN115671123A CN202110826943.XA CN202110826943A CN115671123A CN 115671123 A CN115671123 A CN 115671123A CN 202110826943 A CN202110826943 A CN 202110826943A CN 115671123 A CN115671123 A CN 115671123A
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- clematis root
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Abstract
The invention relates to application of clematis root saponin in preparation of a purine-lowering drug, belonging to the technical field of medicines. The invention provides application of clematis root saponin or a composition containing the clematis root saponin in preparation of purine-lowering medicaments, and researches show that clematis root sugar saponin CP1, clematis root disaccharide saponin CP2, clematis root trisaccharide saponin CP3, clematis root trisaccharide saponin CP4, clematis root trisaccharide saponin CP5, clematis root trisaccharide saponin CP6 and clematis root extracts can effectively improve the metabolic capability of a mouse with hyperpurine syndrome on purine substances, so that the clematis root trisaccharide saponin CP3, the clematis root trisaccharide saponin CP5, the clematis root trisaccharide saponin CP6 and the clematis root extracts can effectively lower purine, and the effects of preventing and/or treating the hyperpurine syndrome are achieved.
Description
Technical Field
The invention relates to application of clematis root saponin in preparing a purine-lowering medicine, belonging to the technical field of medicines.
Background
Statistical data show that the incidence rates of hyperuricemia and gout in China are 13.3% and 1.1% respectively in 2000-2014. Although the number of patients is huge, the drugs that can be selected by patients with hyperuricemia and gout are still few. Currently, the most common drugs for treating hyperuricemia and gout are XOD inhibitors such as allopurinol and febuxostat.
Wherein allopurinol has been the first choice for treating chronic gout since 1963 when being used clinically. Allopurinol and its metabolite oxypurinol can inhibit xanthine oxidase, effectively reduce uric acid production, and reduce uric acid content in blood and urine to below solubility, thereby preventing uric acid from forming crystal and depositing in joints and other tissues. The medicine is also helpful for the re-dissolution of gout nodules and uric acid crystals, and is a uric acid-reducing medicine recommended by the European antirheumatic union in 2006. However, since allopurinol is a purine analog, it inevitably affects other enzymes in purine or pyrimidine metabolism, thereby affecting the metabolism of other organs of the human body, and has many side effects. The side effects of allopurinol administration for the treatment of chronic gout include: 1. the rash has the incidence rate of 3-10 percent, can be pruritic papule or urticaria, can also be vesicular reaction, can also have other anaphylactic reactions in severe cases, such as exfoliative and purpuric lesions, erythema multiforme and the like, and the medicine is stopped once the skin lesion occurs; 2. the incidence rate of gastrointestinal tract diseases such as diarrhea, nausea, vomit, stomachache or paroxysmal abdominal pain is 3-10%, and proper symptomatic treatment should be carried out when severe cases or persistent cases exist; 3. headache, dizziness and rare peripheral neuritis symptoms such as hand and foot numbness, stabbing pain or pain, hypodynamia and the like.
Febuxostat is listed in europe in 2008, in the usa in 2009, and in china in 2013, by virtue of the characteristics of higher uric acid reducing speed, fewer side effects, suitability for long-term treatment and the like, febuxostat at the beginning of the listing rapidly seizes the market share of global anti-gout drugs, has the advantages of strong effect, safety, mild and moderate renal insufficiency without adjusting dosage and the like, is suitable for long-term treatment of hyperuricemia complicated with gout, comprises gout patients with increased uric acid generation and reduced renal clearance, and is particularly suitable for gout patients who cannot tolerate allopurinol or have taboos and cannot reach the uric acid standard after allopurinol treatment. However, with the expansion of the population, various adverse reactions of febuxostat have been reported. Adverse reactions to treat chronic gout with febuxostat include liver dysfunction (3.5%), diarrhea (2.7%), headache (1.8%), nausea (1.7%), rash (1.5%), and the like. In 2017, the FDA issued a warning of the risk of heart-related death of febuxostat, which is likely to increase the risk of heart-related death compared to allopurinol.
In addition, the XOD inhibitors such as allopurinol and febuxostat can obviously increase the content of purine which is an upstream substance for synthesizing uric acid in a patient body while inhibiting the synthesis of uric acid, and the deposition of purine substances in the kidney further aggravates kidney injury. Therefore, there is an urgent need for the development of a drug which has few side effects and is effective for the prevention and/or treatment of hyperpurine syndrome caused by XOD inhibitors, or for the development of a drug which has few side effects and is effective for the prevention and/or treatment of hyperpurine syndrome and, at the same time, is effective for the prevention and/or treatment of hyperuricemia.
According to the record of Chinese pharmacopoeia, clematis root is the dried root and rhizome of clematis root, clematis filamentosa or clematis terniflora of Ranunculaceae, has the efficacy of dispelling wind-damp and dredging the channels and collaterals, and is clinically used for treating rheumatism, numbness of limbs, spasm of tendons and vessels, difficulty in flexion and extension and the like. The clematis root does not cause any toxic or side effect to human bodies when being taken. At present, no report exists about the application of clematis root or an extract thereof in preparing a medicament for preventing and/or treating hyperpurine syndrome.
Disclosure of Invention
In order to solve the above problems, the present invention provides an application of clematis saponins or a composition containing clematis saponins in the preparation of a medicament, wherein the medicament has at least one of the following uses:
(a) Purine reduction;
(b) Reducing uric acid; and/or the presence of a gas in the atmosphere,
(c) Preventing and/or treating kidney injury.
In one embodiment of the invention, the hypopurine refers to prevention and/or treatment of hyperpurine syndrome; the uric acid reduction refers to the prevention and/or treatment of hyperuricemia.
In one embodiment of the invention, the hyperpurine mia is hyperpurine mia caused by XOD inhibitors.
In one embodiment of the present invention, the hyperuricemia is hyperuricemia caused by purine metabolic disorder.
In one embodiment of the present invention, the kidney injury is kidney injury caused by accumulation of purine.
In one embodiment of the invention, the drug is clematis saponin or a composition containing clematis saponin, and conventional auxiliary materials are added to prepare at least one of clinically acceptable tablets, capsules, powder, mixtures, pills, granules, solutions, syrups and sustained and controlled release preparations according to a conventional process.
In one embodiment of the present invention, the clematis saponins include at least one of clematis sugar saponin CP1, clematis disaccharide saponin CP2, clematis trisaccharide saponin CP3, clematis trisaccharide saponin CP4, clematis trisaccharide saponin CP5, and clematis trisaccharide saponin CP6.
In one embodiment of the present invention, the clematis saponins include at least one of clematis root trisaccharide saponin CP3, clematis root trisaccharide saponin CP5 and clematis root trisaccharide saponin CP6.
In one embodiment of the present invention, the clematis chinensis sugar saponin-containing composition is a clematis chinensis extract.
In one embodiment of the present invention, the preparation method of the clematis root extract comprises the steps of: the clematis root is taken as a raw material, and an organic solvent and/or water is taken as an extraction solvent for extraction to obtain the clematis root extract.
In one embodiment of the invention, the extraction solvent is a 50 to 80vt% aqueous alcohol solution.
In one embodiment of the present invention, the preparation method of the clematis root extract comprises the steps of: adding the clematis chinensis into 5-10 times of 50-80 vt% alcohol-water solution by weight for soaking and extracting for 2-3 times, extracting for 5-10 days each time, combining extracting solutions, concentrating to obtain clematis chinensis extractum, and drying to obtain the clematis chinensis extract.
In one embodiment of the present invention, the preparation method of the clematis root extract further comprises the step of subjecting the clematis root extract to macroporous resin chromatography.
In one embodiment of the invention, the macroporous resin chromatography comprises the following steps: and (3) washing impurities of the clematis root extract by adopting 0-5 vt% of alcohol-water solution, then eluting by adopting 60-95 vt% of alcohol-water solution, and collecting eluent.
In one embodiment of the present invention, in the macroporous resin chromatography step, the elution flow rate is controlled to be 2 to 5BV/h, and the elution volume of each elution reagent is controlled to be 2 to 5BV.
In one embodiment of the present invention, the macroporous resin column used in the macroporous resin adsorption step is a D101 macroporous resin column.
In one embodiment of the present invention, the preparation method of clematis root extract further comprises the step of subjecting the eluate to macroporous resin chromatography again.
In one embodiment of the present invention, the performing macroporous resin chromatography again comprises the steps of: and (3) eluting the eluent by adopting 30-95 vt% alcohol-water solution, and collecting the eluent again.
In one embodiment of the present invention, the preparation method of clematis root extract further comprises a step of concentrating the eluate.
In one embodiment of the present invention, the alcohol aqueous solution is a mixed solution of ethanol and water.
The present invention also provides a method for preparing an extract of clematis root, comprising the steps of: radix clematidis is used as a raw material, and an organic solvent and/or water are/is used as an extraction solvent for extraction to obtain the radix clematidis extract.
In one embodiment of the invention, the extraction solvent is a 50 to 80vt% aqueous alcohol solution.
In one embodiment of the invention, the method comprises the steps of: adding the clematis chinensis into 5-10 times of 50-80 vt% alcohol-water solution by weight for soaking and extracting for 2-3 times, extracting for 5-10 days each time, combining extracting solutions, concentrating to obtain clematis chinensis extractum, and drying to obtain the clematis chinensis extract.
In one embodiment of the present invention, the method further comprises the step of subjecting the clematis root extract to macroporous resin chromatography.
In one embodiment of the invention, the macroporous resin chromatography comprises the following steps: and (3) washing impurities of the clematis root extract by adopting 0-5 vt% of alcohol-water solution, then eluting by adopting 60-95 vt% of alcohol-water solution, and collecting eluent.
In one embodiment of the present invention, in the macroporous resin chromatography step, the elution flow rate is controlled to be 2 to 5BV/h, and the elution volume of each elution reagent is controlled to be 2 to 5BV.
In one embodiment of the present invention, the macroporous resin column used in the macroporous resin adsorption step is a D101 macroporous resin column.
In one embodiment of the invention, the method further comprises the step of taking the eluate for macroporous resin chromatography again.
In one embodiment of the present invention, the performing macroporous resin chromatography again comprises the steps of: and (3) eluting the eluent by adopting 30-95 vt% alcohol-water solution, and collecting the eluent again.
In one embodiment of the invention, the method further comprises the step of concentrating the eluate.
In one embodiment of the present invention, the alcohol-water solution is a mixed solution of ethanol and water.
The invention also provides a clematis root extract, which is prepared by adopting the method.
The invention also provides a medicine for reducing purine, which contains the clematis root saponin or a composition containing the clematis root saponin.
In one embodiment of the invention, the purine reduction refers to prevention and/or treatment of hyperpurine syndrome.
In one embodiment of the invention, the hyperpurine syndrome is hyperpurine syndrome caused by an XOD inhibitor.
In one embodiment of the invention, the drug is clematis root saponin or a composition containing clematis root saponin, and conventional auxiliary materials are added according to a conventional process to prepare at least one of clinically acceptable tablets, capsules, powder, mixture, pills, granules, solutions, syrups and sustained and controlled release preparations.
In one embodiment of the present invention, the clematis saponins include at least one of clematis root sugar saponin CP1, clematis root disaccharide saponin CP2, clematis root trisaccharide saponin CP3, clematis root trisaccharide saponin CP4, clematis root trisaccharide saponin CP5, and clematis root trisaccharide saponin CP6.
In one embodiment of the present invention, the clematis saponins include at least one of clematis root trisaccharide saponin CP3, clematis root trisaccharide saponin CP5 and clematis root trisaccharide saponin CP6.
In one embodiment of the present invention, the composition containing clematis root sugar saponin is the clematis root extract.
The invention also provides a medicine for reducing uric acid, which contains clematis saponin or a composition containing the clematis saponin.
In one embodiment of the present invention, the uric acid reduction refers to prevention and/or treatment of hyperuricemia.
In one embodiment of the present invention, the hyperuricemia is hyperuricemia caused by purine metabolic disorder.
In one embodiment of the invention, the drug is clematis root saponin or a composition containing clematis root saponin, and conventional auxiliary materials are added according to a conventional process to prepare at least one of clinically acceptable tablets, capsules, powder, mixture, pills, granules, solutions, syrups and sustained and controlled release preparations.
In one embodiment of the present invention, the clematis saponins include at least one of clematis sugar saponin CP1, clematis disaccharide saponin CP2, clematis trisaccharide saponin CP3, clematis trisaccharide saponin CP4, clematis trisaccharide saponin CP5, and clematis trisaccharide saponin CP6.
In one embodiment of the present invention, the clematis saponins include at least one of clematis root trisaccharide saponin CP3, clematis root trisaccharide saponin CP5 and clematis root trisaccharide saponin CP6.
In one embodiment of the present invention, the composition containing clematis root sugar saponin is the clematis root extract.
The invention also provides a medicine for treating and/or preventing kidney injury, which contains the clematis root saponin or a composition containing the clematis root saponin.
In one embodiment of the present invention, the kidney injury is kidney injury caused by accumulation of purine.
In one embodiment of the invention, the drug is clematis root saponin or a composition containing clematis root saponin, and conventional auxiliary materials are added according to a conventional process to prepare at least one of clinically acceptable tablets, capsules, powder, mixture, pills, granules, solutions, syrups and sustained and controlled release preparations.
In one embodiment of the present invention, the clematis saponins include at least one of clematis sugar saponin CP1, clematis disaccharide saponin CP2, clematis trisaccharide saponin CP3, clematis trisaccharide saponin CP4, clematis trisaccharide saponin CP5, and clematis trisaccharide saponin CP6.
In one embodiment of the present invention, the clematis saponins include at least one of clematis root trisaccharide saponin CP3, clematis root trisaccharide saponin CP5 and clematis root trisaccharide saponin CP6.
In one embodiment of the present invention, the composition containing clematis root sugar saponin is the clematis root extract.
The technical scheme of the invention has the following advantages:
1. the invention provides application of clematis saponins or a composition containing the same in preparing medicines, wherein the application comprises the following steps:
researches show that clematis root sugar saponin CP1, clematis root disaccharide saponin CP2, clematis root trisaccharide saponin CP3, clematis root trisaccharide saponin CP4, clematis root trisaccharide saponin CP5, clematis root trisaccharide saponin CP6 and clematis root extract can effectively improve the metabolic capability of a hyperpurine-induced molding mouse on purine substances, so that the clematis root trisaccharide saponin CP3, the clematis root trisaccharide saponin CP5, the clematis root trisaccharide saponin CP6 and the clematis root extract can effectively reduce purine, and have the effect of preventing and/or treating hyperpurine;
researches show that the clematis root sugar saponin CP1, the clematis root disaccharide saponin CP2, the clematis root trisaccharide saponin CP3, the clematis root trisaccharide saponin CP4, the clematis root trisaccharide saponin CP5, the clematis root trisaccharide saponin CP6 and the clematis root extract can effectively reduce the content of uric acid in a hyperuricemia molding mouse, so that the clematis root trisaccharide saponin CP3, the clematis root trisaccharide saponin CP5, the clematis root trisaccharide saponin CP6 and the clematis root extract can effectively reduce uric acid, and the effects of preventing and/or treating hyperuricemia are achieved;
researches show that clematis root sugar saponin CP1, clematis root disaccharide saponin CP2, clematis root trisaccharide saponin CP3, clematis root trisaccharide saponin CP4, clematis root trisaccharide saponin CP5, clematis root trisaccharide saponin CP6 and clematis root extract can effectively reduce CRE value of blood plasma of a hyperpurine blood disease molding mouse and effectively relieve inflammation of kidney of the hyperpurine disease molding mouse, which indicates that the clematis root trisaccharide saponin CP3, the clematis root trisaccharide saponin CP5, the clematis root trisaccharide saponin CP6 and the clematis root extract can effectively relieve kidney injury caused by purine accumulation, and have the function of preventing and/or treating the kidney injury,
therefore, the clematis root saponin or the composition containing the clematis root saponin can be independently applied to achieve the purposes of treating hyperuricemia and avoiding hyperuricemia and kidney injury, and can also be combined with allopurinol, febuxostat and other XOD inhibitors to achieve the purposes of treating hyperuricemia and avoiding hyperuricemia and kidney injury.
Further, the clematis root saponin or the composition containing the clematis root saponin provided by the invention is applied to the preparation of medicines, 50-80 vt% of alcohol-water solution is selected as an extraction solvent to extract the clematis root, and the extract obtained by extraction has better purine and uric acid reducing capacity and liver injury preventing capacity.
Furthermore, the clematis root saponin or the composition containing the clematis root saponin is applied to the preparation of medicines, the clematis root extract is subjected to impurity washing by adopting 0-5 vt% alcohol-water solution, and then the macroporous resin adsorption step of eluting by adopting 60-95 vt% alcohol-water solution effectively enriches the active ingredients in the clematis root, and the collected eluent has better purine and uric acid reducing and liver injury preventing capabilities.
Further, the clematis root saponin or the clematis root saponin-containing composition is applied to the preparation of medicines, the active ingredients in the clematis root are further enriched by taking the eluent to perform a macroporous resin adsorption step of eluting by adopting 30-95 vt% alcohol-water solution, and the collected eluent has the capabilities of better reducing purine and uric acid and preventing liver injury.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows the kidney dissection of a blank group of mice.
FIG. 2 shows the results of kidney dissection of model mice.
Fig. 3 shows the results of kidney dissection of the test group 3 mice.
FIG. 4 shows the kidney pathological section results (interstitial inflammatory cell infiltration) of blank mice.
FIG. 5 shows the pathological section results of kidney (interstitial inflammatory cell infiltration) in the model group of mice.
FIG. 6 shows the pathological section results of kidney (interstitial inflammatory cell infiltration) of the mice in the test drug group 3.
FIG. 7 shows the pathological section results of kidney (renal tubule type) of the mice in the model group.
FIG. 8 shows the results of pathological section of kidney (renal tubule type) of the test group 3 mice.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The following examples, where specific experimental procedures or conditions are not indicated, can be performed according to the procedures or conditions of the conventional experimental procedures described in the literature in the art. The reagents or instruments used are conventional reagent products which are commercially available, and manufacturers are not indicated.
Example 1: preparation of radix Clematidis extract
The method comprises the following specific steps:
soaking radix Clematidis (purchased from the Bozhou market of Anhui) in 70vt% ethanol aqueous solution 8 times the weight of radix Clematidis, extracting twice, each time for one week, and mixing extractive solutions; concentrating the extractive solution at 60 deg.C under reduced pressure until no alcohol smell exists to obtain radix Clematidis extract 1; carrying out chromatographic purification on the clematis root extract 1 through a D101 macroporous resin column (the diameter of the D101 macroporous resin column is 8cm, the height of the D101 macroporous resin column is 80cm, and the column volume is 4L), sequentially eluting with pure water and 95vt% ethanol water solution, controlling the elution flow rate to be 3BV/h and the elution volume of each elution reagent to be 5BV, collecting the eluent of the 95vt% ethanol water solution, and carrying out reduced pressure concentration at 60 ℃ until no alcohol smell exists, thus obtaining clematis root extract 2; and (3) subjecting the radix clematidis extract 2 to chromatographic purification again by using a D101 macroporous resin column (the diameter of the D101 macroporous resin column is 8cm, the height of the D101 macroporous resin column is 80cm, and the column volume of the D101 macroporous resin column is 4L), sequentially eluting by using 30vt%, 40vt%, 50vt%, 60vt% and 95vt% ethanol water solution, controlling the elution flow rate to be 3BV/h and the elution volume of each elution reagent to be 3BV, respectively collecting the eluates, concentrating under reduced pressure at 60 ℃, and drying to be powdery to obtain radix clematidis extracts E30, E40, E50, E60 and E95.
HPLC analysis of the radix clematidis extracts E30, E40 and E50 was performed using the saponin detection method of pharmacopeia 2015, and the content of clematis saponins in the radix clematidis extract E30 was found to be 24.2%, the content of clematis saponins in the radix clematidis extract E40 was found to be 45.7%, and the content of clematis saponins in the radix clematidis extract E50 was found to be 38.9%. Therefore, the clematis root extracts E30, E40 and E50 all contain rich clematis root saponins.
Experimental example 1: influence of radix Clematidis extract on hyperpurine syndrome of mice
The method comprises the following specific steps:
1. experimental materials
Clematis root sugar saponin CP1, clematis root disaccharide saponin CP2, clematis root trisaccharide saponin CP3, clematis root trisaccharide saponin CP4, clematis root trisaccharide saponin CP5 and clematis root trisaccharide saponin CP6 (the purity is more than or equal to 95%): purchased from sienna grass technologies ltd.
Kunming mouse (SPF grade, 6-8 weeks old, male, weight 17-19 g): purchased from laboratory animal technology, inc. of Wei Tong Li Hua, beijing.
0.5% of CMC-Na aqueous solution by mass fraction: sodium carboxymethylcellulose (CMC-Na, available from national drug group chemical reagents, inc.) was weighed and added with distilled water to prepare a CMC-Na aqueous solution with a mass fraction of 0.5%.
Febuxostat solution with concentration of 0.4 mg/mL: febuxostat (purchased from Wanbang pharmaceutical company) is weighed and added with CMC-Na aqueous solution with the mass fraction of 0.5 percent to prepare febuxostat solution with the concentration of 0.4 mg/mL.
Test substance solution at a concentration of 0.4 mg/mL: weighing a test object, adding a CMC-Na aqueous solution with the mass fraction of 0.5 percent to prepare a test object solution with the concentration of 0.4 mg/mL.
2. Test method
55 mice were taken and randomly divided into 11 groups of 5 mice each, 11 groups were: blank group, model group, test substance group 1-9 for respectively administering test substance 1-9, wherein test substance 1-3 are radix Clematidis extract E30, radix Clematidis extract E40 and radix Clematidis extract E50 prepared in example 1, respectively, and test substance 4-9 are radix Clematidis sugar saponin CP1, radix Clematidis disaccharide saponin CP2, radix Clematidis trisaccharide saponin CP3, radix Clematidis trisaccharide saponin CP4, radix Clematidis trisaccharide saponin CP5 and radix Clematidis trisaccharide saponin CP6, respectively.
After the experiment begins, the mice are subjected to gavage administration, wherein the gavage administration is carried out for 1 time every morning, the continuous molding is carried out for 11 days, the administration is carried out for 1 time every afternoon, and the continuous administration is carried out for 14 days, wherein the test object groups 1-9 of the mice are filled with febuxostat solution with the gavage concentration of 0.4mg/mL at the morning in a metering manner of 8mg/kg every day, and are filled with test object solution with the gavage concentration of 0.4mg/mL at the afternoon in a metering manner of 600 mg/kg; the mice in the model group are gavaged with febuxostat solution with the measuring gastric perfusion concentration of 0.4mg/mL at 8mg/kg every morning and are given CMC-Na aqueous solution with the mass fraction of 0.5% at equal dose (equal dose with the tested group) in the afternoon; the blank mice are given equal doses of CMC-Na water solution with the mass fraction of 0.5 percent in the morning and afternoon (equal doses of the test object group).
And on the 13 th day of administration, transferring each group of mice to a metabolism cage, collecting urine of each group of mice for 24 hours, on the 14 th day of administration, removing eyeballs from each group of mice to collect blood, wherein the blood collection capacity is not lower than 0.5mL, then, killing each group of mice, picking the kidney, weighing the weight of the kidney, photographing, cutting a small piece of kidney tissue to perform homogenate treatment, placing the other small piece of kidney tissue in formalin fixing solution to fix, and storing at 4 ℃ for slice observation. The remaining kidney tissue was rapidly maintained at-20 ℃.
Measuring CRE value of urine of each group of mice by using a full-automatic biochemical analyzer (the CRE value of the urine can effectively represent the kidney function of the mice), measuring Hypoxanthine (HX) and xanthine (X) contents in the urine of each group of mice by using high performance liquid chromatography (the hypoxanthine and xanthine contents in the urine can effectively represent the excretion situation of the mice on hypoxanthine and xanthine), and calculating HX/CRE value and X/CRE value of the urine of each group of mice (the HX/CRE value and the X/CRE value of the urine can effectively represent the metabolic capacity of the mice on hypoxanthine and xanthine), wherein the detection results are shown in Table 3;
wherein, the conditions of the high performance liquid chromatography are as follows:
preparing a mobile phase:
phase D: 10mM ammonium acetate-0.03% (v/v) glacial acetic acid
And C phase: methanol
A chromatographic column: c18 column, 4.6X 250mm,5um, available from YMC
The instrument comprises the following steps: agilent 1260
Flow rate: 1mL/min
Sample injection amount: 5 μ L
Column temperature: 35 deg.C
Detection wavelength: 0-6 min:230nm, 6-30 min:270nm
Gradient of the method: see table 1.
Measuring CRE value of blood plasma of each group of mice by using a full-automatic biochemical analyzer, and measuring the content of Hypoxanthine (HX) and xanthine (X) in the blood plasma of each group of mice by using high performance liquid chromatography, wherein the detection results are shown in Table 4;
wherein, the conditions of the high performance liquid chromatography are as follows:
preparing a mobile phase:
phase D: 10mM ammonium acetate-0.03% (v/v) glacial acetic acid
And C phase: methanol
A chromatographic column: c18 column, 4.6X 250mm,5um, available from YMC
The instrument comprises the following steps: agilent 1260
Flow rate: 1mL/min
Sample introduction amount: 5 μ L
Column temperature: 35 deg.C
Detection wavelength: 0-6 min:230nm, 6-30 min:270nm
Gradient of the method: see table 1.
Determining the content of Hypoxanthine (HX) and xanthine (X) in the kidney homogenate of each group of mice by using high performance liquid chromatography, wherein the detection result is shown in Table 5;
wherein, the conditions of the high performance liquid chromatography are as follows:
preparing a mobile phase:
phase D: 10mM KH2PO4-0.04% (v/v) phosphoric acid
Phase B: 80% (v/v) methanol +20% (v/v) D phase
And (3) chromatographic column: c18 column, 4.6X 250mm,5um, available from YMC
The instrument comprises: agilent 1100
Flow rate: 1mL/min
Sample introduction amount: 5 μ L
Column temperature: 35 deg.C
Detection wavelength: 270nm
Gradient of the method: see table 2.
After the kidney tissues of each group of mice were sectioned and HE-stained, the pathological conditions of the kidney tissues of each group of mice were observed under an optical microscope, and the observation results are shown in fig. 1 to 8.
TABLE 1 method gradients
TABLE 2 method gradients
| Time(min) | C％ | D％ |
| 0 | 100 | 0 |
| 15 | 100 | 0 |
| 16 | 0 | 100 |
| 21 | 0 | 100 |
| 22 | 100 | 0 |
| 29 | 100 | 0 |
3. Test results
As can be seen from Table 3, compared with the blank group, the metabolic capacity of purine is not significantly improved in the model group mice, while the HX/CRE value and the X/CRE value in urine of the mice in the test object groups 1-9 are significantly improved, which indicates that the test objects 1-9 have stronger purine metabolism promoting effect, wherein the test objects 2, 3, 6, 8 and 9 have particularly significant effects.
As can be seen from table 4, after the model was made, CRE, HX and X in the plasma of mice increased significantly, and after the model was administered, CRE, HX and X in the plasma of mice decreased to different degrees, wherein the test substances 2, 3, 6, 8 and 9 decreased hypoxanthine and xanthine in the plasma significantly.
As can be seen from table 5, after the model was made, the HX content in the mouse kidney did not increase, but the X content increased significantly, indicating that HX did not significantly accumulate in the kidney, but X accumulated significantly in the kidney, and after the model was administered, the X content in the mouse kidney was greatly reduced, and the accumulation was significantly improved, among which the test substances 2, 3, 6, 8, and 9 had significant effects.
As can be seen from fig. 1 to 3, after the administration of febuxostat, the mouse kidney shows macroscopic renal lesion caused by purine crystals, and after the administration of the test substance 3, the proportion of the macroscopic lesion caused by purine crystals in the mouse kidney is reduced, and the lesion site is reduced, which indicates that the test substance 3 has a certain recovery effect on renal injury caused by febuxostat.
As can be seen from fig. 4 to 8, after gavage, the mouse kidney shows focal inflammation focus, interstitial inflammatory cell infiltration, part of glomerular cystic fibrosis, renal tubule dilatation, and ductular type in the lumen, after gavage of the test object 3, the mouse kidney interstitial inflammatory cell infiltration is significantly reduced, and the test object 3 has a certain recovery effect on the mouse renal tubule type.
TABLE 3 HX/CRE and X/CRE values of urine from different groups of mice
| Group of | HX/CRE(umol/mmol) | X/CRE(umol/mmol) |
| Blank group | 311.74±51.60 | 129.98±19.70 |
| Model set | 338.26±55.16 | 140.72±34.60 |
| Test object group 1 | 447.96±80.47** | 275.16±50.25*** |
| Test object group 2 | 651.08±115.43*** | 495.26±82.10*** |
| Test group 3 | 559.79±108.06*** | 408.31±72.20*** |
| Test group 4 | 408.66±57.99* | 211.89±48.82** |
| Test group 5 | 465.45±96.30** | 304.49±71.29*** |
| Test group 6 | 604.63±121.85*** | 521.25±86.37*** |
| Test group 7 | 402.49±72.30* | 200.41±48.06** |
| Test object group 8 | 522.21±80.06*** | 368.80±80.76*** |
| Test object group 9 | 596.20±43.12*** | 500.81±58.92*** |
Note: * Represents P <0.05 compared to the model set (t-test); * Denotes P <0.01 compared to model group (t-test); * Denotes P <0.001 compared to model group (t-test).
TABLE 4 CRE value, HX content and X content of the plasma of different groups of mice
Note: * Represents P <0.05 compared to the model set (t-test); * Denotes P <0.01 compared to model group (t-test); * Denotes P <0.001 compared to the model group (t-test).
TABLE 5 HX content and X content of kidney homogenate from different groups of mice
| Group of | HX(μg/g) | X(μg/g) |
| Blank group | 178.98±20.05 | 32.01±9.55 |
| Model set | 167.31±19.47 | 68.66±13.45 |
| Test object group 1 | 164.44±18.75 | 51.70±12.11** |
| Test object group 2 | 161.74±19.31 | 30.32±7.72*** |
| Test object group 3 | 164.95±20.79 | 37.77±8.88*** |
| Test group 4 | 166.72±20.14 | 54.56±11.07* |
| Test group 5 | 166.94±19.51 | 49.07±12.59** |
| Test group 6 | 161.59±17.42 | 33.75±8.00*** |
| Test group 7 | 167.38±24.07 | 57.11±7.45* |
| Test object group 8 | 163.72±9.89 | 45.82±9.91*** |
| Test object group 9 | 161.81±16.74 | 33.86±7.86*** |
Note: * Represents P <0.05 compared to the model set (t-test); * Denotes P <0.01 compared to model group (t-test); * Denotes P <0.001 compared to model group (t-test).
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This need not be, nor should it be exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. The application of clematis root saponin or a composition containing clematis root saponin in preparing a medicine is characterized in that the medicine has at least one of the following applications:
(a) Purine reduction;
(b) Reducing uric acid; and/or the presence of a gas in the gas,
(c) Preventing and/or treating kidney injury.
2. The use according to claim 1, wherein the hypopurine is prevention and/or treatment of hyperpurine syndrome; the uric acid reduction refers to the prevention and/or treatment of hyperuricemia.
3. The use according to claim 1 or 2, wherein the clematis saponins comprise at least one of clematis root sugar saponin CP1, clematis root disaccharide saponin CP2, clematis root trisaccharide saponin CP3, clematis root trisaccharide saponin CP4, clematis root trisaccharide saponin CP5, and clematis root trisaccharide saponin CP 6;
preferably, the clematis saponins include at least one of clematis chinensis trisaccharide saponin CP3, clematis chinensis trisaccharide saponin CP5, and clematis chinensis trisaccharide saponin CP6.
4. The use according to any one of claims 1 to 3, wherein the composition containing clematis root sugar saponin is clematis root extract.
5. The use as claimed in claim 4, wherein the clematis root extract is prepared by a method comprising the steps of: extracting radix Clematidis with organic solvent and/or water as extraction solvent to obtain radix Clematidis extract;
preferably, the extraction solvent is a 50 to 80vt% aqueous alcohol solution.
6. A method for preparing an extract of clematis root, comprising the steps of: extracting radix Clematidis with organic solvent and/or water as extraction solvent to obtain radix Clematidis extract;
preferably, the extraction solvent is a 50 to 80vt% aqueous alcohol solution.
7. An extract of clematis root obtained by the method of claim 6.
8. A medicament for decreasing purine, which comprises clematis root saponin or a composition comprising clematis root saponin.
9. A medicine for reducing uric acid is characterized by comprising clematis root saponin or a composition containing the clematis root saponin.
10. A medicament for treating and/or preventing kidney injury, which comprises clematis saponins or a composition comprising clematis saponins.
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