CN114869923B - Water-soluble extract of national medicine double ginseng, preparation method and application thereof - Google Patents
Water-soluble extract of national medicine double ginseng, preparation method and application thereof Download PDFInfo
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- CN114869923B CN114869923B CN202210668831.0A CN202210668831A CN114869923B CN 114869923 B CN114869923 B CN 114869923B CN 202210668831 A CN202210668831 A CN 202210668831A CN 114869923 B CN114869923 B CN 114869923B
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- water
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- ginseng
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Abstract
The invention provides a national medicine double ginseng water-soluble extract and a preparation method and application thereof, belonging to the technical field of pharmaceutical chemistry. Firstly, alcohol extraction is carried out on national medicine double ginseng to obtain alcohol extract and alcohol extraction residues, then water-soluble crude extracts are obtained through further treatment respectively, specifically, the alcohol extract is concentrated and then extracted by n-butanol, and the alcohol extraction residues are subjected to water extraction and deproteinization treatment; and (3) mixing the water-soluble crude extracts, performing macroporous adsorption resin column chromatography separation by taking lower alcohol-water as an eluent to obtain different elution components, selecting water-soluble parts according to the composition of the eluent, eluting other elution components except the water-eluted components, performing alcohol precipitation, and mixing the water-eluted components with the water-eluted components to obtain the water-soluble extract of the national medicine double ginseng. The national medicine double ginseng water-soluble extract prepared by the method can be applied to preventing and treating diabetes and nephropathy, has high safety and has good development and application prospects.
Description
Technical Field
The invention relates to the technical field of pharmaceutical chemistry, in particular to a national medicine double ginseng water-soluble extract, a preparation method and application thereof.
Background
Diabetes mellitus (Diabetes Mellitus, DM) is a syndrome of disturbed metabolism of sugar, fat and protein due to insulin secretion or underutilization. DM not only affects people's health, but also places a heavy burden on society. Diabetic nephropathy (Diabetic Nephropathy, DN) is one of the common complications of DM patients, a major cause of mortality in end stage renal disease and DM patients, about 30-40% of DM patients develop DN, and the incidence increases with the course of the disease. The early clinical manifestation of DN is increased glomerular filtration rate, increased microalbuminuria in urine, elevated plasma urea nitrogen and creatinine, and finally development of chronic renal insufficiency. The main pathological feature of DN early is glomerular hypertrophy, glomerular and tubular basement membrane thickening and progressive accumulation of extracellular matrix in the mesangial region; later stage is fibrosis of glomeruli, tubular interstitium, and ultimately results in proteinuria and renal failure.
At present, DM is mainly treated by blood sugar reduction, DN is mainly treated by combined medication, and medicines for comprehensively reducing blood sugar, blood pressure, blood fat and the like are mainly used for treating DM, so that the development of DN is mainly delayed, and the effect of radical treatment cannot be achieved. Currently, DN is clinically treated by hormone and cytotoxic drugs, and although a certain curative effect is achieved, adverse reactions such as easy recurrence and easy generation of hormone dependence exist. Angiotensin converting enzyme inhibitors (e.g., captopril, enalapril, etc.) have therapeutic effects on DN. However, angiotensin converting enzyme inhibitors have great side effects and cause symptoms such as hypotension, hyperkalemia, renal insufficiency, cough, etc. So far, there is no ideal treatment. Therefore, aiming at DM and DN, finding a high-efficiency and low-toxicity control drug is a problem to be solved urgently.
Disclosure of Invention
The invention aims to provide a national medicine double ginseng water-soluble extract, a preparation method and application thereof. The national medicine double ginseng water-soluble extract prepared by the method can be applied to preventing and treating diabetes and nephropathy, has high safety and has good development and application prospects.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of a national medicine double ginseng water-soluble extract, which comprises the following steps:
extracting the national medicinal double ginseng with alcohol to obtain alcohol extract and alcohol extract residues;
the alcohol extract is subjected to first concentration to obtain an alcohol extract component A, and the alcohol extract component A is mixed with water and n-butanol for extraction to obtain a water-soluble component A1;
extracting the alcohol extraction residues with hot water, performing second concentration on the obtained water extract to obtain water extract concentrate B, performing protein removal treatment on the water extract concentrate B, and performing third concentration on the obtained protein removal solution to obtain a water extract component B1;
the method comprises the steps of taking lower alcohol-water as an eluent, carrying out macroporous adsorption resin column chromatographic separation on a mixed component obtained by combining a water-soluble component A1 and a water-extracted component B1 under a gradient elution condition, sequentially obtaining a flow AB1, a flow AB2 and a flow AB3, wherein the gradient elution comprises 3 gradients which are sequentially carried out, and the composition of the eluent is sequentially 0 according to the volume ratio of the lower alcohol to the water: 1. 1 to 7:9 to 3 and 9.5:0.5, corresponding to the fraction AB1, the fraction AB2 and the fraction AB3, respectively;
Fourth concentrating the flow AB1 to obtain a water eluting component AB1'; sequentially carrying out fifth concentration and alcohol precipitation on the flow AB2 to obtain a water-soluble alcohol precipitation component AB2'; and combining the water eluting component AB1 'with the water-soluble alcohol precipitating component AB2' to obtain the water-soluble extract of the national drug double ginseng.
Preferably, the ethnic medicine double ginseng is double ginseng or big flower double ginseng.
Preferably, the alcohol extraction is cold leaching extraction, the alcohol extraction reagent adopted in the alcohol extraction is ethanol water solution, and the volume fraction of the ethanol water solution is 60-95%.
Preferably, the deproteinizing treatment is carried out by Sevag method.
Preferably, the macroporous adsorption resin adopted in the macroporous adsorption resin column chromatographic separation is AB-8 macroporous adsorption resin or 101 macroporous adsorption resin.
Preferably, the water eluting component AB1 'and the water soluble alcohol precipitating component AB2' after being combined further comprises: and carrying out fractional alcohol precipitation on the obtained mixed components to obtain supernatant and alcohol precipitation, and carrying out sixth concentration on the supernatant to obtain the first ethnic medicine double-ginseng water-soluble extract, wherein the alcohol precipitation is the second ethnic medicine double-ginseng water-soluble extract.
The invention provides a water-soluble extract of national drug double ginseng, which is prepared by the preparation method of the technical proposal, and comprises water-soluble polysaccharide components and water-soluble micromolecular components, wherein the relative molecular weight of the water-soluble polysaccharide components is in the range of 504-2.0x10 6 The relative molecular weight of the water-soluble small molecules ranges from 214 to 792.
Preferably, the water-soluble polysaccharide component comprisesA high molecular weight polysaccharide and a low molecular weight polysaccharide, the high molecular weight polysaccharide having a relative molecular weight in the range of 5.0X10 4 ～2.0×10 6 The relative molecular weight of the low molecular weight polysaccharide is in the range of 504-4.0X10 4 The method comprises the steps of carrying out a first treatment on the surface of the The water-soluble small molecule is a water-soluble iridoid glycoside compound and/or a derivative thereof.
The invention provides application of the national medicine double ginseng water-soluble extract in preparing medicines for preventing and treating diabetes or nephropathy.
Preferably, the diabetes is type II diabetes; the kidney disease is at least one of diabetic nephropathy, acute renal failure, chronic renal failure, nephrotic syndrome and glomerulonephritis.
The invention provides a preparation method of a water-soluble extract of national drug double ginseng, which comprises the steps of firstly, carrying out alcohol extraction on the national drug double ginseng to obtain alcohol extract and alcohol extraction residues, then respectively carrying out further treatment to obtain a water-soluble crude extract, specifically, concentrating the alcohol extract, extracting the concentrated alcohol extract by n-butanol, and carrying out water extraction and deproteinization treatment on the alcohol extraction residues; and (3) combining the water-soluble crude extracts, performing macroporous adsorption resin column chromatography separation by taking lower alcohol-water as an eluent, removing pigments and other low-polarity micromolecular components to obtain different elution components, selecting water-soluble parts according to the composition of the eluent, eluting the water-soluble parts by water, and combining the other elution components with the water-eluted components after alcohol precipitation to obtain the water-soluble extract of the national medicine double ginseng (the water-soluble total extract of the national medicine double ginseng).
Further, the water-soluble extract of the national drug double ginseng is subjected to fractional alcohol precipitation to obtain supernatant and alcohol precipitation, and active substances in the water-soluble extract of the national drug double ginseng can be further separated to obtain a first water-soluble extract (TGPB) of the national drug double ginseng and a second water-soluble extract (TGPC) of the national drug double ginseng respectively.
According to the embodiment of the invention, the activity screening is carried out by using human glomerular mesangial cells and a diabetes mouse model established by STZ induction, and the result shows that the double-ginseng water-soluble extract provided by the invention has an obvious protection effect on the high-sugar cultured human glomerular mesangial cells, has an obvious protection effect on the blood sugar, glycosylated serum protein and blood lipid disorders of the STZ induced diabetes mouse, and also has an obvious protection effect on the kidneys of the STZ induced diabetes mouse, so that the double-ginseng water-soluble extract can be used for preventing and treating diabetes and kidney diseases and can be used for preparing medicines for preventing and treating diabetes and kidney diseases; toxicity tests prove that the double ginseng water-soluble extract has high safety, can ensure the safety of long-term administration of patients with diabetes and nephropathy, and has good development and application prospects.
Drawings
FIG. 1 is a graph showing the effect of each of the active ingredients of double ginseng in example 4 on Glycated Serum Proteins (GSP) in STZ diabetic mice;
FIG. 2 is a graph showing the effect of each of the active ingredients of double ginseng on serum creatinine (Scr) in STZ diabetic mice in example 4;
FIG. 3 shows the results of the treatment of STZ diabetic mice with superoxide dismutase (T-SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-P) by using the active ingredients of example 4 X ) Is a graph of the influence of (1);
FIG. 4 is an optical micrograph of a kidney section of the mice of example 4;
FIG. 5 is a graph showing the proliferation of HRMC cells (x+ -s) with respect to the blank group at various time points and at various concentrations of high sugar in example 5;
FIG. 6 is a graph showing the cytotoxicity test result of the water-soluble extract of double ginseng in example 5 against human mesangial in the concentration range of 50-800. Mu.g/mL;
FIG. 7 is a graph showing the effect of different concentrations of the active ingredient of double ginseng on proliferation of mesangial cells of high sugar cultured human being in comparison with normal sugar group (NG) in example 5.
Detailed Description
The invention provides a preparation method of a national medicine double ginseng water-soluble extract, which comprises the following steps:
extracting the national medicinal double ginseng with alcohol to obtain alcohol extract and alcohol extract residues;
the alcohol extract is subjected to first concentration to obtain an alcohol extract component A, and the alcohol extract component A is mixed with water and n-butanol for extraction to obtain a water-soluble component A1;
Extracting the alcohol extraction residues with hot water, performing second concentration on the obtained water extract to obtain water extract concentrate B, performing protein removal treatment on the water extract concentrate B, and performing third concentration on the obtained protein removal solution to obtain a water extract component B1;
the method comprises the steps of taking lower alcohol-water as an eluent, carrying out macroporous adsorption resin column chromatographic separation on a mixed component obtained by combining a water-soluble component A1 and a water-extracted component B1 under a gradient elution condition, sequentially obtaining a flow part AB1, a flow part AB2 and a flow part AB3, wherein the gradient elution comprises 3 gradients which are sequentially carried out, and the composition of the eluent is sequentially 0 according to the volume ratio of the lower alcohol to the water: 1. 1 to 7:9 to 3 and 9.5:0.5, corresponding to the fraction AB1, the fraction AB2 and the fraction AB3, respectively;
fourth concentrating the flow AB1 to obtain a water eluting component AB1'; sequentially carrying out fifth concentration and alcohol precipitation on the flow AB2 to obtain a water-soluble alcohol precipitation component AB2'; and combining the water eluting component AB1 'with the water-soluble alcohol precipitating component AB2' to obtain the water-soluble extract of the national drug double ginseng.
The traditional Chinese medicine, the folk medicine and the like belong to natural medicines, and the discovery of the active ingredients with multiple targets for preventing and treating DM and DN from the traditional Chinese medicine and the folk medicine is an important effective way for preventing and treating DM and DN medicine development. The water-soluble components of the traditional Chinese medicine and the folk medicine have the characteristics of low toxicity or no toxicity, no side effect and the like, and have huge demand space in the medical care field. 2 medicinal plants of the genus double ginseng (dipssaceae), in particular double ginseng and double ginseng with big flower, wherein double ginseng (Triplostegia grandiflora Gagnep) with big flower is also called green sheep ginseng or big flower bag bud, is a Yi nationality medicine, and the Yi nationality is not the same color, and is commonly used for treating kidney deficiency lumbago, spermatorrhea, impotence, irregular menstruation, amenorrhea and the like; double ginseng (Triplostegia glandulifera Wall) is also called radish ginseng, p-ginseng, child ginseng, ganla, tu ginseng or Yizhihao, and is commonly used for tonifying kidney, strengthening spleen and qi, promoting blood circulation and regulating menstruation, and is also used for treating asthenia due to long-term illness, enriching blood and tonifying qi. The water-soluble extract of the national drug double ginseng is extracted and separated from the national drug double ginseng (double ginseng or large flower double ginseng), has remarkable curative effect on DM (DM), especially DN, and has the characteristic of no toxicity on human glomerular mesangial cells and a diabetes mouse model established by STZ induction, so that the water-soluble extract of the national drug double ginseng can be applied to preventing and treating DM and DN, and has good development and application prospects. The preparation method of the water-soluble extract of the national medicine double ginseng is described in detail below.
The invention carries out alcohol extraction on the national medicine double ginseng to obtain alcohol extract and alcohol extraction residues. In the invention, the national medicine double ginseng is specifically double ginseng or big flower double ginseng. In the invention, the national drug of radix scrophulariae is preferably washed and dried sequentially before use; the invention preferably takes the underground part (namely rhizome) of the national drug double ginseng for use; the drying mode preferably comprises natural airing or drying, and the drying temperature is preferably 40-50 ℃. The invention preferably cuts the dried national drug double ginseng into small blocks with the length of 1cm for use.
In the invention, the alcohol extraction is preferably cold leaching extraction, specifically, the ethnic medicine double-participation alcohol extraction reagent is mixed, and the extraction is carried out under the condition of room temperature; the number of the alcohol extractions is preferably 2 to 4, more preferably 3; the alcohol extraction reagent is preferably ethanol water solution, and the volume fraction of the ethanol water solution used in each alcohol extraction is preferably 60-95%, more preferably 80-95%; the dosage ratio of the alcohol extraction reagent to the material to be alcohol extracted is independently preferably (3-6) L:2kg, more preferably 2L:1kg; the time of each alcohol extraction is preferably 24 to 36 hours independently, more preferably 24 hours. The invention specifically comprises the steps of filtering out dregs after each alcohol extraction, carrying out the next alcohol extraction, taking the dregs filtered out after the last alcohol extraction as alcohol extraction dregs, and combining the solutions obtained after the dregs are filtered out after each alcohol extraction is finished, thus being taken as alcohol extraction liquid. The invention preferably adopts a cold leaching extraction mode to carry out alcohol extraction, and the effect is to extract small molecular components with lower polarity.
In the invention, after an alcohol extract is obtained, the alcohol extract is subjected to first concentration to obtain an alcohol extract component A, and the alcohol extract component A is mixed with water and n-butanol for extraction to obtain a water-soluble component A1. In the present invention, the first concentration is preferably reduced pressure concentration, and the temperature of the first concentration is preferably 50 to 60 ℃, more preferably 55 ℃; the first concentration is preferably to concentrate the alcohol extract until no alcohol smell exists, so as to obtain an alcohol extract component A; and recovering alcohol in the alcohol extraction reagent in the first concentration process. In the invention, the alcohol extraction component A is preferably mixed with water to obtain an aqueous solution of the alcohol extraction component A; and mixing the aqueous solution of the alcohol extraction component A with n-butanol for extraction. In the invention, the dosage ratio of water to national medicine double ginseng in the aqueous solution of the alcohol extraction component A is preferably (0.5-1) L:2kg, more preferably 1L:2kg. In the present invention, the number of times of the extraction is preferably 2 to 4 times, more preferably 3 times; the volume ratio of the n-butanol to the materials to be extracted is independently preferably (1-2) during each extraction: 1, more preferably (1 to 1.5): 1. the invention removes pigments and low-polarity components dissolved in n-butanol by adopting n-butanol extraction.
In the invention, after alcohol extraction residues are obtained, hot water extraction is carried out on the alcohol extraction residues, the obtained water extraction liquid is subjected to second concentration to obtain water extraction concentrate B, protein removal treatment is carried out on the water extraction concentrate B, and the obtained protein removal solution is subjected to third concentration to obtain water extraction component B1. In the present invention, the number of times of the hot water extraction is preferably 2 to 4 times, more preferably 3 times; the method of each hot water extraction is independently preferably reflux extraction or cooking extraction, and the temperature of the reflux extraction is preferably 90-95 ℃; the mass of water used for each hot water extraction is preferably 2-5 times, more preferably 3 times of the mass of the material to be extracted; the time of each hot water extraction is independently preferably 2 to 3 hours, more preferably 2.5 hours. The invention specifically comprises the steps of filtering out dregs after each hot water extraction, carrying out next hot water extraction, and combining the solutions obtained after the dregs are filtered out after each hot water extraction is finished, thereby being used as water extract. The invention preferably adopts a reflux extraction or cooking extraction mode to carry out hot water extraction, and can completely extract water-soluble components in alcohol extraction residues. In the present invention, the second concentration is preferably reduced pressure concentration, and the temperature of the second concentration is preferably 60 to 70 ℃, more preferably 65 ℃; the second concentration is preferably to concentrate the aqueous extract to 10-20% of the original volume (i.e., the volume of the aqueous extract concentrate B is 10-20% of the volume of the aqueous extract). In the present invention, the method used for the deproteinization is preferably a Sevag method, and the number of times of deproteinization is preferably 2 to 4 times, more preferably 3 times; the Sevag reagent adopted by the Sevag method is preferably chloroform and n-butanol, and the volume ratio of the chloroform to the n-butanol is independently preferably (2-5) during each deproteinization treatment: 1, more preferably 4:1, a step of; the volume ratio of the Sevag reagent to the material to be deproteinized is independently preferably (1-2) at each deproteinization treatment: 1, more preferably 1:1. in the present invention, the third concentration is preferably reduced pressure concentration, and the temperature of the third concentration is preferably 60 to 70 ℃, more preferably 60 to 65 ℃; the third concentration is preferably to concentrate the deproteinized solution to no n-butanol.
After the water-soluble component A1 and the water-extracted component B1 are obtained, the invention uses lower alcohol-water as an eluent, the mixed components after the water-soluble component A1 and the water-extracted component B1 are combined are subjected to macroporous adsorption resin column chromatographic separation under the gradient elution condition, the flow AB1, the flow AB2 and the flow AB3 are sequentially obtained, the gradient elution comprises 3 gradients which are sequentially carried out, and the composition of the eluent is sequentially 0 according to the volume ratio of lower alcohol to water: 1. 1 to 7:9 to 3 and 9.5:0.5, corresponding to fraction AB1, fraction AB2 and fraction AB3, respectively. In the invention, the macroporous adsorption resin used for macroporous adsorption resin column chromatographic separation is preferably AB-8 macroporous adsorption resin or 101 macroporous adsorption resin, more preferably AB-8 macroporous adsorption resin. In the invention, a wet loading mode is preferably adopted when macroporous adsorption resin column chromatography separation is carried out, specifically, the mixed component obtained by combining the water-soluble component A1 and the water-extracted component B1 is directly loaded after a large amount of water is removed by concentration. In the present invention, the lower alcohol is preferably methanol or ethanol, more preferably methanol. In the present invention, the composition of the eluent is preferably 0 in the following order, based on the volume ratio of lower alcohol to water: 1. 6:4 and 9.5:0.5. the invention adopts a gradient elution mode to facilitate the separation of different polarity parts in the national drug double ginseng.
After fraction AB1 is obtained, the invention concentrates said fraction AB1 by a fourth concentration to obtain water eluted fraction AB1'. The fourth concentration is not particularly limited in the present invention, and a large amount of water in the fraction AB1 may be removed.
After the fraction AB2 is obtained, the fraction AB2 is subjected to fifth concentration and alcohol precipitation in sequence to obtain a water-soluble alcohol precipitation component AB2'. The fifth concentration is not particularly limited, and methanol and water in the fraction AB2 can be removed, and the fifth concentration is preferably performed by concentrating the fraction AB2 to 5-10% of the original volume (i.e., the fraction AB2 is subjected to fifth concentration to obtain a fraction AB2 concentrate, and the volume of the fraction AB2 concentrate is 5-10% of the volume of the fraction AB 2). In the present invention, the alcohol precipitation reagent used for the alcohol precipitation is preferably an aqueous alcohol solution, and the alcohol in the aqueous alcohol solution is preferably methanol or ethanol, more preferably ethanol; in the examples of the present invention, industrial alcohol is specifically used. In the present invention, the number of times of the alcohol precipitation is preferably 2 to 4 times, more preferably 3 times; the volume fraction of the alcohol aqueous solution for each alcohol precipitation is independently preferably 90-95%, more preferably 93-95%; the volume ratio of the alcohol precipitation reagent to the material to be alcohol precipitated is preferably (1.5-3): 1, more preferably (1.5 to 2): 1, a step of; the temperature of each alcohol precipitation is preferably 4-8 ℃, more preferably 4 ℃, and in the embodiment of the invention, the alcohol precipitation is particularly carried out in a refrigerator; the time of each alcohol precipitation is independently preferably 12 to 24 hours, more preferably 12 to 18 hours. In the embodiment of the invention, after each alcohol precipitation, the obtained supernatant is decompressed and concentrated until the water-soluble components are just fully dissolved in water, so that the final concentration of ethanol in the system reaches more than 90% (volume fraction) in the last alcohol precipitation, and the precipitates obtained by each alcohol precipitation are combined to obtain the water-soluble alcohol precipitation component AB2'.
After obtaining a water-eluting component AB1 'and a water-soluble alcohol-precipitating component AB2', the invention combines the water-eluting component AB1 'and the water-soluble alcohol-precipitating component AB2' to obtain the national drug double ginseng water-soluble extract (recorded as the national drug double ginseng water-soluble total extract).
In the present invention, the water eluting component AB1 'and the water soluble alcohol precipitating component AB2' preferably further comprise: and carrying out fractional alcohol precipitation on the obtained mixed components to obtain supernatant and alcohol precipitation, and carrying out sixth concentration on the supernatant to obtain a first ethnic medicine double ginseng water-soluble extract (marked as TGPB), wherein the alcohol precipitation is a second ethnic medicine double ginseng water-soluble extract (marked as TGPC). In the invention, the alcohol precipitation reagent used for the fractional alcohol precipitation is preferably alcohol water solution, and the alcohol in the alcohol water solution is preferably ethanol; the number of times of the fractional alcohol precipitation is preferably 2 to 4 times, more preferably 3 times; the volume fraction of alcohol in the system is preferably 30-60%, more preferably 30% during each alcohol precipitation; the temperature of each alcohol precipitation is independently preferably 4-8 ℃, more preferably 5-6 ℃; the time of each alcohol precipitation is independently preferably 12 to 24 hours, more preferably 15 to 18 hours. In the present invention, the sixth concentration is preferably reduced pressure concentration, and the temperature of the sixth concentration is preferably 60 to 70 ℃, more preferably 65 ℃; the sixth concentration is specifically to remove the solvent from the supernatant. After the sixth concentration, the obtained concentrate is preferably dried to obtain the water-soluble extract of the first ethnic medicine double ginseng; the drying is preferably freeze-drying. The alcohol precipitation is preferably dried to obtain a second ethnic medicine double ginseng water-soluble extract; the drying is preferably freeze-drying.
The method provided by the invention preferably further comprises a decoloring treatment, and particularly the decoloring treatment can be carried out in any one of the following two modes:
mode one: decolorizing the water-soluble alcohol precipitation component AB2', mixing the decolorized solution with the fraction AB1, and performing seventh concentration to obtain a mixed concentrated solution; and then carrying out fractional alcohol precipitation on the mixed concentrated solution according to the method to obtain supernatant and alcohol precipitation, and then carrying out corresponding treatment on the supernatant and the alcohol precipitation according to the scheme to obtain the first ethnic medicine double ginseng water-soluble extract and the second ethnic medicine double ginseng water-soluble extract. In the present invention, the decoloring treatment may be performed using a decoloring agent or may be performed using a macroporous adsorbent resin, respectively, as will be described below.
In the invention, when the decoloring agent is adopted for decoloring, the water-soluble alcohol precipitation component AB2', water and the decoloring agent are mixed for decoloring to obtain a decoloring solution; the mass ratio of the water-soluble alcohol precipitation component AB2' to water is preferably 1: (0.5 to 1), more preferably 1:0.5; the decoloring reagent preferably comprises activated carbon or hydrogen peroxide, and the mass of the activated carbon is preferably 2-5% of the mass of the water-soluble alcohol precipitation component AB2', more preferably 4%; the mass concentration of the hydrogen peroxide is preferably 30%, and the volume of the hydrogen peroxide is preferably 5-8% of the total volume of the solution obtained by mixing the water-soluble alcohol precipitation component AB2' with water, and more preferably 7%. In the present invention, the temperature of the decoloring treatment is preferably 50 to 60 ℃, more preferably 55 ℃; when activated carbon is used, the time of the decoloring treatment is preferably 30 to 60 minutes, more preferably 40 minutes; when hydrogen peroxide is used, the time for the decoloring treatment is preferably 1 to 3 hours, more preferably 2 hours.
In the invention, when macroporous adsorption resin is adopted for decolorization treatment, specifically, a water-soluble alcohol precipitation component AB2' is loaded on the macroporous adsorption resin, and elution is carried out by taking alcohol-water as an eluent, so as to obtain a decolorization solution. In the invention, the macroporous adsorption resin is preferably AB-8 macroporous adsorption resin or 101 macroporous adsorption resin; the alcohol in the eluent is preferably methanol; the elution mode is preferably gradient elution, and the volume fraction of the alcohol in the eluent is preferably 0-60%, more preferably 0-40%, namely the volume ratio of the collected water to the alcohol is preferably 1:0 to 4: the decolorized solution obtained in the step 6, more preferably, the volume ratio of the collected water to the alcohol is 1:0 to 6:4, obtaining a decolorized solution.
In the present invention, the seventh concentration is preferably a reduced pressure concentration; the seventh concentration is preferably such that the system obtained after mixing the decolorized solution with fraction AB1 is concentrated until the water-soluble components are just fully soluble in water.
Mode two: the water eluting component AB1 'and the water soluble alcohol precipitating component AB2' are combined, the obtained mixed component is subjected to graded alcohol precipitation, and the obtained supernatant and the obtained alcohol precipitate are respectively subjected to decolorization treatment, so that a first ethnic medicine double ginseng water-soluble extract and a second ethnic medicine double ginseng water-soluble extract are respectively obtained; the method for classifying the alcohol precipitation and decoloring treatment is preferably carried out by referring to the technical scheme, and is not repeated.
The invention provides a water-soluble extract of national drug double ginseng, which is prepared by the preparation method of the technical proposal, and comprises water-soluble polysaccharide components and water-soluble micromolecular components, wherein the relative molecular weight of the water-soluble polysaccharide components is in the range of 504-2.0x10 6 The relative molecular weight of the water-soluble small molecules ranges from 214 to 792. In the present invention, the water-soluble polysaccharide component preferably includes a high molecular weight polysaccharide and a low molecular weight polysaccharide, and the relative molecular weight of the high molecular weight polysaccharide is preferably in the range of 5.0X10 4 ～2.0×10 6 The low molecular weight polysaccharide preferably has a relative molecular weight in the range of 504 to 4.0X10 4 The method comprises the steps of carrying out a first treatment on the surface of the The water-soluble small molecule is preferably a water-soluble iridoid glycoside compound and/or a derivative thereof, and the water-soluble iridoid glycoside compound preferably comprises at least one of strychnine, a strychnine derivative, strychnine and a strychnine derivative.
In the invention, the functional substances in the water-soluble extract of the first ethnic medicine double ginseng comprise low molecular weight polysaccharide, water-soluble iridoid compounds and derivatives thereof; the functional substances in the second ethnic group drug double ginseng water-soluble extract comprise high molecular weight polysaccharide.
The invention provides application of the national medicine double ginseng water-soluble extract in preparing medicines for preventing and treating diabetes or nephropathy. In the present invention, the diabetes is preferably type II diabetes, and the kidney disease is at least one of diabetic nephropathy, acute renal failure, chronic renal failure, nephrotic syndrome and glomerulonephritis, preferably diabetic nephropathy.
In the present invention, the medicament preferably comprises an active ingredient and pharmaceutically acceptable excipients. In the invention, the active ingredient in the medicine is the water-soluble extract of the national medicine double ginseng, and the pharmaceutically acceptable auxiliary materials comprise pharmaceutically acceptable carriers and/or excipients; the content of the active ingredient in the medicament is preferably 0.1 to 99.9wt%, more preferably 1 to 90wt%; the pharmaceutically acceptable carrier is not particularly limited, and pharmaceutically acceptable carriers well known to those skilled in the art can be adopted; the pharmaceutically acceptable excipient is not particularly limited, and may be any pharmaceutically acceptable excipient known to those skilled in the art, and may include mannitol, magnesium stearate, starch or cyclodextrin. In the present invention, the dosage form of the drug preferably includes injection or oral preparation. In the present invention, when the drug is used for preventing and treating diabetes, the effective dose of the drug is preferably 50 to 200mg, and the administration method of the drug is preferably oral; when the medicament is used for preventing and treating diabetic nephropathy, the effective dose of the medicament is preferably 100mg, and the administration method of the medicament is preferably oral.
The national drug double ginseng water-soluble extract is nontoxic to model animal and human glomerular mesangial cells, can reduce Triglyceride (TG), total Cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) of a diabetic mouse, and can raise high density lipoprotein cholesterol (HDL-C), wherein the effect of the first national drug double ginseng water-soluble extract (TGPB) is particularly remarkable, which indicates that the national drug double ginseng water-soluble extract provided by the invention has a certain capacity of regulating blood lipid metabolic disorder. Meanwhile, the national drug double-ginseng water-soluble extract can reduce the urea nitrogen (BUN) and serum creatinine (Scr) content of a diabetic mouse, wherein the TGPB effect is particularly remarkable, which indicates that the national drug double-ginseng water-soluble extract provided by the invention can improve the kidney function of the diabetic mouse. Therefore, the national medicine double ginseng water-soluble extract provided by the invention is suitable for people with diabetes and kidney diseases.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Chloroform, n-butanol, ethanol and methanol used in the following experiments are industrial-grade reagents and are used after re-evaporation.
Example 1
The preparation method of the water-soluble extract of the double ginseng by taking the double ginseng as a raw material comprises the following steps:
(1) Cleaning root and stem of radix Ginseng, air drying, and cutting into 1cm volume 3 The small blocks of the double-reference sample are used for standby; weighing 2kg of double ginseng sample, adding 4L of 95% ethanol water solution, cold leaching at room temperature (25deg.C) for 3 times, each time for 24 hr, filtering to obtain residue, extracting the residue with ethanol, collecting residue, and concentrating the residueFiltering the residue, and mixing the obtained solutions to obtain ethanol extractive solution;
concentrating the ethanol extract under reduced pressure at 50 ℃ until no ethanol smell exists, obtaining an ethanol extract component A, and simultaneously recovering ethanol; mixing the alcohol extraction component A with 1L of distilled water to obtain an aqueous solution of the alcohol extraction component A; mixing the aqueous solution of the alcohol extraction component A with equal volume of n-butanol for extraction, repeating for 3 times to obtain an extraction mother solution (namely a water-soluble component A1) and an n-butanol extract, and concentrating the n-butanol extract under reduced pressure at 70 ℃ to remove n-butanol to obtain an alcohol extraction component A2;
Adding distilled water with the weight of 3 times of that of the ethanol extraction residues into the ethanol extraction residues, heating for steaming and extracting for 3 times, wherein the extracting time is 2.5h each time, filtering out the residues after each extraction, extracting the residues for the next time, and combining the solutions obtained after the filtering out of the residues after each extraction is finished to obtain an aqueous extract; concentrating the water extract to 2L under reduced pressure at 65 ℃ to obtain water extract concentrate B; deproteinizing the aqueous extract concentrate B by a Sevag method, and repeating the deproteinizing treatment 3 times, wherein the Sevag reagent adopted by the Sevag method is chloroform and n-butanol, and the volume ratio of chloroform to n-butanol is 4:1, the volume ratio of the Sevag reagent to the water extraction concentrate B is 1:1, concentrating the aqueous solution obtained after protein removal under reduced pressure at 60 ℃ to obtain an aqueous extract component B1;
combining the water-soluble component A1 and the water-extracted component B1, separating by AB-8 macroporous adsorption resin column chromatography, loading a sample by a wet method, carrying out gradient elution by taking methanol-water as an eluent according to the volume ratio of methanol to water of 0:1, 6:4 and 9.5:0.5 in sequence, wherein the dosage of each gradient eluent is 2-3 column volumes, and detecting by thin-layer chromatography to sequentially obtain a flow AB1, a flow AB2 and a flow AB3;
concentrating the flow AB2 to 1L under reduced pressure, mixing with industrial ethanol (the volume fraction of ethanol is 95% at most), carrying out ethanol precipitation in a refrigerator at 4 ℃ for 3 times, wherein the volume of the industrial ethanol used for each time of ethanol precipitation is 2 times of the volume of the flow AB2, the time of each time of ethanol precipitation is 12 hours, concentrating the obtained supernatant under reduced pressure until water-soluble components are just fully dissolved in water after each time of ethanol precipitation, enabling the final concentration of ethanol in a system to reach more than 90% (volume fraction) in the last time of ethanol precipitation, merging precipitates obtained by each time of ethanol precipitation, and obtaining a water-soluble ethanol precipitation component AB2', wherein the supernatant obtained by the last time of ethanol precipitation is an ethanol-soluble supernatant AB3';
Concentrating the fraction AB1 under reduced pressure to remove solvent to obtain water-eluted component AB1', and combining the water-eluted component AB1' with water-soluble alcohol precipitation component AB2' to obtain water-soluble extract of radix Ginseng (marked as water-soluble total extract of radix Ginseng).
Example 2
Fraction AB1 and water-soluble alcohol-precipitated fraction AB2' were prepared as in example 1;
decolorizing the water-soluble alcohol precipitation component AB2' by adopting an AB-8 macroporous adsorption resin column chromatography, specifically, gradient eluting by taking methanol-water as an eluent, wherein the volume ratio of the collected water to the methanol is 1:0 to 6:4, obtaining a decolorized solution; mixing the decolorized solution with the fraction AB1, concentrating under reduced pressure until the water-soluble components are just fully dissolved in water, then carrying out fractional alcohol precipitation at 5 ℃ by using an ethanol water solution, repeating for 3 times, wherein the final concentration of ethanol in a system is 30% (volume fraction) in each alcohol precipitation process, and the time of each alcohol precipitation is 18h, so as to obtain supernatant and alcohol precipitation; concentrating the supernatant at 65deg.C under reduced pressure, and lyophilizing to obtain first water-soluble extract (TGPB) of radix Ginseng Rubra; the alcohol precipitate is freeze-dried to obtain a second water-soluble extract of ginseng (TGPC).
Comparative example 1
Fraction AB3, alcohol-soluble supernatant AB3 'and alcohol-extracted component A2 were prepared as in example 1, and the fraction AB3, alcohol-soluble supernatant AB3' and alcohol-extracted component A2 were combined and concentrated under reduced pressure at 50 ℃ to remove the solvent, to obtain a total alcohol extract of the ethnic drug ginseng, designated TGPA.
Example 3
Using radix et rhizoma Rhei Palmati as raw material, preparing into flow AB1 and water-soluble alcohol precipitation component AB2' according to the method of example 1; then the first and second water-soluble extracts of ginseng and ginseng are prepared according to the method of example 2.
Example 4
Animal experiment: protective effect of double ginseng water-soluble extract on STZ-induced diabetes mouse kidney
1. In vivo activity study:
male Kunming mice were fed 1W adaptively, fasted overnight for 12h after normal condition, and single intraperitoneal injection of STZ at a dose of 120mg.kg -1 Establishing a diabetes mouse model (tail vein blood sampling to detect Fasting Blood Glucose (FBG), if the FBG value is more than or equal to 11.1 mmol.L -1 Successful molding); taking mice successfully molded, randomly dividing the mice into 11 groups of 20 mice each, wherein the grouping conditions are as follows: model group (Model), tempol group; TGPA low dose group (TGPA-L), medium dose group (TGPA-M), high dose group (TGPA-H); the water-soluble extracts of radix Ginseng Rubra include TGPB low dose group (TGPB-L), medium dose group (TGPB-M), and high dose group (TGPB-H); the water-soluble extracts of radix Ginseng Rubra, TGPC low dose group (TGPC-L), medium dose group (TGPC-M), and high dose group (TGPC-H). The stomach is irrigated in the morning of each day, and the Tempol group administration dosage is 90 mg.kg -1 The high, medium and low doses of each administration group are 30 mg.kg respectively -1 、60mg·kg -1 、120mg·kg -1 . The administration was continued at 4-6W, and distilled water was administered in equal volumes to the normal group and the model group. After administration of 4W and 6W, respectively, half of the mice were bled to determine fasting blood glucose (Fasting blood glucose, FBG), and Serum to determine glycated Serum proteins (Glycosylated serumprotein, GSP), total cholesterol (Total Cholesterol, TC), serum creatinine (Scr), triglycerides (TG), serum nitrogen (Blood urea nitrogen, BUN), low-density lipoprotein cholesterol (Low density lipoprotein cholesterol, LDL-C), and high-density lipoprotein cholesterol (High density lipoprotein cholesterol, HDL-C) were isolated; after taking blood, the cervical dislocation is killed, the liver, thymus, spleen and kidney are taken out, filter paper is used for drying water, and then the filter paper is weighed, and viscera index is calculated. One side of the kidney is put into 4% tissue fixing solution for preparing pathological sections and performing histomorphology examination. Then, 0.1g of tissue was homogenized to prepare 10% liver homogenate and 10% kidney homogenate, and the supernatant was centrifuged to determine superoxide dismutase (T-SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-P) X ) The content is as follows.
2. Results of in vivo experiments:
(1) The three more and a few of the three active components (TGPA, TGPB and TGPC) of the double ginseng 3 improve the fasting blood glucose level of the STZ diabetic mice, and particularly the fasting blood glucose level of the STZ diabetic mice is obviously reduced by the TGPB, wherein the P of the TGPB-H group is less than 0.01, and the double ginseng three active components have obvious difference and better activity. The specific results are shown in Table 1.
TABLE 1 Effect of double ginseng active ingredients on FBG of STZ diabetic mice
| Group | 4W(n＝20) | 6W(n＝10) |
| Normal | 6.80±1.30 | 5.85±2.05 |
| Model | 18.50±1.40 **,△ | 23.15±2.65 **,△△ |
| Tempol | 14.65±2.85 **,# | 11.91±1.45 **,## |
| TGPA-H | 15.05±2.35 **,## | 13.00±1.30 **,##,△ |
| TGPA-M | 16.80±1.70 **,#,△ | 14.25±1.95 **,#,△ |
| TGPA-L | 17.27±1.86 **,#,△△ | 16.63±2.82 **，#,△ |
| TGPB-H | 14.91±2.13 **,## | 13.63±1.17 **,## |
| TGPB-M | 15.55±2.76 **,##,△△ | 11.90±1.29 **,## |
| TGPB-L | 16.79±1.29 **,#,△△ | 13.40±2.20 **,# |
| TGPC-H | 15.78±2.10 **,# | 13.73±1.49 **,# |
| TGPC-M | 14.09±1.84 **,# | 15.36±2.16 **,# |
| TGPC-L | 16.63±1.66 **,#,△ | 14.74±1.17 **,# |
Remarks: in comparison to Normal group (Normal), "x" indicates P <0.01, "x" indicates P <0.05; in comparison to Model group (Model), "#" represents P <0.01, "#" represents P <0.05; in comparison to the positive control group (Tempol), "ΔΔ" means P <0.01, "Δ" means P <0.05. Values are expressed as mean and standard deviation.
(2) The index of each organ of the mice in the Model group was significantly lower than that in the Normal group. The organ index of the mice in the Tempol group and each administration group is close to the Normal group or even higher than the Normal group; the kidney index of the high dose administration group is higher than that of the Normal group, which indicates that the active ingredients of the double ginseng have significant influence on the kidney of the diabetic mice. The specific results are shown in tables 2 and 3.
TABLE 2 influence of active ingredients of double ginseng on liver index and kidney index of STZ diabetic mice n＝10)
Remarks: in comparison to Normal group (Normal), "x" indicates P <0.01, "x" indicates P <0.05; in comparison to Model group (Model), "#" represents P <0.01, "#" represents P <0.05; in comparison to the positive control group (Tempol), "ΔΔ" means P <0.01, "Δ" means P <0.05. Values are expressed as mean and standard deviation.
TABLE 3 influence of the active ingredients of double ginseng on spleen index and thymus index of STZ diabetic micen＝10)
Remarks: in comparison to Normal group (Normal), "x" indicates P <0.01, "x" indicates P <0.05; in comparison to Model group (Model), "#" represents P <0.01, "#" represents P <0.05; in comparison to the positive control group (Tempol), "ΔΔ" means P <0.01, "Δ" means P <0.05. Values are expressed as mean and standard deviation.
(3) TGPB significantly reduced Glycated Serum Proteins (GSP) in model mice compared to model group. The specific results are shown in Table 4 and FIG. 1.
TABLE 4 active ingredients of double ginseng against GSP (mmol.L) in STZ diabetic mice -1 ) Influence of (a)n＝10)
Remarks: in comparison to Normal group (Normal), "x" indicates P <0.01, "x" indicates P <0.05; in comparison to Model group (Model), "#" represents P <0.01, "#" represents P <0.05; in comparison to the positive control group (Tempol), "ΔΔ" means P <0.01, "Δ" means P <0.05. Values are expressed as mean and standard deviation.
(4) The water-soluble extract of the double ginseng can reduce the urea nitrogen (BUN) and serum creatinine (Scr) content of the diabetic mice, wherein the TGPB-H group has remarkable effect, which indicates that the water-soluble extract of the double ginseng can improve the kidney function of the diabetic mice. The specific results are shown in Table 5 and FIG. 2.
TABLE 5 influence of the active ingredients of double ginseng on BUN and Scr in STZ diabetic micen＝10)/>
Remarks: in comparison to Normal group (Normal), "x" indicates P <0.01, "x" indicates P <0.05; in comparison to Model group (Model), "#" represents P <0.01, "#" represents P <0.05; in comparison to the positive control group (Tempol), "ΔΔ" means P <0.01, "Δ" means P <0.05. Values are expressed as mean and standard deviation.
(5) The double ginseng can reduce Triglyceride (TG), total Cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) of diabetic mice, and raise high density lipoprotein cholesterol (HDL-C), and the effect of TGPB-H is particularly remarkable. The active components of the double ginseng have certain capacity of regulating blood lipid metabolic disturbance. The specific results are shown in tables 6 and 7.
TABLE 6 Effect of double ginseng active ingredients on TG and TC in STZ diabetic micen＝10)/>
TABLE 7 Effect of double ginseng active ingredient on HDL-C and LDL-C of STZ diabetic micen＝10)
Remarks: in comparison to Normal group (Normal), "x" indicates P <0.01, "x" indicates P <0.05; in comparison to Model group (Model), "#" represents P <0.01, "#" represents P <0.05; in comparison to the positive control group (Tempol), "ΔΔ" means P <0.01, "Δ" means P <0.05. Values are expressed as mean and standard deviation.
(6) The components of the double ginseng can raise the activity of T-SOD and GSH-PX of the liver of a diabetic mouse, and reduce the MDA content of the diabetic mouse, wherein the antioxidation capability of the TGPB-H group is inferior to that of a positive control Tempol group. The specific results are shown in tables 8 and 9.
TABLE 8 influence of double ginseng active ingredients on T-SOD and MDA of liver of STZ diabetic micen＝10)
TABLE 9 active ingredients of double ginseng against STZ diabetes mouse liver GSH-PX (mmol.L) -1 ) Influence of (a) n＝10)
| Group | 4w | 6w |
| Normal | 335.67±4.08 | 347.95±7.62 |
| Model | 104.07±4.94 **,△△ | 91.43±4.31 **,△△ |
| Tempol | 270.32±7.85 *,## | 278.58±2.87 *,## |
| TGPA-H | 189.59±5.10 **,#,△ | 187.54±4.11 **,##,△ |
| TGPA-M | 164.18±6.01 **,#,△△ | 167.54±5.12 **,#,△ |
| TGPA-L | 160.77±2.50 **,#,△△ | 160.35±3.07 **,#,△ |
| TGPB-H | 260.15±5.81 *,## | 267.71±4.17 *,## |
| TGPB-M | 249.55±4.83 **,## | 251.90±5.95 *,## |
| TGPB-L | 165.01±3.01 **,#,△△ | 163.10±5.17 **,#,△ |
| TGPC-H | 128.87±4.04 **,△△ | 132.12±5.14 **,#,△△ |
| TGPC-M | 117.07±3.98 **,△△ | 123.16±4.06 **,△△ |
| TGPC-L | 101.45±4.68 **,△△ | 106.33±5.05 **,△△ |
Remarks: in comparison to Normal group (Normal), "x" indicates P <0.01, "x" indicates P <0.05; in comparison to Model group (Model), "#" represents P <0.01, "#" represents P <0.05; in comparison to the positive control group (Tempol), "ΔΔ" means P <0.01, "Δ" means P <0.05. The values are represented by mean and standard deviation
(7) The active component of radix Ginseng Rubra can raise T-SOD and GSH-P of kidney of diabetic mouse X Activity, but reduces MDA content, and has concentration dependence, wherein TGPB-H group has most obvious effect, and antioxidant capacity is inferior to that of positive control Tempol group. The specific results are shown in Table 10, table 11 and FIG. 3.
TABLE 10 influence of double ginseng active ingredients on T-SOD and MDA of kidney of STZ diabetic micen＝10)
TABLE 11 active ingredients of double ginseng against STZ diabetes mouse kidney GSH-P X (mmol·L -1 ) Influence of (a) n＝10)/>
Remarks: in comparison to Normal group (Normal), "x" indicates P <0.01, "x" indicates P <0.05; in comparison to Model group (Model), "#" represents P <0.01, "#" represents P <0.05; in comparison to the positive control group (Tempol), "ΔΔ" means P <0.01, "Δ" means P <0.05. Values are expressed as mean and standard deviation.
(8) Results of in vivo experiments
(a) TGPB can improve the kidney index of STZ diabetic mice; combining liver, spleen and thymus index results, it is found that TGPC has a certain effect of improving immunity.
(b) The three more and one less of the 3 active components of the double ginseng can be improved for a certain degree, and the FBG level is reduced; can improve the antioxidant capacity of livers and kidneys, and is characterized by improving the activities of T-SOD and GSH-Px and reducing the MDA content; the active component of the double ginseng can correct blood sugar and blood lipid disorders to a certain extent, and is shown as a trend of decreasing GSP, TC, TG, LDL-C level in serum, and the HDL-C content is influenced.
(c) TGPB can improve kidney function to some extent, and is shown to reduce the content of Scr and BUN.
(9) Pathological section results
Fig. 4 is an optical micrograph (magnification 400) of a kidney section of a mouse, and as can be seen from fig. 4, the kidney structure of the "normal group (Control)" is not changed newly, the glomerulus has no adhesion membranous cells and matrix proliferation, the glomerulus volume is not changed significantly, the epithelial cells of the renal tubule have no edema, the renal interstitial tissue has no fibrosis and mononuclear cell infiltration.
The glomerular volume of the Model is obviously increased, and the glomerular mesangial cells and stroma are locally focus, segmental hyperplasia, renal tubular epithelial cell edema and renal interstitial are not obviously changed.
In the treatment group (TGPA, TGPB, TGPC) -high (H), medium (M), low (L) dose groups, the glomerular volume of the high dose (-H) was reduced, mesangial cells and stromal local focal proliferation were reduced, and tubular epithelial cell edema was reduced, as compared to the model group. Consistent with the positive control (Tempol) group. The 3 active components of the double ginseng have protective effect on the kidneys of model mice, and particularly the high-dose group effect of the TGPB component is excellent.
3. Conclusion:
(1) The three more or less of the 3 active components of the double ginseng improve the STZ diabetic mice, reduce the fasting blood glucose level and the Glycosylated Serum Protein (GSP), and particularly the water-soluble component of the double ginseng TGPB obviously reduces the fasting blood glucose level and the Glycosylated Serum Protein (GSP) of the STZ diabetic mice.
(2) The active component of the double ginseng has a certain kidney protecting effect on STZ diabetic mice, and the mechanism of the active component of the double ginseng is possibly related to the effect that the active component of the double ginseng can improve the oxidation resistance of the kidneys and livers of the STZ diabetic mice, regulate the metabolic disorder of glycolipids and improve the renal functions.
Example 5
Cell experiment: protective effect of water-soluble extract of radix Ginseng Rubra on high sugar cultured human mesangial cells
1. High sugar modeling: the MTT method is adopted to detect the proliferation condition of the high-sugar stimulated HMC cells, and the result shows that: culturing the cells for 12-72 h with the concentration of 25mmol/L, The culture medium of 27.5mmol/L, 30mmol/L and 32.5mmol/L glucose shows obvious proliferation state for HMC cells in 12-48 h, and the cells grow to the limit in 48 h; the proliferation of the culture medium containing 30mmol/L glucose is most obvious, and when the cells are cultured for 64-72 h, the cells are in a slow growth state. HMC cells were cultured for 48h using 30mmol/L high sugar medium as the best model for high sugar stimulation of HMC cells. The specific results are shown in fig. 5, wherein, * P﹤0.05， ** P﹤0.01。
2. component cytotoxicity experiments: the water-soluble extract of the double ginseng is nontoxic to human glomerular mesangial cells in the concentration range of 50-800 mug/mL, so that the double ginseng active component in the concentration range is adopted to further verify the proliferation inhibition effect of the sugar-stimulated human glomerular mesangial cells (HMC). The specific results are shown in FIG. 6.
3. Cell experiment: detecting the proliferation of human glomerular mesangial cells (Human renalmesangial Cell, HRMC) induced by high sugar with double ginseng active component by MTT method, uniformly seeding HRMC cells in 96-well plate to obtain cell density of 2.0X10 4 A/hole; after the cells are attached, the serum-free culture medium is changed for continuous culture for 24 hours, and 100 mu L of normal sugar culture medium, 100 mu L of high sugar complete culture medium and different concentration administration groups are added according to groups, and 5 compound holes are formed in each group for continuous culture for 48 hours. Adding 20 mu L of MTT solution and CO 2 After incubation in the incubator for 4h, the supernatant was decanted, 150. Mu.L of DMSO was added, and after shaking the plate for 2min, the OD was measured at 490 nm. Setting normal control group (NG, 5.5 mmol.L) -1 Glucose), high sugar group (HG, 30 mmol.L -1 Glucose), tempol group (100. Mu. Mol.L -1 ) Three component pharmaceutical compositions of the active ingredients TGPA, TGPB and TGPC of double ginseng (50. Mu.g.L) -1 、200μg·L -1 、800μg·L -1 ) A group; the intervention time of each drug was 48h, the precipitated cells were obtained by repeated centrifugation, and then disrupted by sonication. The influence of each experimental group on the proliferation and the activity of the high sugar culture HMC cells is detected by an MTT method; ROS content was then assayed and T-SOD and MDA content in each group of HRMC cell supernatants was determined by biotin double antibody sandwich ELISA (Enzyme-linked immunosorbent assaym).
(1) Inhibition of high sugar induced HRMC cell proliferation by double ginseng active ingredient
The water-soluble extract of the double ginseng has no cytotoxicity, and both TGPB and TGPC have certain inhibition effect on the proliferation of the HRMC induced by high sugar. Specific results are shown in fig. 7 (TGPA for TGA, TGPB for TGB, TGPC for TGC), where "×" indicates P <0.01, "×" indicates P <0.05; the "#" indicates P <0.01 and "#" indicates P <0.05, compared to the high sugar group (HG).
(2) The influence of the active component of double ginseng on the content of MDA and T-SOD in high sugar-induced HRMC cells is obviously increased compared with that of the group NG (P < 0.05), the effect of the active component of double ginseng on the intracellular MDA of HG (P < 0.05) is better than that of other administration groups, and a certain drug concentration dependence appears.
And NG (P)<0.05 Compared with the HG group, the T-SOD content in the high sugar induction HRMC cells is obviously reduced, the T-SOD content of each dose group of TGPA and TGPB is improved, and the TGPC group only has the highest dose group of TGC-800 (800 mug.L) -1 ，P<0.05 Slightly raised, the other two dose groups almost agree with the HG content. The specific results are shown in Table 12.
TABLE 12 influence of active ingredients of double ginseng on MDA and T-SOD content in high sugar induced HRMC cells n＝6)
| Group | MDA(nmol·mL -1 ) | T-SOD(U·L -1 ) |
| NG | 6.12±0.26 | 94.22±1.24 |
| HG | 8.35±0.62 ** | 62.00±2.73 ** |
| TGPA-50 | 8.85±0.30 ** | 76.44±1.00 **,# |
| TGPA-200 | 8.75±0.40 * | 79.64±1.57 **,# |
| TGPA-800 | 8.09±0.66 ** | 79.52±0.84 **,# |
| TGPB-50 | 7.72±0.56 **,## | 80.74±1.41 **,# |
| TGPB-200 | 7.40±0.42 **,# | 80.33±2.38 *,## |
| TGPB-800 | 7.22±0.66 **,## | 82.56±1.73 *,## |
| TGPC-50 | 8.78±0.76 *,# | 62.78±1.48 **,# |
| TGPC-200 | 8.39±0.39 *,# | 62.78±1.73 *,# |
| TGPC-800 | 8.12±0.16 *,# | 66.44±1.89 * |
Remarks: in comparison to Normal Glycogroup (NG), "x" means P <0.01, "x" means P <0.05; in comparison to the high sugar group (HG), "#" indicates P <0.01, "#" indicates P <0.05.
(3) Detection of ROS content in high sugar-induced HRMC cells after action of double ginseng active components
Comparison of the extent of ROS reduction in high sugar-induced HRMC cells by three active ingredients of bistort rhizome: TGPB-800> TGPB-200> TGPA-800> TGPA-50> TGPB-50> TGPC-800> TGPC-200> TGPC-50, so that the influence of TGPB on intracellular ROS content is greatly influenced by the administration dosage, the effect of TGPA is obvious, and the reduction degree of different administration dosages is equivalent. The specific results are shown in Table 13.
TABLE 13 Effect of double ginseng active ingredients on the ROS content in high sugar-induced HRMC cellsn＝6)
| Group | ROS |
| NG | 529.00±4.24 |
| HG | 1373.00±25.46 ** |
| TGPA-50 | 820.00±11.31 *,## |
| TGPA-200 | 807.50±2.12 *,## |
| TGPA-800 | 817.00±7.07 *,## |
| TGPB-50 | 878.50±3.54 **,## |
| TGPB-200 | 791.00±11.31 *,## |
| TGPB-800 | 770.00±5.66 *,# |
| TGPC-50 | 1185.50±47.38 |
| TGPC-200 | 1164.00±38.18 **,# |
| TGPC-800 | 1148.50±4.95 * |
Remarks: in comparison to Normal Glycogroup (NG), "x" means P <0.01, "x" means P <0.05; in comparison to the high sugar group (HG), "#" indicates P <0.01, "#" indicates P <0.05.
(4) The result shows that: the water-soluble extract of the double ginseng increases T-SOD in high sugar cultured HRMC cells, and reduces the content of MDA and ROS; in particular, TGPA and TGPB have protective effects on human mesangial cells in high sugar culture, and their mechanism may be related to their ability to increase the antioxidant capacity of HRMC cells.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A preparation method of water-soluble extract of radix Ginseng Rubra of ethnic group medicine comprises the following steps:
extracting the national medicinal double ginseng with alcohol to obtain alcohol extract and alcohol extract residues;
the alcohol extract is subjected to first concentration to obtain an alcohol extract component A, and the alcohol extract component A is mixed with water and n-butanol for extraction to obtain a water-soluble component A1; the extraction times are 2-4 times, and the volume ratio of n-butanol to the material to be extracted is (1-2) independently during each extraction: 1, a step of;
Extracting the alcohol extraction residues with hot water, performing second concentration on the obtained water extract to obtain water extract concentrate B, performing protein removal treatment on the water extract concentrate B, and performing third concentration on the obtained protein removal solution to obtain a water extract component B1;
the method comprises the steps of taking lower alcohol-water as an eluent, carrying out macroporous adsorption resin column chromatographic separation on a mixed component obtained by combining a water-soluble component A1 and a water-extracted component B1 under a gradient elution condition, sequentially obtaining a flow AB1, a flow AB2 and a flow AB3, wherein the gradient elution comprises 3 gradients which are sequentially carried out, and the composition of the eluent is sequentially 0 according to the volume ratio of the lower alcohol to the water: 1. 1 to 7:9 to 3 and 9.5:0.5, corresponding to the fraction AB1, the fraction AB2 and the fraction AB3, respectively;
fourth concentrating the flow AB1 to obtain a water eluting component AB1'; sequentially carrying out fifth concentration and alcohol precipitation on the flow AB2 to obtain a water-soluble alcohol precipitation component AB2'; and combining the water eluting component AB1 'with the water-soluble alcohol precipitating component AB2' to obtain the water-soluble extract of the national drug double ginseng.
2. The method according to claim 1, wherein the ethnic group of medicinal herbs is ginseng or ginseng.
3. The preparation method according to claim 1, wherein the alcohol extraction is cold leaching extraction, the alcohol extraction reagent adopted in the alcohol extraction is ethanol aqueous solution, and the volume fraction of the ethanol aqueous solution is 60-95%.
4. The method according to claim 1, wherein the deproteinizing treatment is performed by Sevag method.
5. The preparation method according to claim 1, wherein the macroporous adsorption resin used for the macroporous adsorption resin column chromatographic separation is AB-8 macroporous adsorption resin or 101 macroporous adsorption resin.
6. The method according to any one of claims 1 to 5, wherein the combination of the water-eluting component AB1 'and the water-soluble alcohol-precipitating component AB2' further comprises: and carrying out fractional alcohol precipitation on the obtained mixed components to obtain supernatant and alcohol precipitation, and carrying out sixth concentration on the supernatant to obtain the first ethnic medicine double-ginseng water-soluble extract, wherein the alcohol precipitation is the second ethnic medicine double-ginseng water-soluble extract.
7. The water-soluble extract of national drug double ginseng prepared by the preparation method of any one of claims 1 to 6, which comprises water-soluble polysaccharide components and water-soluble small molecular components, wherein the relative molecular weight of the water-soluble polysaccharide components is in the rangeThe circumference is 504-2.0x10 6 The relative molecular weight of the water-soluble small molecules ranges from 214 to 792.
8. The water-soluble extract of national drug double ginseng according to claim 7, wherein the water-soluble polysaccharide component comprises a high molecular weight polysaccharide and a low molecular weight polysaccharide, and the relative molecular weight of the high molecular weight polysaccharide is in the range of 5.0 x 10 4 ～2.0×10 6 The relative molecular weight of the low molecular weight polysaccharide is in the range of 504-4.0X10 4 The method comprises the steps of carrying out a first treatment on the surface of the The water-soluble small molecule is a water-soluble iridoid glycoside compound and/or a derivative thereof.
9. Use of the water-soluble extract of national drug double ginseng according to claim 7 or 8 for preparing a medicament for preventing and treating diabetes or diabetic nephropathy.
10. Use of a water-soluble extract of the ethnic group of ginseng in claim 7 or 8 for the preparation of a medicament for kidney protection.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK170290D0 (en) * | 1988-11-18 | 1990-07-16 | Biopharm Kutato Fejleszto Kft | PROCEDURE FOR THE PREPARATION OF WATER SOLUBLE PRIMYCIN AND ITS COMPONENT AND PHARMACEUTICAL PREPARATIONS CONTAINING THEM |
| JPH07228539A (en) * | 1994-02-18 | 1995-08-29 | Itouen:Kk | Extrct from leaf lagerstroemia speciosa and antidiabetic agent |
| KR20040069415A (en) * | 2003-01-29 | 2004-08-06 | 손달훈 | Pharmaceutical composition comprising the extract or the polyphenol fraction isolated from Ganoderma applanatum for treating or preventing diabetes |
| CN1903251A (en) * | 2005-07-26 | 2007-01-31 | 成都精晶天然药物开发有限公司 | Shuangshen phenol glucoside injection, prepn. method and use thereof |
| CN108732266A (en) * | 2018-04-18 | 2018-11-02 | 吉林修正药业新药开发有限公司 | A kind of construction method of the HPLC characteristic spectrums of capsule for radix astragalis and red-rooted salvia root |
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| US20110300246A1 (en) * | 2010-06-03 | 2011-12-08 | Qinheng Huang | Herbal Extracts for Treatment of Diabetic Complications and Oxidative Stress |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK170290D0 (en) * | 1988-11-18 | 1990-07-16 | Biopharm Kutato Fejleszto Kft | PROCEDURE FOR THE PREPARATION OF WATER SOLUBLE PRIMYCIN AND ITS COMPONENT AND PHARMACEUTICAL PREPARATIONS CONTAINING THEM |
| JPH07228539A (en) * | 1994-02-18 | 1995-08-29 | Itouen:Kk | Extrct from leaf lagerstroemia speciosa and antidiabetic agent |
| KR20040069415A (en) * | 2003-01-29 | 2004-08-06 | 손달훈 | Pharmaceutical composition comprising the extract or the polyphenol fraction isolated from Ganoderma applanatum for treating or preventing diabetes |
| CN1903251A (en) * | 2005-07-26 | 2007-01-31 | 成都精晶天然药物开发有限公司 | Shuangshen phenol glucoside injection, prepn. method and use thereof |
| CN108732266A (en) * | 2018-04-18 | 2018-11-02 | 吉林修正药业新药开发有限公司 | A kind of construction method of the HPLC characteristic spectrums of capsule for radix astragalis and red-rooted salvia root |
Non-Patent Citations (1)
| Title |
|---|
| 不同分子量双参多糖抗氧化活性研究;李彩艺等;《 中国民族民间医药》;第35页左栏2.1提取与分离和第35页右栏倒数第2段 * |
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