CN114869923A - National medicine double ginseng water-soluble extract and preparation method and application thereof - Google Patents
National medicine double ginseng water-soluble extract and preparation method and application thereof Download PDFInfo
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- CN114869923A CN114869923A CN202210668831.0A CN202210668831A CN114869923A CN 114869923 A CN114869923 A CN 114869923A CN 202210668831 A CN202210668831 A CN 202210668831A CN 114869923 A CN114869923 A CN 114869923A
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- soluble
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- extract
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a ethnic medicine-radix codonopsis water-soluble extract, a preparation method and application thereof, belonging to the technical field of medicinal chemistry. Firstly, carrying out alcohol extraction on the ethnic medicine double ginsengs to obtain an alcohol extract and alcohol extraction dregs, and then respectively carrying out further treatment to obtain a water-soluble crude extract, specifically, carrying out n-butyl alcohol extraction on the alcohol extract after concentration, and carrying out water extraction and protein removal on the alcohol extraction dregs; mixing the water-soluble crude extracts, separating with macroporous adsorbent resin column chromatography with lower alcohol-water as eluent to obtain different eluting components, selecting water-soluble part according to eluent composition, precipitating with ethanol and eluting with water to obtain components except water-soluble component, and mixing with water-soluble component to obtain water-soluble extract of radix Ginseng Indici of ethnic group. The ethnic medicine double-ginseng water-soluble extract prepared by the method can be applied to the prevention and treatment of diabetes and nephropathy, has high safety and good development and application prospects.
Description
Technical Field
The invention relates to the technical field of medicinal chemistry, in particular to a national medicine-radix codonopsis water-soluble extract and a preparation method and application thereof.
Background
Diabetes Mellitus (DM) is a syndrome of disturbances of sugar, fat and protein metabolism due to insufficient secretion or utilization of insulin. DM not only affects the health of people, but also places a heavy burden on society. Diabetic Nephropathy (DN) is one of the common complications of DM patients, and is the leading cause of end-stage Nephropathy and death of DM patients, with about 30-40% of DM patients developing DN and the incidence increasing with the course of disease. The clinical manifestations of DN in early stage include high glomerular filtration rate, increase of microalbumin in urine, increase of urea nitrogen and creatinine in blood plasma, and finally development of chronic renal insufficiency. The main pathological features of the early stage of DN are glomerular hypertrophy, thickening of glomerular and tubular basement membranes and progressive accumulation of extracellular matrix in the mesangial region; the later stage is fibrosis of the glomerulus, the tubulointerstities, and ultimately, proteinuria and renal failure.
At present, DM is mainly treated by reducing blood sugar, DN is mainly treated by combined medication, and medicines in aspects of reducing blood sugar, blood pressure, blood fat and the like are comprehensively treated, so that the development of DN is mainly delayed, and the effect of radical treatment cannot be achieved. Currently, hormone and cytotoxic drugs are clinically used for treating DN, and although a certain curative effect is achieved, adverse reactions such as easy relapse 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 a large side effect and cause symptoms such as hypotension, hyperkalemia, renal insufficiency, and cough. To date, there is no ideal treatment. Therefore, aiming at DM and DN, the problem to be solved is urgently needed to find a high-efficiency and low-toxicity prevention and treatment medicine.
Disclosure of Invention
The invention aims to provide a ethnic medicine-radix codonopsis water-soluble extract and a preparation method and application thereof. The ethnic medicine double-ginseng water-soluble extract prepared by the method can be applied to the prevention and treatment of diabetes and nephropathy, has high safety and 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-radix codonopsis water-soluble extract, which comprises the following steps:
performing alcohol extraction on the national medicine double ginseng to obtain alcohol extract and alcohol extraction dregs;
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, carrying out second concentration on the obtained water extract to obtain a water extraction concentrated solution B, carrying out deproteinization treatment on the water extraction concentrated solution B, and carrying out third concentration on the obtained deproteinized solution to obtain a water extraction component B1;
using lower alcohol-water as an eluent, carrying out macroporous adsorption resin column chromatography separation on a mixed component obtained by combining the water-soluble component A1 with the water-extracted component B1 under a gradient elution condition to obtain a flow part AB1, a flow part AB2 and a flow part AB3 in sequence, wherein the gradient elution comprises 3 gradients which are carried out in sequence, and the composition of the eluent is 0: 1. 1-7: 9 to 3 and 9.5:0.5, corresponding to fraction AB1, fraction AB2 and fraction AB3, respectively;
subjecting the fraction AB1 to fourth concentration to obtain water eluent AB 1'; sequentially carrying out fifth concentration and alcohol precipitation on the fraction AB2 to obtain a water-soluble alcohol precipitation component AB 2'; and mixing the water elution component AB1 'with the water-soluble alcohol precipitation component AB2' to obtain the ethnic drug-containing water-soluble extract of the ginseng.
Preferably, the ethnic medicine ginseng is ginseng or American ginseng.
Preferably, the alcohol extraction is cold leaching extraction, an alcohol extraction reagent adopted by the alcohol extraction is an ethanol water solution, and the volume fraction of the ethanol water solution is 60-95%.
Preferably, the method used for protein removal is the Sevag method.
Preferably, the macroporous adsorption resin used for macroporous adsorption resin column chromatography separation is AB-8 macroporous adsorption resin or 101 macroporous adsorption resin.
Preferably, the water elution component AB1 'and the water-soluble alcohol precipitation component AB2' are combined to further include: and carrying out graded 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, wherein the alcohol precipitation is a second ethnic medicine double ginseng water-soluble extract.
The invention provides a national medicine double ginseng water-soluble extract prepared by the preparation method of the technical scheme, which comprises water-soluble polysaccharide components and water-soluble micromolecule components, wherein the relative molecular weight range of the water-soluble polysaccharide components is 504-2.0 multiplied by 10 6 The relative molecular weight range of the water-soluble small molecules is 214-792.
Preferably, the water-soluble polysaccharide component includes 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 5.0X 10 4 ～2.0×10 6 The relative molecular weight range of the low molecular weight polysaccharide is 504-4.0 multiplied by 10 4 (ii) a The water-soluble micromolecules are water-soluble iridoid glycoside compounds and/or derivatives thereof.
The invention provides application of the national medicine double ginseng water-soluble extract in preparing a medicine or a health-care product for preventing and treating diabetes or nephropathy.
Preferably, the diabetes is type II diabetes; the nephropathy 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 ethnic medicine double ginseng water-soluble extract, which comprises the steps of firstly carrying out alcohol extraction on ethnic medicine double ginseng to obtain alcohol extract and alcohol extraction dregs, then respectively carrying out further treatment to obtain water-soluble crude extracts, specifically, extracting the alcohol extract by n-butyl alcohol after concentration, and carrying out water extraction and protein removal treatment on the alcohol extraction dregs; mixing the water-soluble crude extracts, performing macroporous adsorption resin column chromatography with lower alcohol-water as eluent to remove pigment and other low-polarity small molecular components to obtain different eluting components, selecting water-soluble parts according to the composition of the eluent, and mixing the water-soluble parts except the water-eluted components with the ethanol-precipitated and water-eluted components to obtain the water-soluble extract of the national medicine of the double ginseng (the total water-soluble extract of the national medicine of the double ginseng).
Further, the water-soluble extract of the ethnic medicine double ginseng is further subjected to fractional alcohol precipitation to obtain supernatant and alcohol precipitation, and active substances in the water-soluble extract of the ethnic medicine double ginseng can be further separated to respectively obtain a first water-soluble extract (TGPB) of the ethnic medicine double ginseng and a second water-soluble extract (TGPC) of the ethnic medicine double ginseng.
The result of the active screening of the diabetes mouse model established by the human mesangial cells and the STZ induction shows that the water-soluble extract of the ginseng provided by the invention has an obvious protective effect on the human mesangial cells cultured by high sugar, has a correction effect on blood sugar, glycated serum protein and blood lipid disorder of the STZ-induced diabetes mouse, and has an obvious protective effect on the kidney, so that the water-soluble extract of the ginseng can prevent and treat diabetes and nephropathy and can be used for preparing medicines or health care products for preventing and treating diabetes and nephropathy; and toxicity tests prove that the water-soluble extract of the holothuria japonica has high safety, can ensure the safety of long-term administration for 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 fractions of the Bishen on Glycated Serum Protein (GSP) in STZ diabetic mice in example 4;
FIG. 2 is a graph showing the effect of each of the active fractions of the Bingshen in example 4 on blood creatinine (Scr) in STZ diabetic mice;
FIG. 3 shows the active fractions of each Panax schinseng of example 4 against superoxide dismutase (T-SOD), Malondialdehyde (MDA) and glutathione peroxidase (GSH-P) in kidney tissue of STZ diabetic mice X ) Influence graph of (2);
FIG. 4 is an optical micrograph of a kidney section of a mouse obtained in example 4;
FIG. 5 is a graph comparing the proliferation (x. + -.s) of HRMC cells at different time points and concentrations of high sugar in example 5 with that of a blank control group;
FIG. 6 is a graph showing the results of the test of the toxicity of the water-soluble extract of Panax schinseng in example 5 to human mesangial cells at a concentration of 50-800. mu.g/mL;
FIG. 7 is a graph showing the effect of different concentrations of the active ingredient of the ginseng on proliferation of mesangial cells in high-sugar cultured human in example 5 in comparison with that of Normal Glycome (NG).
Detailed Description
The invention provides a preparation method of a national medicine-radix codonopsis water-soluble extract, which comprises the following steps:
performing alcohol extraction on the national medicine double ginseng to obtain alcohol extract and alcohol extraction dregs;
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, carrying out second concentration on the obtained water extract to obtain a water extraction concentrated solution B, carrying out deproteinization treatment on the water extraction concentrated solution B, and carrying out third concentration on the obtained deproteinized solution to obtain a water extraction component B1;
using lower alcohol-water as an eluent, carrying out macroporous adsorption resin column chromatography separation on a mixed component obtained by combining the water-soluble component A1 with the water-extracted component B1 under a gradient elution condition to obtain a flow part AB1, a flow part AB2 and a flow part AB3 in sequence, wherein the gradient elution comprises 3 gradients which are carried out in sequence, and the composition of the eluent is 0: 1. 1-7: 9 to 3 and 9.5:0.5, corresponding to fraction AB1, fraction AB2 and fraction AB3, respectively;
subjecting the fraction AB1 to fourth concentration to obtain water eluent AB 1'; sequentially carrying out fifth concentration and alcohol precipitation on the fraction AB2 to obtain a water-soluble alcohol precipitation component AB 2'; and mixing the water elution component AB1 'with the water-soluble alcohol precipitation component AB2' to obtain the ethnic drug-containing water-soluble extract of the ginseng.
Traditional Chinese medicines, national folk medicines and the like belong to natural medicines, and discovery of active ingredients with multiple target points for preventing and treating DM and DN from the traditional Chinese medicines and the national folk medicines is an important effective way for research and development of medicines for preventing and treating DM and DN. The water soluble components in traditional Chinese medicine and national folk medicine have the characteristics of low toxicity or no toxicity, no side effect and the like, and have huge demand space in the field of medical care. Teasel root family (Diplacaceae) radix codonopsis pilosulae (Triplostegia) medicinal plants are 2 in total, specifically radix codonopsis pilosulae and radix codonopsis pilosulae, wherein the radix codonopsis pilosulae (Triplostegiagdirandiflora Gagnep) is also called as Qingyang ginseng or big flower bud flower, is a Yi medicine, is of non-color and color, and is commonly used for treating lumbago due to kidney deficiency, spermatorrhea, impotence, menstrual disorder, amenorrhea and the like; radix Ginseng alba (Triplostegian ginseng Wall), also known as radish ginseng, radix Ginseng, Tongzi ginseng, Dula, American ginseng or herba Achilleae, is commonly used for tonifying kidney, strengthening spleen and qi, promoting blood circulation and regulating menstruation. The water-soluble extract of the ethnic medicine double ginseng is obtained by extracting and separating the ethnic medicine double ginseng (double ginseng or large-flower double ginseng), has obvious curative effect on DM, particularly DN, and has the characteristic of no toxicity on a diabetes mouse model established by human glomerular mesangial cells and STZ induction, thereby showing that the water-soluble extract of the ethnic medicine double ginseng can be applied to preventing and treating DM and DN and has good development and application prospects. The preparation method of the ethnic drug-containing water-soluble extract of ginseng will be described in detail below.
The invention carries out alcohol extraction on the national medicine double ginseng to obtain alcohol extract and alcohol extraction dregs. In the invention, the ethnic medicine ginseng is concretely ginseng or ginseng with large flowers. In the invention, the ethnic medicine ginseng is preferably washed and dried in sequence before use; the invention is preferably used by taking underground parts (namely rhizomes) of ethnic medicine ginseng; the drying mode preferably comprises natural airing or drying, and the drying temperature is preferably 40-50 ℃. The invention preferably cuts the dried ethnic medicine double ginseng into small blocks with the length of 1cm for use.
In the invention, the alcohol extraction is preferably cold-soaking extraction, and specifically, the ethnic medicine double ginseng and the alcohol extraction reagent are mixed and extracted at room temperature; the number of times of alcohol extraction is preferably 2-4, and more preferably 3; the ethanol extraction reagent is preferably ethanol water solution, and the volume fraction of the ethanol water solution used in each ethanol extraction is preferably 60-95% independently, and more preferably 80-95%; the dosage ratio of the alcohol extraction reagent to the material to be extracted by alcohol is preferably (3-6) L independently during each alcohol extraction: 2kg, more preferably 2L: 1 kg; the time for each alcohol extraction is preferably 24-36 h independently, and more preferably 24 h. The invention particularly filters dregs after each alcohol extraction to carry out the next alcohol extraction, filters dregs after the last alcohol extraction to serve as alcohol extraction dregs, and combines solutions obtained after filtering dregs after each alcohol extraction to serve 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 invention, the first concentration is preferably reduced pressure concentration, and the temperature of the first concentration is preferably 50-60 ℃, and 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. Preferably, the ethanol-extracted component A is mixed with water to obtain an ethanol-extracted component A water solution; and mixing the ethanol extraction component A water solution with n-butyl alcohol for extraction. In the invention, the dosage ratio of water to ethnic medicine double ginseng in the alcohol extraction component A water solution is preferably (0.5-1) L: 2kg, more preferably 1L: 2 kg. In the invention, the extraction times are preferably 2-4 times, and more preferably 3 times; the volume ratio of n-butanol to the material to be extracted is preferably (1-2) independently during each extraction: 1, more preferably (1 to 1.5): 1. the invention removes the pigment and low-polarity components dissolved in the n-butyl alcohol by adopting the n-butyl alcohol for extraction.
In the invention, after alcohol extraction dregs are obtained, the alcohol extraction dregs are extracted by hot water, the obtained water extract is subjected to second concentration to obtain a water extract concentrated solution B, the water extract concentrated solution B is subjected to deproteinization treatment, and the obtained deproteinized solution is subjected to third concentration to obtain a water extract component B1. In the invention, the number of times of hot water extraction is preferably 2-4, and more preferably 3; the mode of each hot water extraction is independently and preferably reflux extraction or cooking extraction, and the temperature of the reflux extraction is preferably 90-95 ℃; the mass of the water used for each hot water extraction is preferably 2-5 times, and more preferably 3 times of the mass of the material to be extracted; the time of each hot water extraction is preferably 2-3 hours independently, and more preferably 2.5 hours. The invention particularly filters out dregs after each hot water extraction for the next hot water extraction, and combines the obtained solution after filtering out the dregs after each hot water extraction to be used as the water extract. The invention preferably adopts a reflux extraction or cooking extraction mode to carry out hot water extraction, and can completely extract the water-soluble components in the alcohol extraction medicine residue. In the invention, the second concentration is preferably reduced pressure concentration, and the temperature of the second concentration is preferably 60-70 ℃, and more preferably 65 ℃; the second concentration is preferably to concentrate the aqueous extract to 10 to 20% of the original volume (i.e., the volume of the aqueous extract concentrate B is 10 to 20% of the volume of the aqueous extract). In the invention, the method adopted by the protein removing treatment is preferably a Sevag method, and the number of times of the protein removing treatment is preferably 2-4 times, and more preferably 3 times; the Sevag reagent adopted by the Sevag method is preferably chloroform and n-butyl alcohol, and the volume ratio of the chloroform to the n-butyl alcohol is preferably (2-5) independently during each deproteinization treatment: 1, more preferably 4: 1; the volume ratio of the Sevag reagent to the material to be deproteinized is preferably (1-2) independently during each deproteinization treatment: 1, more preferably 1: 1. in the invention, the third concentration is preferably reduced pressure concentration, and the temperature of the third concentration is preferably 60-70 ℃, and more preferably 60-65 ℃; the third concentration is preferably to concentrate the deproteinized solution to be free of n-butanol.
After the water-soluble component A1 and the water-extracted component B1 are obtained, the mixed component obtained by combining the water-soluble component A1 and the water-extracted component B1 is subjected to macroporous adsorption resin column chromatography separation under the condition of gradient elution by using lower alcohol-water as an eluent, so that a flow part AB1, a flow part AB2 and a flow part AB3 are sequentially obtained, the gradient elution comprises 3 gradients which are sequentially performed, and the composition of the eluent is 0: 1. 1-7: 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 chromatography separation is preferably AB-8 macroporous adsorption resin or 101 macroporous adsorption resin, and 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, and specifically, a mixed component obtained by combining the water-soluble component A1 and the water-extracted component B1 is concentrated to remove a large amount of water and then directly loaded. 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: 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 ethnic medicine double ginseng.
After obtaining the part AB1, the invention carries out fourth concentration on the part AB1 to obtain a water elution component AB 1'. The fourth concentration is not particularly limited in the present invention, and a large amount of water in fraction AB1 may be removed.
After obtaining the part AB2, the invention sequentially carries out fifth concentration and alcohol precipitation on the part AB2 to obtain a water-soluble alcohol precipitation component AB 2'. The fifth concentration is not particularly limited, and the methanol and water in the part AB2 can be removed, and the fifth concentration is preferably to concentrate the part AB2 to 5-10% of the original volume (namely, the part AB2 is subjected to the fifth concentration to obtain a part AB2 concentrated solution, and the part AB2 concentrated solution is 5-10% of the part AB2 volume). In the invention, the alcohol precipitation reagent adopted in the alcohol precipitation is preferably an alcohol aqueous solution, and the alcohol in the alcohol aqueous solution is preferably methanol or ethanol, and more preferably ethanol; in the embodiment of the invention, industrial alcohol is specifically adopted. In the invention, the number of times of alcohol precipitation is preferably 2-4 times, and more preferably 3 times; the volume fraction of the alcohol aqueous solution subjected to alcohol precipitation at each time is preferably 90-95% independently, and more preferably 93-95%; the volume ratio of the alcohol precipitation reagent to the material to be subjected to alcohol precipitation in each alcohol precipitation is preferably (1.5-3): 1, more preferably (1.5 to 2): 1; the temperature of each alcohol precipitation is preferably 4-8 ℃ independently, more preferably 4 ℃, and in the embodiment of the invention, the alcohol precipitation is carried out in a refrigerator specifically; the time of each alcohol precipitation is preferably 12-24 hours independently, and more preferably 12-18 hours independently. In the embodiment of the invention, after each alcohol precipitation, the obtained supernatant is specifically concentrated under reduced pressure until the water-soluble components are just completely dissolved in water, so that the final concentration of ethanol in the system during the last alcohol precipitation reaches more than 90% (volume fraction), and the precipitates obtained by each alcohol precipitation are combined to obtain the water-soluble alcohol precipitation component AB 2'.
After a water eluting component AB1 'and a water-soluble alcohol precipitation component AB2' are obtained, the water eluting component AB1 'and the water-soluble alcohol precipitation component AB2' are combined to obtain the ethnic drug double ginseng water-soluble extract (marked as ethnic drug double ginseng water-soluble total extract).
In the invention, the water elution component AB1 'and the water-soluble alcohol precipitation component AB2' are preferably combined to further comprise: and carrying out graded alcohol precipitation on the obtained mixed components to obtain supernate and alcohol precipitation, wherein the supernate is subjected to sixth concentration to obtain a first ethnic medicine double ginseng water-soluble extract (marked as TGPB), and the alcohol precipitation is a second ethnic medicine double ginseng water-soluble extract (marked as TGPC). In the invention, the alcohol precipitation reagent adopted in the fractional alcohol precipitation is preferably an alcohol aqueous solution, and the alcohol in the alcohol aqueous solution is preferably ethanol; the number of times of grading alcohol precipitation is preferably 2-4 times, and more preferably 3 times; the volume fraction of the alcohol in the system is preferably 30-60% and more preferably 30% during each alcohol precipitation; the temperature of each alcohol precipitation is preferably 4-8 ℃ independently, and more preferably 5-6 ℃; the time of each alcohol precipitation is preferably 12-24 hours independently, and more preferably 15-18 hours independently. In the invention, the sixth concentration is preferably reduced pressure concentration, and the temperature of the sixth concentration is preferably 60-70 ℃, and 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 a water-soluble extract of the first ethnic medicine, namely the double ginseng; the drying is preferably freeze drying. Preferably, the alcohol precipitate is dried to obtain a water-soluble extract of the second ethnic group Chinese medicinal ginseng; the drying is preferably freeze drying.
The method provided by the invention preferably further comprises decolorization treatment, and specifically, the decolorization treatment can be carried out according to any one of the following two ways:
the first method is as follows: decolorizing the water-soluble alcohol precipitation component AB2', mixing the obtained decolorized solution with the fluid AB1, and performing seventh concentration to obtain a mixed concentrated solution; and then carrying out grading 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 a first ethnic medicine double ginseng water-soluble extract and a second ethnic medicine double ginseng water-soluble extract. In the present invention, the decoloring treatment may be performed by using a decoloring agent, or may be performed by using a macroporous adsorbent resin, which will be described below.
In the invention, when a decoloring reagent is adopted for decoloring, the water-soluble alcohol precipitation component AB2' and water are mixed with the decoloring reagent 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 decolorizing reagent preferably comprises activated carbon or hydrogen peroxide, and the mass of the activated carbon is preferably 2-5% of that of a water-soluble alcohol precipitation component AB2', and 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 a solution obtained by mixing the water-soluble alcohol precipitation component AB2' with water, and more preferably 7%. In the invention, the temperature of the decolorization treatment is preferably 50-60 ℃, and more preferably 55 ℃; when activated carbon is adopted, the time of the decoloring treatment is preferably 30-60 min, and more preferably 40 min; when hydrogen peroxide is adopted, the time of the decoloring treatment is preferably 1-3 hours, and more preferably 2 hours.
In the invention, when macroporous adsorption resin is adopted for decoloring, a water-soluble alcohol precipitation component AB2' is loaded on the macroporous adsorption resin, and alcohol-water is used as an eluant for elution to obtain a decoloring 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, the volume fraction of the alcohol in the eluent is preferably 0-60%, more preferably 0-40%, namely the volume ratio of collected water to alcohol is preferably 1: 0-4: 6, more preferably collecting the decolorized solution having a water to alcohol volume ratio of 1: 0-6: 4 time the resulting decolorized solution.
In the present invention, the seventh concentration is preferably concentration under reduced pressure; the seventh concentration is preferably such that the system obtained after mixing the decolourised solution with the fraction AB1 is concentrated until the water soluble components are just completely soluble in water.
The second method comprises the following steps: mixing the water elution component AB1 'and the water-soluble alcohol precipitation component AB2' to obtain a mixed component, carrying out graded alcohol precipitation on the mixed component, and respectively carrying out decoloration treatment on the obtained supernatant and the alcohol precipitation to obtain a first ethnic drug double ginseng water-soluble extract and a second ethnic drug double ginseng water-soluble extract; the grading alcohol precipitation and decolorization treatment method is preferably carried out according to the technical scheme, and is not repeated.
The invention provides a national medicine double ginseng water-soluble extract prepared by the preparation method of the technical scheme, which comprises water-soluble polysaccharide components and water-soluble micromolecule components, wherein the relative molecular weight range of the water-soluble polysaccharide components is 504-2.0 multiplied by 10 6 The relative molecular weight range of the water-soluble small molecules is 214-792. 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 range of the high-molecular-weight polysaccharide is preferably 5.0X 10 4 ～2.0×10 6 The relative molecular weight range of the low molecular weight polysaccharide is preferably 504-4.0 × 10 4 (ii) a The water-soluble micromolecules are preferably water-soluble iridoid glycoside compounds and/or derivatives thereof, and the water-soluble iridoid glycoside compounds preferably comprise at least one of strychnic acid, strychnic acid derivatives, strychnine and strychnine glycoside derivatives.
In the invention, the functional substances in the water-soluble extract of the first ethnic medicine, namely the double ginseng, comprise low-molecular-weight polysaccharide, water-soluble iridoid compounds and derivatives thereof; the functional substance in the water-soluble extract of the second ethnic group Chinese medicinal ginseng comprises high molecular weight polysaccharide.
The invention provides application of the national medicine double ginseng water-soluble extract in preparing a medicine or a health-care product 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 medicament is the ethnic medicine double ginseng water-soluble extract, and the pharmaceutically acceptable auxiliary materials comprise pharmaceutically acceptable carriers and/or excipients; the content of the active ingredients in the medicine is preferably 0.1-99.9 wt%, and more preferably 1-90 wt%; the pharmaceutically acceptable carrier is not particularly limited in the present invention, and pharmaceutically acceptable carriers well known to those skilled in the art may be used; the pharmaceutically acceptable excipient is not particularly limited, and pharmaceutically acceptable excipients well known to those skilled in the art can be used, and specifically, mannitol, magnesium stearate, starch or cyclodextrin can be included. In the present invention, the dosage form of the drug preferably includes an injection or an oral preparation. In the invention, when the medicine is used for preventing and treating diabetes, the effective dose of the medicine is preferably 50-200 mg, and the administration method of the medicine is preferably oral administration; when the medicine is used for preventing and treating diabetic nephropathy, the effective dose of the medicine is preferably 100mg, and the medicine is preferably taken orally.
The application of the ethnic medicine double ginseng water-soluble extract in preparing the health care product for preventing and treating diabetes or nephropathy is not particularly limited, and the method well known by the technical personnel in the field can be adopted.
The water-soluble extract of the ethnic medicine double ginseng is non-toxic to model animals and human glomerular mesangial cells, can reduce Triglyceride (TG), Total Cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) of diabetic mice, and can increase high-density lipoprotein cholesterol (HDL-C), wherein the effect of the water-soluble extract (TGPB) of the ethnic medicine double ginseng is particularly remarkable, and the water-soluble extract of the ethnic medicine double ginseng provided by the invention has certain capacity of regulating blood lipid metabolism disorder. Meanwhile, the water-soluble extract of the national medicine double ginseng can reduce the urea nitrogen (BUN) and the serum creatinine (Scr) contents of the diabetic mice, wherein the effect of TGPB is particularly remarkable, which shows that the water-soluble extract of the national medicine double ginseng provided by the invention can improve the renal function of the diabetic mice. Therefore, the ethnic medicine shuangshen water-soluble extract provided by the invention is suitable for people with diabetes and nephropathy.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Chloroform, n-butanol, ethanol and methanol used in the following experiments are industrial reagents and are used after being re-steamed.
Example 1
The method for preparing the water-soluble extract of the double ginseng by using the double ginseng as a raw material comprises the following steps:
(1) cleaning rhizome of Ginseng radix, air drying, and cutting into 1cm 3 The small blocks are used as double-ginseng samples for standby; weighing 2kg of a double ginseng sample, adding 4L of 95% ethanol aqueous solution by volume fraction, carrying out cold leaching extraction at room temperature (25 ℃), repeating for 3 times, wherein the extraction time is 24h each time, filtering out dregs after each extraction for the next extraction, taking dregs filtered out after the last extraction as alcohol extraction dregs, and combining the obtained solutions after each extraction and dregs filtering out to obtain an alcohol extract;
concentrating the alcohol extract at 50 deg.C under reduced pressure until no alcohol smell exists to obtain alcohol extract component A, and recovering ethanol; mixing the ethanol-extracted component A with 1L of distilled water to obtain an ethanol-extracted component A water solution; mixing the ethanol extraction component A water solution with n-butanol of the same volume, extracting, repeating for 3 times to obtain extraction mother liquor (namely water-soluble component A1) and n-butanol extract, and concentrating the n-butanol extract at 70 deg.C under reduced pressure to remove n-butanol to obtain ethanol extraction component A2;
adding distilled water 3 times the weight of the ethanol extraction residue into the ethanol extraction residue, heating for steam extraction, repeating for 3 times, wherein the extraction time is 2.5h each time, filtering out the residue after each extraction for the next extraction, and mixing the solutions obtained after each extraction is finished and filtering out the residue to obtain a water extract; concentrating the water extract to 2L under reduced pressure at 65 ℃ to obtain a water extract concentrated solution B; and (2) carrying out protein removal treatment on the water extract concentrated solution B by adopting a Sevag method, repeating for 3 times, wherein Sevag reagents adopted by the Sevag method are chloroform and n-butyl alcohol, and the volume ratio of the chloroform to the n-butyl alcohol is 4: 1, the volume ratio of the Sevag reagent to the water extraction concentrated solution B is 1: 1, concentrating the aqueous solution obtained after protein removal under reduced pressure at the temperature of 60 ℃ to obtain a water extraction component B1;
combining the water-soluble component A1 and the water-soluble component B1, separating by AB-8 macroporous adsorption resin column chromatography, loading a sample by a wet method, taking methanol-water as an eluent, performing gradient elution according to the volume ratio of methanol to water of 0:1, 6:4 and 9.5:0.5 in sequence, wherein the using amount of each gradient eluent is 2-3 column volumes, and detecting by thin-layer chromatography to obtain a flow part AB1, a flow part AB2 and a flow part AB3 in sequence;
concentrating the fraction AB2 under reduced pressure to 1L, mixing with industrial ethanol (ethanol volume fraction is 95% at most), precipitating with ethanol in a refrigerator at 4 deg.C for 3 times, wherein the volume of industrial ethanol used in each precipitation is 2 times of that of fraction AB2, the precipitation time is 12h, after each precipitation, concentrating the obtained supernatant under reduced pressure until the water-soluble components are just completely dissolved in water, so that the final concentration of ethanol in the system reaches above 90% (volume fraction) in the last precipitation, mixing the precipitates obtained in each precipitation to obtain water-soluble precipitation components AB2', and collecting the supernatant obtained in the last precipitation to obtain alcohol-soluble supernatant AB 3';
concentrating the fraction AB1 under reduced pressure to remove solvent to obtain water eluate AB1', and mixing the water eluate AB1' with water soluble ethanol precipitation fraction AB2' to obtain water soluble extract of radix Ginseng Indici (as water soluble total extract of radix Ginseng Indici).
Example 2
The fraction AB1 and the water-soluble alcohol-precipitated fraction AB2' were prepared according to the method of example 1;
and (2) decoloring the water-soluble alcohol precipitation component AB2' by adopting an AB-8 macroporous adsorption resin column chromatography, specifically performing gradient elution by using methanol-water as an eluent, wherein the volume ratio of collected water to methanol is 1: 0-6: 4, the obtained decolorized solution; mixing the decolorized solution with the fraction AB1, concentrating under reduced pressure until the water-soluble components are just completely dissolved in water, performing fractional alcohol precipitation with ethanol water solution at 5 deg.C, repeating for 3 times, wherein the final concentration of ethanol in the system in each alcohol precipitation process is 30% (volume fraction), and the alcohol precipitation time is 18h, to obtain supernatant and alcohol precipitation; concentrating the supernatant at 65 deg.C under reduced pressure, and freeze drying to obtain a first radix Codonopsis water soluble extract (TGPB); the alcohol precipitate is lyophilized to obtain a second water soluble extract (TGPC) of radix Ginseng Indici.
Comparative example 1
The method comprises the steps of preparing a fluid fraction AB3, an alcohol-soluble supernatant AB3 'and an alcohol-soluble component A2 according to the method of example 1, combining the fluid fraction AB3, the alcohol-soluble supernatant AB3' and the alcohol-soluble component A2, and removing a solvent by vacuum concentration at 50 ℃ to obtain a national medicine double-ginseng total alcohol extract which is marked as TGPA.
Example 3
Preparing a fluid part AB1 and a water-soluble alcohol precipitation component AB2' by using the honeysuckle flower and the holothurian as raw materials according to the method in the embodiment 1; then, the first and second water-soluble extracts of the ginseng dahua and the ginseng were prepared according to the method of example 2.
Example 4
Animal experiments: protection effect of water-soluble extract of radix codonopsis pilosulae on kidney of STZ-induced diabetic mouse
1. In vivo activity studies:
adaptively feeding male Kunming mouse with 1W, fasting overnight for 12h, and performing single intraperitoneal injection of STZ at a dose of 120 mg/kg -1 Establishing a diabetes mouse model (tail vein blood sampling for detecting Fasting Blood Glucose (FBG), if the FBG value is more than or equal to 11.1 mmol.L -1 Successfully molding); mice which are successfully modeled are taken and randomly divided into 11 groups, each group comprises 20 mice, and the grouping condition is 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 extract of radix Codonopsis Lanceolatae comprises TGPB low dose group (TGPB-L), middle dose group (TGPB-M), and high dose group (TGPB-H); TGPC low dose group (TGPC-L), middle dose group (TGPC-M), and high dose group (TGPC-H) are extracted from radix Codonopsis Lanceolatae water soluble extract. The administration is carried out by gavage every morning, and the administration dosage of Tempol group is 90mg kg -1 The high, medium and low doses of each administration group are respectively 30 mg/kg -1 、60mg·kg -1 、 120mg·kg -1 . Continuous administration of 4-6W, administration of normal group and model groupEqual volume of distilled water. After 4W and 6W administration, respectively, half of the mouse eyeballs were bled to determine Fasting plasma glucose (FBG), Serum-measured Glycated Serum Protein (GSP), Total Cholesterol (TC), Serum creatinine (Scr), Triglyceride (TG), Blood Urea Nitrogen (BUN), Low density lipoprotein Cholesterol (LDL-C) and High density lipoprotein Cholesterol (HDL-C) were isolated; after taking blood, taking out the liver, thymus, spleen and kidney after killing the animal by dislocation of cervical vertebra, weighing after absorbing water by filter paper, and calculating the index of the viscera. One kidney was loaded with 4% tissue fixative for making pathological sections for histomorphological examination. Then 0.1g of tissue is respectively taken to prepare 10% liver homogenate and 10% kidney homogenate, and the supernatant is centrifuged to determine superoxide dismutase (T-SOD), Malondialdehyde (MDA) and glutathione peroxidase (GSH-P) X ) And (4) content.
2. Results of in vivo experiments:
(1) the three active components (TGPA, TGPB and TGPC) of the ginseng radix et rhizoma Rhei and the ginseng radix et rhizoma Rhei improve the 'three more and one less' of STZ diabetic mice, reduce the fasting blood glucose level, particularly obviously reduce the fasting blood glucose level of the STZ diabetic mice by the TGPB, wherein the P of the TGPB-H group is less than 0.01, and the active components have significant difference and better activity. Specific results are shown in table 1.
TABLE 1 Effect of Bisheng active ingredients on FBG in 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 **,# |
Remarking: compared to Normal group (Normal), "x" indicates P <0.01, "x" indicates P < 0.05; in comparison to the Model set (Model), "##" indicates P <0.01, "#" indicates P < 0.05; compared to the positive control group (Tempol), ". DELTA." indicates P <0.01 and "DELTA." indicates P < 0.05. Values are expressed as means and standard deviations.
(2) The index of each organ was significantly reduced in the Model group mice compared to the Normal group. The organ indexes of mice in the Tempol group and each administration group are close to or even higher than the level of the Normal group; wherein, 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 obvious influence on the kidney of the diabetic mouse. Specific results are shown in tables 2 and 3.
TABLE 2 Effect of active fractions of Bishen on liver index and Kidney index of STZ diabetic mice: (
n＝10)
Remarking: compared to Normal group (Normal), "x" indicates P <0.01, "x" indicates P < 0.05; in comparison to the Model set (Model), "##" indicates P <0.01, "#" indicates P < 0.05; compared to the positive control group (Tempol), ". DELTA." indicates P <0.01 and "DELTA." indicates P < 0.05. Values are expressed as means and standard deviations.
TABLE 3 Effect of Bisheng active fractions on spleen index and thymus index of STZ diabetic mice ((II))
n＝10)
Remarking: compared to Normal group (Normal), "x" indicates P <0.01, "x" indicates P < 0.05; in comparison to the Model set (Model), "##" indicates P <0.01, "#" indicates P < 0.05; compared to the positive control group (Tempol), ". DELTA." indicates P <0.01 and "DELTA." indicates P < 0.05. Values are expressed as means and standard deviations.
(3) Compared to the model group, TGPB significantly reduced Glycated Serum Proteins (GSP) in the model mice. Specific results are shown in table 4 and fig. 1.
TABLE 4 active ingredient pairs of Bishen in STZ diabetic miceGSP(mmol·L -1 ) Influence of (A), (B)
n＝10)
| Group | 4W | 6W |
| Normal | 2.09±0.11 | 2.07±0.10 |
| Model | 3.02±0.09 **,△ | 3.45±0.16 **,△ |
| Tempol | 2.89±0.16 **,# | 2.88±0.10 **,# |
| TGPA-H | 2.56±0.08 **,##,△ | 2.34±0.08 *,##,△△ |
| TGPA-M | 2.99±0.03 **,#,△ | 2.71±0.01 **,##,△ |
| TGPA-L | 3.10±0.08 ** ,△ | 2.95±0.15 **,## |
| TGPB-H | 2.38±0.131 *,##,△△ | 2.29±0.121 *,##,△△ |
| TGPB-M | 2.51±0.11 *,#,△ | 2.42±0.09 *,#,△ |
| TGPB-L | 2.90±0.05 **,# | 2.71±0.14 **,## |
| TGPC-H | 2.87±0.13 **,# | 2.60±0.02 **,## |
| TGPC-M | 2.93±0.08 ** | 2.62±0.02 **,## |
| TGPC-L | 3.09±0.17 ** | 2.66±0.07 **,# |
Remarking: compared to Normal group (Normal), "×" indicates P <0.01, "×" indicates P < 0.05; in comparison to the Model set (Model), "##" indicates P <0.01, "#" indicates P < 0.05; compared to the positive control group (Tempol), ". DELTA." indicates P <0.01 and "DELTA." indicates P < 0.05. Values are expressed as mean and standard deviation.
(4) The water-soluble extract of the holothuria dichotoma can reduce the urea nitrogen (BUN) and the serum creatinine (Scr) contents of diabetic mice, wherein the effect of the TGPB-H group is obvious, which indicates that the water-soluble extract of the holothuria dichotoma can improve the renal function of the diabetic mice. Specific results are shown in table 5 and fig. 2.
TABLE 5 Effect of Bisheng active fractions on BUN and Scr in STZ diabetic mice ((S))
n＝10)
Remarking: compared to Normal group (Normal), "x" indicates P <0.01, "x" indicates P < 0.05; in comparison to the Model set (Model), "##" indicates P <0.01, "#" indicates P < 0.05; compared to the positive control group (Tempol), ". DELTA." indicates P <0.01 and "DELTA." indicates P < 0.05. Values are expressed as means and standard deviations.
(5) The ginseng can reduce Triglyceride (TG), Total Cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) of a diabetic mouse and increase high-density lipoprotein cholesterol (HDL-C), wherein the effect of TGPB-H is particularly obvious. The active components of the double ginseng have certain capacity of regulating blood lipid metabolism disorder. Specific results are shown in tables 6 and 7.
TABLE 6 Effect of Bishen active fractions on TG and TC in STZ diabetic mice (II)
n＝10)
TABLE 7 Effect of Bisheng active ingredients on HDL-C and LDL-C in STZ diabetic mice: (
n＝10)
Remarking: compared to Normal group (Normal), "x" indicates P <0.01, "x" indicates P < 0.05; in comparison to the Model set (Model), "##" indicates P <0.01, "#" indicates P < 0.05; compared to the positive control group (Tempol), ". DELTA." indicates P <0.01 and "DELTA." indicates P < 0.05. Values are expressed as means and standard deviations.
(6) The components of the ginseng can increase the activity of T-SOD and GSH-PX of the liver of a diabetic mouse and reduce the MDA content, wherein the oxidation resistance of the TGPB-H group is only inferior to that of a positive control Tempol group. Specific results are shown in tables 8 and 9.
TABLE 8 Effect of active fractions of Ginseng radix and Ginseng radix on T-SOD and MDA in STZ diabetic mice liver: (
n＝10)
TABLE 9 active fractions of Ginseng radix and Ginseng radix for STZ diabetic mouse liver GSH-PX (mmol. L) -1 ) Influence of (A), (B)
n＝10)
Remarking: compared to Normal group (Normal), "x" indicates P <0.01, "x" indicates P < 0.05; in comparison to the Model set (Model), "##" indicates P <0.01, "#" indicates P < 0.05; compared to the positive control group (Tempol), "Δ" indicates P <0.01 and "Δ" indicates P < 0.05. The values are expressed as mean and standard deviation
(7) The active components of radix Ginseng Indici can increase T-SOD and GSH-P of kidney of diabetic mouse X The activity is reduced, the MDA content is reduced, and a concentration dependence relationship exists, wherein the TGPB-H group has the most obvious effect, and the oxidation resistance is only inferior to that of a positive control Tempol group. The specific results are shown in table 10, table 11 and fig. 3.
TABLE 10 Effect of active fractions of Ginseng radix and Ginseng radix on Kidney T-SOD and MDA in STZ diabetic mice: (
n＝10)
TABLE 11 active ingredient of Bishen on kidney GSH-P of STZ diabetic mice X (mmol·L -1 ) Influence of (A), (B)
n＝10)
| Group | 4W | 6W |
| Normal | 73.34±2.68 | 73.22±3.50 |
| Model | 52.77±1.17 **,△△ | 51.01±2.05 **,△△ |
| Tempol | 67.82±2.93 *,## | 68.51±2.08 *,## |
| TGPA-H | 59.55±2.10 **,#,△ | 60.54±3.11 **,##,△ |
| TGPA-M | 57.67±1.12 **,#,△ | 56.64±2.01 **,#,△ |
| TGPA-L | 51.60±2.30 **,△△ | 52.60±1.07 **,△△ |
| TGPB-H | 61.49±2.03 *,##,△ | 62.31±1.50 *,##,△ |
| TGPB-M | 59.67±1.17 **,#,△ | 60.60±2.15 **,#,△ |
| TGPB-L | 53.65±2.01 **,△△ | 53.36±1.10 **,△△ |
| TGPC-H | 54.14±1.52 **,△△ | 55.25±2.40 **,#,△ |
| TGPC-M | 53.57±2.70 **,△△ | 52.53±1.06 **,△△ |
| TGPC-L | 51.66±3.50 **,△△ | 51.61±2.04 **,#,△△ |
Remarking: compared to Normal group (Normal), "x" indicates P <0.01, "x" indicates P < 0.05; in comparison to the Model set (Model), "##" indicates P <0.01, "#" indicates P < 0.05; compared to the positive control group (Tempol), ". DELTA." indicates P <0.01 and "DELTA." indicates P < 0.05. Values are expressed as means and standard deviations.
(8) Results of in vivo experiments
(a) TGPB can improve kidney index of STZ diabetic mice; the results of liver, spleen and thymus index are combined, and TGPC is found to have a certain function of improving the immunity.
(b) The active components of the ginseng 3 can improve more than three and one less than three of STZ diabetic mice to a certain extent, and reduce the FBG level; can improve the oxidation resistance of liver and kidney, and has the effects of improving the activity of T-SOD and GSH-Px and reducing the content of MDA; the active components of the double ginseng can correct blood sugar and blood fat disorders to a certain extent, and show that the levels of GSP, TC, TG and LDL-C in serum tend to be reduced, so that the content of HDL-C is influenced.
(c) TGPB can improve renal function to a certain extent, and is expressed in reducing Scr and BUN content.
(9) Pathological section results
Fig. 4 is an optical micrograph (400 times magnification) of a mouse kidney section, and it can be seen from fig. 4 that there is no new change in the kidney structure in the "normal group (Control)", there is no proliferation of mesangial cells and stroma in the glomerular capsule, there is no significant change in the volume of the glomerulus, there is no edema in the tubular epithelial cells, and there is no fibrosis and mononuclear infiltration in the renal interstitium.
The ' Model group ' (Model) ' glomerulus volume is obviously increased, local focus of mesangial cells and stroma, segmental hyperplasia, renal tubular epithelial cell edema and renal interstitium are not obviously changed.
In the treatment groups (TGPA, TGPB, TGPC) -high (H), medium (M) and low (L) dose groups, the glomerular volume of the high dose (-H) is reduced, the proliferation of mesangial cells and stroma local lesions is reduced, and the edema of renal tubular epithelial cells is reduced, compared with the model group. Consistent with the positive control (Tempol) group. The results show that the active components of the double ginseng 3 have a protective effect on the kidney of a model mouse, and particularly the high-dose component of the TGPB has an excellent effect.
3. And (4) conclusion:
(1) the active components of the ginseng, namely 3, all improve more than three and one less than three of STZ diabetic mice, reduce the fasting blood glucose level and the Glycated Serum Protein (GSP), and particularly, the water-soluble component TGPB of the ginseng obviously reduces the fasting blood glucose level and the Glycated Serum Protein (GSP) of the STZ diabetic mice.
(2) The active components of the double ginseng have certain kidney protection effect on STZ diabetic mice, and the mechanism of the active components of the double ginseng is probably related to the effect that the active components of the double ginseng can improve the oxidation resistance of the kidney and the liver of the STZ diabetic mice, regulate glycolipid metabolic disorder and improve kidney functions.
Example 5
Cell experiments: protection effect of water-soluble extract of radix Ginseng Indici on human glomerular mesangial cells cultured with high sugar
1. High sugar modeling: the MTT method is adopted to detect the proliferation condition of HMC cells stimulated by high sugar, and the result shows that: culturing the cells for 12-72 h, wherein a culture medium containing 25mmol/L, 27.5mmol/L, 30mmol/L and 32.5mmol/L glucose is in an obvious proliferation state for HMC cells after 12-48 h, and the cells grow to the utmost point after 48 h; the culture medium containing 30mmol/L glucose has the most obvious proliferation, and when the cells are cultured for 64-72 h, the cells are in a slow growth state. Therefore, the HMC cells were cultured for 48h in 30mmol/L high-sugar medium as the best model for high-sugar stimulation of HMC cells. The results are shown in fig. 5, in particular, where, * P﹤0.05， ** P﹤0.01。
2. component pair cytotoxicity assay: the water-soluble extract of the ginseng radix sophorae flavescentis has no toxicity to human mesangial cells within the concentration range of 50-800 mu g/mL, so that the sugar-stimulated Human Mesangial Cell (HMC) proliferation inhibition effect is further verified by adopting the active components of the ginseng radix sophorae flavescentis within the concentration range. The specific results are shown in FIG. 6.
3. Cell experiments: detecting the effect of Ginseng radix extract on inhibiting proliferation of Human renal glomerulus Cell (HRMC) induced by high glucose by MTT method, uniformly seeding HRMC Cell in 96-well plate to make Cell density of 2.0 × 10 4 Per well; and after the cells adhere to the wall, changing a serum-free culture medium for continuous culture for 24 hours, adding 100 mu L of a normal sugar culture medium, 100 mu L of a high-sugar complete culture medium and administration groups with different concentrations according to groups, and continuously culturing for 48 hours after each group of 5 compound holes. 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 MSO was added, and after shaking the plate for 2min, the OD was measured at 490 nm. Setting a normal control group (NG, containing 5.5 mmol. multidot.L) -1 Glucose), high saccharide group (HG, containing 30 mmol. multidot.L) -1 Glucose), Tempol group (100. mu. u.)mol·L -1 ) And active components TGPA, TGPB and TGPC of Ginseng radix, and three medicinal components (50 μ g. L) -1 、 200μg·L -1 、800μg·L -1 ) Group (d); the drug intervention time was 48h, and the precipitated cells were obtained by repeated centrifugation and subsequently disrupted by sonication. Detecting the influence of each experimental group on the proliferation and the cell activity of the HMC cells cultured by high sugar by using an MTT method; then, ROS content detection is carried out, and the content of T-SOD and MDA in the supernatant of each group of HRMC cells is determined by adopting a biotin-linked immunosorbent assay (ELISA).
(1) Inhibition of high-sugar induced HRMC cell proliferation by active components of radix Ginseng Indici
The water-soluble extract of radix Ginseng Indici has no cytotoxicity, and TGPB and TGPC have certain inhibiting effect on high sugar induced HRMC proliferation. Specific results are shown in fig. 7(TGPA corresponds to TGA, TGPB corresponds to TGB, TGPC corresponds to TGC), wherein "×" indicates P <0.01, "×" indicates P < 0.05; in comparison to the high saccharide group (HG), "###" indicates P <0.01, and "#" indicates P < 0.05.
(2) Compared with the NG (P <0.05) group, the HG (P <0.05) has obviously increased intracellular MDA, the TGPB (P <0.05) has better effect than other administration groups, and certain drug concentration dependence appears.
And NG (P)<0.05) compared with the group, the content of T-SOD in HRMC cells induced by high sugar is obviously reduced, the T-SOD content of each dose group of TGPA and TGPB is increased compared with that of HG group, and only the highest dose group of TGC-800(800 mug. L) is included in the TGPC group -1 ，P<0.05) slightly increased back, and the other two dose groups were almost identical in content to the HG group. The specific results are shown in Table 12.
TABLE 12 Effect of active fractions of Ginseng radix and Ginseng radix on MDA and T-SOD levels 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 * |
Remarking: compared to the normal sugar group (NG), "x" indicates P <0.01, "x" indicates P < 0.05; in comparison to the high saccharide group (HG), "###" indicates P <0.01, and "#" indicates P < 0.05.
(3) Detection of ROS content in high-sugar-induced HRMC (high-glucose inducible protein kinase) cells after action of active components of double-ginseng
The three active components of the double ginseng are compared with the reduction degree of the ROS in the high-sugar induced HRMC cells: TGPB-800, TGPB-200, TGPA-800, TGPA-50, TGPB-50, TGPC-800, TGPC-200, TGPC-50, therefore, the influence of TGPB on the 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 results are shown in Table 13.
TABLE 13 Effect of active fractions of Bishen on ROS levels in high-sugar induced HRMC cells ((R))
n＝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 * |
Remarking: compared to the normal sugar group (NG), "×" indicates P <0.01, "×" indicates P < 0.05; in comparison to the high saccharide group (HG), "###" indicates P <0.01, and "#" indicates P < 0.05.
(4) As a result, it was found that: the water-soluble extract of radix Codonopsis Lanceolatae increases T-SOD in high sugar cultured HRMC cell, and reduces the content of MDA and ROS; especially, TGPA and TGPB have protective effect on human mesangial cells cultured with high sugar, and the mechanism of the TGPA and TGPB is probably related to the capability of improving the antioxidant capacity of HRMC cells.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for preparing a water-soluble extract of national medicine, shuangshen, comprises the following steps:
performing alcohol extraction on the national medicine double ginseng to obtain alcohol extract and alcohol extraction dregs;
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, carrying out second concentration on the obtained water extract to obtain a water extraction concentrated solution B, carrying out deproteinization treatment on the water extraction concentrated solution B, and carrying out third concentration on the obtained deproteinized solution to obtain a water extraction component B1;
using lower alcohol-water as an eluent, carrying out macroporous adsorption resin column chromatography separation on a mixed component obtained by combining the water-soluble component A1 with the water-extracted component B1 under a gradient elution condition to obtain a flow part AB1, a flow part AB2 and a flow part AB3 in sequence, wherein the gradient elution comprises 3 gradients which are carried out in sequence, and the composition of the eluent is 0: 1. 1-7: 9 to 3 and 9.5:0.5, corresponding to fraction AB1, fraction AB2 and fraction AB3, respectively;
subjecting the fraction AB1 to fourth concentration to obtain water eluent AB 1'; sequentially carrying out fifth concentration and alcohol precipitation on the fraction AB2 to obtain a water-soluble alcohol precipitation component AB 2'; and mixing the water elution component AB1 'with the water-soluble alcohol precipitation component AB2' to obtain the ethnic drug-containing water-soluble extract of the ginseng.
2. The method according to claim 1, wherein the ethnic group ginseng is ginseng radix or ginseng radix et rhizoma Rhei Palmati.
3. The preparation method of claim 1, wherein the alcohol extraction is cold leaching extraction, an alcohol extraction reagent adopted in the alcohol extraction is an ethanol water solution, and the volume fraction of the ethanol water solution is 60-95%.
4. The method according to claim 1, wherein the deproteinizing treatment is carried out by Sevag method.
5. The preparation method of claim 1, wherein the macroporous adsorption resin used for the macroporous adsorption resin column chromatography separation is AB-8 macroporous adsorption resin or 101 macroporous adsorption resin.
6. The preparation method according to any one of claims 1 to 5, wherein the water eluting component AB1 'and the water soluble alcohol precipitation component AB2' are combined to further comprise: and carrying out graded 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, wherein the alcohol precipitation is a second ethnic medicine double ginseng water-soluble extract.
7. The water-soluble extract of national herb ginseng prepared by the preparation method of any one of claims 1 to 6, which comprises a water-soluble polysaccharide component and a water-soluble small molecular component, wherein the relative molecular weight of the water-soluble polysaccharide component is in the range of 504 to 2.0 x 10 6 The relative molecular weight range of the water-soluble small molecules is 214-792.
8. The water-soluble extract of national herb of the genus Panax as claimed in claim 7, wherein the water-soluble polysaccharides component comprises high molecular weight polysaccharides and low molecular weight polysaccharides, and the relative molecular weight of the high molecular weight polysaccharides is in the range of 5.0X 10 4 ～2.0×10 6 The relative molecular weight range of the low molecular weight polysaccharide is 504-4.0 multiplied by 10 4 (ii) a The water-soluble micromolecules are water-soluble iridoid glycoside compounds and/or derivatives thereof.
9. The use of the water-soluble extract of the ethnic medicine salvia miltiorrhiza bunge as claimed in claim 7 or 8 in the preparation of a medicine or health product for preventing and treating diabetes or nephropathy.
10. The use according to claim 9, wherein the diabetes is type II diabetes; the nephropathy is at least one of diabetic nephropathy, acute renal failure, chronic renal failure, nephrotic syndrome and glomerulonephritis.
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