CN107349244B - Extraction method of malonyl ginsenoside - Google Patents
Extraction method of malonyl ginsenoside Download PDFInfo
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- CN107349244B CN107349244B CN201710763361.5A CN201710763361A CN107349244B CN 107349244 B CN107349244 B CN 107349244B CN 201710763361 A CN201710763361 A CN 201710763361A CN 107349244 B CN107349244 B CN 107349244B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
- A61K36/258—Panax (ginseng)
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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
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
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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
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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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
Abstract
The invention provides an extraction method of malonyl ginsenoside and application in treating diabetic nephropathy, which takes fresh ginseng or American ginseng as a raw material, and comprises the steps of enzymolysis treatment by cellulase, ultrasonic extraction, decompression recovery of a solvent, concentration to obtain an aqueous solution, separation by NKA-12 adsorption resin, gradient elution by ethanol and phosphate buffer salt solution, and desalting by sephadex to obtain the malonyl ginsenoside. The method effectively utilizes cellulase to degrade cell walls of ginseng and American ginseng, thereby improving the extraction rate of malonyl ginsenoside, and has the characteristics of simple and rapid operation, low cost, high extraction efficiency and the like; animal experiments prove that the malonyl ginsenoside has remarkable prevention and treatment effects on type 2 diabetic nephropathy, and can be applied to preparation of health food or medicines.
Description
Technical Field
The invention relates to a preparation method of a traditional Chinese medicine extract and application thereof in preparing a medicament, in particular to an extraction method of malonyl ginsenoside, and also provides application thereof in preparing medicaments for preventing and treating diabetic nephropathy, belonging to the technical field of traditional Chinese medicines.
Background
Diabetic Nephropathy (DN) is one of the most serious complications of diabetes and also one of the leading causes of death in clinical diabetic patients. Approximately 20% to 40% of patients with type i or type ii diabetes have been reported to develop nephropathy; in China, the incidence of diabetic nephropathy is about 40%. The onset of diabetic nephropathy is a result of a combination of multiple factors: sugar metabolism disorders, alterations in renal hemodynamics, various cytokines, and genetic background play a very important role. When persistent proteinuria clinically occurs, the kidney lesion is not early, irreversible lesion of the kidney occurs, and no effective measure exists so far to prevent the occurrence and the development of the kidney lesion. At present, the treatment measures for diabetic nephropathy mainly include: control of blood glucose, control of blood pressure, restriction of protein intake, control of blood lipids, dialysis, kidney transplantation, etc. However, these treatments have unsatisfactory results and are not effective in inhibiting the development of diabetic nephropathy.
The ginseng and the American ginseng are the most valuable Chinese herbal medicines in China, have various biological activities and have long-term medicine in ChinaIt has effects in tonifying body and protecting health. Ginsenoside is the main ingredient of the two medicinal plants and can be divided into neutral saponin (ginsenoside-Rb)1, -Rb2 , -Rc, -Rd, -Rg1, -Re, -Rg3, -Rg2, -Rh1Etc.) and acidic saponins (malonyl-ginsenosides)-Rb1, -Rb2, -Rc, -Rd, -Rg1and-Re, etc.). More than 100 ginsenosides have been isolated and identified from ginseng and American ginseng. Malonyl ginsenosides are acidic saponins with a melting point (mp) of typically 150~At 161 deg.C, it is easily soluble in water and methanol, insoluble in ethanol and n-butanol, and insoluble in chloroform and diethyl ether. The content of malonyl ginsenoside is very high, and accounts for about 50% of total ginsenoside in ginseng, however, because of strong polarity of the ginsenoside, the ginsenoside is difficult to separate and identify, and the research on chemical components and pharmacological activity of the ginsenoside is less reported at present.
At present, a great number of reports relating to the treatment of diabetic nephropathy by ginseng and American ginseng have been reported. The ginseng has a protective effect on renal damage caused by oxidative stress of diabetic rats or AGEs formation; ginseng not only reduces the blood glucose and glycosylation protein levels in diabetic rats, but also reduces creatinine and urine protein levels (biol. pharm. Bull.2016,29(8): 1678-. The American ginseng leaf 20 s-protopanaxadiol saponin can reduce the blood sugar of a diabetic nephropathy rat, reduce the matrix of kidney cells, reduce the proliferation of mesangial cells, protect the ultrastructure of the kidney cells and improve the pathological damage of the kidney caused by the diabetes of the rat, and the mechanism of the panaxadiol saponin can be related to the function of reducing the GLUT-1 of the kidney (the Proc. Gillen university: medical edition, 2007, 33 (5): 845, 848.) the J. pathophysiology of China, 2008,24 (6): 1237, 1239). Monomeric ginsenoside Rb1Can improve the kidney function of DN rat and reduce the pathological damage of kidney, and the specific mechanism of the polypeptide can be related to the down regulation of MCP-1 mRNA and protein expression level of rat kidney tissue. (J.Reptile & Kidney disease, 2008(7): 578-. Ginsenoside Rg1Can reduce the levels of TNF-alpha, ED-1, TGF-beta 1 and MCP-1, improve the pathological damage of podocytes and kidneys of diabetic nephropathy rats and can showThe composition can reduce 24h urine protein and blood creatinine of diabetic rats and has certain kidney protection effect (the Proc. Sichuan university: medical edition 2009, 40 (3): 466-. Ginsenoside Rg3Can significantly reduce the blood sugar, the blood creatinine and the 24h urine protein of the diabetic rat, reduce the thickening degree of the glomerular basement membrane, reduce the accumulation of extracellular matrix and improve the pathological damage of the kidney of the diabetic rat. (progress in modern biomedicine 2014, 36 7015 and 7018).
However, no research report on the therapeutic effect of malonyl ginsenoside on diabetic nephropathy has been found so far.
In the preparation of malonyl ginsenoside, chinese patent application No. 200510016845.0 discloses a method for preparing malonyl ginsenoside from fresh ginseng, which comprises cold sulfur extraction with high concentration ethanol, extraction with water saturated n-butanol, and separation with macroporous resin. However, most of the effective components in ginseng are wrapped by cell walls rich in cellulose, so that the extraction efficiency of ginsenoside is low and the extraction time is long. In addition, because malonyl ginsenoside is an acidic saponin, it is difficult to separate malonyl ginsenoside from neutral saponin using a conventional ethanol-water solvent system, resulting in low purity and low yield of the preparation.
Disclosure of Invention
The invention discloses a method for extracting and purifying malonyl ginsenoside, which solves the defects of low extraction rate, difficult separation and purification, low yield of high-purity malonyl ginsenoside and the like of the existing preparation process.
The invention further provides the medical application of the malonyl ginsenoside in treating diabetes and nephropathy caused by the diabetes, the cell walls of ginseng and American ginseng can be enzymolyzed by cellulase, the malonyl ginsenoside can be efficiently and green extracted and purified, and the technical support is provided for the development and utilization of the effective components of ginseng and American ginseng.
The invention relates to a method for extracting and purifying malonyl ginsenoside, which comprises the following steps:
1) cutting fresh Ginseng radix or radix Panacis Quinquefolii, and adding 1-10 times of distilled water;
2) adding cellulase accounting for 1-20% of the mass of the sample, and oscillating in a water bath at a constant temperature of 20-60 ℃ for 1-12 h;
3) adding ethanol and water after enzymolysis to make ethanol concentration reach 0-80%, ultrasonic extracting for 3-5 times, each time for 0.5-4 hr, with 10-50 times of extraction solvent;
4) recovering solvent from the extractive solution under reduced pressure at 40 deg.C, and concentrating to obtain water solution;
5) separating the extract obtained in the step 4) by NKA-12 adsorption resin, performing gradient elution by using ethanol and phosphate buffer salt solution as eluent, respectively collecting elution fractions, detecting by adopting a thin-layer chromatography, collecting and combining the fractions with the same Rf value as the malonyl ginsenoside positive reference substance, recovering the solvent under reduced pressure, desalting the concentrated solution by using sephadex, and freeze-drying to obtain malonyl ginsenoside powder; the purity of the malonyl ginsenoside is above 70%, and the yield can reach 60%.
The malonyl ginsenoside preparation method of the invention preferably comprises the following steps (1): cutting fresh Ginseng radix or radix Panacis Quinquefolii, adding 3-5 times of distilled water; cellulase was then added at 5% of the sample mass.
The preparation method of the malonyl ginsenoside preferably comprises the following step (2): the temperature of the cellulase enzymolysis is 40 ℃, and the constant temperature oscillating water bath is carried out for 4 h.
The preparation method of the malonyl ginsenoside preferably comprises the following step (3): adding ethanol and water after enzymolysis to make the concentration of ethanol reach 0-20% and the dosage of solvent be 20-30 times, and then extracting for 3 times with ultrasound, each time for 1 h.
The preparation method of the malonyl ginsenoside preferably comprises the following step (5): the phosphate buffered saline solution is selected from: NaH2PO4、 KH2PO4、Na2HPO4、K2HPO4。
A medical application of malonyl ginsenoside in preparing medicine for treating diabetes is provided.
A medical application of malonyl ginsenoside in preparing medicine for treating nephropathy caused by diabetes is disclosed.
The invention also provides a malonyl ginsenoside preparation with strong pharmacological activity.
The medicine of malonyl ginsenoside of the invention contains the extract with effective treatment amount as the active component and one or more pharmaceutically acceptable carriers.
In addition, it should be expressly noted that one or more natural or synthetic additional ingredients which have a synergistic or auxiliary effect on the active substances may be added to the pharmaceutical combinations of the invention as desired, these possible additional natural or synthetic auxiliary ingredients being known and conceivable to the person skilled in the art. The composition can be formulated into oral dosage forms, such as tablet, pill, granule, capsule, powder, etc.
For the preparation of tablets, pills, granules, capsules and the like suitable for oral administration, sucrose, galactose, corn starch, gelatin, microcrystalline cellulose, talc and the like may be used as carriers or excipients. In these orally administered dosage forms, suitable other additives such as disintegrants, lubricants, fillers, binders, flavoring agents, preservatives, dispersants, surfactants, coloring agents, and the like may also be included.
The most preferred route of administration of the pharmaceutical compositions of the invention is in various orally administered dosage forms, such as tablets, granules, capsules or pills, which generally contain 10 to 1000mg of the active substance per daily dose.
The invention has the positive effects that: the new method is adopted to extract and purify malonyl ginsenoside in fresh ginseng and American ginseng. Compared with the existing preparation process, the extraction rate of malonyl ginsenoside can be increased by 1 time by adopting the method of cellulase enzymolysis extraction; meanwhile, the NKA-12 adsorption resin and an ethanol-phosphate buffer salt elution system are adopted in the separation and purification process, so that the defect that the separation of the malonyl ginsenoside and the neutral saponin is difficult is overcome, and the purity and the yield of the malonyl ginsenoside are improved. The invention fully utilizes ginseng resources, extracts and enriches malonyl ginsenoside to the maximum extent, achieves the aims of simple, quick, environment-friendly and low-cost enrichment, and provides a method guarantee for industrial production and new drug preparation.
Drawings
FIG. 1: influence of enzymolysis temperature on the extraction rate of malonyl ginsenoside;
FIG. 2: influence of enzyme dosage on extraction rate of malonyl ginsenoside;
FIG. 3: influence of enzymolysis time on the extraction rate of malonyl ginsenoside;
FIG. 4: influence of ethanol concentration on the extraction yield of malonyl-ginsenoside.
Detailed Description
The present invention is further illustrated by the following examples, which do not limit the present invention in any way, and any modifications or changes that can be easily made by a person skilled in the art to the present invention will fall within the scope of the claims of the present invention without departing from the technical solution of the present invention.
Example 1
Cutting fresh Ginseng radix (fibrous root, rootlet, rhizome, main root, etc.) as raw material, adding 2 times of distilled water; then adding cellulase accounting for 1 percent of the mass of the ginseng sample, and oscillating the water bath at the constant temperature of 20 ℃ for 2 hours. Adding appropriate amount of distilled water after enzymolysis to make the amount of solvent 10 times, and performing ultrasonic extraction for 3 times, each time for 0.5 h. Recovering solvent from the extractive solution under reduced pressure at 40 deg.C, and concentrating to obtain water solution; then separating with NKA-12 adsorbent resin, eluting with ethanol and 0.01mol/L K2HPO4Gradient eluting, respectively collecting eluate, detecting by thin layer chromatography, collecting and mixing eluate solutions with same Rf value as malonyl ginsenoside positive control, recovering solvent under reduced pressure, desalting the concentrated solution with dextran gel, and freeze drying to obtain malonyl ginsenoside powder.
Example 2
Cutting the fibrous root, branch root and main root of fresh ginseng, and adding 3 times of distilled water; then adding cellulase accounting for 5 percent of the mass of the ginseng sample, and oscillating the water bath at the constant temperature of 40 ℃ for 4 hours. After enzymolysis, addAdding appropriate amount of ethanol and water to make ethanol concentration 20% and solvent amount 30 times, and ultrasonic extracting for 4 times, each time for 1 hr. Recovering solvent from the extractive solution under reduced pressure at 40 deg.C, concentrating to obtain water solution, and separating with NKA-12 adsorbent resin to obtain eluate containing ethanol and 0.02mol/L KH2PO4Gradient eluting, respectively collecting eluate, detecting by thin layer chromatography, collecting and mixing eluate solutions with same Rf value as malonyl ginsenoside positive control, recovering solvent under reduced pressure, desalting the concentrated solution with dextran gel, and freeze drying to obtain malonyl ginsenoside powder.
Example 3
Cutting fresh radix Panacis Quinquefolii, and adding 5 times of distilled water; then adding cellulase accounting for 10 percent of the mass of the American ginseng sample, and oscillating the mixture in a water bath at the constant temperature of 60 ℃ for 6 hours. Adding appropriate amount of ethanol and water after enzymolysis to make ethanol concentration 40% and solvent amount 50 times, and performing ultrasonic extraction for 5 times, each time for 2 hr. Recovering solvent from the extractive solution under reduced pressure at 40 deg.C, and concentrating to obtain water solution; then separating with NKA-12 adsorbent resin, eluting with ethanol and 0.05mol/L Na2HPO4Gradient eluting, respectively collecting eluate, detecting by thin layer chromatography, collecting and mixing eluate solutions with same Rf value as malonyl ginsenoside positive control, recovering solvent under reduced pressure, desalting the concentrated solution with dextran gel, and freeze drying to obtain malonyl ginsenoside powder.
Example 4
Cutting fresh radix Panacis Quinquefolii rhizome, and adding 1 times of distilled water; then adding cellulase accounting for 20 percent of the mass of the American ginseng sample, and oscillating the mixture in water bath at constant temperature of 30 ℃ for 8 hours. Adding appropriate amount of ethanol and water after enzymolysis to make the concentration of ethanol 60% and the dosage of solvent 20 times, and then extracting for 3 times with ultrasound, each time for 4 h. Recovering solvent from the extractive solution under reduced pressure at 40 deg.C, and concentrating to obtain water solution; then separating with NKA-12 adsorbent resin, eluting with ethanol and 0.03mol/L NaH2PO4Gradient eluting, respectively collecting eluate, detecting by thin layer chromatography, collecting and mixing eluate with same Rf value as malonyl ginsenoside positive controlRecovering solvent under reduced pressure, desalting the concentrated solution with dextran gel, and freeze drying to obtain malonyl ginsenoside powder.
Example 5
Cutting stems, leaves, buds and fruits of Ginseng radix, and adding 5 times of distilled water; then adding cellulase accounting for 3 percent of the mass of the ginseng sample, and oscillating the water bath at the constant temperature of 35 ℃ for 5 hours. Adding appropriate amount of ethanol and water after enzymolysis to make ethanol concentration 5% and solvent amount 40 times, and performing ultrasonic extraction for 5 times, each time for 2 hr. Recovering solvent from the extractive solution under reduced pressure at 40 deg.C, and concentrating to obtain water solution; then separating with NKA-12 adsorbent resin, eluting with ethanol and 0.04mol/L KH2PO4Gradient eluting, respectively collecting eluate, detecting by thin layer chromatography, collecting and mixing eluate solutions with same Rf value as malonyl ginsenoside positive control, recovering solvent under reduced pressure, desalting the concentrated solution with dextran gel, and freeze drying to obtain malonyl ginsenoside powder.
Example 6
Cutting fresh stems, leaves, buds and fruits of Panax quinquefolium L, and adding 2 times of distilled water; then adding cellulase accounting for 8 percent of the mass of the American ginseng sample, and oscillating in a water bath at the constant temperature of 45 ℃ for 3 hours. Adding appropriate amount of ethanol and water after enzymolysis to make ethanol concentration 15% and solvent amount 30 times, and performing ultrasonic extraction for 3 times, each time for 1 hr. Recovering solvent from the extractive solution under reduced pressure at 40 deg.C, and concentrating to obtain water solution; then separating with NKA-12 adsorbent resin, eluting with ethanol and 0.1mol/L NaH2PO4Gradient eluting, respectively collecting eluate, detecting by thin layer chromatography, collecting and mixing eluate solutions with same Rf value as malonyl ginsenoside positive control, recovering solvent under reduced pressure, desalting the concentrated solution with dextran gel, and freeze drying to obtain malonyl ginsenoside powder.
Example 7
Taking 1kg of malonyl total saponins prepared by the scheme 1, the scheme 2 or the scheme 5, adding 10kg of medical cyclodextrin serving as an excipient, a proper amount of dry starch, a proper amount of carboxymethyl cellulose, a proper amount of talcum powder and a proper amount of cane sugar. The sucrose and the talcum powder are used for coating sugar, mixed evenly, granulated and tabletted to obtain tablets with the weight of 0.50g and each tablet contains 100mg of malonyl total saponin.
Example 8
Taking 1kg of the malonyl total saponin prepared in the scheme 3, the scheme 4 or the scheme 6, adding 20kg of corn starch and a proper amount of cyclodextrin, uniformly mixing, and encapsulating, wherein each tablet contains 100mg of the malonyl total saponin.
In order to further illustrate the extraction and purification process of malonyl ginsenoside and the application of the malonyl ginsenoside in treating diabetic nephropathy in the method of the present invention, the following experiments further demonstrate the preparation process and pharmacological effects of the drug of the present invention:
test example 1
1 test materials and reagents
Fresh ginseng (Panax ginseng C.a.meyer) five-year old roots, purchased in the pacifying panacea market; cellulase was purchased from boao biotechnology limited, shanghai; STZ is a Sigma product, and the blood sugar detection kit is purchased from Beijing Lidman Biochemical technology GmbH; the microalbuminuria albuminase detection-free kit is purchased from Nanjing Xinfan Biotech limited. Ginsenoside Rb1、Rb2Rc, Rd, Re and Rg1Purchased from the institute of food and drug testing, China; malonyl ginsenoside-Rb1、-Rb2and-Rc and-Rd are prepared with the purity of 99 percent by HPLC, NMR, MS and the like. Chromatographic grade acetonitrile (Fisher corporation, USA), other reagents for analytical purification.
2 laboratory animals
Healthy SD rats 100, male, with a weight of 200- & 220g, were provided by the Experimental animals center of Jilin university.
Experimental methods
2.1 treatment of Ginseng samples with cellulase
After fresh ginseng roots are cut up, a plurality of 5g samples are precisely weighed and placed in a 250ml triangular flask, and the influence of the temperature (20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃), the enzyme adding amount (0, 1%, 5%, 10%, 20%), the enzymolysis time (1 h, 2h, 4h, 6h, 8 h) and the ethanol concentration (0, 20%, 40%, 60%, 80%) on the extraction rate of malonyl ginsenoside is respectively considered.
2.2 preparation of Standard solutions
Precisely weighing appropriate amount of each monomer ginsenoside standard, placing in the same volumetric flask, adding 80% ethanol for dissolving and fixing volume, preparing into ginsenoside reference substance with concentration of 1 mg/ml, and shaking to obtain mixed standard stock solution. Precisely sucking 0.2 ml of stock solution, placing in a 1ml volumetric flask, diluting with 80% ethanol water solution to scale to obtain mixed reference solution, and storing at 4 deg.C.
2.3 preparation of test samples to be tested
Carrying out enzymolysis treatment on a fresh ginseng sample by cellulase, carrying out ultrasonic extraction for 3 times, extracting for 0.5h each time, concentrating under reduced pressure at 40 ℃, then using 80% methanol to be dissolved in a 25mL volumetric flask, shaking up, passing through a 0.45 mu m filter membrane, and using for HPLC quantitative analysis.
2.4 chromatographic conditions
Chromatographic column Cosmosil 5C18MS (250 mm. times.4.6 mm, 5 μm); mobile phase: acetonitrile (A) -0.05 mol/l potassium dihydrogen phosphate aqueous solution (B); elution gradient: 0-20 min, 22% A; 20-25 min, 22% -29% of A; 25-45 min, 29% A; 45-55 min, 29-35% A; 55-60 min, 35-50% A; column temperature: 25 ℃; detection wavelength: 203 nm; flow rate: 1 ml/min; sample introduction amount: 20 mu l of the mixture; analysis time: and (5) 60 min.
2.5 drawing of Standard Curve
Precisely sucking appropriate amount of stock solution of Ginseng radix reference, diluting to 7 mixed reference solutions with different concentrations, precisely sucking 20 μ l, performing sample injection analysis according to the above conditions, recording peak area of each peak, and taking peak areaYTo mass concentrationX (mg/ml) linear regression is carried out, the linear relation of each monomer saponin in the range of 0.010 mg/ml-0.640 mg/ml is good, and a regression equation, a linear range and a correlation coefficient are obtained.
2.6 purification of malonyl-ginsenosides
Extracting the ginseng sample treated by the cellulase by an ultrasonic method, concentrating under reduced pressure at 40 ℃, separating the concentrated solution by NKA-12 adsorption resin, and inspecting the separation and purification of malonyl saponin by different solvent systems (ethanol-water and ethanol-potassium dihydrogen phosphate aqueous solution).
2.7 Effect of malonyl-ginsenoside on type 2 diabetic nephropathy rats
SD rats, male, after 5 days of adaptive balance rearing, were used for the experiments. After the experimental rats are fasted for 12 hours, the left side kidney ligation is performed under the abdominal cavity anesthesia of 10% chloral hydrate by the method that: the artery and vein and ureter of kidney are ligated along the kidney association. After 2 weeks of operation, the rats were fasted for 24h, and were injected with 50mg/kg Streptozotocin (STZ) (0.1 mol/L in 0.1mol/L pH4.4 citric acid-sodium citrate buffer solution) via intraperitoneal injection once, and were allowed to take food freely after the injection. The non-fasting tail vein blood sugar level of 72h of model making is more than or equal to 13.8mmol/L as the selection standard of the success of the diabetic nephropathy rat model. The molded DN rats were randomly divided into 4 groups, a blank control group, a model group, and a malonyl-ginsenoside high-low dose group (purified dry powder prepared in example 1, 50, 100 mg/kg/d); each group had 12, each group was administered intragastrically. In the experiment, the weight, the amount of water fed, the urine volume, the hair and the like of each group of rats are observed, after 8 weeks of continuous administration, the rats are placed in a metabolism cage for 12h of urine volume, the rats are anesthetized by intraperitoneal injection with 10% chloral hydrate, blood is taken from an abdominal aorta, the left kidney is taken out of a capsule, and the capsule is removed and weighed to calculate the organ coefficient (kidney weight/body weight).
Results and discussion
4.1 Single factor test to investigate the influence of cellulase on the extraction yield of malonyl ginsenoside
4.1.1 Effect of enzymolysis temperature on the extraction yield of malonyl-ginsenoside
Precisely weighing 15 parts of fresh ginseng root samples, each 5g of fresh ginseng root samples are respectively placed in a 250ml triangular flask, adding 25ml of distilled water, adding cellulase accounting for 5 percent of the mass of the samples, and performing enzymolysis for 1 hour at 20 ℃, 30 ℃, 40 ℃, 50 ℃ and 60 ℃ respectively; after enzymolysis, adding appropriate amount of ethanol and distilled water to make ethanol concentration reach 80%, and ultrasonic extracting for 3 times, each time for 0.5 hr. The extracted sample was contained in a 25mL volumetric flask for HPLC quantitative analysis. The effect of the enzymolysis temperature on the extraction rate of malonyl-ginsenoside is shown in figure 1. As can be seen from fig. 1, the malonyl ginsenoside extraction effect was the best at 40 ℃, and the malonyl ginsenoside extraction rate tended to decrease instead as the medium temperature increased.
4.1.2 Effect of enzyme dosage on the extraction yield of malonyl-ginsenoside
Weighing 15 parts of 5g fresh ginseng root sample, placing the sample in a 250ml triangular flask, adding 25ml of distilled water, respectively adding cellulase with the mass of 0,1%, 5%, 10% and 20% of the sample, and performing enzymolysis for 1h at 40 ℃; after enzymolysis, adding appropriate amount of ethanol and distilled water to make ethanol concentration reach 80%, and ultrasonic extracting for 3 times, each time for 0.5 hr. The extracted samples were used for HPLC quantitative analysis. The result is shown in figure 2, the extraction rate is increased with the increase of the enzyme adding amount, but when the enzyme adding amount reaches 10%, the extraction rate is not obviously increased, and the enzyme adding amount is selected to be 5% of the quality of the ginseng sample for reducing the cost.
4.1.3 Effect of enzymolysis time on the extraction yield of malonyl-ginsenoside
Weighing 15 parts of fresh ginseng root sample, each 5g, placing in a 250ml triangular flask, adding 25ml of distilled water, adding cellulase accounting for 5% of the mass of the sample, and performing enzymolysis for 1h, 2h, 4h, 6h and 8h at 40 ℃ respectively; after enzymolysis, adding appropriate amount of ethanol and distilled water to make ethanol concentration reach 80%, and ultrasonic extracting for 3 times, each time for 0.5 hr. The extracted samples were used for HPLC quantitative analysis. The results show that the extraction rate of the malonyl ginsenoside increases continuously at the beginning of a period of time along with the extension of the enzymolysis time, and the extraction rate is not obviously increased along with the extension of the extraction time after the enzymolysis time is 4h, so the optimal enzymolysis time is 4h (figure 3).
4.1.4 Effect of ethanol concentration on malonyl ginsenoside extraction yield
Weighing 15 parts of fresh ginseng root sample, each 5g, placing in a 250ml triangular flask, adding 25ml of distilled water, adding cellulase accounting for 5% of the mass of the sample, and performing enzymolysis for 4 hours at 40 ℃; after enzymolysis, adding a proper amount of ethanol and distilled water to make the concentration of ethanol reach 0, 20%, 40%, 60% and 80%, respectively, and performing ultrasonic extraction for 3 times, wherein each extraction time is 0.5 h. The extracted samples were used for HPLC quantitative analysis. The results show that high concentration of ethanol favors the extraction of neutral saponins, while low concentration of ethanol favors the extraction of malonyl ginsenosides; the optimal extraction solvent is therefore 0-20% ethanol (FIG. 4).
4.2 Effect of different solvent systems on the isolation and purification of malonyl saponins
The influence of different solvent systems (ethanol-water, ethanol-potassium dihydrogen phosphate aqueous solution) on the separation and purification of malonyl saponin was examined by using NKA-12 adsorption resin. The result shows that the malonyl ginsenoside and the neutral saponin can be separated by the gradient elution of the ethanol-100 mmol/l potassium dihydrogen phosphate aqueous solution, the purity of the malonyl ginsenoside can reach 78.6 percent, the yield is 62.4 percent, and the malonyl ginsenoside and the neutral saponin are difficult to separate by the gradient elution of the ethanol-water solvent system.
4.3 therapeutic Effect of malonyl-ginsenoside on diabetic nephropathy in rats
The experimental results are shown in table 1, and the indexes of blood sugar, urine albumin, HbAlC, creatinine, BUN, kidney weight/body weight and the like of the DN model group rat are obviously increased compared with those of a normal control group, and have significant difference (p is less than 0.01); compared with the model group, the malonyl ginsenoside high-low dose group can obviously reduce the fasting blood glucose of rats (p is less than 0.01 or p is less than 0.05) after being treated for 8 weeks, and the total output of urine albumin is also obviously reduced (p is less than 0.01). In addition, the high dose group of malonyl ginsenosides significantly decreased the amount of HbAlC and creatinine in the rats, and there was a tendency to decrease BUN and kidney weight/body weight. These results indicate that malonyl-ginsenoside has a superior preventive and therapeutic effect on diabetic nephropathy.
TABLE 1 amelioration of diabetic nephropathy in rats (+ -s) by malonyl ginsenosides
Compared with normal control group##p＜0.01,#p < 0.05, p < 0.01, p < 0.05 compared to model control.
Claims (5)
1. A method for extracting and purifying malonyl ginsenoside comprises the following steps:
(1) Cutting fresh Ginseng radix or radix Panacis Quinquefolii, and adding 1-10 times of distilled water;
(2) Adding cellulase accounting for 1-20% of the mass of the sample, and oscillating in a water bath at a constant temperature of 20-60 ℃ for 1-12 h;
(3) Adding ethanol and water after enzymolysis to make ethanol concentration reach 0-80%, ultrasonic extracting for 3-5 times, each time for 0.5-4 hr, with 10-50 times of extraction solvent;
(4) Recovering solvent from the extractive solution under reduced pressure at 40 deg.C, and concentrating to obtain water solution;
(5) Separating the extract obtained in the step (4) by NKA-12 adsorption resin, performing gradient elution by using ethanol and phosphate buffer solution as eluent, respectively collecting elution fractions, detecting by adopting thin-layer chromatography, collecting and combining the fractions with the same Rf value as the malonyl ginsenoside positive reference substance, recovering the solvent under reduced pressure, desalting the concentrated solution by using sephadex, and freeze-drying to obtain malonyl ginsenoside powder.
2. The method for preparing malonyl-ginsenoside according to claim 1, wherein: chopping the fresh ginseng or American ginseng in the step (1), and adding 3-5 times of distilled water; cellulase was then added at 5% of the sample mass.
3. The method for preparing malonyl-ginsenoside according to claim 1, wherein: and (3) carrying out enzymolysis on the cellulase in the step (2) at the temperature of 40 ℃, and carrying out constant-temperature oscillation water bath for 4 hours.
4. The method for preparing malonyl-ginsenoside according to claim 1, wherein: and (3) adding ethanol and water after enzymolysis to enable the concentration of the ethanol to reach 0-20% and the dosage of the solvent to be 20-30 times, and then carrying out ultrasonic extraction for 3 times, wherein each extraction time is 1 hour.
5. The method for preparing malonyl-ginsenoside according to claim 1, wherein: the phosphate buffered saline solution in step (5) is selected from: NaH2PO4、 KH2PO4、Na2HPO4、K2HPO4。
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