CN103211828B - Rhizoma Panacis Japonici saponin IV is preparing the application in blood lipid-lowering medicine - Google Patents
Rhizoma Panacis Japonici saponin IV is preparing the application in blood lipid-lowering medicine Download PDFInfo
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- CN103211828B CN103211828B CN201310165941.6A CN201310165941A CN103211828B CN 103211828 B CN103211828 B CN 103211828B CN 201310165941 A CN201310165941 A CN 201310165941A CN 103211828 B CN103211828 B CN 103211828B
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- panacis japonici
- rhizoma panacis
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- japonici saponin
- extract
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- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
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- BPAWXSVOAOLSRP-UHFFFAOYSA-N oleanane Natural products CCCCCCCCCCCCCCCC(=O)OC1CCC2(C)C(CCC3(C)C2CC=C4C5CC(C)(C)CCC5(C)C(O)CC34C)C1(C)C BPAWXSVOAOLSRP-UHFFFAOYSA-N 0.000 description 1
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- Medicines Containing Plant Substances (AREA)
- Steroid Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses Rhizoma Panacis Japonici saponin IV and prepare the application in blood lipid-lowering medicine.Effect experiment shows that Rhizoma Panacis Japonici saponin IV significantly can reduce TG, TC and LDL-C content in egg-nog hyperlipemia in mice serum, raises HDL-C content; Safety is high, and effect for reducing blood fat affirmative, for clinical treatment hyperlipidaemic conditions provides a kind of selection newly.
Description
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to a kind of Rhizoma Panacis Japonici saponin IV and preparing the application in blood lipid-lowering medicine.
Background technology
Hyperlipidemia is a breakneck disease, can cause coronary heart disease and apoplexy; Prevalence is high, and global hyperlipidemia prevalence is 7.6%, and U.S.'s prevalence is up to 50%, and Chinese prevalence is up to 30%, only just reaches 1.6 hundred million more than at Chinese number of patients; The prevalence of hyperlipidemia is rising year by year, and treatment drops into huge.
An existing clinical line lipid lowerers mainly statins (simvastatin etc.).Statins is by the rate-limiting enzyme HMG-CoA reductase of T suppression cell inner cholesterol synthesis commitment, make hepatic cholesterol biosynthesis block, reduce endocellular liberation cholesterol, then feedback raises the expression of cell surface low density lipoprotein, LDL (LDL) receptor, make cell ldl receptor increased number and increased activity, accelerate the removing of low-density lipoprotein cholesterol (LDL-C) in blood, thus reduce blood fat; But statins has serious untoward reaction, can hepar damnification be caused, cause transaminase to raise, myalgia, serum creatine creatase (CK) raise, severe patient can cause rhabdomyolysis, acute renal failure.
Therefore, research at present has the new Chinese medicine that effect for reducing blood fat is definite, safety is high, quality controllable, has important social meaning and economic implications.
Summary of the invention
Rhizoma Panacis Japonici is Araliaceae Radix Ginseng, belongs to the dry root of herbaceos perennial Panax japonicus C.A.Mey., containing multiple saponin, volatile oil, saccharide and several amino acids.Former is a kind of famous and precious Chinese herbal medicine among the people, has now recorded in the Pharmacopoeia of the People's Republic of China, the similar Radix Ginseng of medicinal function and Radix Notoginseng.Rhizoma Panacis Japonici is born in by hillside limes marginis, dark and damp area or rock ditch ravine, is mainly distributed in the ground such as Guizhou, Hunan, Yunnan, Anhui, Jiangxi, Hubei.Annual autumn excavates, and removing stem and leaf, separates fleshy tap root, rhizome is removed crust, dry or dry in the shade and can be used as medicine.Begin to be loaded in supplementary Amplifications of the Compendium of Materia Medica.Sweet, the micro-hardship of Rhizoma Panacis Japonici, temperature.Have the multiple efficacies such as blood circulation promoting and blood stasis dispelling, reducing swelling and alleviating pain, strengthening by means of tonics, the strengthening by means of tonics effect of existing similar Radix Ginseng, has again the eliminating stasis to stop pain of similar Radix Notoginseng, hemostasis, phlegm-dispelling functions, is mainly used in weakness, chronic cough hemoptysis, cough with copious phlegm, traumatic injury after being ill.
The object of this invention is to provide a kind of Rhizoma Panacis Japonici saponin IV and prepare the application in blood lipid-lowering medicine.
The object of the invention is to be achieved through the following technical solutions:
Rhizoma Panacis Japonici saponin IV is Oleanolic triterpenic glycoside, and its chemical constitution is clear and definite, as formula I;
Formula I Rhizoma Panacis Japonici saponin IV structure
The invention provides Rhizoma Panacis Japonici saponin IV as the application of active component in the medicine of preparation blood fat reducing, purity >=90% of the Rhizoma Panacis Japonici saponin IV preferably used.
Above-mentioned Rhizoma Panacis Japonici saponin IV prepares by the following method:
A, medicinal material extract: get Rhizoma Panacis Japonici medical material, the ethanol or the methanol eddy that add Rhizoma Panacis Japonici medical material 6-12 times amount extract 1-3 hour, filter, obtain filtrate, medicinal residues use the ethanol of Rhizoma Panacis Japonici medical material 6-12 times amount or methanol eddy to extract again 1-3 time, each 1-3 hour, filter, merging filtrate, reclaim under reduced pressure alcohol, to without alcohol taste, is mixed with the extracting solution containing 0.05-0.5g medical material/ml;
The HPD722 macroporous resin column enrichment of b, extracting solution: HPD722 macroporous resin column on extracting solution, wash with water and do not permitted reaction to without vertical, mass concentration is the neutral alcohol solution remove impurity of 5%, the mass concentration of 5-8 times amount column volume is the neutral alcohol eluant solution of 60%, collect the neutral alcohol eluent that mass concentration is 60%, recycling design is near dry, and drying under reduced pressure, obtains crude extract;
The precipitate and separate of c, crude extract: get crude extract and add the methanol solution that mass concentration is 8%, dissolving is mixed with 0.067g/ml lysate, acid adding adjustment lysate pH3.5, precipitant dichloromethane is added in lysate, abundant mixing, leaves standstill more than 24h, filters, collecting precipitation, drying under reduced pressure obtains the separator of crude extract;
Prepared by d, Rhizoma Panacis Japonici saponin IV compound ODS column purification: SunChrom filler, particle diameter 10 μm, 50mm × 250mm; Mobile phase: the volume ratio of methanol-0.5% aqueous formic acid=65:35(and methanol and mass percent concentration 0.5% aqueous formic acid is 65:35); Flow velocity: 20ml/min, determined wavelength: 203nm; Collect corresponding fraction, after decompression and solvent recovery, drying under reduced pressure obtains Rhizoma Panacis Japonici saponin IV monomer.
The ethanol of above-mentioned reflux, extract, or the mass concentration of methanol are 70-90%, and the mass concentration of preferred alcohol or methanol is 90%.
The part by weight of above-mentioned lysate and precipitant is 1-2:1-2, and the part by weight of preferred dissolution liquid and precipitant is 1:1.
The dosage form of medicine of the present invention can be any one dosage form pharmaceutically approved.
Rhizoma Panacis Japonici saponin IV of the present invention preferably prepares by the following method:
A, medicinal material extract: get Rhizoma Panacis Japonici medical material, pulverize, the mass concentration adding Rhizoma Panacis Japonici medical material 10 times amount is 90% soak with ethanol 2h, heating and refluxing extraction 2h, filter, obtain filtrate, medicinal residues are 90% alcohol reflux 2h, 2h by Rhizoma Panacis Japonici medical material 9 times amount, 8 times amount mass concentrations respectively, filter, obtain filtrate respectively, merge three filtrates, decompression recycling ethanol is extremely without alcohol taste, thin up is to 0.1g crude drug/ml, and this is the sample solution of macroporous resin column enrichment;
The HPD722 macroporous resin column enrichment of b, extracting solution: the sample solution 200ml getting above-mentioned 0.1g crude drug/ml, is added to (the heavy 60g of wet resin, column volume is 75ml) in HPD722 macroporous resin column, is washed to and is not permitted without molish(is vertical) reaction; Use 5% neutral alcohol remove impurity instead, the amount of 5% neutral alcohol used is the column volume of 10 times amount; Use 60% neutral alcohol eluting again instead, the amount of 60% neutral alcohol used is the column volume of 6 times amount; Collect 60% neutral alcohol eluent, decompression recycling ethanol and too much moisture are near dry, and drying under reduced pressure obtains crude extract.
The precipitate and separate of c, crude extract: get crude extract 6.7g, adding concentration is 8% methanol aqueous solution 100ml, ultrasonic dissolution, be mixed with 0.067g/ml lysate, add dilute hydrochloric acid, the pH=3.5 of adjustment lysate, in lysate: precipitant=1:1 ratio adds precipitant dichloromethane, abundant mixing, leave standstill 24h, sucking filtration, collecting precipitation, drying under reduced pressure at 70 DEG C, obtains the separator of crude extract.
Prepared by d, Rhizoma Panacis Japonici saponin IV compound ODS column purification: Chinese nation high performance liquid chromatograph; NP7000 double pump; NU3000UV/Vis detector; DAC-HB50 dynamic axial compression column (SunChrom filler, particle diameter 10 μm, 50mm × 250mm); Mobile phase: methanol-containing 0.5% aqueous formic acid=65:35; Flow velocity: 20ml/min, sample size: 5ml, determined wavelength: 203nm; Collect corresponding fraction, after decompression and solvent recovery, drying under reduced pressure obtains Rhizoma Panacis Japonici saponin IV monomer.
Repeatedly prepare, q.s Rhizoma Panacis Japonici saponin IV compound can be prepared.
Above-mentioned Rhizoma Panacis Japonici saponin IV can be prepared into the compositions of blood fat reducing with pharmaceutically acceptable carrier or customary adjuvant as active component.Above-mentioned pharmaceutically acceptable carrier comprises the adjuvant of oral formulations adjuvant or Parenteral.Route of administration can be oral, injection, topical etc.According to technical scheme of the present invention, said composition can be oral formulations or injection preparation.Wherein oral formulations comprises capsule, soft capsule, granule, oral liquid, tablet, drop pill etc.Adjuvant used comprises: the customary adjuvant such as starch, sucrose, lactose, Icing Sugar, glucose, mannitol, xylitol, Polyethylene Glycol, isopropyl alcohol, tween 80, glycerol, propylene glycol, microcrystalline Cellulose sodium, dextrin, cyclodextrin, sodium chloride, vitamin C, cysteine, citric acid, sodium thiosulfate, sodium sulfite, stearate and gelatin, preparation later stage preparation technology and equipment all belong to the routine techniques of pharmaceutical field, the present invention is not construed as limiting this, therefore will not describe in detail at this.
The specification of Rhizoma Panacis Japonici saponin IV hard capsule is: 0.21g/ grain (No. 2 capsules, wherein starch-containing adjuvant 5%, i.e. 0.01g); Consumption per day: each 2-4 grain, every day 6-12 grain.
Rhizoma Panacis Japonici saponin IV of the present invention extracts to obtain from natural Chinese medicine Rhizoma Panacis Japonici, and effect experiment shows that Rhizoma Panacis Japonici saponin IV significantly can reduce TG, TC and LDL-C content in egg-nog hyperlipemia in mice serum, raises HDL-C content; Safety is high, and effect for reducing blood fat certainly.
Beneficial effect of the present invention compared with the prior art: the present invention shows that Rhizoma Panacis Japonici saponin IV has the effect of significant blood fat reducing first, therefore, is developed to blood lipid-lowering medicine by Rhizoma Panacis Japonici saponin IV and has broad application prospects.
Accompanying drawing explanation
Fig. 1 is the mass spectrum of Rhizoma Panacis Japonici saponin VI.
Fig. 2 is the hydrogen spectrogram of Rhizoma Panacis Japonici saponin VI.
Fig. 3 is the carbon spectrogram of Rhizoma Panacis Japonici saponin VI.
Fig. 4 is the HPLC chromatogram of Rhizoma Panacis Japonici saponin IV.
Fig. 5 respectively organizes cell oil red O stain figure.
Detailed description of the invention
Embodiment 1: the preparation of Rhizoma Panacis Japonici saponin IV, Structural Identification and purity testing thereof
The preparation of Rhizoma Panacis Japonici saponin IV compound:
Medicinal material extract: get Rhizoma Panacis Japonici medical material, pulverize, the mass concentration adding Rhizoma Panacis Japonici medical material 10 times amount is 90% soak with ethanol 2h, heating and refluxing extraction 2h, filter, obtain filtrate, medicinal residues are 90% alcohol reflux 2h, 2h by Rhizoma Panacis Japonici medical material 9 times amount, 8 times amount mass concentrations respectively, filter, obtain filtrate respectively, merge three filtrates, decompression recycling ethanol is extremely without alcohol taste, thin up is to 0.1g crude drug/ml, and this is the sample solution of macroporous resin column enrichment;
The HPD722 macroporous resin column enrichment of extracting solution: the sample solution 200ml getting above-mentioned 0.1g crude drug/ml, is added to (the heavy 60g of wet resin, column volume is 75ml) in the HPD722 macroporous resin column handled well according to a conventional method, is washed to and reacts without molish; Use 5% neutral alcohol remove impurity instead, the amount of 5% neutral alcohol used is the column volume of 10 times amount; Use 60% neutral alcohol eluting again instead, the amount of 60% neutral alcohol used is the column volume of 6 times amount.Collect 60% neutral alcohol eluent, decompression recycling ethanol and too much moisture are near dry, and transfer in container, drying under reduced pressure obtains crude extract.Repeated multiple times preparation, obtains the crude extract of q.s.
The precipitate and separate of crude extract: get crude extract 6.7g, adding concentration is 8% methanol aqueous solution 100ml, ultrasonic dissolution, be mixed with 0.067g/ml lysate, add dilute hydrochloric acid, the pH=3.5 of adjustment lysate, in lysate: precipitant=1:1 ratio adds precipitant dichloromethane, abundant mixing, leave standstill 24h, sucking filtration, collecting precipitation, drying under reduced pressure at 70 DEG C, obtains the separator of crude extract.Repeated multiple times preparation, obtains the crude extract separator of q.s.
Prepared by Rhizoma Panacis Japonici saponin IV compound ODS column purification: Chinese nation high performance liquid chromatograph; NP7000 double pump; NU3000UV/Vis detector; DAC-HB50 dynamic axial compression column (SunChrom filler, particle diameter 10 μm, 50mm × 250mm); Mobile phase: methanol-0.5% aqueous formic acid=65:35; Flow velocity: 20ml/min, sample size: 5ml, determined wavelength: 203nm.Collect corresponding fraction, after decompression and solvent recovery, transfer in container, drying under reduced pressure obtains Rhizoma Panacis Japonici saponin IV monomer.Repeatedly prepare, prepare q.s Rhizoma Panacis Japonici saponin IV compound.
The Structural Identification of Rhizoma Panacis Japonici saponin IV compound:
Compound 1, white powder (methanol), Liebermann-Burchard and Molish reaction is all positive.The colour developing of silica gel thin-layer ethanol solution of sulfuric acid is aubergine, shows for triterpenoid compound.ESI-MS m/z:925.3[M-H]
-。
1h-NMR(300MHz, Pyr) spectrum in: δ 0.83 ~ 1.28(each 3H, s) be 7 methyl signals, in conjunction with
13c-NMR(75MHz, Pyr) in spectrum, δ 176.39 points out is likely carbonyl on ester bond, δ 172.45 is a carbonyl carbon signals, prompting is likely the carbonyl on carboxylic acid key, δ 144.12,123.87(C-12,13) double key carbon signal, δ 108.65,107.02 and 95.74 points out on this compound structure and is connected with 3 sugar, infers that this compound is be connected with three sugared triterpene saponin componds, sees Fig. 1.(Fujioka N in reference literature, Kohda H, Yamasaki K, Deng .Dammarane and oleanane saponins from callus tissue of Panax japonicus [J] .Phytochemistry, 1989,28 (7): 1855-1858.) spectral data, identifies that this compound is Rhizoma Panacis Japonici saponin VI (Chikusetsusaponin VI), sees formula I.
Formula I Rhizoma Panacis Japonici saponin IV structure
Hydrogen spectrum and carbon modal data as follows, spectrogram is shown in Fig. 2 and Fig. 3.
1H NMR(300MHz,Pyr)δ6.31(d,J=7.8Hz,1H),6.12(s,1H),5.41(s,1H),3.35(d,J=7.7Hz,1H),3.19(d,J=10.8Hz,1H),1.28(3H,s,2×CH
3),1.08,0.96,0.91,0.89,0.83(3H,s,5×CH
3)。
13C NMR(75MHz,Pyr)δppm:38.64(C-1),26.54(C-2),89.19(C-3),39.48(C-4),55.74 (C-5),18.48(C-6),33.11(C-7),39.91(C-8),48.00(C-9),36.94(C-10),23.74(C-11),123.87(C-12),144.12(C-13),42.13(C-14),28.16(C-15),23.64(C-16),46.99(C-17),41.74(C-18),46.20(C-19),30.75(C-20),34.01(C-21),32.54(C-22),28.25(C-23),16.91(C-24),15.49(C-25),17.46(C-26),26.09(C-27),176.39(C-28),33.11(C-29),23.75(C-30),3-O-Glc A,107.02(C-1’),75.32(C-2’),76.02(C-3’),78.91(C-4’),76.64(C-5’),172.45(C-6’),Ara,108.65(C-1’’),82.47(C-2’’),87.81(C-3’’),74.13(C-4’’),62.66(C-5’’),28-O-Glc,95.74(C-1’’’),74.13(C-2’’’),79.28(C-3’’’),71.16(C-4’’’),78.81(C-5’’’),62.25(C-6’’’)。
The purity testing of Rhizoma Panacis Japonici saponin IV compound:
Shimadzu LC-20A high performance liquid chromatograph; SPD-20A UV/Vis detector; Chromatographic column: Boston ODS chromatographic column (4.6 × 150mm, 5 μm); Mobile phase: acetonitrile-mass percent 0.05% phosphoric acid water=34:66(volume ratio); Determined wavelength: 203nm; Flow velocity: 1ml/min; Column temperature: 30 DEG C; Sample size: 10 μ l, acquisition time: 19min.
Get and prepare Rhizoma Panacis Japonici saponin IV monomer 10mg, put in 10ml volumetric flask, add mobile phase and dissolve, standardize solution, obtain Rhizoma Panacis Japonici saponin IV solution that concentration is 1mg/ml, sample introduction 10 μ l carries out purity testing.
Measurement result: appearance time: 13.782min; Peak area: 351704.594; Content: 94.27%(calculates by area normalization method).HPLC spectrogram is shown in Fig. 4.
Embodiment 2: the preparation of Rhizoma Panacis Japonici saponin IV
The preparation of Rhizoma Panacis Japonici saponin IV compound:
Medicinal material extract: get Rhizoma Panacis Japonici medical material, pulverizes, and the mass concentration adding Rhizoma Panacis Japonici medical material 12 times amount is that 90% methanol soaks 2h, heating and refluxing extraction 3h, filter, obtain filtrate, medicinal residues respectively with Rhizoma Panacis Japonici medical material 9 times amount, 10 times amount mass concentrations be 90% methanol eddy extraction 2h, 2h, filter, obtain filtrate respectively, merge three filtrates, reclaim under reduced pressure methanol is extremely without alcohol taste, thin up is to 0.1g crude drug/ml, and this is the sample solution of macroporous resin column enrichment;
The HPD722 macroporous resin column enrichment of extracting solution: the sample solution 200ml getting above-mentioned 0.1g crude drug/ml, is added to (the heavy 60g of wet resin, column volume is 75ml) in the HPD722 macroporous resin column handled well according to a conventional method, is washed to and reacts without molish; Use 5% neutral alcohol remove impurity instead, the amount of 5% neutral alcohol used is the column volume of 10 times amount; Use 60% neutral alcohol eluting again instead, the amount of 60% neutral alcohol used is the column volume of 6 times amount.Collect 60% neutral alcohol eluent, decompression recycling ethanol and too much moisture are near dry, and transfer in container, drying under reduced pressure obtains crude extract.Repeated multiple times preparation, obtains the crude extract of q.s.
The precipitate and separate of crude extract: get crude extract 6.7g, adding concentration is 8% methanol aqueous solution 100ml, ultrasonic dissolution, be mixed with 0.067g/ml lysate, add dilute hydrochloric acid, the pH=3.5 of adjustment lysate, in lysate: precipitant=1:1 ratio adds precipitant dichloromethane, abundant mixing, leave standstill 24h, sucking filtration, collecting precipitation, drying under reduced pressure at 70 DEG C, obtains the separator of crude extract.Repeated multiple times preparation, obtains the crude extract separator of q.s.
Prepared by Rhizoma Panacis Japonici saponin IV compound ODS column purification: Chinese nation high performance liquid chromatograph; NP7000 double pump; NU3000UV/Vis detector; DAC-HB50 dynamic axial compression column (SunChrom filler, particle diameter 10 μm, 50mm × 250mm); Mobile phase: methanol-0.5% aqueous formic acid=65:35; Flow velocity: 20ml/min, sample size: 5ml, determined wavelength: 203nm.Collect corresponding fraction, after decompression and solvent recovery, transfer in container, drying under reduced pressure obtains Rhizoma Panacis Japonici saponin IV monomer.
The external blood fat reducing experiment of embodiment 3 Rhizoma Panacis Japonici saponin IV
1. sample is to the preparation of drug solns
Take Rhizoma Panacis Japonici saponin IV (preparing according to embodiment 1 method) 0.41mg in 15ml centrifuge tube by 100,000/electronic balance precision, add 10ml DMEM in high glucose culture medium and dissolve, obtain Rhizoma Panacis Japonici saponin IV sample solution that concentration is 41 μ g/ml.
2. cell inoculation
Get the cell of 80-90% bottom the good covering culture bottle of growth conditions, become cell suspension through 0.25% trypsinization and count, adjustment cell concentration about 70,000 cells/ml, are inoculated in 24 orifice plates, every hole adds 1ml cell suspension, i.e. every hole inoculation about 70,000 cells.The cell inoculated is put in calorstat and continues to hatch.
3. experiment grouping
After 24 orifice plates of inoculating cell hatch 24h, take out, discard former culture medium, be divided into matched group, model group, dosing group, often group establishes 6 parallel multiple holes.Select 100,10,1,0.1 μm of ol/L as the adding consistency of compound.Matched group adds the DMEM in high glucose culture medium containing BSA, and model group adds the DMEM in high glucose culture medium containing final concentration 1mmol/L oleic acid, and each dosing group adds the DMEM in high glucose culture medium containing final concentration 1mmol/L oleic acid and each acute drug, and often group is 6 parallel holes.After pharmaceutical intervention 24h, suck culture supernatants, lower floor's cell per well adds cell pyrolysis liquid 50 μ l, cracking 30min on ice, by TG content in TG kit measurement cell (the every porocyte of cell counting about 170,000).
TG concentration=A sample/A mark × concentration of standard solution; Concentration of standard solution is 2mg/ml.
4. data statistic analysis
By Excel software to data analysis, experimental result is with mean+/-standard error
represent, adopt t method of inspection to compare group difference, P < 0.05 has significant difference, and P < 0.01 has pole significant difference.
5. oil red O stain: adopt conventional method to carry out oil red O stain.
The preparation of oil red O working solution:
Take 0.5g oil red dry powder, be dissolved in 100ml isopropyl alcohol, solution is heated to 100 DEG C 5 minutes, and ceaselessly stir, filtered while hot, refilter once after cooling, be made into the oil red storage liquid of 0.5%, the preservation of brown bottle sealing (or masking foil parcel lucifuge) 4 DEG C, is storage liquid, can preserves for a long time.Used time gets 6ml oil red storing solution and adds tri-distilled water 4ml and mix, and qualitative filter paper filters, and is finished after dilution in a few hours.
Staining procedure:
Carefully light and slowly remove culture fluid; With the light and slow rinsing 1-2 time of PBS; With the alcohol fixation 30min of 75%, cell membrane is fixed; With 60% isopropyl alcohol, mordant dyeing 1-2min is carried out to cell; At the bottom of the dyeing of oil red working solution 30min, liquid feeding 0.5-1ml overlay; With distilled water rinsing 2 times, removing excess dyestuff; Inverted light microscope is observed and is taken pictures, and preserves photo.6. experimental result
TG content in 6.1 administration group cells
In each group of cell, TG assay result is as shown in the table.
The content of table 1 administration group cell TG (n=6,
Note: compared with blank group, * represents that P < 0.05, * * represents P < 0.01;
Compared with model group,
▲represent P < 0.05,
▲ ▲represent P < 0.01.
Compared with matched group, model group cell TG content is that significance increases (P<0.01), illustrates that cell hyperlipidemia model is successfully established; Compared with model group, Rhizoma Panacis Japonici saponin IV administration group (100 μMs, 10 μMs) TG content significantly reduces (P<0.01, P<0.05), illustrates that Rhizoma Panacis Japonici saponin IV has cell effect for reducing fat.
6.2 oil red coloration results
Each group of cell dyeing situation as shown in Figure 5.
Can be found out clearly by Fig. 5: compared with matched group, model group has can be dyed red fat by oil red O in a large number and drip existence, shows that HepG2 cell hyperlipidemia model is successfully established; Administration group is compared with model group, and under high concentration (100 μMs), Rhizoma Panacis Japonici saponin IV administration group significantly can alleviate fatty heaped-up condition in cell, and in cell, red fat drips and all obviously reduces, and this is basically identical with TG value in the cell recorded.
Blood fat reducing experiment in the body of embodiment 4 Rhizoma Panacis Japonici saponin IV
1. experiment material
1.1 medicines and reagent: No. 12, mouse stomach syringe needle, plastic centrifuge tube 1.5ml, Fresh Egg some, Simvastatin Tablets (Shanghai Xinyi Wanxiang Pharmaceutical Co., Ltd., lot number: 080201), (Beijing WBL Peking University Biotech Co., Ltd assisted by Xuezhikang glue, lot number: 20080708), triglyceride determination test kit (the safe clinical reagent company limited of Beijing Northization, lot number: 20090318), T-CHOL test kit (the safe clinical reagent company limited of Beijing Northization, lot number: 20090318), low-density LP determination reagent box (the safe clinical reagent company limited of Beijing Northization, lot number: 20090318).
1.2 experimental facilitiess: low speed centrifuge (Anting Scientific Instrument Factory, Shanghai, model: 80-2B), microplate reader (Nanjing Huadong Electronic Group Medical Equipment LLC, model: DG5033A), high speed desktop refrigerated centrifuger (Changsha Xiang Yi centrifuge Instrument Ltd., model: TGL-16).
1.3 laboratory animals: Kunming mouse, 72, male and female half and half, body weight 18-22g.
2. modeling method
Lumbar injection 75% egg-nog normal saline solution 0.2ml/10g.75% egg yolk Emulsion: Fresh Egg some, bottom is opened an osculum and is vacated Ovum Gallus domesticus album, and egg yolk has put into beaker. and needle tubing probes into egg yolk thin film, draw egg yolk liquid, put in the graduated cylinder of 100ml, get 75ml egg yolk liquid with normal saline dilution to 100ml, be made into the egg-nog solution of 75%.(during modeling, use magnetic stirrer egg-nog, make egg-nog even, reduce error).
3. experimental technique
Kunming mouse 72,18-22g, male and female half and half, are divided into 6 groups at random, often organize 12.Be divided into Normal group, hyperlipidemia model group, Xuezhikang group, simvastatin group, Rhizoma Panacis Japonici saponin IV (preparing according to embodiment 1 method) high dose group (IV-H), Rhizoma Panacis Japonici saponin IV low dose group (IV-L).Gastric infusion, Normal group and hyperlipidemia model group gavage 0.5%CMCNa every day, gavage volume is 20ml/kg; Administration group gavage every day gives the medicine of same volume, altogether administration 14 days.The dosage of each administration group is respectively: Xuezhikang matched group dosage 1g/kg(concentration is 50mg/ml), simvastatin matched group dosage 0.014g/kg(concentration is 0.7mg/ml), to be respectively 0.18g/kg(concentration be 9mg/ml for the dosage of Rhizoma Panacis Japonici saponin IV high dose group and Rhizoma Panacis Japonici saponin IV low dose group), 0.06g/kg(concentration is 3mg/ml).Last administration is after 2 hours, and the equal lumbar injection 75% egg-nog normal saline solution 0.2ml/10g of each group, causes experimental hyperlipidemia.Mice modeling, after 20 hours, adopts and plucks eyeball blood collection method, centrifugalize serum, and according to T-CHOL, triglyceride, low density lipoprotein, LDL, high density lipoprotein test kit description operates, and measures the content of TC, TG, LDL-C, HDL-C.
4. experimental result:
Table 2 each treated animal blood lipid level table (n=12,
Note: compared with blank group, * represents that P < 0.05, * * represents P < 0.01;
Compared with model group,
▲represent P < 0.05,
▲ ▲represent P < 0.01.
Compared with Normal group, in model group mice serum, TG, TC, LDL-C content significantly raises, (P<0.01, P<0.01, P<0.01), HDL-C content significantly reduces (P<0.05);
Compared with model group, simvastatin group TG, TC, LDL-C content significantly reduce (P<0.01, P<0.01, P<0.01), and HDL-C content significantly increases (P<0.05); Compared with model group, Xuezhikang group TG, TC, LDL-C content significantly reduce (P<0.01, P<0.05, P<0.01), and HDL-C content is without significant change.
Compared with model group, Rhizoma Panacis Japonici saponin IV high dose group (IV-H) TG, TC, LDL-C content significantly reduces (P<0.01, P<0.01, P<0.05), HDL-C content significantly increases (P<0.01); Compared with model group, Rhizoma Panacis Japonici saponin IV low dose group (IV-L) TG, TC, LDL-C content significantly reduces (P<0.01, P<0.01, P<0.05), and HDL-C content is without significant change; Rhizoma Panacis Japonici saponin IV high dose effect for reducing fat is better than low dosage.
Embodiment 5 Rhizoma Panacis Japonici saponin IV tablet
Get the Rhizoma Panacis Japonici saponin IV obtained by embodiment 1 method, add tablet and commonly use adjuvant, preparation technology is prepared into tablet routinely.
Embodiment 6 Rhizoma Panacis Japonici saponin IV capsule
Get the Rhizoma Panacis Japonici saponin IV obtained by embodiment 1 method, add capsule and commonly use adjuvant, preparation technology is prepared into capsule routinely.
Embodiment 7 Rhizoma Panacis Japonici saponin IV oral liquid
Get the Rhizoma Panacis Japonici saponin IV obtained by embodiment 1 method, add oral liquid and commonly use adjuvant, preparation technology is prepared into oral liquid routinely.
Claims (8)
1. Rhizoma Panacis Japonici saponin IV is as the application of active component in the medicine of preparation reduction cholesterol, reduction low density lipoprotein, LDL and high density lipoprotein increasing content.
2. application according to claim 1, is characterized in that purity >=90% of described Rhizoma Panacis Japonici saponin IV.
3. application according to claim 1, is characterized in that described Rhizoma Panacis Japonici saponin IV prepares by the following method:
A, medicinal material extract: get Rhizoma Panacis Japonici medical material, the ethanol or the methanol eddy that add Rhizoma Panacis Japonici medical material 6-12 times amount extract 1-3 hour, filter, obtain filtrate, medicinal residues use the ethanol of Rhizoma Panacis Japonici medical material 6-12 times amount or methanol eddy to extract again 1-3 time, each 1-3 hour, filter, merging filtrate, reclaim under reduced pressure alcohol, to without alcohol taste, is mixed with the extracting solution containing 0.05-0.5g medical material/ml;
The HPD722 macroporous resin column enrichment of b, extracting solution: HPD722 macroporous resin column on extracting solution, wash with water and do not permitted reaction to without vertical, concentration is the neutral alcohol solution remove impurity of 5%, the concentration of 5-8 times amount column volume is the neutral alcohol eluant solution of 60%, collect the neutral alcohol eluent that concentration is 60%, recycling design is near dry, and drying under reduced pressure, obtains crude extract;
The precipitate and separate of c, crude extract: get crude extract and add the methanol solution that concentration is 8%, dissolving is mixed with 0.067g/ml lysate, acid adding adjustment lysate pH3.5, precipitant dichloromethane is added in lysate, abundant mixing, leaves standstill more than 24h, filters, collecting precipitation, drying under reduced pressure obtains the separator of crude extract;
Prepared by d, Rhizoma Panacis Japonici saponin IV compound ODS column purification: SunChrom filler, particle diameter 10 μm, 50mm × 250mm; Mobile phase: methanol-0.5% aqueous formic acid=65:35; Flow velocity: 20ml/min, determined wavelength: 203nm; Collect corresponding fraction, after decompression and solvent recovery, drying under reduced pressure.
4. application according to claim 3, is characterized in that the ethanol of described reflux, extract, or the concentration of methanol are 70-90%.
5. application according to claim 4, is characterized in that the ethanol of described reflux, extract, or the concentration of methanol are 90%.
6. application according to claim 3, is characterized in that the part by weight of described lysate and precipitant is 1-2:1-2.
7. application according to claim 6, is characterized in that the part by weight of described lysate and precipitant is 1:1.
8. application according to claim 1, is characterized in that the dosage form of described medicine is any one dosage form pharmaceutically approved.
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CN102247391A (en) * | 2010-05-19 | 2011-11-23 | 杨小林 | Application of panax japonicus saponin V to reduction of blood fat |
CN102247400A (en) * | 2010-05-19 | 2011-11-23 | 杨小林 | Blood lipid-reducing effect of panax japonicus polysaccharide |
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CN102247416A (en) * | 2010-05-19 | 2011-11-23 | 杨小林 | Blood lipid lowering application of total saponins of panax japonicus |
CN102247391A (en) * | 2010-05-19 | 2011-11-23 | 杨小林 | Application of panax japonicus saponin V to reduction of blood fat |
CN102247400A (en) * | 2010-05-19 | 2011-11-23 | 杨小林 | Blood lipid-reducing effect of panax japonicus polysaccharide |
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