CN102875615B - Extraction method and application of falcate dolichos root or leaf glucoside A and total saponins of falcate dolichos root or leaf - Google Patents
Extraction method and application of falcate dolichos root or leaf glucoside A and total saponins of falcate dolichos root or leaf Download PDFInfo
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- CN102875615B CN102875615B CN 201210320436 CN201210320436A CN102875615B CN 102875615 B CN102875615 B CN 102875615B CN 201210320436 CN201210320436 CN 201210320436 CN 201210320436 A CN201210320436 A CN 201210320436A CN 102875615 B CN102875615 B CN 102875615B
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Abstract
The invention discloses an extraction method of falcate dolichos root or leaf glucoside A. The method comprises the following steps: (1) taking falcate dolichos root or leaf herbs, adding alcohol aqueous solution for reflux extraction, wherein the weight of the alcohol aqueous solution is 6-12 times of that of falcate dolichos root or leaf herbs; (2) recovering the falcate dolichos root or leaf extract solution obtained from the step 1 under reduced pressure to alcohol till no alcohol taste exists, and adding water or alcohol to liquid medicine; (3) adding hydrochloric acid into the liquid medicine obtained from the step 2 till the pH value is 1-6, and adding sodium chloride into the liquid medicine till the salt concentration to 0.1-2mol/L; (4) placing the liquid medicine obtained from the step (3) on a macroporous adsorption resin column for separation; (5) cleaning and purifying with alcohol aqueous solution, of which the volume is 4-10 times of the column volume of the macroporous adsorption resin column, eluting with alcohol aqueous solution, of which the volume is 4-10 times of the column volume of the macroporous adsorption resin column, collecting the eluant, and drying to obtain falcate dolichos root or leaf glucoside A. The invention further discloses an extraction method of total saponins of falcate dolichos root or leaf, and the application of the falcate dolichos root or leaf glucoside A, the total saponins of falcate dolichos root or leaf, falcate dolichos root or leaf glucoside A hydrolysate and salt thereof and falcate dolichos root or leaf glucoside A derivate in preparing immune-inflammation resistant drugs.
Description
Technical field
The present invention relates to preparation method and the application thereof of a kind of Root or leaf of Falcate dolichos glycosides A and Root or leaf of Falcate dolichos total saponins.
Background technology
Root or leaf of Falcate dolichos is root or the leaf of leguminous plants sickle French beans (Dolichos falcatus Kalcatus Klein), and another name has fiber crops in fiber crops, three sections of fiber crops, beans leaf Euphorbia sessiliflora Roxb etc.This moral character is hot, fiber crops, warm.In Yunnan Province of China, Sichuan, all there is distribution on the ground such as Gansu, and abundant plant resources is especially arranged in Yunnan.Has expelling wind and activating blood circulation, the effects such as hemostasis and pain-relieving.Fracture, wound, the rheumatalgia of being mainly used among the people in Yunnan, the external curing traumatic hemorrhage, also have anticancer, the effect of diuresis.Main chemical compositions about Root or leaf of Falcate dolichos is triterpene saponin and alkaloid compound at present.In prior art, the extraction process of these compounds is complicated, and extract purity is not high.
The immune inflammations such as rheumatoid arthritis are common clinical and frequently-occurring disease, belong to the immune inflammation category.The population of the nearly 1-2% in the whole world suffers from rheumatoid arthritis, and according to Ministry of Health's statistics, China arthritic sum surpasses 100,000,000, wherein has 400 all creation rheumatic arthritis patients at least.At present mainly adopt non-steroidal anti-inflammatory drugs (NSAIDs), improve antirheumatic (DMARDs), glucocorticosteroid medicine and the plant amedica of the state of an illness for the treatment of rheumatoid arthritis.Though the first-line drug of NSAIDs Chang Zuowei treatment rheumatoid arthritis all has obvious untoward reaction, its relief of symptoms is mainly due to its anti-inflammatory action, and can not prevent tissue injury, destruction of joint and deformity.Can improve symptom although improve the DMARDs of the state of an illness, all there is obvious untoward reaction in these medicines, cause peptide ulceration as Gastroduodenal lesion, liver injury, fash etc.Therefore, develop safe and effectively, quality controllable, the anti-immune inflammation medicine of specific chemical components will have tempting market outlook and exploitation is worth.
Summary of the invention
In order to solve the problems of the technologies described above, the invention provides the extracting method of a kind of Root or leaf of Falcate dolichos glycosides A, comprise the steps:
1) get the Root or leaf of Falcate dolichos medicinal material, after adding the alcohol solution refluxing extraction of 6 ~ 12 times of weight of Root or leaf of Falcate dolichos medicinal material, filter, obtain Root or leaf of Falcate dolichos medicinal substances extract solution;
2) the Root or leaf of Falcate dolichos medicinal substances extract solution that obtains of step 1), the reclaim under reduced pressure alcohols is to nothing alcohol flavor, add water or alcohol, be mixed with liquid, every milliliter of liquid contains the Root or leaf of Falcate dolichos medicinal substances extract that extracts from 0.01g ~ 2g Root or leaf of Falcate dolichos medicinal material, wherein, when adding alcohol, alcohol in described liquid volume percent less than 30%;
3) liquid that step 2) obtains is transferred pH to 1 ~ 6 with hydrochloric acid, transfers salt concn as 0.1 ~ 2mol/L take sodium-chlor;
4) on the liquid that step 3) is obtained, macroporous adsorptive resins separates, applied sample amount take the weighing scale of Root or leaf of Falcate dolichos medicinal material with the weight resin ratio as 1:0.25 ~ 4;
5) after loading, macroporous adsorptive resins rinses removal of impurities with the alcohol solution of 4 ~ 10 times of column volumes, and then with the alcohol solution wash-out of 4 ~ 10 times of column volumes, collects elutriant, concentrates, dry, namely gets Root or leaf of Falcate dolichos glycosides A.
Preferably, the alcohol solution in step 1) is methyl alcohol or the aqueous ethanolic solution of volumetric concentration 10% ~ 90%; Step 2) alcohol in is methyl alcohol or ethanol; Methyl alcohol take volumetric concentration as 10% ~ 40% in step 5) or aqueous ethanolic solution rinse removal of impurities, and flow velocity is 1 ~ 4 BV/h, and then methyl alcohol or the aqueous ethanolic solution take volumetric concentration as 45% ~ 90% rinses, and flow velocity is 0.5 ~ 4 BV/h.
Preferably, step 1) is got the Root or leaf of Falcate dolichos medicinal material, adds the alcohol solution refluxing extraction 1 ~ 3 hour of 6 ~ 12 times of weight of Root or leaf of Falcate dolichos medicinal material, refluxing extraction 1 ~ 3 time, and merging filtrate namely obtains Root or leaf of Falcate dolichos medicinal substances extract solution.
Preferably, the macroporous adsorbent resin in step 4) is polar macroporous adsorption resin.
Preferably, the blade diameter length ratio of macroporous adsorptive resins described in step 4) is 1:4 ~ 8, and the loading flow velocity is 0.5 ~ 2 BV/h.
The present invention also provides a kind of extracting method of Root or leaf of Falcate dolichos total saponins, comprises the steps:
1) get the Root or leaf of Falcate dolichos medicinal material, add the alcohol solution refluxing extraction of 6 ~ 12 times of weight of Root or leaf of Falcate dolichos medicinal material, filter, get filtrate;
2) filtrate that obtains of step 1), reclaim under reduced pressure alcohol is to without after the alcohol flavor, drying.
Preferably, step 1) is got the Root or leaf of Falcate dolichos medicinal material, adds the alcohol solution refluxing extraction 1 ~ 3 hour of 6 ~ 12 times of weight of Root or leaf of Falcate dolichos medicinal material, refluxing extraction 1 ~ 3 time, and merging filtrate, wherein said alcohol solution are that volumetric concentration is 10 ~ 90% methyl alcohol or aqueous ethanolic solution.
The present invention also provides the application of Root or leaf of Falcate dolichos glycosides A in preparation anti-immune inflammation medicine: described Root or leaf of Falcate dolichos glycosides A is prepared into medicine or the pharmaceutical composition of anti-immune inflammation as activeconstituents and pharmaceutically acceptable carrier or auxiliary material.
Preferably, described anti-immune inflammation medicine is resisting rheumatoid disease arthritis drug or antigout arthritis drug.That is, Root or leaf of Falcate dolichos glycosides A is preferably for the preparation of resisting rheumatoid disease sacroiliitis and urarthritis medicine.
The present invention also provides the hydrolysate of Root or leaf of Falcate dolichos glycosides A and the application of salt in preparation anti-immune inflammation medicine thereof: the hydrolysate of described Root or leaf of Falcate dolichos glycosides A is prepared into anti-immune inflammation medicine or pharmaceutical composition as activeconstituents and pharmaceutically acceptable carrier or auxiliary material.
The application of the derivative that the present invention also provides Root or leaf of Falcate dolichos glycosides A in preparation anti-immune inflammation medicine: the derivative of described Root or leaf of Falcate dolichos glycosides A is prepared into anti-immune inflammation medicine or pharmaceutical composition as activeconstituents and pharmaceutically acceptable carrier or auxiliary material, and the structural formula of the derivative of wherein said Root or leaf of Falcate dolichos glycosides A is as follows:
Wherein, R is the alkyl of C2 ~ C4.
The present invention also provides the application of Root or leaf of Falcate dolichos total saponins in preparation anti-immune inflammation medicine: described Root or leaf of Falcate dolichos total saponins is prepared into medicine or the pharmaceutical composition of anti-immune inflammation as activeconstituents and pharmaceutically acceptable carrier or auxiliary material.
Preferably, described anti-immune inflammation medicine is resisting rheumatoid disease arthritis drug or antigout arthritis drug.That is, the hemp total saponins is preferably for the preparation of resisting rheumatoid disease sacroiliitis and urarthritis medicine.
The present invention also provide Root or leaf of Falcate dolichos glycosides A and Root or leaf of Falcate dolichos total saponins arbitrarily than the application of mixture in preparation anti-immune inflammation medicine: Root or leaf of Falcate dolichos glycosides A and Root or leaf of Falcate dolichos total saponins be prepared into arbitrarily medicine or the pharmaceutical composition of anti-immune inflammation as activeconstituents and pharmaceutically acceptable carrier or auxiliary material than mixture.
Preferably, described anti-immune inflammation medicine is resisting rheumatoid disease arthritis drug or antigout arthritis drug.That is, hemp total saponins and Root or leaf of Falcate dolichos total saponins are preferably for the preparation of resisting rheumatoid disease sacroiliitis and urarthritis medicine.
Above-mentioned pharmaceutically acceptable carrier comprises the auxiliary material of oral preparations auxiliary material, parenteral administration or topical administration.Route of administration can be oral, injection, local external use's administration etc.According to technical scheme of the present invention, said composition is oral preparations or external preparation, and wherein oral preparations comprises capsule, granule, oral liquid, tablet, dripping pill etc.Auxiliary material used comprises: the conventional auxiliary materials such as starch, sucrose, lactose, Icing Sugar, cyclodextrin, Microcrystalline Cellulose sodium, propylene glycol, glycerine, Virahol, tween-80, vitamins C, acetylcysteine, S-WAT, Magnesium Stearate, citric acid and gelatin.External preparation can be bar cloth subsides etc.Preparation later stage preparation technology and equipment all belong to the routine techniques of pharmacy field, the present invention is not construed as limiting this, therefore will not describe in detail.
The present invention has beneficial effect compared with the prior art:
1, the invention provides effective extracting method of Root or leaf of Falcate dolichos glycosides A and Root or leaf of Falcate dolichos total saponins, utilize extracting method of the present invention can access purity higher than 90% Root or leaf of Falcate dolichos glycosides A and Root or leaf of Falcate dolichos glycosides A content higher than 50% Root or leaf of Falcate dolichos total saponins.
2, the present invention has proposed the Root or leaf of Falcate dolichos extract take Root or leaf of Falcate dolichos glycosides A as main active ingredient first, the test of pesticide effectiveness shows, Root or leaf of Falcate dolichos total saponins and Root or leaf of Falcate dolichos glycosides A have significant anti-immune inflammatory effect, the particularly application in preparation resisting rheumatoid disease sacroiliitis and urarthritis medicine, and toxic side effect is little, determined curative effect, safety is controlled, cheap.
Description of drawings
Fig. 1 is the mass spectrum of Root or leaf of Falcate dolichos glycosides A.
Fig. 2 is Root or leaf of Falcate dolichos glycosides A's
1H-NMR figure.
Fig. 3 is Root or leaf of Falcate dolichos glycosides A's
13C-NMR figure.
Embodiment
The invention will be further described below in conjunction with the drawings and specific embodiments, can be implemented so that those skilled in the art can better understand the present invention also, but illustrated embodiment is not as a limitation of the invention.
The invention provides the extracting method of a kind of Root or leaf of Falcate dolichos glycosides A, comprise the steps:
1) get the Root or leaf of Falcate dolichos medicinal material, add the alcohol solution refluxing extraction of 6 ~ 12 times of weight of Root or leaf of Falcate dolichos medicinal material, obtain Root or leaf of Falcate dolichos medicinal substances extract solution.When alcohol solution was the methyl alcohol of volumetric concentration 10% ~ 90% or aqueous ethanolic solution, Root or leaf of Falcate dolichos medicinal substances extract solubleness was higher, is easy to be extracted.Root or leaf of Falcate dolichos medicinal material refluxing extraction 1 ~ 3 time (that is: refluxing extraction once after, filter, filter residue carries out refluxing extraction again, filtrate merging is Root or leaf of Falcate dolichos medicinal substances extract solution), each 1 ~ 3 hour, can guarantee the complete stripping of effective constituent, can not cause the waste of solvent alcohols simultaneously.
2) the Root or leaf of Falcate dolichos medicinal substances extract solution that obtains of step 1), the reclaim under reduced pressure alcohols adds water or alcohol to without after the alcohol flavor, be mixed with liquid, every milliliter of liquid contains the extract that extracts from 0.01g ~ 2g Root or leaf of Falcate dolichos medicinal material, wherein, when adding alcohol, alcohol in liquid volume percent less than 30%.For impurity component the least possible in the preparation that guarantees extract, the alcohol in this step is preferably methyl alcohol or ethanol, and the addition of alcohol is unsuitable too much, otherwise can affect macroporous adsorbent resin in the later separation step to the fractionation by adsorption ability of effective constituent.
3) liquid that step 2) obtains is transferred pH to 1 ~ 6 with hydrochloric acid, take sodium-chlor accent salt concn as 0.1 ~ 2mol/L(namely, adds sodium-chlor or its aqueous solution in liquid, and making the concentration of sodium-chlor in liquid is 0.1 ~ 2mol/L).Therefore because the saponins compounds such as Root or leaf of Falcate dolichos glycosides A are slightly acidic, more easily be adsorbed in acidic solution, and the saponin(e such as Root or leaf of Falcate dolichos glycosides A is water-soluble strong especially, after adding suitable salt, the solubleness that is purified composition is descended, thereby be conducive to resin absorption, adsorptive capacity is increased.
4) on the liquid that step 3) is obtained, macroporous adsorptive resins separates, the amount of liquid medicine of applied sample amount take the weighing scale of Root or leaf of Falcate dolichos medicinal material and weight resin ratio as 1:0.25 ~ 4(loading be with the weighing scale of its corresponding Root or leaf of Falcate dolichos medicinal material, be from weight resin than obtaining liquid for the Root or leaf of Falcate dolichos medicinal material of 1:0.25 ~ 4).The macroporous adsorbent resin that is used for macroporous adsorptive resins in this step can be ion-exchange macroporous adsorbent resin, nonpolar macroporous adsorption resin or polar macroporous adsorption resin.From adsorption effect, be preferably polar macroporous adsorption resin, more preferably large Polar Adsorbent Resin or intermediate-polarity macroporous adsorption resin.Wherein, the ion-exchange macroporous adsorbent resin can be D900 or ADS-7 type macroporous adsorbent resin, nonpolar macroporous adsorption resin can be D101, HPD-700, HPD-200A, Semi-polarity macroporous absorption or large polar macroporous adsorption resin can be HPD400, HPD-100 or DM-130, preferred HPD-400, HPD-100, D101, most preferably HPD-400 type macroporous adsorbent resin.The blade diameter length ratio of macroporous adsorptive resins is 1:4 ~ 8, and the loading flow velocity is 0.5 ~ 2 BV/h.
5) after loading, macroporous adsorptive resins is that 10 ~ 40% methyl alcohol or aqueous ethanolic solution rinse removal of impurities with the volumetric concentration of 4 ~ 10 times of column volumes, flow velocity is 1 ~ 4 BV/h, and then be 45 ~ 90% methyl alcohol or aqueous ethanolic solution wash-out with the volumetric concentration of 4 ~ 10 times of column volumes, flow velocity is 0.5 ~ 4 BV/h, collecting mass percent concentration is 45 ~ 90% elutriants, concentrated, dry, namely gets Root or leaf of Falcate dolichos glycosides A.
The present invention also provides a kind of extracting method of Root or leaf of Falcate dolichos total saponins, comprises the steps:
1) get the Root or leaf of Falcate dolichos medicinal material, add the alcohol solution refluxing extraction of 6 ~ 12 times of weight of Root or leaf of Falcate dolichos medicinal material, filter, get filtrate; For guaranteeing the complete stripping of effective constituent, step 1) can be: get the Root or leaf of Falcate dolichos medicinal material, add the alcohol solution refluxing extraction 1 ~ 3 hour of 6 ~ 12 times of weight of Root or leaf of Falcate dolichos medicinal material, refluxing extraction 1 ~ 3 time, merging filtrate, wherein alcohol solution is that volumetric concentration is 10 ~ 90% ethanol or methanol aqueous solution.
2) filtrate that obtains of step 1), reclaim under reduced pressure alcohol is to without after the alcohol flavor, drying.
The present invention also provides the application in preparation anti-immune inflammation medicine of Root or leaf of Falcate dolichos glycosides A, its hydrolysate and salt.
The present invention also provides the application of Root or leaf of Falcate dolichos total saponins in preparation anti-immune inflammation medicine: the Root or leaf of Falcate dolichos total saponins is prepared into medicine or the pharmaceutical composition of anti-immune inflammation, the particularly application in preparing resisting rheumatoid disease sacroiliitis and urarthritis medicine as activeconstituents and pharmaceutically acceptable carrier or auxiliary material.
The below enumerates a preferred embodiment of the present invention so that technical scheme of the present invention is described.
The preparation of Root or leaf of Falcate dolichos total saponins, Root or leaf of Falcate dolichos glycosides A
The preparation of Root or leaf of Falcate dolichos total saponins: get Root or leaf of Falcate dolichos medicinal material particle 250g, be placed in the round-bottomed flask of 5L, the volumetric concentration that adds 10 times of weight of Root or leaf of Falcate dolichos medicinal material particle is 60% ethanol, soak after 2 hours, the water-bath refluxing extraction, extract altogether three times, extraction time was respectively 2,1.5,1 hours, merging filtrate.Decompression filtrate recycling ethanol to doing, gets approximately 55 grams (measuring the content of Root or leaf of Falcate dolichos glycosides A 〉=52% through the HPLC method) of Root or leaf of Falcate dolichos total saponins through 60 ℃ of drying under reduced pressure to without after the alcohol flavor.
Root or leaf of Falcate dolichos glycosides A preparation method: get Root or leaf of Falcate dolichos medicinal material particle 250g, be placed in the round-bottomed flask of 5L, the volumetric concentration that adds 10 times of weight of Root or leaf of Falcate dolichos medicinal material particle is 60% ethanol, soak after 2 hours, the water-bath refluxing extraction, extract altogether three times, extraction time was respectively 2,1.5,1 hours, merging filtrate.Decompression filtrate recycling ethanol is configured to contain the liquid (being that every milliliter of liquid contains the extract that extracts from 0.01g ~ 2g Root or leaf of Falcate dolichos) of 0.05g raw medicinal herbs/mL to without after the alcohol flavor; It is 5 that liquid is transferred PH with hydrochloric acid, and it is 0.8 mol/L that sodium-chlor is transferred salt concn; With the HPD400 type macroporous adsorptive resins of having handled well on the liquid for preparing, the blade diameter length ratio of post is 1:6, applied sample amount take medicinal material amount and weight resin than limiting as the amount of the 1:2(loading liquid weight with its corresponding Root or leaf of Falcate dolichos medicinal material particle, be from obtaining liquid with the weight resin ratio for the Root or leaf of Falcate dolichos medicinal material particle of 1:2), the loading flow velocity is 1 BV/h.After loading, concentration with 8 times of column volumes is 30% ethanol or washed with methanol removal of impurities, flow velocity is 2.5 BV/h, concentration with 7 times of column volumes is 60% ethanol or methanol-eluted fractions again, flow velocity is 2 BV/h, collecting concentration is 60% elutriant, concentrated, dry, namely gets Root or leaf of Falcate dolichos glycosides A(HPLC method and measures purity 〉=90%).
Root or leaf of Falcate dolichos glycosides A, this compound molecule formula is C
37H
58O
11, white powder is soluble in methyl alcohol, ethanol and water; Fusing point: m.p.295-297 ℃;
Mass-spectrometric data (in conjunction with Fig. 1): EI-MS (70eV) m/z, [M]
-677.4 (100); IR:3400,2930,1695,1445,1260,1165 cm
-1, showing has phenolic hydroxyl group, carbonyl and two keys;
UV: carry out all wave band scanning under ultraviolet 190-400nm, finding has maximum absorption under 195nm;
Nucleus magnetic hydrogen spectrum (in conjunction with Fig. 2):
1H-NMR (DMSO-d6,500MHz) δ: 5.5654 (1H, m, J=10.08Hz, H-12), 4.7265 (1H, d, J=4.12, H-3), 4.5022 (1H, m, J=9.95Hz, H-2), 2.0242 (s, 2H, H-2), (1.6229 1H, s, H-6), 1.0279,0.8144,0.9455;
Nuclear-magnetism carbon spectrum:
13C-NMR (DMSO-d6,500MHz) data see Table 1;
According to the spectroscopic data data of above physico-chemical property and Fig. 1 ~ 3, in conjunction with existing document (Chongqing, Hunan, Pu, Wu Dagang, Yang Chongren, Zhou Jun, 1984: the triterpene saponin component of Root or leaf of Falcate dolichos (1).Yunnan plant research, 6(3) 321-324.) report, authenticating compound is Root or leaf of Falcate dolichos glycosides A.
Root or leaf of Falcate dolichos glycosides A structural formula
Table 1 Root or leaf of Falcate dolichos glycosides A carbon-13 nmr spectra chemical displacement value
| 13C | Carbon No. | Literature [1] | Experiment |
| ? | 1 | 44.2 | 43.49 |
| ? | 2 | 69.9 | 68.76 |
| ? | 3 | 85.4 | 83.33 |
| ? | 4 | 50.2 | 51.04 |
| ? | 5 | 52.1 | 51.10 |
| ? | 6 | 21.0 | 19.90 |
| ? | 7 | 33.2 | 32.06 |
| ? | 8 | 40.1 | 40.00 |
| ? | 9 | 48.6 | 47.49 |
| ? | 10 | 36.6 | 35.76 |
| ? | 11 | 23.7 | 22.59 |
| ? | 12 | 122.5 | 121.46 |
| ? | 13 | 144.7 | 143.79 |
| ? | 14 | 42.0 | 40.80 |
| ? | 15 | 28.0 | 27.06 |
| ? | 16 | 23.4 | 21.44 |
| ? | 17 | 46.5 | 45.70 |
| ? | 18 | 42.3 | 41.42 |
| ? | 19 | 46.5 | 45.40 |
| ? | 20 | 30.0 | 30.33 |
| ? | 21 | 34.4 | 33.30 |
| ? | 22 | 33.4 | 32.78 |
| ? | 23 | 179.1 | 178.56 |
| ? | 24 | 14.0 | 13.35 |
| ? | 25 | 16.6 | 16.04 |
| ? | 26 | 17.3 | 16.72 |
| ? | 27 | 26.0 | 25.57 |
| ? | 28 | 180.9 | 180.05 |
| ? | 29 | 33.2 | 32.12 |
| ? | 30 | 23.7 | 22.97 |
| ? | 1’ | 103.8 | 102.73 |
| ? | 2’ | 74.6 | 73.68 |
| ? | 3’ | 77.1 | 76.76 |
| ? | 4’ | 70.6 | 70.05 |
| ? | 5’ | 77.1 | 76.46 |
| ? | 6’ | 61.7 | 61.02 |
| ? | COOMe | 52.1 | 51.10 |
The assay of Root or leaf of Falcate dolichos glycosides A and Root or leaf of Falcate dolichos total saponins
1. chromatographic condition
Agilent 1100 high performance liquid chromatographs, chromatographic column are Agilent TC-C18(4.6 * 250 nm, 5 μ m); Detect wavelength: 203 nm; Moving phase is: A is CH
3CN, B are 0.1% phosphate aqueous solution, gradient elution [0 ~ 8 min, 30%～40% A, 8～15 min, 40%～65% A, 15～20 min, 65%～75% A], and flow velocity is: 1.0 mLmin
-1, column temperature: 30 ℃, sample size is 20 μ L.
2. the preparation of test liquid
2.1 the configuration of standard solution: precision takes Root or leaf of Falcate dolichos glycosides A standard substance 2.4 mg that are dried to constant weight, adds in methanol constant volume to 10 mL volumetric flask, shakes up, and namely gets 0.24 mgmL
-1Root or leaf of Falcate dolichos glycosides A standard substance.
2.2 the preparation of Root or leaf of Falcate dolichos glycosides A and Root or leaf of Falcate dolichos total saponins sample solution:
Precision takes Root or leaf of Falcate dolichos total saponins sample 12.8 mg, puts in 25 mL volumetric flasks, to scale, is Root or leaf of Falcate dolichos total saponins sample solution with methanol constant volume.
Precision takes Root or leaf of Falcate dolichos glycosides A sample 5.5 mg, puts in 25 mL volumetric flasks, to scale, is Root or leaf of Falcate dolichos glycosides A sample solution with methanol constant volume.
3. the assay of Root or leaf of Falcate dolichos glycosides A and Root or leaf of Falcate dolichos total saponins sample:
Precision absorbs each 20 μ L of above-mentioned Root or leaf of Falcate dolichos glycosides A sample solution, Root or leaf of Falcate dolichos total saponins sample solution and reference substance solution respectively, injecting chromatograph, measure by above-mentioned chromatographic condition, to divide area as calculated value, the peak area that records reference substance Root or leaf of Falcate dolichos glycosides A is 257.65, the peak area that records Root or leaf of Falcate dolichos glycosides A in Root or leaf of Falcate dolichos total saponins sample is 358.22, records that in Root or leaf of Falcate dolichos glycosides A sample, Root or leaf of Falcate dolichos glycosides A peak area is 221.85.Be 65.3% by the percentage composition that calculates Root or leaf of Falcate dolichos glycosides A in Root or leaf of Falcate dolichos total saponins sample, in Root or leaf of Falcate dolichos glycosides A sample, the percentage composition of Root or leaf of Falcate dolichos glycosides A is 93.8%.
Root or leaf of Falcate dolichos glycosides A(doliroside A is called for short DA) or/and anti-immune inflammation (being specially resisting rheumatoid disease sacroiliitis) experiment in the body of Root or leaf of Falcate dolichos total saponins (being called for short DZ)
1. tested medicine
Root or leaf of Falcate dolichos total saponins sample and the Root or leaf of Falcate dolichos glycosides A sample of the described method preparation of administration sample: embodiment 1.Control drug: Chinese medicine Pa Fulin (Ningbo LiHua Pharmaceutical Co., Ltd, lot number: 100411).
2. laboratory animal
Laboratory animal is SPF level SD rat, and body weight 200 ± 20 grams are male female half and half, and credit number SCXK (Hubei Province) 2004-0007, laboratory animal conformity certification No00009567 are provided by Tongji Medical College, Huazhong Science and Technology Univ..Animal is used for experiment after 25 ℃, relative humidity are to raise for 1 week under the 60-75% condition.
3. main agents and equipment
Physiological saline: Shijiazhuang Siyao Co., Ltd produces, lot number: 110628352; Sartorius analytical balance: Beijing Sai Duolisi balance company limited.
4. experimental technique
Heat-inactivated mycobacterium butyricum is added in autoclaved whiteruss, fully grind evenly, be mixed with the suspension that concentration is 10mg/mL, use as complete Freund's adjuvant.Before causing inflammation, after 10% chloral hydrate anesthesia rat, with whole sole of the foot position after each Rat Right of alcohol wipe, avoid the rat paw vascular site, the 1mL injection needles enters Rat Right metapedes sole of the foot intracutaneous 3/4 left and right, and injection Freund's complete adjuvant 0.1mL causes inflammation.
4.1 rat (causing scorching front) is surveyed the left back sufficient volume of rat with the glass volumetric method, right foot is injected Freund's complete adjuvant afterwards, 21 days left back sufficient volumes of measurement rat after Yu Zhiyan, by the body weight random packet, every group 8, this day is given the rat oral gavage administration, experimental group gives Root or leaf of Falcate dolichos total saponins of the present invention (50,100 mg/kg) and Root or leaf of Falcate dolichos glycosides A(25,50 mg/kg), control group administration Pa Fulin (150 mg/kg), once a day, be administered to and cause scorching rear the 31st day, measure left back sufficient sole of the foot volume, and calculate its swelling rate (%) and inhibitory rate of intumesce (%).Each organizes t check between data work group, the results are shown in Table 2.
Swelling rate=(cause scorching metapedes sole of the foot volume-cause scorching front foot sole of the foot volume)/cause scorching front foot sole of the foot volume * 100%
Inhibitory rate of intumesce=(the average swelling rate of control group-average swelling rate of administration the group)/average swelling rate of control group * 100%
The impact (x ± s, n=8) on adjuvanticity rat Secondary cases pedal swelling of table 2 Root or leaf of Falcate dolichos glycosides A and Root or leaf of Falcate dolichos total saponins
Compare with model group:
*P＜0.05,
*P＜0.01,
* *P＜0.001.
Compare with the Pa Fulin group:
#P＜0.05,
##P＜0.01,
###P＜0.001.
4.2 rat random packet, every group 8, experimental group gives Root or leaf of Falcate dolichos total saponins of the present invention (50,100 mg/kg) and Root or leaf of Falcate dolichos glycosides A(25,50 mg/kg), control group administration Pa Fulin (150 mg/kg), once a day, successive administration 10 days, in the 10th day (before causing inflammation), survey Rat Right metapedes volume with the glass volumetric method, in the last administration after 30 minutes, Rat Right metapedes intradermal injection 0.1mL complete Freund's adjuvant causes inflammation, measures Rat Right metapedes sole of the foot volume after 18 hours, and calculates its swelling rate (%) and inhibitory rate of intumesce (%).Each organizes t check between data work group, the results are shown in Table 3.
The impact (x ± s, n=8) on adjuvanticity rat primary pedal swelling of table 3 Root or leaf of Falcate dolichos glycosides A and Root or leaf of Falcate dolichos total saponins
Compare with model group:
*P＜0.05,
*P＜0.01,
* *P＜0.001.
Compare with the Pa Fulin group:
#P＜0.05,
##P＜0.01,
###P＜0.001.
5. experimental result
By table 2,3 as seen, Root or leaf of Falcate dolichos glycosides A and Root or leaf of Falcate dolichos total saponins are at two dosage of height, and successive administration 10 days all can significantly suppress the former of adjuvant rat arthritis and swell swollen and secondary swelling, alleviate the secondary arthritis of rat paw, and Root or leaf of Falcate dolichos glycosides A high dose group to obviously be better than the Pa Fulin group.
Root or leaf of Falcate dolichos glycosides A(doliroside A is called for short DA) or/and anti-immune inflammation (being specially antigout sacroiliitis) experiment in the body of Root or leaf of Falcate dolichos total saponins (being called for short DZ)
1. tested medicine
Root or leaf of Falcate dolichos total saponins sample and the Root or leaf of Falcate dolichos glycosides A sample of the described method preparation of administration sample: embodiment 1.Control drug: colchicine sheet (Jilin Province Huinan Huifa Pharmacy stock Co., Ltd, lot number: 110516).
2. laboratory animal
Laboratory animal is Kunming mouse, and body weight 20 ± 2 grams are male female half and half, and credit number SCXK (Hubei Province) 2004-0007, laboratory animal conformity certification No00009567 are provided by Tongji Medical College, Huazhong Science and Technology Univ..Animal is used for experiment after 25 ℃, relative humidity are to raise for 1 week under the 60-75% condition.
3. main agents and equipment
Physiological saline: Shijiazhuang Siyao Co., Ltd produces, lot number: 110628352; Sartorius analytical balance: Beijing Sai Duolisi balance company limited; Uric acid sodium crystallization: self-control.
4. experimental technique
With aseptic, without endotoxic uric acid sodium crystallization 0.5mg, be suspended in 20 microlitre physiological saline, then inject in the mouse ankle joint chamber of each administration group and cause inflammation, blank group injection equivalent physiological saline.
4.1 mouse is by the body weight random packet, every group 12, before modeling, the continuous gastric infusion of mouse is 4 days, experimental group gives Root or leaf of Falcate dolichos total saponins of the present invention (50,100 mg/kg) and Root or leaf of Falcate dolichos glycosides A(25,50 mg/kg), control group gives colchicine sheet (20 mg/kg), once a day, be administered to after modeling the 3rd day.Measure mouse ankle joint diameter with vernier callipers before causing inflammation, uric acid sodium crystallization afterwards causes inflammation, detects 1,2,3 day mouse ankle joint diameter respectively at same position, and calculates swelling rate.Each organizes t check between data work group, the results are shown in Table 4.
Swelling rate=(cause scorching after diameter-cause scorching before diameter)/cause scorching before diameter * 100%
The impact (x ± s, n=10) on uric acid sodium induced mice urarthritis of table 4 Root or leaf of Falcate dolichos glycosides A and Root or leaf of Falcate dolichos total saponins
Compare with model group:
*P＜0.05,
*P＜0.01,
* *P＜0.001.
5. experimental result
By as seen from Table 4, Root or leaf of Falcate dolichos glycosides A and Root or leaf of Falcate dolichos total saponins be at two dosage of height, and successive administration 7 days all can significantly suppress the swelling of the mouse urarthritis due to uric acid sodium, alleviates the mouse arthropathy.
The preparation of Root or leaf of Falcate dolichos glycosides A hydrolysate and salt thereof
In Root or leaf of Falcate dolichos glycosides A, with ester group, after hydrolysis, the structural formula of hydrolysate is as follows:
The preparation method of Root or leaf of Falcate dolichos glycosides A hydrolysate is as follows: with Root or leaf of Falcate dolichos glycosides A 10g, add in the NaOH solution of 1L 30%, 60 ℃ of reactions were filtered after 4 hours; The filtrate concentrated hydrochloric acid is transferred PH to 6.7 left and right, filters, and the gained precipitation is the hydrolysate of Root or leaf of Falcate dolichos glycosides A.
The structural formula of the salt of Root or leaf of Falcate dolichos glycosides A hydrolysate is as follows
Wherein, M
n+Can be Na
+, K
+, Mg
2+, Fe
3+, NH
4 +, Zn
2+In nominal price ion etc.
The preparation method of the salt of Root or leaf of Falcate dolichos glycosides A hydrolysate: after being hydrolyzed, gained Root or leaf of Falcate dolichos glycosides A hydrolysate adds corresponding NaOH, KOH, NH
3OH etc. generate the salt of corresponding Root or leaf of Falcate dolichos glycosides A hydrolysate.
The preparation of Root or leaf of Falcate dolichos glycosides A derivative
The structural formula of the glycosides of Root or leaf of Falcate dolichos described in the present embodiment A derivative is as follows:
Wherein, R is the alkyl of C2 ~ C4.
The preparation method of Root or leaf of Falcate dolichos glycosides A derivative: get appropriate Root or leaf of Falcate dolichos glycosides A hydrolysate and add corresponding C
2H
5OH, C
3H
7OH, C
4H
9OH carries out esterification, reacts products therefrom through the column chromatography purifying and get final product.
The extracorporeal anti-inflammatory experiment of Root or leaf of Falcate dolichos glycosides A hydrolysate and salt thereof, Root or leaf of Falcate dolichos glycosides A
1 materials and methods
1. 1 material
RAW264. 7 cell strains are available from Chinese Academy of Sciences's Shanghai school of life and health sciences; The DMEM cell culture medium is Hyclone company product; Trypsinase is U.S. Gibco company product; Foetal calf serum (fetal bovine serum, FBS) is German Biochrom AG company product; E. coli lipopolysaccharide (Lipopolysaccharide, LPS) is Sigma company product; Each compound is self-control.
1. 2 cell cultures
RAW264. 7 cell cultures are used the DMEM substratum contain 10 % FBS and 2 mmol/L glutamine, cultivate in 37 ℃, 5 % CO2 incubators.
1. 3 cells are processed
The RAW264.7 cell that will be in logarithmic phase adds 96 porocyte culture plates, 1 * 10
6Cell hole
-1The compound 1,2,3,4,5 difference 7.5,15,30,60, the 90 μ M that add different final concentrations, hatch 24 h in incubator, change fresh culture, after using LPS (10 μ g/mL) inducing cell 6h, according to typical curve, the Griess method is measured the content of NO in cell conditioned medium liquid.
2 experimental results
In the scavenger cell supernatant liquor NO level mensuration adopt Griess (nitrate reductase method) method to detect the scavenger cell that each compound induces LPS to produce the NO impact.The experimental result demonstration, after 10 μ g/mL LPS acted on scavenger cell, in nutrient solution, the level of NO was significantly higher than control group; Compare with the LPS group, each compound all can significantly suppress the generation of NO, its IC under 7.5,15,30,60,90 μ M dosage
50The results are shown in Table 1.
Each chemical combination of table 1 suppresses the scavenger cell NO release IC that LPS induces
50
As can be seen from Table 1, after Root or leaf of Falcate dolichos glycosides A mild hydrolysis, products therefrom 1 its anti-inflammatory activity outline is better than former compound; After salify, compound 4,5 anti-inflammatory activities are better, may be water-soluble relevant with its improvement.
Press the described method of embodiment 1, the Root or leaf of Falcate dolichos glycosides A sample for preparing adds tablet to commonly use auxiliary material, and preparation technology is prepared into tablet routinely.
Press the described method of embodiment 1, the Root or leaf of Falcate dolichos total saponins sample for preparing adds tablet to commonly use auxiliary material, and preparation technology is prepared into tablet routinely.
Also the Root or leaf of Falcate dolichos total saponins sample for preparing by the described method of embodiment 1 can be mixed in any proportion with Root or leaf of Falcate dolichos glycosides A sample and add tablet to commonly use auxiliary material, preparation technology is prepared into tablet routinely.
Press the described method of embodiment 6, the Root or leaf of Falcate dolichos glycosides A sample of hydrolysate for preparing adds tablet to commonly use auxiliary material, and preparation technology is prepared into tablet routinely.
Press the described method of embodiment 6, the salt sample of the Root or leaf of Falcate dolichos glycosides A hydrolysate for preparing adds tablet to commonly use auxiliary material, and preparation technology is prepared into tablet routinely.
Press the described method of embodiment 7, the Root or leaf of Falcate dolichos glycosides A derivative sample for preparing adds tablet to commonly use auxiliary material, and preparation technology is prepared into tablet routinely.
Press the described method of embodiment 1, the Root or leaf of Falcate dolichos glycosides A sample for preparing adds capsule to commonly use auxiliary material, and preparation technology is prepared into capsule routinely.
Press the described method of embodiment 1, the Root or leaf of Falcate dolichos total saponins sample for preparing adds capsule to commonly use auxiliary material, and preparation technology is prepared into capsule routinely.
Also the Root or leaf of Falcate dolichos total saponins sample for preparing by the described method of embodiment 1 can be mixed in any proportion with Root or leaf of Falcate dolichos glycosides A sample and add capsule to commonly use auxiliary material, preparation technology is prepared into capsule routinely.
Press the described method of embodiment 6, the Root or leaf of Falcate dolichos glycosides A sample of hydrolysate for preparing adds capsule to commonly use auxiliary material, and preparation technology is prepared into capsule routinely.
Press the described method of embodiment 6, the salt sample of the Root or leaf of Falcate dolichos glycosides A hydrolysate for preparing adds capsule to commonly use auxiliary material, and preparation technology is prepared into capsule routinely.
Press the described method of embodiment 7, the Root or leaf of Falcate dolichos glycosides A derivative sample for preparing adds capsule to commonly use auxiliary material, and preparation technology is prepared into capsule routinely.
Embodiment 11
Press the described method of embodiment 1, the Root or leaf of Falcate dolichos glycosides A sample for preparing adds cataplasma to commonly use auxiliary material, and preparation technology is prepared into cataplasma routinely.
Press the described method of embodiment 1, the Root or leaf of Falcate dolichos total saponins sample for preparing adds cataplasma to commonly use auxiliary material, and preparation technology is prepared into cataplasma routinely.
Also the Root or leaf of Falcate dolichos total saponins sample for preparing by the described method of embodiment 1 can be mixed in any proportion with Root or leaf of Falcate dolichos glycosides A sample and add cataplasma to commonly use auxiliary material, preparation technology is prepared into cataplasma routinely.
Press the described method of embodiment 6, the Root or leaf of Falcate dolichos glycosides A sample of hydrolysate for preparing adds cataplasma to commonly use auxiliary material, and preparation technology is prepared into cataplasma routinely.
Press the described method of embodiment 6, the salt sample of the Root or leaf of Falcate dolichos glycosides A hydrolysate for preparing adds cataplasma to commonly use auxiliary material, and preparation technology is prepared into cataplasma routinely.
Press the described method of embodiment 7, the Root or leaf of Falcate dolichos glycosides A derivative sample for preparing adds cataplasma to commonly use auxiliary material, and preparation technology is prepared into cataplasma routinely.
The above embodiment is the preferred embodiment that proves absolutely that the present invention lifts, and protection scope of the present invention is not limited to this.Being equal to that those skilled in the art do on basis of the present invention substitutes or conversion, all within protection scope of the present invention.Protection scope of the present invention is as the criterion with claims.
Claims (2)
1. the application of Root or leaf of Falcate dolichos glycosides A in preparation anti-immune inflammation medicine, described Root or leaf of Falcate dolichos glycosides A is prepared into anti-immune inflammation medicine or pharmaceutical composition as activeconstituents and pharmaceutically acceptable carrier or auxiliary material.
2. the application of Root or leaf of Falcate dolichos glycosides A according to claim 1 in preparation anti-immune inflammation medicine is characterized in that described anti-immune inflammation medicine is resisting rheumatoid disease arthritis drug or antigout arthritis drug.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210320436 CN102875615B (en) | 2012-09-03 | 2012-09-03 | Extraction method and application of falcate dolichos root or leaf glucoside A and total saponins of falcate dolichos root or leaf |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210320436 CN102875615B (en) | 2012-09-03 | 2012-09-03 | Extraction method and application of falcate dolichos root or leaf glucoside A and total saponins of falcate dolichos root or leaf |
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| CN102875615A CN102875615A (en) | 2013-01-16 |
| CN102875615B true CN102875615B (en) | 2013-11-06 |
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| CN 201210320436 Expired - Fee Related CN102875615B (en) | 2012-09-03 | 2012-09-03 | Extraction method and application of falcate dolichos root or leaf glucoside A and total saponins of falcate dolichos root or leaf |
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| CN107375309A (en) * | 2017-08-09 | 2017-11-24 | 武汉翼博济生生物科技有限公司 | The O β D glucopyranosides of medicagenic acid 3 are preparing the application in treating ischemic brain damage medicine |
| CN110684071B (en) * | 2018-07-06 | 2022-02-11 | 云南民族大学 | A kind of triterpenoid compound and its preparation method and application |
| CN109336946B (en) * | 2018-11-27 | 2020-08-28 | 中南民族大学 | Cannabis sativa glycoside A crystal and preparation method thereof |
| CN109336947B (en) * | 2018-11-27 | 2020-08-28 | 中南民族大学 | Method for separating and purifying cannabinoids glycoside A from cannabis sativa |
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