CN103145790B - Method for simultaneously preparing oleanolic acid and echinocystic acid from fructus gleditsiae - Google Patents
Method for simultaneously preparing oleanolic acid and echinocystic acid from fructus gleditsiae Download PDFInfo
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Abstract
The invention discloses a method for simultaneously preparing oleanolic acid and echinocystic acid from fructus gleditsiae. The method comprises the following steps of: (1), extracting fructus gleditsiae saponin extract; (2), hydrolyzing the fructus gleditsiae saponin; and (3), preparing thin layers of oleanolic acid and echinocystic acid. According to the method using the fructus gleditsiae as the material disclosed by the invention, the oleanolic acid and echinocystic acid can be simultaneously prepared through alcohol extraction, hydrolysis and prepared thin-layer separation. For the two prepared components, according to the oleanolic acid and echinocystic acid reference product contrastive analysis and content measurement provided by National Institute for the Control of Pharmaceutical and Biological Products, the content of the oleanolic acid prepared by the method disclosed by the invention is 97.8%, and the content of the echinocystic acid is 98.2%; the purities of the oleanolic acid and echinocystic acid are equal to those of the oleanolic acid and echinocystic acid reference products, so that the quality requirements of traditional Chinese medicine chemical reference samples are reached. Besides, the method is free of valuable apparatuses, simple and easy to carry out, low in cost and capable of meeting the quality requirements of traditional Chinese medicine chemical reference samples.
Description
Technical field
The present invention relates to a kind of method simultaneously preparing Oleanolic Acid and Echinocystic acid from Fructus Gleditsiae Abnormalis.
Background technology
Fructus Gleditsiae Abnormalis is the sterile fruit of drying of leguminous plants Chinese honey locust Gledisia Sinensis Lam., and main containing pentacyclic triterpene type Gleditschiasaponin constituents, Oleanolic Acid and Echinocystic acid are the main aglycons (Fig. 1 and Fig. 2 be shown in chemical structural formula) of Gleditschiasaponin.Oleanolic Acid and Echinocystic acid not only have important physiologically active to human body, and are chemical reference substances the most frequently used in Chinese medicine study and analytical test, therefore, prepare Oleanolic Acid and Echinocystic acid has important using value.At present preparative high performance liquid chromatography is adopted to the preparation of Oleanolic Acid and Echinocystic acid reference substance more, though reference substance purity prepared by the method is higher, need large-scale expensive equipment and special chromatography column, there is the defects such as cost is high, preparation cycle is long.
Summary of the invention
For above-mentioned prior art, the invention provides a kind of method simultaneously preparing Oleanolic Acid and Echinocystic acid from Fructus Gleditsiae Abnormalis, present method take Fructus Gleditsiae Abnormalis as raw material, can prepare Oleanolic Acid and Echinocystic acid through ethanol-extracted, hydrolysis and preparative Thin-layer separation simultaneously.To two kinds of compositions of preparation, the Oleanolic Acid that warp and Nat'l Pharmaceutical & Biological Products Control Institute provide and the comparative analysis of Echinocystic acid reference substance, show Oleanolic Acid prepared by employing this law and Echinocystic acid, its purity is substantially suitable with two kinds of reference substances, reaches the specification of quality of traditional Chinese chemical contrast.Present method is without the need to expensive instrument, simple and easy to do, and cost is low, and can reach the specification of quality of traditional Chinese chemical contrast.
The present invention is achieved by the following technical solutions:
From Fructus Gleditsiae Abnormalis, prepare a method for Oleanolic Acid and Echinocystic acid, step is as follows simultaneously:
(1) extraction of Fructus Gleditsiae Abnormalis total saponin extracts: take Fructus Gleditsiae Abnormalis 100g, uses 600mL75%(percent by volume) alcohol reflux 2h, filter, residue uses 600mL75% extraction using alcohol 2 times again, each 1h, filters, merges 3 alcohol extracts, reclaim ethanol, be condensed into medicinal extract, 60 ~ 65 DEG C of drying under reduced pressure, pulverize, obtain Fructus Gleditsiae Abnormalis total saponin extracts, for subsequent use;
(2) hydrolysis of Fructus Gleditsiae Abnormalis total saponins: get Fructus Gleditsiae Abnormalis total saponin extracts 10g, add diatomite 3 ~ 5g, mix thoroughly, put in apparatus,Soxhlet's, add methyl alcohol 200mL, reflux 3 hours, let cool, evaporate to dryness, residue adds 10% hydrochloric acid (mass percent) 200mL makes dissolving, be transferred in round-bottomed flask, heating in water bath hydrolysis 2h, let cool, put in separating funnel, with chloroform extraction, 3 times (the chloroform consumption of 3 times is followed successively by 100mL, 100mL, 60mL), combined chloroform extracting solution, wash 2 times with water, each 150mL, discard water liquid, reclaim chloroform, evaporate to dryness, residue dissolve with methanol is also settled in 25mL measuring bottle, shake up, 0 ~ 4 DEG C of placement, adularescent crystallization, leach crystallization, mother liquor concentrations is to about 10ml, the same placement, there is crystallization again, leach crystallization again, twice crystallization is merged, with a small amount of methanol wash crystallization, 55 ~ 60 DEG C of dryings, obtain drying crystalline, for subsequent use, this crystallization is mainly containing Oleanolic Acid and Echinocystic acid,
(3) the thin layer preparation of Oleanolic Acid and Echinocystic acid:
1. the preparation of thin layer need testing solution: get above-mentioned drying crystalline, makes the solution that concentration is 26mg/ml, as thin layer need testing solution with methyl alcohol;
2. thin layer condition:
Chromatoplate: the silica gel g thin-layer plate (20cm × 20cm) of chromatoplate to be thickness be 0.6mm, 105 DEG C of activation 30 minutes, for subsequent use;
Developping agent: cyclohexane-ethyl acetate-formic acid (volume ratio is 15:4:2) upper strata;
Developer: 10% sulfuric acid ethanol (mass percent), heat gun is clear to developing the color;
Exhibition distance: 15cm;
3. point sample and preparation: with miniature sample applicator pipette samples liquid 800 μ l, with strip or point format continuity point on thin layer plate, be placed in the chromatography cylinder filling developping agent, exhibition journey 15cm, take out, dry, develop the color in thin layer plate both sides, chromatoplate Oleanolic Acid (top colour developing spot) and Echinocystic acid (bottom colour developing spot) the non-color development area in centre that the spot that develops the color is corresponding are carefully scraped respectively, the Oleanolic Acid scraped and Echinocystic acid silica-gel powder are respectively put in a 250mL Erlenmeyer flask, add methyl alcohol 150mL, supersound process 30min, centrifugal, leach supernatant liquor, precipitation adds methyl alcohol 150ml again, the same ultrasonic, centrifugal repetitive operation 2 times, merge three ultrasonic filtrates containing Oleanolic Acid and Echinocystic acid respectively, reclaim methyl alcohol, evaporate to dryness, namely obtain respectively prepare Oleanolic Acid and Echinocystic acid (this preparation method can according to the requirement of preparation amount, adopt aforesaid method can carry out separation preparation with time point in polylith thin layer chromatography board).
The method simultaneously preparing Oleanolic Acid and Echinocystic acid from Fructus Gleditsiae Abnormalis of the present invention, take Fructus Gleditsiae Abnormalis as raw material, Oleanolic Acid and Echinocystic acid is prepared through ethanol-extracted, hydrolysis and preparative Thin-layer separation, and carried out the preparation test of three batches of two kinds of compositions, shown by the analysis of two kinds of compositions to preparation, the Oleanolic Acid adopting this law to prepare and Echinocystic acid, its purity is substantially suitable with two kinds of reference substances, reaches the requirement of traditional Chinese chemical contrast.This preparation method compares with the preparation method of Echinocystic acid with current Oleanolic Acid, has without the need to expensive instrument, and simple and easy to do, cost is low, and can reach the advantages such as the specification of quality of chemical reference substance, is the comparatively ideal method simultaneously preparing two kinds of traditional Chinese chemical contrasts.Through looking into newly about the research of this preparation method has no bibliographical information.
Accompanying drawing explanation
Fig. 1: the chemical structural formula of Oleanolic Acid.
Fig. 2: the chemical structural formula of Echinocystic acid.
Fig. 3: be hydrolyzed crystallization TLC figure.
Fig. 4: thin layer preparation signal all develops the color figure.
Fig. 5: thin layer prepares only both sides colour developing schematic diagram.
Fig. 6: the TLC figure of Oleanolic Acid prepared by thin layer and Echinocystic acid, wherein, band 1: Echinocystic acid contrasts; Echinocystic acid prepared by band 2:TLC; Band 3: Oleanolic Acid contrasts; Oleanolic Acid prepared by band 4:TLC.
Fig. 7-1: Echinocystic acid reference substance HPLC schemes.
Fig. 7-2: Oleanolic Acid reference substance HPLC schemes.
Fig. 7-3: Echinocystic acid HPLC prepared by thin layer schemes.
Fig. 7-4: Oleanolic Acid HPLC prepared by thin layer schemes.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
Embodiment 1 prepares Oleanolic Acid and Echinocystic acid from Fructus Gleditsiae Abnormalis simultaneously
Step is as follows:
(1) extraction of Fructus Gleditsiae Abnormalis total saponin extracts: take Fructus Gleditsiae Abnormalis 100g, with the alcohol reflux 2h of 600mL75%, filters, residue uses 600mL75% extraction using alcohol 2 times again, each 1h, filters, merge 3 alcohol extracts, reclaim ethanol, be condensed into medicinal extract, 60 ~ 65 DEG C of drying under reduced pressure, pulverize, obtain Fructus Gleditsiae Abnormalis total saponin extracts, for subsequent use, three batches of total saponin extracts preparations the results are shown in Table 1.
Table 1 three batches of total saponin extracts prepare result
* yield is in the Fructus Gleditsiae Abnormalis medicinal material that feeds intake.
(2) hydrolysis of Fructus Gleditsiae Abnormalis total saponins: get Fructus Gleditsiae Abnormalis total saponin extracts 10g, add diatomite 4g, mix thoroughly, put in apparatus,Soxhlet's, add methyl alcohol 200mL, reflux 3 hours, let cool, evaporate to dryness, residue adds 10% hydrochloric acid (mass percent) 200mL makes dissolving, be transferred in round-bottomed flask, heating in water bath hydrolysis 2h, let cool, put in separating funnel, with chloroform extraction, 3 times (the chloroform consumption of 3 times is followed successively by 100mL, 100mL, 60mL), combined chloroform extracting solution, wash 2 times with water, each 150mL, discard water liquid, reclaim chloroform, evaporate to dryness, residue dissolve with methanol is also settled in 25mL measuring bottle, shake up, 0 ~ 4 DEG C of placement, adularescent crystallization, leach crystallization, mother liquor concentrations is to about 10ml, the same placement, there is crystallization again, leach crystallization again, twice crystallization is merged, with a small amount of methanol wash crystallization, 55-60 DEG C of drying, obtain drying crystalline, for subsequent use, this crystallization is mainly containing Oleanolic Acid and Echinocystic acid, three batches of Fructus Gleditsiae Abnormalis total saponins results of hydrolysis are in table 2.
Table 2 three batches of Fructus Gleditsiae Abnormalis total saponins results of hydrolysis
* yield is in the total saponins that feeds intake.
Tlc is adopted to analyze (thin layer condition is identical with following preparation condition) gained crystallization, and contrast with Oleanolic Acid and Echinocystic acid, find this crystallization mainly containing Oleanolic Acid and Echinocystic acid two colour developing spot, as shown in Figure 3 (in Fig. 3, prepare many batch samples and contrasted with Echinocystic acid, Oleanolic Acid reference substance, band 1,2,5,6,7 is sample, band 3 is Echinocystic acid reference substance, and band 4 is Oleanolic Acid reference substance; Result shows in every batch sample all mainly containing Oleanolic Acid and Echinocystic acid);
(3) the thin layer preparation of Echinocystic acid and Oleanolic Acid:
1. the preparation of thin layer need testing solution: get above-mentioned drying crystalline, makes the solution that concentration is 26mg/ml, as Preparative TLC sample liquid with methyl alcohol;
2. thin layer condition:
Chromatoplate: the silica gel g thin-layer plate (20cm × 20cm) of chromatoplate to be thickness be 0.6mm, 105 DEG C of activation 30 minutes, for subsequent use;
Developping agent: cyclohexane-ethyl acetate-formic acid (volume ratio is 15:4:2) upper strata;
Developer: 10% sulfuric acid ethanol (mass percent), heat gun is clear to developing the color;
Exhibition distance: 15cm;
3. point sample and preparation: with miniature sample applicator pipette samples liquid 800 μ l, with point format continuity point on thin layer plate, be placed in the chromatography cylinder filling developping agent, Zhan Cheng 15cm, take out, dry, launch colour developing rear as shown in Fig. 4 (entirely develop the color, determine whether Oleanolic Acid can be separated completely with Echinocystic acid), can find out in figure, there are obvious two developed band, represent that Oleanolic Acid can be separated completely with Echinocystic acid.And then with miniature sample applicator pipette samples liquid 800 μ l, with bar form continuity point on 20 blocks of thin layer plates, be placed in the chromatography cylinder filling developping agent, exhibition journey 15cm, take out, dry, develop the color in thin layer plate both sides, launch colour developing rear as Fig. 5 (only both sides colour developing, for the preparation of Oleanolic Acid and Echinocystic acid) shown in, chromatoplate Oleanolic Acid (top colour developing spot) and Echinocystic acid (bottom colour developing spot) the non-color development area in centre that the spot that develops the color is corresponding are carefully scraped respectively, the Oleanolic Acid scraped and Echinocystic acid silica-gel powder are placed in 250mL Erlenmeyer flask respectively, add methyl alcohol 150mL, supersound process 30min, centrifugal, leach supernatant liquor, precipitation adds methyl alcohol 150ml again, the same ultrasonic, centrifugal repetitive operation 2 times, merge three ultrasonic filtrates containing Oleanolic Acid and Echinocystic acid respectively, reclaim methyl alcohol, evaporate to dryness, obtain Oleanolic Acid and Echinocystic acid.
Aforesaid method is adopted to prepare three batches of Oleanolic Acids and Echinocystic acid, often criticize each preparation 20 blocks of thin layer plates, the silica-gel powder containing Oleanolic Acid and Echinocystic acid scraped by each chromatoplate merges respectively, add methyl alcohol as stated above, supersound extraction, filter, reclaim, drying, obtains three batches of Oleanolic Acids and the Echinocystic acid sample of preparation, and each batch sample preparation the results are shown in Table 3,4:
The preparation result of table 3 three batches of Oleanolic Acids
* yield is with the suitable crystallization gauge of point sample volume.
The preparation result of table 4 three batches of Echinocystic acid
* yield is with the suitable crystallization gauge of point sample volume.
Embodiment 2 is to the Oleanolic Acid of preparation and the qualification of Echinocystic acid and determination study:
For identifying whether the two kinds of compositions adopting the method for embodiment 1 to prepare are Oleanolic Acid and Echinocystic acid, and analyze its purity, again with Oleanolic Acid and Echinocystic acid reference substance for contrast, carry out the fusing point test of two kinds of compositions respectively, thin-layer chromatography and HPLC comparative analysis, result shows, the two kinds of compounds adopting preparative thin layer to get have substantially identical fusing point with Oleanolic Acid with Echinocystic acid reference substance respectively, Rf value and HPLC retention time, the reference substance that its purity provides with Nat'l Pharmaceutical & Biological Products Control Institute is substantially identical, reach the specification of quality of traditional Chinese chemical contrast, its every test method and result as follows:
(1) fusing point mixes test and tests: carried out fusing point test respectively to the Oleanolic Acid be separated and Echinocystic acid, the fusing point recording Oleanolic Acid is 307-309 DEG C (bibliographical information 306-308 DEG C or 308-310 DEG C), the fusing point of Echinocystic acid be 308-310 DEG C (bibliographical information 305-312 DEG C, basically identical with two kinds of reference substance bibliographical informations; And after respectively the Echinocystic acid got and Echinocystic acid reference substance being ground well with the Oleanolic Acid got and Oleanolic Acid reference substance, load in kapillary, carry out the mixed survey of fusing point, record fusing point 307-309 DEG C and the 308-310 DEG C respectively of Oleanolic Acid and Echinocystic acid mixture, show that the mixed melting point of two kinds of compositions measures and be showed no obvious decline, show that the purity of getting Oleanolic Acid and Echinocystic acid two kinds of compositions is substantially suitable with reference substance.
(2) tlc is to preparing the analytical results of Oleanolic Acid with Echinocystic acid: by the Oleanolic Acid that is separated and Echinocystic acid and Oleanolic Acid and Echinocystic acid reference substance, the solution of every 1ml containing 1mg is made into methyl alcohol, put respectively on same thin laminate, the same expansion, colour developing (the expansion of embodiment 1, coloration method) after, with on the correspondence position of reference substance Oleanolic Acid and Echinocystic acid chromatogram, all only there is the single main colour developing point of Oleanolic Acid and Echinocystic acid, see Fig. 6, this shows that Oleanolic Acid prepared by the method for embodiment 1 and Echinocystic acid have higher purity, from the result of thin layer colour developing, its purity and reference substance are substantially suitable.
(3) Oleanolic Acid of HPLC to preparation and the analytical results of Echinocystic acid:
High performance liquid chromatograph (Waters2695Separations Module, Waters2996Photodiode Array Detector).Chromatographic column is Di Ma company Diamonsil
tM(diamond) C18,5 μ, 150 × 4.6r, ultrapure water (Millipore company water purifior), moving phase acetonitrile is chromatographically pure, and other reagent are analytical pure; Oleanolic Acid and Echinocystic acid reference substance, provided, for assay by Chinese pharmaceutical biological product inspection institute, Oleanolic Acid lot number 0709 ~ 9803, Echinocystic acid lot number 110756 ~ 2011010, the purity of its reference substance is by the requirement of relevant national standard, and its content should more than 97.0%.
Chromatographic condition and system suitability:
Be weighting agent with octadecylsilane chemically bonded silica; Acetonitrile: water (90:10) is moving phase; Flow velocity 1.0mL.min-1; Determined wavelength is 210nm; Temperature: room temperature.Number of theoretical plate calculates should be not less than 2500 by Oleanolic Acid and Echinocystic acid peak.
The preparation of need testing solution: get Oleanolic Acid prepared by thin layer and Echinocystic acid in right amount each, make every 1ml about containing the solution of 0.5mg by moving phase (acetonitrile: water=90:10, V/V), shake up, as need testing solution.
The preparation of reference substance solution: get Echinocystic acid reference substance with appropriate in 2mL measuring bottle, accurately weighed is 2.60mg; Get Oleanolic Acid reference substance in right amount in 2mL measuring bottle, accurately weighed is 2.12mg.Respectively add moving phase (acetonitrile: water=90:10, V/V) make dissolving in right amount and be diluted to scale, shake up, as two reference substance storing solutions, (its concentration is respectively 1.30mg.mL
-1, 1.06mg.mL
-1); Again in each 1mL to the 2mL measuring bottle of above-mentioned two storing solution of accurate absorption, shake up, in contrast product mixing solutions, the concentration of its Oleanolic Acid and Echinocystic acid reference substance is respectively 0.65mg.mL
-1, 0.53mg.mL
-1.
Test-results:
Draw each 10 μ l of 2 kinds of need testing solutions of Echinocystic acid reference substance, Oleanolic Acid reference substance, preparation respectively, injection liquid chromatography, obtains each HPLC color atlas, as shown in Fig. 7-1 ~ 7-4.
1. to the analysis of Echinocystic acid HPLC collection of illustrative plates: adopt above-mentioned HPLC condition to show Echinocystic acid analytical results prepared by thin layer, thin layer prepares the HPLC color atlas (Fig. 7-3) of Echinocystic acid, with Echinocystic acid reference substance (Fig. 7-1) identical retention time (5.459) place, there is the major absorbance peak of obvious Echinocystic acid, two HPLC figure contrasts, except Echinocystic acid main peak, the sample of reference substance and preparation all has small impurity peaks, but totally it seems, Echinocystic acid prepared by thin layer has had higher purity, its content preparing Echinocystic acid in sample should be substantially suitable with Echinocystic acid reference substance, reach the specification of quality of traditional Chinese chemical contrast to Echinocystic acid.
2. to the analysis of Oleanolic Acid HPLC collection of illustrative plates: adopt above-mentioned HPLC condition to show Oleanolic Acid analytical results prepared by thin layer, the Oleanolic Acid HPLC color atlas (Fig. 7-4) prepared of thin layer has the major absorbance peak of obvious Oleanolic Acid with Oleanolic Acid reference substance (Fig. 7-2) identical retention time (12.602) place, two HPLC figure contrasts, except Oleanolic Acid main peak, the sample of reference substance and preparation all has small impurity peaks, and the impurity peaks in the Oleanolic Acid color atlas prepared of thin layer is obviously less than reference substance, this shows that thin layer prepares the content of Oleanolic Acid in sample higher than Oleanolic Acid reference substance used, reach the specification of quality of traditional Chinese chemical contrast to Oleanolic Acid.
(4) assay of Oleanolic Acid and Echinocystic acid in sample is prepared: to Oleanolic Acid and the Echinocystic acid of preparation, according to (3) HPLC, assay is carried out to the analysis condition of Oleanolic Acid and Echinocystic acid and measuring method, recording the content preparing Oleanolic Acid is 97.8%, the content preparing Echinocystic acid is 98.2%, and reaching the traditional Chinese chemical contrast that national correlation department specifies should lower than the technical requirements of 97.0%.
The record " HPLC measures the content of Echinocystic acid and Oleanolic Acid in Fructus Gleditsiae Abnormalis total saponins " of embodiment 6 in the application for a patent for invention 201210594743.7 that above-mentioned concrete method is submitted in 2012.12.31 with reference to applicant.
Brief summary and discussion
(1) method of the present invention take Fructus Gleditsiae Abnormalis as raw material, prepare Oleanolic Acid and Echinocystic acid reference substance through ethanol-extracted, hydrolysis and preparative Thin-layer separation simultaneously, and carried out the preparation test of three batches of two kinds of compositions, by showing the analysis of preparation two kinds of compositions, the content adopting this law to prepare Oleanolic Acid is 97.8%, the content of Echinocystic acid is 98.2%, and reaching the traditional Chinese chemical contrast that national correlation department specifies should lower than the technical requirements of 97.0%.
(2) this method preparing Oleanolic Acid and Echinocystic acid is compared with current preparation method, the method has without the need to expensive instrument, simple and easy to do, and cost is low, and the advantages such as the specification of quality of traditional Chinese chemical contrast can be reached, be the comparatively ideal method simultaneously preparing two kinds of traditional Chinese chemical contrasts.Through looking into newly about the research of this preparation method has no bibliographical information.
(3) the preparation amount Oleanolic Acid of present method reaches more than 100mg, Echinocystic acid reaches more than 200mg, in preparation process, and also can according to the expense to two kinds of compositions, proceed the preparation of magnification level again, its preparation amount even can reach level prepared by gram level.
Claims (6)
1. from Fructus Gleditsiae Abnormalis, prepare a method for Oleanolic Acid and Echinocystic acid simultaneously, it is characterized in that: step is as follows:
(1) extraction of Fructus Gleditsiae Abnormalis total saponin extracts: take Fructus Gleditsiae Abnormalis 100g, with the alcohol reflux 2h of 75%, filters, residue uses 75% extraction using alcohol 2 times again, each 1h, filters, merges 3 alcohol extracts, reclaim ethanol, be condensed into medicinal extract, 60 ~ 65 DEG C of drying under reduced pressure, pulverize, obtain Fructus Gleditsiae Abnormalis total saponin extracts, for subsequent use;
(2) hydrolysis of Fructus Gleditsiae Abnormalis total saponins: get Fructus Gleditsiae Abnormalis total saponin extracts 10g, add diatomite 3 ~ 5g, mix thoroughly, put in apparatus,Soxhlet's, add methyl alcohol, reflux 3 hours, let cool, evaporate to dryness, residue adds dilute hydrochloric acid makes dissolving, be transferred in round-bottomed flask, heating in water bath hydrolysis 2h, let cool, put in separating funnel, with chloroform extraction 3 times, combined chloroform extracting solution, wash 2 times with water, discard water liquid, reclaim chloroform, evaporate to dryness, residue dissolve with methanol, 0 ~ 4 DEG C of placement, adularescent crystallization, leach crystallization, mother liquor concentrations is to 10ml, the same placement, there is crystallization again, leach crystallization again, twice crystallization is merged, use methanol wash crystallization, 55 ~ 60 DEG C of dryings, obtain drying crystalline,
(3) the thin layer preparation of Oleanolic Acid and Echinocystic acid:
1. the preparation of thin layer need testing solution: get above-mentioned drying crystalline, makes the solution that concentration is 26mg/ml, as thin layer need testing solution with methyl alcohol;
2. point sample and preparation: with miniature sample applicator pipette samples liquid, with strip or point format continuity point on polylith thin layer plate, be placed in the chromatography cylinder filling developping agent, take out, dry, develop the color in thin layer plate both sides, the non-color development area in centre that the spot that chromatoplate Oleanolic Acid and Echinocystic acid developed the color is corresponding scrapes respectively, the Oleanolic Acid scraped and Echinocystic acid silica-gel powder are placed in Erlenmeyer flask respectively, add methyl alcohol, supersound process 30min, centrifugal, leach supernatant liquor, precipitation adds methyl alcohol again, the same ultrasonic, centrifugal repetitive operation 2 times, merge three ultrasonic filtrates, reclaim methyl alcohol, evaporate to dryness, namely Oleanolic Acid and the Echinocystic acid of preparation is obtained respectively,
In described step (3), developping agent is cyclohexane-ethyl acetate-formic acid, and the volume ratio of hexanaphthene, ethyl acetate, formic acid three is 15:4:2.
2. the method simultaneously preparing Oleanolic Acid and Echinocystic acid from Fructus Gleditsiae Abnormalis according to claim 1, is characterized in that: in described step (2), and dilute hydrochloric acid refers to that mass percent is the hydrochloric acid of 10%.
3. the method simultaneously preparing Oleanolic Acid and Echinocystic acid from Fructus Gleditsiae Abnormalis according to claim 1, is characterized in that: in described step (2), and the chloroform consumption of 3 times is followed successively by 100mL, 100mL, 60mL.
4. the method simultaneously preparing Oleanolic Acid and Echinocystic acid from Fructus Gleditsiae Abnormalis according to claim 1, is characterized in that: in described step (3), the silica gel g thin-layer plate of the thin layer plate of thin-layer chromatography to be thickness be 0.6mm.
5. the method simultaneously preparing Oleanolic Acid and Echinocystic acid from Fructus Gleditsiae Abnormalis according to claim 1, it is characterized in that: in described step (3), developer to be mass percent be 10% sulfuric acid ethanol, colour developing mode is: heat gun is clear to developing the color.
6. the method simultaneously preparing Oleanolic Acid and Echinocystic acid from Fructus Gleditsiae Abnormalis according to claim 1, is characterized in that: in described step (3), and the exhibition distance of thin-layer chromatography is 15cm.
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| CN110927292A (en) * | 2019-12-20 | 2020-03-27 | 成都普思生物科技股份有限公司 | Characteristic spectrum based on gleditsia sinensis extract and construction method and application thereof |
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| CN1216896C (en) * | 2003-01-21 | 2005-08-31 | 吉林天药科技股份有限公司 | Echinocystic acid preparation, medicinal preparation and new use as medicine |
| JP5671524B2 (en) * | 2009-05-15 | 2015-02-18 | 成都康弘制薬有限公司 | Pharmaceutical composition for the treatment of cardiovascular disorders and use thereof |
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