CN102940666A - Method for preparing cortex albiziae saponin through adopting macroporous resin separation - Google Patents
Method for preparing cortex albiziae saponin through adopting macroporous resin separation Download PDFInfo
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- CN102940666A CN102940666A CN2012105127809A CN201210512780A CN102940666A CN 102940666 A CN102940666 A CN 102940666A CN 2012105127809 A CN2012105127809 A CN 2012105127809A CN 201210512780 A CN201210512780 A CN 201210512780A CN 102940666 A CN102940666 A CN 102940666A
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- cortex albiziae
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Abstract
The invention relates to a method for preparing cortex albiziae saponin through adopting macroporous resin separation. The method is characterized in that cortex albiziae ethanol extract is prepared firstly, after the separation of the D101 type macroporous adsorption resin, the diameter height ratio is 1:5, the sample adsorbing time is 2 hours, distilled water, 30% ethanol and 70% ethanol solution are respectively adopted to elute sequentially, the elution flow velocity is 1.5 milliliters/minute, the elution flow of the 70% ethanol solution is collected, the ethanol is recovered, and after freezing and drying, the cortex albiziae total saponins freeze-dried powder is obtained. The method provides references for the technological parameters and scale production of cortex albiziae saponin separation and purification.
Description
Technical field
The present invention relates to a kind of macroporous resin that adopts and separate the method for preparing the Cortex Albiziae saponin.
Background technology
Albizzia Albizzia is pulse family Mimosoideae plant, is the bark of Albizzia julibrissin Durazz, is the Chinese medicine that we commonly use, effect with resolving stagnation for tranquilization, promoting blood circulation and detumescence is used for the treatment of irritability traditionally, melancholy insomnia, traumatic pain, the lung abscess skin ulcer is swollen etc.
Present stage, people carry out pharmaceutical research to Cortex Albiziae different chemical component, find the multiple pharmacological effect of Cortex Albiziae: 1. tranquilizing effect: Cortex Albiziae belongs to medicine for nourishing the heart and tranquilizing the mind, and Long-term clinical uses proof, and it has significant mind tranquilizing and the heart calming effect, and is better to direct stimulation institute induced insomnia curative effect.2. antidepressant effect: clinical prescription Herba Albiziae soup has the resolving depression effect, and its mechanism of action is relevant with excited cerebral cortex.3. antifertility action: the water extract of its peel of stem has the rhythm and pace of moving things of enhancing and shrinks many animals and people's uterus muscle bar, antiearly pregnancy and midtrimester pregnancy termination and the effect of Killing Sperm.4. antitumor action: can effectively suppress the Yoshida tumor in its peel of stem alcohol extract, external have a stronger tumor cytotoxic activity.Wherein, according to bibliographical information, extract Cortex Albiziae with the alcohol reflux method, contain a large amount of saponins materials in the extract, a lot of pharmacological experiments prove, the extract that the method extracts (main component is saponin) has the effect that suppresses the growth of mouse entity tumor and extracorporeal suppression tumor cell growth.The present invention is on the basis of previous experiments, and macroporous adsorbent resin separates the technological parameter of saponin behind the discussion Cortex Albiziae alcohol reflux, determines rationally effectively purification route.
Macroporous adsorbent resin is a class organic polymer adsorbent, and its characteristics show: good stability, and be subjected to pH, temperature and other factor affecting little, fractional dose is large, and resin can reuse, the aspects such as the easy amplification of technique.The macroporous adsorbent resin partition method in to Chinese medicine and compound recipe thereof in the extraction of effective site or effective ingredient, separation, purification, the enrichment process, demonstrate unique effect, be widely used among the preparation technology of the effective ingredient such as saponins, flavonoid, alkaloids of Chinese medicine or effective site.The factor that affects the macroporous adsorbent resin separation efficiency is a lot, comprising: the kind of separated chemical compound polarity power and molecular size range difference, eluent and concentration, elution flow rate etc. in the concentration of resin model, resin demand, resin preprocess method, resin column blade diameter length ratio, suction type, pH value, adsorption sample, the extracting solution.
Summary of the invention
The purpose of this invention is to provide a kind of macroporous resin chromatography that uses and separate the method for preparing the Cortex Albiziae saponin.
Technical scheme of the present invention:
Described utilization macroporous resin separates the method for preparing saponin, and the preparation Extract of alcoholized Albizia cortex is through D
101The model macroporous adsorbent resin separates, its blade diameter length ratio is 1: 5, the sample adsorption time is 2h, adopt successively distilled water, 30% ethanol, each 3 times of volume eluting of 70% alcoholic solution, elution flow rate is 1.5mL/min, collect 70% ethanol elution stream part, Recycled ethanol, lyophilization namely gets the Cortex Albiziae triterpenes freeze-dried powder.
Description of drawings
Fig. 1 is different model macroporous resin Staticadsorption experiment results; Fig. 2 is the static eluting experimental result of different model macroporous resin; Fig. 3 investigates the result static adsorption time; Fig. 4 is that dynamic adsorption is resolved the investigation result; Fig. 5 is that the ethanol elution volume is investigated the result; Fig. 6 is that the ethanol elution agent concentration is investigated the result; Fig. 7 is that flow velocity is investigated experimental result; Fig. 8 is that blade diameter length ratio is investigated experimental result.
The specific embodiment
Embodiment 1: the preparation of Extract of alcoholized Albizia cortex
Take by weighing Cortex Albiziae, add 60 times of amount 70% ethanol, heating and refluxing extraction 2 times, each 2.5h, the concentrated Cortex Albiziae extracting solution that to get of merging, lyophilization is for subsequent use.
Embodiment 2: the determining of macroreticular resin
Take by weighing the Extract of alcoholized Albizia cortex lyophilized powder and be dissolved in distilled water, preparation 1.617mg/mL solution.Macroporous adsorbent resin AB-8, D
101, D
201, D
301Four kinds of models are used respectively 95% soak with ethanol 24h, wet method dress post, and 95% ethanol drip washing 5BV, deionized water drip washing 5BV, for subsequent use.Take by weighing respectively the good resin of 2g pretreatment, place the 100mL triangular flask, at room temperature add respectively Cortex Albiziae alcohol reflux thing lyophilized powder solution (1.617mg/mL) 7mL, shake up and make it reach abundant absorption, leave standstill under the room temperature.Draw supernatant respectively at 4,8,12,16,20,24h, reclaim solvent, again use dissolved in distilled water, detect each total saponin content.Be calculated as follows Adsorbent rate:
The mensuration of total saponin content: equal-volume absolute ether extraction three times, merge water, equal-volume water-saturated n-butanol extraction three times merges the n-butyl alcohol phase, reclaims solvent, and the dissolve with methanol residue also is settled to 10mL, and 4 ℃ save backup.Accurately draw 0.1mL solution in color comparison tube, add 0.3mL 5% vanillin-glacial acetic acid solution and 0.8mL perchloric acid, 60 ℃ of reaction 30min add glacial acetic acid again and are settled to 5mL, measure solution 0D
560The oleanolic acid reference substance is good linear relationship with absorbance in 0.02～0.2mg scope, regression equation is y=8.9742x+0.0551, (R
2=0.9971), calculates the total saponin content of respectively organizing sample.
Above sample solution removes by filter supernatant, and resin all soaks 2h under the soak with ethanol room temperature with 20mL 95%, every 5min jolting 10s, filters after fully resolving and obtains supernatant, detects its total saponin content.Be calculated as follows desorption efficiency:
By Fig. 1 and 2 as seen, four kinds of resins have higher static adsorption rate, all can reach about 80% behind the 12h, and D
101Resolution factor reaches 91.6% behind the absorption with macroporous adsorbent resin 4h.Consider the factors such as price, range of application, adopt D
101The type macroporous adsorbent resin carries out the separation and purification of Cortex Albiziae.
Embodiment 3: experiment of single factor is optimized D
101Macroporous adsorbent resin separates total saponins
The investigation of adsorption time: accurately take by weighing respectively pretreated D
101Macroporous adsorbent resin places 6 100mL triangular flasks, at room temperature adds respectively the aqueous solution that contains 1.5mg Cortex Albiziae alcohol reflux thing, difference standing adsorption 0.5h, 1h, 2h, 4h, 6h, 8h.Filter, the Recycled ethanol solvent, precipitation is used dissolved in distilled water, gets supernatant, detects total saponin content, calculates the static adsorption rate.
By shown in Figure 3, adsorption time reaches before the 2h, and the adsorption rate of Cortex Albiziae total saponins obviously improves, after the 2h then without obviously increase, but so adsorption time be that 2h reaches higher adsorption efficiency within a short period of time.
The investigation of dynamic adsorption amount: Cortex Albiziae alcohol reflux thing solution is that to splash into continuously column volume be the pretreated D of 50mL to 1.5mL/min by flow velocity
101Macroporous adsorptive resins, every 10mL are collected 1 stream part, and the Recycled ethanol solvent dissolves again with distilled water, detects total saponin content in the saturated rear effluent of resin column, draws dynamic seepage curve.
As shown in Figure 4, increase along with applied sample amount, the accumulation amount of total saponins also increases thereupon in the effluent, when the application of sample amount reaches 160mL, when being 25.638mg, the total saponins quality reaches stable in the effluent, and namely the macroporous resin dynamic adsorption reaches capacity, and continues loading and can cause total saponins all to lose with the effluent outflow.But do not consider the factor of static adsorption time because of dynamic adsorption, therefore, actual maximum applied sample amount can be greater than the maximum applied sample amount of dynamic adsorption.
The eluant consumption is investigated: pretreated D
101Resin wet method dress post, column volume is 60mL, Cortex Albiziae alcohol reflux thing applied sample amount is 56.773mg, and adsorption time is 2h, behind distilled water eluting 6BV, adopt 70% alcoholic solution eluting 6BV, elution flow rate is 1.5mL/min, collects 70% ethanol elution stream part, and every 20mL collects one, detect the content of each Cortex Albiziae total saponins, calculate the eluting rate of Cortex Albiziae total saponins.
As seen from Figure 5, the Cortex Albiziae total saponins concentrates in front 3 eluting column volumes and is resolved, and eluting rate reaches 64.20%.Therefore, for raising the efficiency, save solvent, select 70% ethanol of 3BV to resolve the elution efficiency that can reach higher.
The ethanol elution volume fraction is to the investigation of eluting: pretreated D
101Resin wet method dress post, column volume is 50mL, Cortex Albiziae total saponins applied sample amount is 84.7mg, adsorption time is 2h, uses successively distilled water, 30%, 50%, each 3BV eluting of 70%, 95% alcoholic solution, elution flow rate is 1.5mL/min, every 30mL collects a stream part, collect altogether 25 parts, measure the content of each Cortex Albiziae total saponins, and calculate its eluting rate.
As seen from Figure 6, the Cortex Albiziae total saponins concentrates in 50% and 70% ethanol elution, therefore considers from saving commercial production cost aspect, at first uses distilled water, the successively eluting remove impurity of 30% ethanol after the absorption, then select the direct eluting of 70% ethanol, obtain total saponin extracts.
Elution speed is investigated: pretreated D
101Resin wet method dress post, column volume is 50mL, Cortex Albiziae total saponins applied sample amount is 55.76mg, adsorption time is 2h, use successively each 3BV eluting of distilled water and 70% alcoholic solution, elution flow rate is respectively 1.5,3,5,7,10mL/min, collects eluent, detect the content of each Cortex Albiziae total saponins, and calculate its eluting rate.
By shown in Figure 7, along with the increase of elution flow rate, eluting rate has obvious reduction, is reduced to gradually 51.19% from 75.98%, and therefore, unilateral increase flow velocity can not improve the elution efficiency of Cortex Albiziae total saponins, should select 1.5mL/min comparatively suitable.
D
101The blade diameter length ratio of macroporous adsorbent resin is investigated: choose that (glass chromatography column of 3cm * 30cm) is filled pretreated D101 resin, and the chromatographic column blade diameter length ratio was respectively 1: 3,1: 5,1: 7,1: 10, each added maximum applied sample amount, adsorption time is 2h, use successively each 3BV eluting of 0 and 70% alcoholic solution, elution flow rate is 1.5mL/min, collects 0 and 70% concentration of alcohol ethanol elution, and carry out the assay of Cortex Albiziae total saponins, and calculate the eluting rate of Cortex Albiziae total saponins.
By shown in Figure 8, be 1: 5 o'clock at blade diameter length ratio, Cortex Albiziae total saponins resolution factor reach 89.52%, so blade diameter length ratio is selected 1: 5 apparently higher than other each groups.
Embodiment 4: process certification
By above process optimization experimental result, in three days, carry out respectively the repeated experiment of different batches, separation process parameters is: use D
101Model macroporous adsorbent resin dress post, blade diameter length ratio is 1: 5, column volume is about 60mL, the applied sample amount of Extract of alcoholized Albizia cortex lyophilized powder is 782mg (containing total saponins 32.4mg), the loading adsorption time is 2h, adopt distilled water, 30% ethanol, 70% ethanol, each 3BV eluting of 95% alcoholic solution, elution flow rate is 1.5mL/min.Collect 70% ethanol elution stream part, extract, drying, the preparation lyophilized powder carries out the assay of Cortex Albiziae total saponins, and calculates the eluting rate of Cortex Albiziae total saponins.
Experimental result is as shown in table 1, and the result that the three batches of experiments obtain is relatively stable, and in the 70% ethanol elution flow point, the eluting rate of Cortex Albiziae total saponins on average reaches 86.06 ± 5.14%; Total saponin content is 63.18 ± 2.17% in the stream part, and only is 4.14% during loading.
Table 1 process certification result (n=3)
The present invention is described in conjunction with most preferred embodiment, yet after having read foregoing of the present invention, those skilled in the art can understand and do many changes also can obtain same or similar result in disclosed embodiment, and do not exceed design of the present invention, spirit and scope.More particularly, obviously some chemistry and alternative reagent disclosed herein of physiological related reagent and obtain same or similar result.All similarly replace and modify for a person skilled in the art, obviously think all that namely all above-mentioned these equivalent form of values all fall within claims limited range of the present invention equally in spirit of the present invention, scope and design and the claim scope.The application of this technique is used for the Cortex Albiziae total saponins and separates preparation and large-scale production, makes multi-form product (such as pharmaceutical preparation, food and health product) and all belongs in the claim scope of the present invention.
Claims (3)
1. one kind is adopted macroporous resin to separate the method for preparing the Cortex Albiziae saponin.It is characterized in that, at first prepare the Cortex Albiziae alcohol extracts, through D
101The model macroporous adsorbent resin separates, its blade diameter length ratio is 1: 5, the sample adsorption time is 2h, adopt successively distilled water, 30% ethanol, each 3 times of volume eluting of 70% alcoholic solution, elution flow rate is 1.5mL/min, collect 70% ethanol elution stream part, Recycled ethanol, lyophilization namely gets the Cortex Albiziae total saponins.
2. described according to claim 1, its application comprises separation preparation and the large-scale production of Cortex Albiziae total saponins.
3. described with 2 according to claim 1, its application comprises for the preparation of pharmaceutical preparation, food and health product.
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| CN110702843A (en) * | 2019-09-30 | 2020-01-17 | 佛山市欧若拉生物科技有限公司 | Non-calibration amount determination kit and determination method for soapberry saponin standard substance |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110702843A (en) * | 2019-09-30 | 2020-01-17 | 佛山市欧若拉生物科技有限公司 | Non-calibration amount determination kit and determination method for soapberry saponin standard substance |
| CN110702843B (en) * | 2019-09-30 | 2022-05-17 | 东莞微领生物科技有限公司 | Non-calibration amount determination kit and determination method for soapberry saponin standard substance |
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Application publication date: 20130227 |