CN110882305A

CN110882305A - Method for purifying total alkaloids of dicranostigma leptopodum by using acidic cation resin

Info

Publication number: CN110882305A
Application number: CN201911132624.8A
Authority: CN
Inventors: 王廷璞; 王鹏; 王霞; 马伟超; 杨玲娟; 邵兵
Original assignee: Tianshui Normal University
Current assignee: Tianshui Normal University
Priority date: 2019-11-19
Filing date: 2019-11-19
Publication date: 2020-03-17

Abstract

The invention discloses a method for purifying total alkaloids of dicranostigma leptopodum by utilizing acidic cation resin, which comprises the steps of adding dicranostigma leptopodum powder into an ethanol solution with the volume fraction of 95%, carrying out immersion and boiling extraction for 3 times at 60 ℃, carrying out suction filtration for 1 hour each time, and combining filtrates for 3 times; concentrating the filtrate at 60 deg.C under reduced pressure to obtain extract, dissolving the extract with 0.05moL/L hydrochloric acid to obtain alkaloid hydrochloride stock solution, and vacuum filtering with diatomaceous earth to obtain filtrate as sample solution; adsorbing the alkaloid from the sample solution by LSD-001 cation exchange resin, washing with distilled water until the effluent is colorless, eluting with ammonia water-ethanol solution, collecting eluate, concentrating and drying to obtain total alkaloids extract of herba Rabdosiae Glaucocalycis, wherein the total alkaloids content of effective components can reach 70%. The total alkaloids of the dicranostigma leptopodum separated by the method have the advantages of few impurities, high content of total alkaloids of active ingredients, simple and convenient operation and low cost, and are suitable for large-scale industrial application.

Description

Method for purifying total alkaloids of dicranostigma leptopodum by using acidic cation resin

Technical Field

The invention relates to the technical field of extraction of effective components of Chinese herbal medicines, in particular to a method for purifying total alkaloids of dicranostigma leptopodum by utilizing acidic cation resin.

Background

Dicranostigma leptopodum (Maxim.) Fedde also called bald sore flower, bald seed flower, herba Lespedezae Reynaudianae (Gansu, Shaanxi) is a plant of bald sore of Papaveraceae, and root and whole herb can be used as medicines. It is bitter and astringent in taste, cool in nature, and has effects of clearing heat and detoxicating, detumescence and relieving pain, and killing parasite etc. and is used for treating tinea pedis, furuncle, scabies, carbuncle, toothache, sore throat, tonsillitis, and lymphoid tuberculosis. The main chemical component and active component of herba Rabdosiae Lophanthoidis are alkaloids with isoquinoline skeleton, including isocorydine, corydaline, protopine, etc. The herba Rabdosiae Glaucocalycis extract and its alkaloid have antibacterial, antitumor, antiviral, antiinflammatory, antioxidant, immunity improving, myocardial protecting, liver injury protecting, and hemolysis resisting effects, and can be used for treating tuberculosis and wound.

At present, the extraction process of the total alkaloids of the dicranostigma leptopodum mainly comprises a water extraction and alcohol precipitation method, an alcohol extraction method and an acid water extraction method. The process has poor selectivity, and the obtained total alkaloid has many impurities and low content of effective components. The invention patent with the publication number of 'CN 105250391B' discloses a preparation method of effective fractions of anabasine of ana. The method obviously improves the purity of the alkaloid, but has complex steps, increases the operation difficulty and the preparation cost, takes volatile and toxic dichloromethane or chloroform as an extracting agent, has poor safety and pollutes the environment, and limits the application of the method in large-scale extraction.

The alkaloid is dissolved in dilute acid to easily generate organic ammonium ions, and the ammonium ions can be selectively exchanged and adsorbed by a cation resin, so that the alkaloid is separated from other unadsorbed chemical components. The cation resin separation technology has the advantages of good selectivity, low cost, simple and convenient operation, environmental protection, regeneration, recycling and the like, and is widely applied to effective enrichment, separation and purification of alkaloid in the traditional Chinese medicine. However, the cationic resin is used for separating and purifying the total alkaloids of the dicranostigma leptopodum, and no public report is found yet. In order to make up the defects of the existing extraction process of the red clover, effectively separate and purify the total alkaloids, improve the content of the total alkaloids and enhance the pharmacological activity of the total alkaloids, the method for purifying the total alkaloids of the red clover by using the acidic cation resin is provided.

Disclosure of Invention

The invention aims to: the method for purifying the total alkaloids of the red cyclopentadienyl grass by using the acidic cation resin is provided, the total alkaloids of the effective components of the red cyclopentadienyl grass obtained by separation have high content, the operation is simple and convenient, the cost is low, and the method is suitable for large-scale industrial application.

The technical scheme adopted by the invention is as follows:

in order to achieve the above object, the present invention provides a method for purifying total alkaloids of dicranostigma leptopodum by using acidic cation resin, wherein dicranostigma leptopodum is collected from wild dicranostigma leptopodum in the Qin state of Tianshui city, Gansu, and is dried in the shade, crushed and sieved into powder. The purification step comprises resin pretreatment and the following steps:

(1) preparing a sample solution: adding herba Rabdosiae Glaucocalycis powder into ethanol, soaking and decocting at 60 deg.C, and vacuum filtering to obtain filtrate; concentrating the filtrate at 60 deg.C under reduced pressure to obtain extract, dissolving the extract with 0.05moL/L hydrochloric acid to obtain alkaloid hydrochloride stock solution, adjusting pH to 1.7, and vacuum filtering the alkaloid hydrochloride stock solution with diatomaceous earth to obtain filtrate as sample solution;

(2) adsorption and elution: adsorbing the alkaloid with strong acid cation exchange resin, washing with distilled water until the effluent is colorless, eluting with ammonia water-ethanol solution, collecting eluate, concentrating, and drying to obtain total alkaloids extract of herba Rabdosiae Glaucocalycis with total alkaloids content of effective components up to 70%.

Preferably, the digestion extraction in the step (1) is repeated for 3 times, each time for 1h, and the filtrates are combined by suction filtration for 3 times.

Preferably, the feed liquid ratio of the red cyclopentadienyl grass powder to the ethanol is that 1g of red cyclopentadienyl grass powder is added into every 10mL of ethanol.

Preferably, the concentration of the sample loading solution is 35mg of extract added into every 1mL of hydrochloric acid. Experiments prove that the resin can adsorb a sample to the maximum extent and the sample is easy to elute at the concentration, the specific adsorption capacity of the resin is maximum, and the elution rate is 90.73%.

Preferably, the sample loading volume of the sample loading solution in the step (2) is 25BV, and the sample loading flow rate is 2 BV/h. Experiments prove that by selecting the parameters, the leakage rate of resin adsorption can be reduced to below 10%, and the adsorption rate reaches 89.26%, so that the adsorption effect is ensured.

Preferably, in the ammonia-ethanol solution, the volume ratio of ammonia to ethanol is 1:3, and ethanol with the volume fraction of 70% is selected as ethanol. Experiments prove that the ammonia water and the ethanol solution with the proportion can ensure that the elution rate reaches the highest.

Preferably, the flow rate of the ammonia-ethanol solution in the step (2) is 6BV/h, and the elution volume of the eluent is 12 BV. Experiments prove that under the parameter, higher elution efficiency can be maintained under the condition of saving time.

The resin pretreatment comprises the following specific steps: fully soaking the strong acid cation exchange resin in distilled water, repeatedly stirring and washing until the distilled water is colorless and transparent; firstly, adding a sodium chloride solution with the mass fraction of 10 percent and the volume of the resin, soaking for 24 hours, and then repeatedly washing with a large amount of distilled water; adding ethanol with the mass fraction of 50% and the volume 2 times of the resin, soaking for 24h, repeatedly stirring and washing with distilled water until no ethanol smell exists, adding 1moL/L hydrochloric acid solution with the volume 2 times of the resin, soaking for 2h, stirring at any time, and repeatedly stirring and washing with distilled water until the effluent is nearly neutral; adding 1moL/L sodium hydroxide solution with 2 times of resin volume, soaking for 2h, stirring at any time, and repeatedly stirring and washing with distilled water until the effluent is nearly neutral; adding 2moL/L hydrochloric acid solution 2 times of the resin volume, soaking for 2h, stirring at any time, repeatedly stirring and washing with distilled water until the effluent is nearly neutral, and obtaining activated resin for later use.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

1. the invention prepares the sample liquid through alcohol boiling and acid dissolution, and uses LSD-001 acidic cation exchange resin to separate and purify the chromocor biobase, avoids using toxic solvent chloroform or dichloromethane for extraction, has simpler and more convenient purification operation and lower cost compared with the neutral macroporous adsorption resin, and is suitable for large-scale industrial application.

2. Compared with the purification method of water extraction and alcohol precipitation, alcohol extraction and acid extraction, the purification method of the invention has the advantages that the impurities of the total alkaloids of the dicranostigma leptopodum are less, the content of the total alkaloids of the effective components can reach 70%, and the process repeatability is good.

3. The purification method used by the invention can ensure that the resin has maximum adsorption capacity and simultaneously the sample is easy to elute by optimizing various process parameters, thereby improving the adsorption effect and the elution rate, ensuring that the specific adsorption capacity of the resin can reach 51.65mg/mL and the elution rate can reach 90 percent, and ensuring that the extraction rate of the total alkaloids of the red cyclopentadienyl grass is high.

Drawings

The invention will now be described, by way of example, with reference to the accompanying drawings, in which:

FIG. 1 is a graph showing the effect of loading solution concentration on adsorption and elution;

FIG. 2 is a graph showing the leakage of total alkaloids from Dicranopteris indica;

FIG. 3 is a graph of adsorption rates for different sample flow rates;

FIG. 4 is the elution curve of total alkaloids from dicranostigma leptopodum.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Parameter selection

(1) Determination of the concentration of the sample solution (definition: mass (mg) of extract per 1mL of hydrochloric acid)

The experimental method comprises the following steps: 9 portions of 100mL samples with concentrations of 5mg/mL, 10mg/mL, 15mg/mL, 20mg/mL, 25mg/mL, 30mg/mL, 35mg/mL, 40mg/mL and 45mg/mL respectively were passed through 5.0mL (diameter to height ratio of 1:5) of LSD-001 cation exchange resin at a flow rate of 2BV/h for adsorption, and eluted with ammonia water and 95% ethanol solution at a volume ratio of 3:7 as eluent at a flow rate of 2BV/h, taking specific adsorption and elution rate as examination indexes.

And (4) analyzing results: the influence of the sample solution concentration on adsorption and elution is plotted on the abscissa of the sample solution concentration and the ordinate of the elution rate and the specific adsorption amount (see FIG. 1). As is clear from FIG. 1, when the concentration of the loading solution was increased from 5mg/mL to 35mg/mL, the specific adsorption amount of LSD-001 resin to total alkaloids increased with the increase in the concentration of the loading solution. When the concentration of the sample solution was 35mg/mL, the specific adsorption amount of the resin reached 53.7mg/mL at the maximum. Subsequently, the loading concentration is increased, the specific adsorption amount is basically unchanged, and the adsorption of the resin on the total alkali is saturated. When the concentration of the sample loading solution is 20mg/mL, the maximum elution rate is 94.92%, the concentration of the sample loading solution is increased to 35mg/mL, and the elution rate is reduced to 90.73%. To maximize the adsorption of the resin to the sample and to make the sample easier to elute, 35mg/mL was determined as the optimal loading concentration.

(2) Determination of sample loading volume

The experimental method comprises the following steps: 200mL of the sample solution with the optimal concentration is flowed through 5mL of LSD-001 cationic resin at the flow rate of 2BV/h, one portion of the effluent solution is collected for every 5mL, 28 portions of the effluent solution are collected, the absorbance of each portion is measured, and the leakage rate is calculated. And (4) taking the leakage rate as a vertical coordinate and the accumulated sample loading volume as a horizontal coordinate to draw a leakage curve.

And (4) analyzing results: as can be seen from the leakage curve of FIG. 2, when the sample loading volume is 5 mL-55 mL, there is almost no leakage; when the sample loading volume is more than 60mL, the leakage rate is obviously increased; when the cumulative sample loading volume is 125mL (25BV), the leakage rate reaches 9.56 percent; when the upper volume is 130mL (26BV), the leakage rate reaches 10.64 percent; the sample volume 125mL with a leak rate of no more than 10% was selected as the final sample volume, i.e. 25 BV.

(3) Determination of sample flow rate

The experimental method comprises the following steps: and (3) loading the determined sample loading liquid with the optimal concentration according to the maximum sample loading volume determined by the leakage curve, performing dynamic adsorption through 5mL of LSD-001 cationic resin at the flow rates of 2BV/h, 3BV/h, 4BV/h, 5BV/h and 6BV/h, collecting effluent liquid, measuring the absorbance of the sample loading liquid and each effluent liquid, and calculating the adsorption rate.

And (4) analyzing results: the results are shown in FIG. 3, which is a graph based on the adsorption rates measured at the respective flow rates, with the flow rates being plotted on the abscissa and the corresponding adsorption rates being plotted on the ordinate. As can be seen from FIG. 3, the influence of the sample loading flow rate on the adsorption rate is significant, when the sample loading flow rate is 2BV/h, the adsorption rate of the sample loading liquid can reach 89.26% at most, and when the sample loading flow rate is 3BV/h, the adsorption rate is significantly reduced, and in order to ensure the adsorption effect, 2BV/h is selected as the optimal sample loading flow rate.

TABLE 1 Absorbance of effluent at different flow rates

Sample flow rate

Stock solution

2BV

3BV

4BV

5BV

6BV

Absorbance of the solution

1.956

0.21

0.307

0.568

0.656

0.734

Adsorption rate

/

89.26％

84.30％

70.96％

66.46％

62.47％

(4) Determination of the volume ratio of the eluents

The experimental method comprises the following steps: respectively eluting with ammonia water-ethanol solutions with different proportions, setting the ammonia water volume fractions to be 5%, 10%, 15%, 20%, 25% and 30%, collecting the eluate, removing ammonia alcohol under reduced pressure, measuring alkaloid content in the eluate by acid dye colorimetry, and calculating the elution rate. The results are shown in Table 2.

And (4) analyzing results: as can be seen from table 2, when the volume ratio of ammonia water to 95% ethanol is 1: at 3, the highest elution rate reached 91.14%.

TABLE 2 Effect of eluents of different volume ratios on elution Rate

(5) Determination of ethanol concentration

The experimental method comprises the following steps: after the proportion of the eluent is determined, the eluent is composed of ethanol and ammonia water with the volume fractions of 90%, 80%, 70%, 60%, 50% and 40% respectively, the eluent is collected, the ammonia alcohol is removed under reduced pressure, the alkaloid content in the eluent is measured by an acid dye colorimetric method, and the elution rate is calculated. The results are shown in Table 3.

And (4) analyzing results: as can be seen from table 3, when the volume fraction of 25% ammonia-70% ethanol was selected for elution, the highest elution rate reached 95.51%.

TABLE 3 Effect of different volume fractions of ethanol on elution Rate

Eluent	Elution Rate (%)
		25% ammonia-90% ethanol	90.55％
25% ammonia-80% ethanol	90.19％
		25% ammonia-70% ethanol	95.51％
25% ammonia-60% ethanol	90.15％
		25% ammonia-50% ethanol	73.07％
25% ammonia-40% ethanol	69.76％

(6) Determination of elution flow Rate

The experimental method comprises the following steps: and (3) by utilizing the determined sample loading conditions and elution proportion, enabling the sample loading liquid with the maximum sample loading amount of 125mL and the concentration of 35mg/mL to pass through 5mL of LSD-001 cationic resin at the flow rate of 2BV/h, eluting by using the previously screened eluent, collecting the eluent, measuring the alkaloid content, comparing the elution rates at different elution flow rates, and determining the optimal elution flow rate, wherein the result is shown in Table 4.

And (4) analyzing results: as can be seen from the data in Table 4, the elution rates at 2BV/h, 4BV/h and 6BV/h were 90.71%, 89.79% and 89.90%, respectively, with little difference. However, when the elution flow rate was increased to 8BV/h, the elution rate dropped significantly to 77.69%, and 6BV/h was selected as the elution flow rate in order to save time and maintain high elution efficiency.

TABLE 4 Effect of different elution flow rates on elution Rate

(7) Determination of elution volume

The experimental method comprises the following steps: the eluent (5mL, i.e. 1BV) was collected in 15 portions according to the adsorption and elution conditions established above. And (4) calculating the alkaloid content in the eluent by using an acid dye colorimetric method. The alkaloid content in the eluent is taken as the ordinate, the accumulated elution volume is taken as the abscissa, and an elution curve is drawn, as shown in fig. 4.

And (4) analyzing results: as can be seen from the elution curve of FIG. 4, when the elution solution was eluted to 12BV, the total alkaloids of the organism had been substantially completely eluted, the total alkaloids co-eluted was 228.34mg, the elution rate was close to 90%, and the elution volume was determined to be 12BV in consideration of the effects of both elution amount and elution time.

Second, example

Example 1

Adding 100g of herba Rabdosiae Lophanthoidis into 1L of 95% ethanol by volume, extracting for 3 times at 60 ℃ for 1h each time, filtering to obtain extractive solution, mixing the three extractive solutions, and concentrating the extractive solution under reduced pressure to obtain 17.5g of extract containing total alkaloids 1.13g and 6.46%. The extract is fully dissolved by 500mL of hydrochloric acid with the concentration of 0.05moL/L to generate alkaloid hydrochloride stock solution, and the pH value of the stock solution is 1.7. The stock solution is filtered by suction through diatomite, the filtrate flows through a 20mL LSD-001 strong acid cation exchange resin column at the flow rate of 2BV/h, the maximum sample loading is 25BV, and the measured specific adsorption capacity is 51.0 mg/mL. Then the resin is firstly washed by distilled water until effluent is colorless, and then eluted by 25 percent ammonia water-70 percent ethanol solution, the elution flow rate is 6BV/h, the elution volume is 12BV, and the elution rate is 88 percent. Collecting eluate, concentrating the eluate until no alcohol smell exists, drying to obtain black powder 1.30g, and measuring the content of total alkaloids in effective component by acid dye colorimetry to be 69%, and the purification multiple to be 11 times.

Example 2

Adding 1kg of herba Rabdosiae Lophanthoidis into 10L of 95% ethanol by volume, soaking and decocting at 60 deg.C for 3 times, each for 1h, vacuum filtering to obtain extractive solution, mixing the extractive solutions, and concentrating under reduced pressure to obtain 175g extract containing total alkaloids 11.6g and 6.63%. The extract was fully dissolved with 5L hydrochloric acid of 0.05moL/L concentration to produce a stock solution of alkaloid hydrochloride, the pH of the stock solution being 1.7. The stock solution is filtered by suction through diatomite, the filtrate flows through a 200mL LSD-001 strong acid cation exchange resin column at the flow rate of 2BV/h, the maximum sample loading is 25BV, and the specific adsorption capacity is 51.6 mg/mL. Then the resin is firstly washed by distilled water until effluent liquid is colorless, and then eluted by 25 percent ammonia water-70 percent ethanol solution, the elution flow rate is 6BV/h, the elution volume is 12BV, and the elution rate is 90 percent. Collecting eluate, concentrating the eluate until no alcohol smell exists, drying to obtain black powder 13.2g, and measuring the content of total alkaloids in effective components by acid dye colorimetry to be 70%, wherein the purification multiple is 11 times.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; these modifications and substitutions do not cause the essence of the corresponding technical solution to depart from the scope of the technical solution of the embodiments of the present invention, and are intended to be covered by the claims and the specification of the present invention.

Claims

1. A method for purifying total alkaloids of dicranostigma leptopodum by utilizing acidic cation resin comprises resin pretreatment, and is characterized by also comprising the following steps:

2. The method for purifying total alkaloids of dicranostigma leptopodum by using acidic cation exchange resin as claimed in claim 1, wherein the leaching and extraction in step (1) are repeated for 3 times, each time for 1h, and the filtrates are combined by suction filtration for 3 times.

3. The method for purifying total alkaloids of dicranostigma leptopodum by using acidic cationic resin as claimed in claim 1, wherein the ratio of the feed solution of dicranostigma leptopodum powder and ethanol is 1g of dicranostigma leptopodum powder added in each 10mL of ethanol.

4. The method for purifying total alkaloids of dicranostigma leptopodum by using acidic cationic resin as claimed in claim 1, wherein the concentration of the loading solution is 35mg of extract added to 1mL of hydrochloric acid.

5. The method for purifying total alkaloids of dicranostigma leptopodum by using acidic cation exchange resin as claimed in claim 1, wherein the sample loading volume of the sample loading solution in the step (2) is 25BV, and the sample loading flow rate is 2 BV/h.

6. The method for purifying total alkaloids of dicranostigma leptopodum by using acidic cation resin as claimed in claim 1, wherein the volume ratio of ammonia to ethanol in the ammonia-ethanol solution is 1:3, and ethanol with the volume fraction of 70% is selected as ethanol.

7. The method for purifying total alkaloids of dicranostigma leptopodum by using acidic cation exchange resin as claimed in claim 1, wherein the elution flow rate of the ammonia-ethanol solution in step (2) is 6BV/h, and the elution volume of the eluent is 12 BV.