CN107050161B - Method for extracting, separating and purifying total alkaloids of zanthoxylum dissitum Hemsl - Google Patents

Method for extracting, separating and purifying total alkaloids of zanthoxylum dissitum Hemsl Download PDF

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CN107050161B
CN107050161B CN201710306850.8A CN201710306850A CN107050161B CN 107050161 B CN107050161 B CN 107050161B CN 201710306850 A CN201710306850 A CN 201710306850A CN 107050161 B CN107050161 B CN 107050161B
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王平
周韬
孙吉康
李猛
戴明龙
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Central South University of Forestry and Technology
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Abstract

The invention discloses a method for extracting, separating and purifying total alkaloids of zanthoxylum dissitum Hemsl, which optimizes the extraction, separation and purification of the total alkaloids of zanthoxylum dissitum Hemsl by a response method, is quick and economical, improves the extraction and preparation process of the total alkaloids of zanthoxylum dissitum Hemsl, and has higher efficiency and higher purity compared with the reported extraction process.

Description

Method for extracting, separating and purifying total alkaloids of zanthoxylum dissitum Hemsl
Technical Field
The invention relates to an extraction preparation method for extracting total alkaloids of zanthoxylum dissitum Hemsl by a response surface method and purifying and separating products by using macroporous resin, belonging to the technical field of extraction of effective components of plants.
Background
Zanthoxylum nitidum (Zanthoxylum Dissitium Hemsl) is also called Zanthoxylum nitidum. The root, stem and leaf can be used as medicine, has the effects of removing blood stasis, relieving pain, removing toxic substance and relieving swelling clinically, and is mainly used for treating toothache, lumbago, menorrhagia, postpartum irregular menstruation and the like; research shows that the active alkaloid in the zanthoxylum dissitum Hemsl is the main medicinal component, wherein the alkaloid contained in the known zanthoxylum dissitum Hemsl contains dictamnine, nitidine, zanthoxylum bungeanum alkali, skimmianine, sanguinarine, zanthoxylum bungeanum alkali and other alkaloids which have the effects of inhibiting inflammation and tumor. In recent years, as the production scale of Chinese patent medicines is gradually enlarged, the natural reproduction rate of the single-face acupuncture is low, and the growth is slow, so that the natural resources are gradually poor, the supply is not adequate, and the normal production of related pharmaceutical factories is gradually threatened. Therefore, the search for a proper extraction and preparation process of the active ingredients is crucial to the full utilization of resources.
The extraction optimization of the response surface method is an effective method for optimizing process conditions, a continuous variable curved surface model and a regression equation are established to fit the relationship between a plurality of response variables and a series of test variables, and the influence of different test factors and the interaction thereof on a target response value is inspected. The method can economically, effectively and quickly determine the optimal combination condition of multiple test factors. The research takes mixed extraction solution as a solvent, examines the influence rule of different volume fractions of the mixed solution, liquid-material ratio, microwave extraction time and temperature on the total alkaloids of zanthoxylum dissitum Hemsl, and utilizes a response surface method to analyze and obtain the optimal extraction process.
The macroporous resin is a polymer adsorbent, and is a high molecular polymer with concentrating and separating effects on organic matters, the separation and purification effects of specific organic matters can be effectively realized by selecting a proper resin type, and the influences of pH, volume fraction of mixed extracting solution, flow rate of sample loading liquid, flow rate of eluent and dosage of eluent on the adsorption rate and desorption of the selected macroporous resin are examined in the separation and purification process. The optimized process for microwave extraction of the clam shell pepper is obtained, and the separation and purification process is also perfected. And the bacteriostatic experiments of the separated and purified active alkaloid are compared, and the purification process is verified to be capable of effectively obtaining the high-purity total alkaloids of zanthoxylum dissitum Hemsl.
Disclosure of Invention
The invention aims at the existing technology for extracting the effective component alkaloid of zanthoxylum dissitum Hemsl: low extraction efficiency, low activity of effective components, high impurity content, etc., and provides a microwave extraction method for optimizing total alkaloids of Zanthoxylum dissitum Hemsl by response surface method, and a separation and purification process.
A method for extracting, separating and purifying total alkaloids from Zanthoxylum dissitum Hemsl comprises mixing root and stem of Zanthoxylum dissitum Hemsl, grinding into powder, adding ethanol-acetone mixed solution, and performing microwave extraction; and separating and purifying the obtained extract by using resin to obtain the total alkaloids of zanthoxylum dissitum Hemsl.
As a further improvement, the single-side needle roots and the single-side needle stems are mixed according to the mass ratio of 1:2 in the method.
As a further improvement, the method comprises mixing and powdering single-side needle roots and stems, and sieving with a 60-mesh sieve to obtain rhizome powder.
As a further improvement, the volume ratio of the ethanol to the acetone in the method is 1: 1.
As a further improvement, in the method, during microwave extraction, the volume percentage of the ethanol-acetone mixed solution is 70-90%, and the balance is water; the ratio of the material to the liquid is 15: 1-25: 1mL-1(ii) a The extraction temperature is 50-70 ℃; the extraction time is 15-25 min.
As a further improvement, the above methodPreferably, the volume percentage of the ethanol-acetone mixed solution is 89 percent, and the rest is water; the ratio of material to liquid is 21:1mL.g-1(ii) a The extraction temperature is 70 ℃; the extraction time is 20 min.
As a further improvement, the microwave power in the method is 300W.
As a further improvement, the microwave extraction times in the method are 2 times, and the second extraction is carried out after the first extraction system is returned to the room temperature. The sum of the time of the two extractions, excluding the time of intermediate cooling, was 40 minutes.
As a further improvement, NKA-9 macroporous adsorption resin is selected as the resin in the method.
As a further improvement, the specific conditions for separation and purification in the method are as follows: the mass ratio of crude drug to resin is 1:1, the pH of the sample loading solution is adjusted to 7.0, the sample loading flow rate is 2BV/h, and the elution is carried out at the speed of 2BV/h by using 6BV 80% ethanol-acetone mixed solution with the volume of 1: 1.
The method comprises the steps of airing and smashing perennial single-side needle rhizome, sieving the smashed single-side needle rhizome with a 60-mesh sieve, mixing the powder according to the ratio of rhizome to root of 1:2, processing to obtain mixed rhizome powder, weighing a certain amount of single-side needle rhizome powder, adding the single-side needle rhizome powder into a container, respectively adding mixed solutions with different volume fractions, and performing microwave extraction by setting different temperatures and material-liquid ratios. Adsorbing and desorbing the obtained extract by using macroporous resin to achieve the purpose of separating and purifying the total alkaloids of zanthoxylum dissitum Hemsl, and obtaining the purified extract of the total alkaloids of zanthoxylum dissitum Hemsl;
the above response surface method sets the extracted variable parameters through the following regression equation: y-43.05333 +0.93982A +0.89240B +13.24900C-0.36270D +3.49700AB +0.049500AC +7.197AD-6.997BC-2.496BD +0.011500CD-9.15250A2-0.027560B2-4.1010C2-1.912D 2.
In the above, Y is response value and the extraction amount of total alkaloids from Zanthoxylum dissitum Hemsl; setting a variable A as the volume fraction of the mixed extracting solution and a variable B as the ratio of the material to the liquid; c is microwave time; d is the extraction temperature; the A accounts for 70-90%; the B is 15: 1-25: 1mL-1(ii) a C is 15-25 min; the D is 50-70 ℃.
Measuring content of total alkaloids from radix Zanthoxyli, measuring absorbance value after diluting corresponding volume of total alkaloids extractive solution, comparing with dictamnine standard curve to obtain total alkaloids concentration in extractive solution, calculating total alkaloids quality, and calculating total alkaloids extraction amount of radix Zanthoxyli. The calculation formula is as follows;
Figure BDA0001285959890000041
obtaining the standard curve and regression equation of dictamnine: precisely weighing 1.2mg of dictamnine standard substance, dissolving in 25mL of absolute ethyl alcohol to prepare a standard solution with the concentration of 48 mu g/mL, precisely sucking 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL and 2.5mL of dictamnine standard solution respectively, placing in a 10mL volumetric flask, and metering the volume of the absolute ethyl alcohol to the scale. The absorbance of each solution was measured at 310nm using absolute ethanol as a blank, and a linear regression equation a was obtained by plotting a standard curve with the concentration C (ug/ml) as the abscissa and the absorbance a as the ordinate, i.e., 32.5C-0.0085 and r-0.9991.
The invention has the beneficial effects that: compared with the prior art, the invention adopts an optimized simulation equation obtained by extracting the total alkaloids of the zanthoxylum dissitum Hemsl by a microwave method: y-43.05333 +0.93982A +0.89240B +13.24900C-0.36270D +3.49700AB +0.049500AC +7.197AD-6.997BC-2.496BD +0.011500CD-9.15250A2-0.027560B2-4.1010C2-1.912D 2. The optimal extraction process conditions of the optimized total alkaloids from Zanthoxylum dissitum Hemsl obtained by response surface analysis include 89% volume fraction of ethanol-acetone (1:1) mixed solution, 21mL/g material-liquid ratio, 20min microwave time, and 70 deg.C extraction temperature, and the total alkaloids from Zanthoxylum dissitum Hemsl amount is 12.35mg/g under the process conditions, and only 0.032mg/g difference from the theoretical value predicted by model. Later-stage NKA-9 macroporous resin is used for separating and purifying the total alkaloids of the zanthoxylum dissitum Hemsl, the mass ratio of crude drug to resin is 1:1(g/g), the pH of a sample loading solution is adjusted to be 7.0, the sample loading flow rate is 2BV/h, during elution, 6BV 80% ethanol-acetone (1:1) mixed solution is used for elution at the speed of 2BV/h, the average purity of the obtained total alkaloids is 70.9%, and bacteriostasis experiments show that the extracted alkaloids have good bacteriostasis to specific bacteria. The extraction, separation and purification of the zanthoxylum dissitum Hemsl are rapid and economical, the extraction and preparation process of the zanthoxylum dissitum Hemsl total alkaloids is perfected, and the extraction and purification process is more efficient and rapid compared with the reported extraction process.
Drawings
FIG. 1 is the effect of the concentration of extraction solvent on the extraction efficiency in example 3;
FIG. 2 is the effect of the ratio of the feed liquid to the total alkaloid extraction in example 4;
FIG. 3 shows the effect of microwave time on total alkaloid extraction in example 5;
FIG. 4 is the effect of temperature on the total alkaloid extraction yield in example 6;
FIG. 5 shows the effect of pairwise interactions between the extraction factors on the total alkaloid extraction in example 7;
FIG. 6 is the influence of pH of the sample solution on the adsorption rate of resin in the separation and purification process of total alkaloids in example 9;
FIG. 7 shows the effect of the ratio of crude drug to resin on the adsorption rate of resin in the separation and purification process of total alkaloids in example 10;
FIG. 8 is the effect of volume fraction of mixed solution on the desorption rate of resin in the process of separating and purifying total alkaloids in example 11;
FIG. 9 shows the effect of flow rate of sample solution on resin adsorption rate during separation and purification of total alkaloids in example 12;
FIG. 10 is the effect of eluent flow rate on resin desorption rate in the process of separating and purifying total alkaloids in example 13;
FIG. 11 is the effect of the amount of eluent used in the separation and purification of total alkaloids in example 14 on the desorption rate of resin.
Detailed Description
The invention will be further elucidated with reference to the drawings and to specific embodiments, which are intended to be illustrative only and not limiting.
The following example 1 is solvent selection by microwave extraction, and example 2 is influence of rhizome mixing ratio and particle size selection on the extraction of total alkaloids from Zanthoxylum dissitum Hemsl, and the most suitable rhizome mixing ratio and particle size are selected according to actual industrial operation to ensure extraction effect and reduce cost. Examples 3 to 6 were conducted by selecting different concentrations of extractive solutions, ratios of materials to liquids, microwave times and temperatures, respectively, to perform a single factor experiment, and examining the influence of the single factor on the extraction amount of total alkaloids from Zanthoxylum dissitum Hemsl. Example 7 on the basis of single factor, Design-expert.v8.0.6 software is applied to experimental Design and analysis of response surface method, and the extraction process of total alkaloids from zanthoxylum dissitum is optimized by taking the extraction amount (Y) of total alkaloids as a response value and taking the concentration (a) of mixed liquor, the ratio of material to liquid (B), the extraction time (C) and the extraction temperature (D) as variable parameters. Examples 8 to 14 were conducted to separate and purify total alkaloids by screening an optimum macroporous resin from 5 kinds of resin materials, and optimum adsorption and resolution of the macroporous resin, that is, optimum purification and separation conditions were gradually established by pH of a primary extract, crude drug-to-resin ratio, volume fraction of a mixed solution, flow rate of a sample, flow rate of an eluent, and a dose, and example 15 was conducted to perform an in vitro bacteriostatic test on the purified total alkaloids from zanthoxylum dissitum Hemsl, and the bacteriostatic effect was verified.
Example 1
Influence of different solvents on microwave extraction efficiency
Weighing 10 parts of single-side needle root and stem mixed crude drug powder which is dried in vacuum, respectively weighing 10g of each mixed crude drug powder, respectively adding 20mL of 1% (v/v) ammonia water for infiltration, then taking distilled water, absolute ethyl alcohol, n-butyl alcohol, chloroform, acetone, ethyl acetate, ethanol-ethyl acetate (v: v ═ 1:1), ethanol-n-butyl alcohol (v: v ═ 1:1), ethanol-chloroform (v: v ═ 1:1) and ethanol-acetone (v: v ═ 1:1) as extraction solvents, putting the crude drug powder and 10 times of solvents into a flask, uniformly mixing, soaking for 15h, then carrying out microwave extraction at 70 ℃ for 20min, concentrating the extraction solution to obtain an extract, and calculating the extraction amount of water-soluble alkaloids, wherein the results are shown in Table 1. Therefore, ethanol-acetone (1:1) mixed solution is selected as the extractant.
TABLE 1 Effect of microwave extraction of Zanthoxylum nitidum with different solvents
Figure BDA0001285959890000071
Example 2
Influence of rhizome powder with different particle size and mixing ratio on microwave extraction effect
Weighing 5 parts of dry single-side needle root and stem powder respectively, sieving with 40, 60, 80 and 100 mesh sieves respectively, and mixing according to the proportion of 1:1, 1:2 and 1:3 to obtain 5g of mixed sample. Respectively adding 50mL of 80% mixed extraction solvent with a material-liquid ratio of 20g/mL, soaking for 15h, performing microwave extraction at 70 ℃ for 20min, filtering, repeatedly extracting the filter residue once by the same method, combining the two filtrates, and calculating the total alkaloid extraction amount, wherein the results are shown in Table 2, and different particle sizes and different rhizome mixing ratios can also influence the extraction effect of the total alkaloids of Zanthoxylum dissitum Hemsl.
TABLE 2 different rhizome mixing ratio and sieving number versus extract single-side acupuncture effect
Mixing ratio of rhizome to root is 1:1 The mixing ratio of the rhizome is 1:2 Mixing ratio of rhizome to root is 1:3
40 mesh sieve 9.02mg/g 8.11mg/g 8.21mg/g
60 mesh sieve 11.25mg/g 11.21mg/g 8.24mg/g
80 mesh sieve 11.31mg/g 11.23mg/g 8.52mg/g
100 mesh sieve 11.56mg/g 11.32mg/g 8.54mg/g
In the process of powdering single-side needle rootstalks, the hardness of roots is higher, the root powder can be obtained by powdering for many times, and the alkaloid content in the roots is relatively higher than that in the stalks. In order to meet the actual industrial extraction, the mixing proportion of the rhizome powder is adjusted, the utilization rate of the stems is improved as much as possible, the consumption of materials can be saved, the waste is reduced, meanwhile, in order to obtain higher total alkaloid extraction rate, the microwave extraction efficiency can also be improved due to the proper particle size, the factors are reduced, the material loss and the extraction cost are reduced, meanwhile, the rhizome powder which is sieved by a 60-mesh sieve and the rhizome powder with the particle size of about 250 micrometers are selected for mixing and extraction according to the proportion of 1:2, although the mixing proportion is not the optimal alkaloid extraction proportion, the cost is relatively lowest and is close to the optimal value compared with other mixing proportions and particle sizes, and the material consumption is required.
Example 3
Effect of extraction solvent concentration on extraction Effect
5 parts of dried root and rhizome powder of zanthoxylum dissitum Hemsl, 5g each, were weighed. Respectively adding 50mL of mixed extraction solvent with a material-liquid ratio of 20g/mL, soaking for 15h, microwave extracting at 70 deg.C for 20min, filtering, extracting the residue with the same method for one time, mixing the filtrates, and calculating total alkaloid extraction amount. When the volume fraction of the mixed extract is 50-90%, the total alkaloid extraction rate gradually increases along with the increase of the volume fraction of the mixed extract. When the volume fraction of the mixed extract is more than 80%, the total alkaloid extraction rate is reduced (figure 1). Therefore, when the volume fraction of the mixed solution is 80%, the polarity of the extracting solution is closest to that of the total alkaloids of zanthoxylum dissitum Hemsl, and the dissolution rate of the alkaloids is highest.
Example 4
Influence of feed liquid on extraction effect of total alkaloids
Weighing 5 parts of dried root and rhizome powder of zanthoxylum dissitum Hemsl, adding 5g of each part of dried root and rhizome powder of zanthoxylum dissitum Hemsl into 80% of mixed extract of 25mL, 50mL, 75 mL, 100 mL and 125mL respectively, soaking for 15h, ultrasonically extracting at 70 deg.C for 20min, filtering, repeatedly extracting the residue once by the same method, mixing the filtrates, and calculating the total alkaloid extraction amount. With the increase of the ratio of the material to the liquid, the concentration difference between the two phases is increased, which is beneficial to the diffusion and dissolution of the total alkaloids, and the extraction rate of the total alkaloids is continuously increased; when the ratio of material to liquid reaches 1:20, the total alkaloid dissolution tends to be complete, the extraction amount does not change greatly along with the increasing trend of the ratio of material to liquid (figure 2), and the excessive ratio of material to liquid can cause the production cost to be increased, the subsequent filtration and concentration time is long, the impurities are increased, the total alkaloid loss is high, and the like, so the proper ratio of material to liquid is selected to be 1: 20.
Example 5
Influence of microwave time on total alkaloid extraction effect
Weighing 5 parts of dried root and rhizome powder of zanthoxylum dissitum Hemsl, adding 50mL of 80% mixed extract solution respectively, soaking for 15h, microwave extracting at 70 deg.C for 5, 10, 15, 20, 25min, filtering, extracting residue once again with the same method, mixing filtrates, and calculating total alkaloid extraction amount. The extraction rate of the total alkaloids is increased along with the increase of the extraction time, when the extraction time exceeds 20min, the increase of the extraction amount is not obvious, at the moment, the total alkaloids are basically dissolved out, the extraction time is too long, the alkaloid is unstable under higher microwave energy, the components are gradually heated and destroyed by microwave, the total extraction amount of the alkaloid is reduced (figure 3), and the microwave energy for a longer time can break the cells to a greater extent, so that more contents are released to cause the improvement of impurities, and the later purification is not easy, so the ultrasonic time is optimal for 20 min.
Example 6
Influence of temperature on extraction rate of total alkaloids
Weighing 5 parts of dried root and rhizome powder of zanthoxylum dissitum Hemsl, adding 50mL of 80% mixed solvent respectively into 5g of each part, soaking for 15h, microwave extracting at 50, 60, 70, 80 and 90 deg.C for 15min, filtering, extracting the residue with the same method for one time, mixing the two filtrates, and calculating the total alkaloid extraction. With the increase of the temperature, the extraction amount of the total alkaloids increases with the increase of the temperature; the temperature is higher than 50 deg.C, the total alkaloid extraction amount is not increased significantly, and the peak value is reached at 60 deg.C (figure 4). The total alkaloid extraction is basically complete under the condition. The excessive high temperature can accelerate the volatilization of the ethanol-acetone to reduce the solubility of the alkaloid, influence the extraction effect, and cause the overheating decomposition of the alkaloid to reduce the extraction amount. Therefore, the optimum extraction temperature of 60 ℃ was selected.
Example 7
Response surface method optimized extraction process of total alkaloids of zanthoxylum dissitum Hemsl
On the basis of a single-factor experiment, Design-expert.V8.0.6 software is adopted to process data. Selecting 4 factors of volume fraction of mixed liquor, material-liquid ratio, microwave extraction time and extraction temperature as experimental factors, and designing an experiment by taking total alkaloid extraction amount as a response value. The experimental design and results of the response surface are shown in table 3.
TABLE 3 response surface test results
Figure BDA0001285959890000101
Figure BDA0001285959890000111
Note: the data in the table are the average of 3 replicates.
Note：The date in table is the mean of three repeated experiments.
Taking A (volume fraction percent of mixed extracting solution), B (material-liquid ratio mL/g), C (microwave time min) and D (temperature ℃) as independent variables and taking the extraction amount of the total alkaloids of the zanthoxylum dissitum Hemsl as a response value, and totally performing 29 groups of response surface analysis experiments, wherein 5 central experiments are used for estimating errors, and 24 experiments are used as analytical cause and analyze test results through response surface software to obtain a regression equation taking the extraction amount of the total alkaloids of the zanthoxylum dissitum Hemsl as a response: y-43.05333 +0.93982A +0.89240B +13.24900C-0.36270D +3.49700AB +0.049500AC +7.197AD-6.997BC-2.496BD +0.011500CD-9.15250A2-0.027560B2-4.1010C2-1.912D 2. The data in table 2 are analyzed, the P value of the regression model is 0.0319 less than 0.05, the coefficient of determination R2 is 0.8369, the pure error term is not significant, and the fitting equation has significant influence and is consistent with the actual situation.
TABLE 4 response surface test analysis of variance
Figure BDA0001285959890000112
Figure BDA0001285959890000121
Note: indicates significance, indicates extreme significance.
Note：*and**indicated significance at 0.05and 0.01levels,respectively
The mismatching term P value is larger than 0.1, which indicates that the error of the orthogonal experimental result and the mathematical model is small. The fitting equation can fully reflect the relationship between the response value and each factor, and the influence of the fitting equation is not a simple linear relationship. There is a certain interaction among all the factors, wherein the P value is less than 0.01 when the P value is greatly influenced by A, B2 and C2, and the P value is obviously influenced by C, AC, AD, BC and A2. Furthermore, the influence sequence is shown to be A (volume fraction of mixed extract liquid) > C (microwave time min) > B (material-liquid ratio mL/g) > D (temperature ℃) from the single factor level. In the presence of interaction, the total alkaloid extraction amount of Zanthoxylum dissitum Hemsl is influenced by AC > BC > AD > AB > CD > BD.
The optimal process conditions for extracting the total alkaloids from the single-side needles are obtained by performing systematic analysis by using Design-expert.V8.0.6 software, wherein the volume fraction of a mixed extracting solution is 88.87%, the material-liquid ratio is 21.23mL/g, the microwave time is 20min, and the extracting amount of the total alkaloids from the single-side needles at the extracting temperature of 70 ℃ can reach 12.382mg/g, and the optimal process conditions are finally obtained by combining the limitations in actual operation, wherein the volume fraction of a mixed solution is 89%, the material-liquid ratio is 21mL/g, the microwave time is 20min, and the extracting temperature is 70 ℃, 3 repeated experiments are performed according to the optimized extracting process, the average value is 12.35mg/g of the total alkaloids extracting amount, and the difference from the theoretical value is 0.032mg/g, and the response surface optimized extracting process conditions are reliable.
Example 8
Selection of 5 macroporous resins
Respectively placing 1g of treated 5 model macroporous resin into 50mL conical flasks, respectively adding 30mL of the stock solution of total alkaloids prepared by the method, and shaking for 24h at room temperature with 120r/min until adsorption balance. Filtering, measuring the concentration of total alkaloids in the filtrate, and calculating the adsorption rate of macroporous resin. And (3) after the filtered macroporous resin is sucked to be dry by using filter paper, putting the dried macroporous resin into a 50mL conical flask, respectively adding 30mL of 70% ethanol-acetone (1:1) mixed solution, and shaking the mixture for 24 hours at room temperature at 120r/min until desorption balance. And (4) measuring the concentration of the total alkaloids in the desorption solution, and calculating the desorption rate. And (3) screening out the resin type most suitable for separating and purifying the total alkaloids by taking the adsorption rate and the desorption rate of the total alkaloids as indexes.
The method for calculating the adsorption rate and the desorption rate comprises the following steps: adsorption rate P ═ ((C0-C1) × 100%)/C0 desorption rate D ═ C × V0)/(Q × M) × 100% adsorption amount ═ ((C0-C1) × V)/M
In the above formula: q is adsorption capacity (mg/g), C0 is total alkaloid extract initial concentration (mg/mL), C1 is total alkaloid solution concentration after adsorption, V is total alkaloid solution volume, M is resin mass, C is desorption solution concentration (mg/mL), and V0 is desorption solution volume. The results are shown in Table 5
TABLE 55 results of static adsorption and desorption of total alkaloids by macroporous resin
Figure BDA0001285959890000131
As can be seen from the results in Table 4, 3 indexes of specific adsorption capacity, adsorption rate and desorption rate of the NKA-9 type resin are obviously higher than those of other 4 types of resin, which indicates that the NKA-9 type resin is suitable for separation and purification of total alkaloids of zanthoxylum dissitum Hemsl, so the NKA-9 type resin is selected for separation and purification of the total alkaloids of zanthoxylum dissitum Hemsl.
Example 9
Influence of sample solution pH on resin adsorption Rate
Precisely weighing 5 parts of 1.0g of pretreated NKA-9 resin, respectively placing in 50mL conical flasks, respectively adding 30mL of total alkaloid extract with pH values (adjusted by hydrochloric acid and ammonia water) of 4.0, 5.0, 6.0, 7.0 and 8.0, and shaking at room temperature for 24h at 120r/min until adsorption is balanced. Filtering, measuring the concentration of total alkaloids in the filtrate, and calculating the adsorption rate of macroporous resin. As shown in FIG. 6, the pH value of the sample solution significantly affects the adsorption of the total alkaloids by the macroporous resin, and the total alkaloids in the sample solution have different existing states and different binding capacities with the resin under different pH conditions, thereby affecting the adsorption of the resin. At the pH range of 4.0-7.0, the adsorption rate of the macroporous resin is gradually increased along with the increase of pH, and the adsorption rate is reduced when the pH is 8.0, which indicates that the adsorption of the macroporous resin on the total alkaloids is most favorable when the pH is 7.0, so the pH of the sample loading solution is determined to be 7.0.
Example 10
Influence of crude drug to resin ratio on resin adsorption Rate
Precisely weighing 5 parts of 1.0g of pretreated NKA-9 resin, respectively placing in conical flasks, respectively adding 10, 20, 30, 40 and 50mL (corresponding to the mass ratio of crude drug to resin of 0.5, 1, 1.5, 2 and 2.5g/g), respectively, adjusting pH to 7.0, and shaking for 24h at room temperature with 120r/min to reach adsorption balance. Filtering, measuring the concentration of total alkaloids in the filtrate, and calculating the adsorption rate of macroporous resin. As shown in FIG. 7, when the ratio of crude drug to resin is 0.5-1, the resin has high adsorption rate to total alkaloids, and almost completely adsorbs alkaloids in extractive solution; the alkaloid adsorption rate decreased significantly with increasing ratio, and the alkaloid adsorption rate was only 54.3% at a crude drug to resin ratio of 2.5, so the crude drug to resin ratio was determined to be 1:1 for optimal adsorption.
Example 11
Influence of volume fraction of ethanol-acetone mixed solution on resin desorption rate
And (3) flushing the NKA-9 resin saturated in adsorption with 20mL of distilled water, placing the resin in a 50mL conical flask, respectively adding 30mL of ethanol-acetone (1:1) mixed solution with volume fractions of 50%, 60%, 70%, 80% and 90%, and shaking the mixture at room temperature for 24h at 120r/min until desorption balance. And (4) measuring the concentration of the total alkaloids in the desorption solution, and calculating the desorption rate. When macroporous resin is used for separating and purifying the total alkaloid desorption, the desorption rate generally increases along with the increase of the ethanol concentration, and the alkaloid in the pepper mainly exists in 70-90% of ethanol-acetone mixed liquid eluent. As can be seen from fig. 8, the desorption rate of the resin was the highest when the concentration of the ethanol-acetone mixture was 80%, and the content of impurities in the analysis solution increased when the concentration was too high, and therefore, the 80% ethanol-acetone mixture was selected as the eluent for the experiment.
Example 12
Influence of sample loading liquid flow velocity on resin adsorption rate
And (3) taking 5 parts of pretreated NKA-9 resin 1.0g, and filling the resin into a column by a wet method. The sample liquid flows through the resin column at 1, 2, 3, 4 and 5BV/h respectively for dynamic adsorption. Collecting effluent liquid, measuring the concentration of the total alkaloids, and calculating the adsorption rate. As can be seen from FIG. 9, the adsorption rate of total alkaloids tends to decrease with the increase of the flow rate of the sample, which indicates that the large flow rate is not favorable for the adsorption of alkaloids, probably because the alkaloids can not contact with the resin sufficiently at the large flow rate, resulting in the decrease of the adsorption rate. The adsorption rate and the time cost are integrated, and the flow rate of the sample loading liquid is selected to be 2 BV/h.
Example 13
Effect of eluent flow Rate on resin desorption Rate
5BV of 80% ethanol-acetone mixed solution is taken to pass through a resin column (washed by 2BV of distilled water to remove impurities) which is saturated by adsorption at the flow rate of 1BV, 2BV, 3 BV, 4 BV and 5BV/h respectively for dynamic desorption, and the influence of the flow rate of an eluent on the desorption rate is examined. As can be seen from FIG. 10, the flow rate of the eluent has a certain influence on the desorption rate of the resin, the desorption rate is the highest when the flow rate of the eluent is 2BV/h, and the resin elution rate is gradually reduced with the increase of the flow rate, so that the flow rate of the eluent is selected to be 2 BV/h.
Example 14
Effect of eluent dosage on resin desorption
Eluting with 2BV distilled water to remove impurities, eluting with 80% ethanol-acetone (1:1) mixture at 2BV/h, collecting eluate every 1BV to determine alkaloid concentration, and determining the amount of eluent. As can be seen from fig. 11, the cumulative desorption rate of the resin gradually increased with the increase in the amount of the eluent, and when the amount of the eluent reached 6BV, the cumulative desorption rate reached 86.6%, the cumulative desorption rate hardly increased any more, and the alkaloid was hardly detected in the collected eluent, indicating that the desorption was substantially completed, and therefore, the amount of the eluent was determined to be 6 BV.
Example 15
In-vitro antibacterial research on total alkaloids of purified zanthoxylum dissitum Hemsl
After the zanthoxylum dissitum total alkaloids extracted by the optimal extraction process obtained from the response surface are separated and purified, the concentrated solution is diluted with sterile distilled water to 7 concentration gradients of 0.10, 0.15, 0.2, 0.25, 0.30, 0.35 and 0.40mg/mL, and positive and negative controls are performed for determination, and the results are shown in Table 6.
TABLE 6 minimum inhibitory concentration and minimum bactericidal concentration of concentrated solution of total alkaloids from Zanthoxylum dissitum Hemsl
Figure BDA0001285959890000171
Note: the data in the table are the average of 3 replicates. "+" indicates the presence of bacteria; "+ +" indicates that the bacteria grew more vigorously; "+ + + +" indicates that the bacteria are flourishing; "-" indicates sterile growth.
The minimum inhibitory concentrations of the separated and purified total alkaloids of the zanthoxylum dissitum Hemsl on 3 test bacteria of Escherichia coli, Staphylococcus aureus and Candida albicans are 0.35, 0.20 and 0.25mg/mL respectively, and the minimum bactericidal concentrations are 0.30, 0.25 and 0.30mg/mL respectively. Candida albicans in the 3 bacteria has stronger anti-drug biofilm characteristics, the previous antibiotics and bactericides are difficult to penetrate through fungal biofilms to contact the surfaces of the Candida albicans to play a role, and dictamnine has an inhibiting effect on the Candida albicans biofilm to stop the growth of the Candida albicans, so that the content and the purity of the dictamnine in the total alkaloids of the zanthoxylum dissitum Hemsl extracted under the condition of response surface optimization process are indirectly shown to be higher, and the extraction and purification process is stable and reliable.
The above embodiments are only for illustrating the invention and are not to be construed as limiting the invention, and those skilled in the relevant art can make various changes and modifications without departing from the spirit and scope of the invention, so that all equivalent technical solutions also belong to the scope of the invention, and the scope of the invention is defined by the claims.

Claims (8)

1. A method for extracting, separating and purifying total alkaloids from zanthoxylum dissitum Hemsl is characterized in that:
firstly, mixing and grinding roots and stems of zanthoxylum dissitum hemsl, adding a mixed solution of ethanol and acetone in a volume ratio of 1:1, and performing microwave extraction at 50-70 ℃; separating and purifying the obtained extract by using resin to obtain zanthoxylum dissitum total alkaloids; NKA-9 macroporous adsorption resin is selected as the resin.
2. The method for extracting, separating and purifying zanthoxylum dissitum total alkaloids as claimed in claim 1, wherein the method comprises the following steps: mixing the single-side needle root and the single-side needle stem according to the mass ratio of 1: 2.
3. The method for extracting, separating and purifying zanthoxylum dissitum total alkaloids as claimed in claim 1, wherein the method comprises the following steps: mixing and powdering the roots and stems of the zanthoxylum dissitum Hemsl, and sieving with a 60-mesh sieve to obtain the root and stem powder.
4. The method for extracting, separating and purifying zanthoxylum dissitum total alkaloids as claimed in claim 1, wherein the method comprises the following steps: during microwave extraction, the volume percentage of the ethanol-acetone mixed solution is 70-90%, and the balance is water; the ratio of the material to the liquid is 15: 1-25: 1mL-1(ii) a The extraction time is 15-25 min.
5. The method for extracting, separating and purifying zanthoxylum dissitum total alkaloids as claimed in claim 4, wherein the method comprises the following steps: during microwave extraction, the volume percentage of the ethanol-acetone mixed solution is 89%, and the balance is water; the ratio of material to liquid is 21:1mL.g-1(ii) a The extraction temperature is 70 ℃; the extraction time is 20 min.
6. The method for extracting, separating and purifying zanthoxylum dissitum total alkaloids as claimed in claim 1, wherein the method comprises the following steps: the microwave power is 300W.
7. The method for extracting, separating and purifying zanthoxylum dissitum total alkaloids as claimed in any of claims 1-6, wherein: the microwave extraction frequency is 2 times, and the second extraction is carried out after the first extraction system is recovered to the room temperature; the sum of the time of the two extractions, excluding the time of the intermediate cooling, was 40 minutes.
8. The method for extracting, separating and purifying zanthoxylum dissitum total alkaloids as claimed in claim 1, wherein the method comprises the following steps: the specific conditions for separation and purification are as follows: the mass ratio of crude drug to resin is 1:1, the pH of the sample loading solution is adjusted to 7.0, the sample loading flow rate is 2BV/h, and the elution is carried out at the speed of 2BV/h by using 6BV 80% ethanol-acetone mixed solution with the volume of 1: 1.
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