CN116715703A - Method for extracting and purifying total alkaloids of sheep horn cotton - Google Patents
Method for extracting and purifying total alkaloids of sheep horn cotton Download PDFInfo
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- 229930013930 alkaloid Natural products 0.000 title claims abstract description 129
- 229920000742 Cotton Polymers 0.000 title claims abstract description 99
- 241001494479 Pecora Species 0.000 title claims abstract description 88
- 238000000034 method Methods 0.000 title claims abstract description 38
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 104
- 238000000605 extraction Methods 0.000 claims abstract description 86
- 239000007788 liquid Substances 0.000 claims abstract description 48
- 239000011347 resin Substances 0.000 claims abstract description 38
- 229920005989 resin Polymers 0.000 claims abstract description 38
- 238000002474 experimental method Methods 0.000 claims abstract description 14
- 238000000746 purification Methods 0.000 claims abstract description 10
- 238000002137 ultrasound extraction Methods 0.000 claims abstract description 7
- 150000003797 alkaloid derivatives Chemical class 0.000 claims description 82
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 45
- 238000011068 loading method Methods 0.000 claims description 22
- 238000001179 sorption measurement Methods 0.000 claims description 22
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 18
- 241000594310 Randia aculeata Species 0.000 claims description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 12
- 239000003480 eluent Substances 0.000 claims description 11
- 238000010828 elution Methods 0.000 claims description 9
- 239000002253 acid Substances 0.000 claims description 8
- 238000001704 evaporation Methods 0.000 claims description 7
- 239000000284 extract Substances 0.000 claims description 5
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- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07G—COMPOUNDS OF UNKNOWN CONSTITUTION
- C07G5/00—Alkaloids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/24—Apocynaceae (Dogbane family), e.g. plumeria or periwinkle
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/35—Extraction with lipophilic solvents, e.g. Hexane or petrol ether
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention belongs to the technical field of green process chemistry, and particularly discloses a method for extracting and purifying total alkaloids of sheep horn cotton, which takes branches and leaves of sheep horn cotton as raw materials, utilizes an ultrasonic extraction method to obtain the optimal feed liquid ratio, extraction temperature and ethanol concentration by fitting a functional relation between response values and factors on the basis of single factor experiments from the influence of factors such as feed liquid ratio, extraction temperature, ethanol concentration and extraction time on the total alkaloids content of the sheep horn cotton, and adopts macroporous resin to purify the total alkaloids of the sheep horn cotton, so that the problem of too little total alkaloids of the sheep horn cotton obtained by the traditional extraction method is solved, and the activity of the total alkaloids of the sheep horn cotton is obviously improved by the purification method.
Description
Technical Field
The invention belongs to the technical field of green process chemistry, and particularly relates to a method for extracting and purifying total alkaloids of sheep horn cotton.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
The sheep horn cotton (Alstonia mailei) is a plant of Apocynaceae (Apocynaceae), the Hedyotis plant is a special species of China, and the sheep horn cotton is distributed in the Yunnan area. Has effects in clearing away heat, relieving pain, stopping bleeding, removing toxic materials, and promoting granulation; can be used for treating traumatic hemorrhage, skin sore, etc. The alkaloid rich in structural diversity is an important reason for the good biological activity of the sheep horn cotton.
The traditional method for extracting the total alkaloids of the sheep horn cotton mainly adopts an organic solvent for extraction or reflux extraction; the purification method adopts macroporous adsorption resin. However, the amount of the total alkaloids of the sheep horn cotton obtained by the traditional extraction method is small, the purification process can further reduce the amount of the total alkaloids of the sheep horn cotton, and the purification method can influence the activity of the total alkaloids of the sheep horn cotton.
Disclosure of Invention
In order to overcome the problems, the invention provides a method for extracting and purifying the total alkaloids of the sheep horn cotton. According to the invention, the sheep horn cotton branches and leaves are used as raw materials, an ultrasonic extraction method is used, on the basis of a single factor experiment, the influence of factors such as feed liquid ratio, extraction temperature, ethanol concentration, extraction time and the like on the alkaloid content in the sheep horn cotton is utilized, a function relation between a response face value and the factors is fitted through a multiple quadratic regression equation, so that the optimal feed liquid ratio, extraction temperature and ethanol concentration are obtained, the extracted sheep horn cotton total alkaloids are purified through macroporous resin, and the activity of the purified sheep horn cotton total alkaloids is obviously improved.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a method for extracting and purifying total alkaloids from sheep horn cotton, the method comprising:
s1, grinding the sheep horn cotton branches and leaves by using a grinder to obtain sheep horn cotton powder;
s2, selecting ethanol as an extracting agent, carrying out ultrasonic extraction on the sheep horn cotton powder in the step S1, and determining the optimal values of the concentration, the feed-liquid ratio, the extraction temperature and the extraction time of the extracting agent through a single-factor experiment;
s3, optimizing an ultrasonic-assisted goat horn cotton alkaloid extraction method by adopting a response surface method, and establishing a mathematical model by using Design-Expert8 software according to a single factor experimental result by taking a feed-liquid ratio A, an extraction temperature B and an ethanol concentration C as independent variables and the content of alkaloid as a response value Y to obtain a regression equation;
s4, calculating a regression equation in the step S3 to obtain the optimal feed-liquid ratio, extraction temperature and ethanol concentration;
s5, carrying out ultrasonic extraction on the wool powder under the conditions of the optimal feed-liquid ratio, the extraction temperature and the ethanol concentration in the S4 to obtain a wool alkaloid crude extract;
s6, evaporating the coarse extract of the goat horn cotton alkaloids in the S5 to obtain coarse goat horn cotton alkaloids, carrying out acid extraction, and then carrying out adsorption purification on the coarse extract by adopting macroporous resin, and determining the type of the macroporous adsorption resin, the pH value of the sample loading liquid and the optimal value of the sample loading quantity by single factor experiments; analyzing macroporous resin for adsorbing the sheep horn cotton alkaloid, and determining the optimal value of the ethanol concentration and the elution amount of the eluent through a single factor experiment;
and S7, alkalizing the parsed goat horn cotton alkaloid by using a NaOH solution, leading the pH value of the alkalized solution to be 10-11, and then extracting by using dichloromethane and evaporating to obtain the pure goat horn cotton alkaloid.
In one or more embodiments, the particle size of the pulverizer in step S1 is 80 to 100 mesh.
In one or more embodiments, in the step S2, the concentration of the extractant (mass fraction of solute) is 80%, the optimal value of the feed liquid ratio is 1:25g/mL, the optimal value of the extraction temperature is 40 ℃, and the optimal value of the extraction time is 50min.
In one or more embodiments, in the step S3, the regression equation is: y=199.75+14.43a-3.75b+28.58c-1.83ab+2.27ac-13.19 BC-2.95A 2 -4.84B 2 -5.91C 2 。
In one or more embodiments, in the step S4, the optimal feed-to-liquid ratio of 23.41g/mL, the extraction temperature of 30.16 ℃ and the ethanol concentration of 89.77% are obtained through calculation.
In one or more embodiments, in the step S5, the conditions for the acid extraction are: acid extraction is carried out by adopting hydrochloric acid solution with the mass fraction of 1-3%; preferably, the mass fraction of the hydrochloric acid is 2%; the volume ratio of the crude goat horn cotton alkaloid to the hydrochloric acid solution is 1mg: 1.34-4 mL; preferably 1mg:2mL.
In one or more embodiments, in the step S6, the macroporous adsorbent resin is a macroporous resin of model X-5.
In one or more embodiments, in the step S6, the pH of the loading solution has an optimum value of ph=5.
In one or more embodiments, in the step S6, the loading amount is expressed by a mass ratio of the crude sheep' S horn alkaloid to the macroporous resin, and the optimal value of the loading amount is 0.063:1.
In one or more embodiments, in the step S6, the optimal value of the ethanol concentration of the eluent is 70%.
In one or more embodiments, in the step S6, the elution amount is expressed as a mass ratio of the volume of the eluent to the macroporous resin, and the optimal value of the elution amount is 44mL:1g.
The invention has the beneficial effects that:
the invention provides a method for extracting and purifying total alkaloids of sheep horn cotton, which takes branches and leaves of sheep horn cotton as raw materials, adopts an ultrasonic extraction method to obtain the optimal feed liquid ratio, extraction temperature and ethanol concentration by fitting a function relation between response values and factors through a multiple quadratic regression equation on the basis of single factor experiments from the influence of factors such as feed liquid ratio, extraction temperature, ethanol concentration and extraction time on the content of alkaloids in the sheep horn cotton, and purifies the extracted total alkaloids of the sheep horn cotton through macroporous resin, so that the activity of the purified total alkaloids of the sheep horn cotton is obviously improved.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a standard curve of berberine hydrochloride;
FIG. 2 is a graph showing the effect of feed liquid ratio on alkaloid extraction;
FIG. 3 is a graph showing the effect of extraction temperature on alkaloid extraction;
FIG. 4 is a graph showing the effect of ethanol concentration on alkaloid extraction;
FIG. 5 is a graph showing the effect of extraction time on alkaloid extraction;
FIG. 6 is a graph showing the response curves of alkaloid extraction under interaction of extraction temperature and feed-liquid ratio;
FIG. 7 is a graph showing the response curves of alkaloid extraction under the interaction of ethanol concentration and feed-liquid ratio;
FIG. 8 is a graph showing the response curves of alkaloid extraction under the interaction of ethanol concentration and extraction temperature;
FIG. 9 shows the effect of pH of the alkaloid sample solution on the amount of alkaloid extracted from the adsorption solution;
FIG. 10 shows the effect of different concentrations of ethanol eluate on the amount of alkaloid extracted from the adsorption solution;
FIG. 11 is an alkaloid adsorption leakage curve;
FIG. 12 shows the alkaloid analytical curve for X-5 macroporous resin;
FIG. 13 shows IC of total alkaloids of sheep horn cotton before and after purification 50 Values.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
The content of the total alkaloids in the sheep horn cotton is determined by an acid dye colorimetric method, and the principle is that a complex formed by the reaction of the acid dye and the alkaloids can be absorbed by ultraviolet at 415.5nm, and berberine hydrochloride is an alkaloid, so that the berberine hydrochloride is selected as a reference substance to determine the quantification of the total alkaloids in the sheep horn cotton.
Example 1
Precisely weighing 10mg of berberine hydrochloride, placing into a beaker, adding 20mL of dichloromethane for dissolution, transferring into a 100mL volumetric flask, adding dichloromethane solution for volume fixing to scale, precisely transferring into a 25 mL-50 mL volumetric flask, and adding dichloromethane for volume fixing to scale to obtain 50 mug/mL reference substance solution.
Respectively taking 50 mug/mL of reference substance solutions 0, 1, 2, 3, 4 and 5mL in a test tube, supplementing to 5mL, precisely adding 10mL of dichloromethane, adding 4mL of bromocresol green solution, transferring to a separating funnel, shaking up and down for 60 times, standing for 30min, separating a water layer and a dichloromethane layer, taking the first part as a blank control, measuring absorbance at 415.5nm, and drawing a standard curve. The content of berberine hydrochloride is taken as an abscissa, the measured absorbance is taken as an ordinate, a standard curve is drawn, a regression equation is calculated to be y=0.0183 x-0.0252, and R is calculated 2 = 0.9996, showing that the berberine hydrochloride concentration is in good linear relationship with absorbance at 10-50 μg/mL, as shown in fig. 1.
Example 2 (one-factor experiment)
Determination of absorbance of the sample solution: taking 1g of ground sheep horn cotton powder, adding a certain amount of ethanol for ultrasonic treatment, centrifuging, taking supernatant, steaming to dryness by rotary evaporation, adding 25mL of 2% HCL for dissolution, transferring the obtained acid water into a 50mL centrifuge tube, centrifuging, alkalizing the supernatant by NaOH solution to a pH value of 10-11, extracting 3 times by using dichloromethane (25 mL each time), combining dichloromethane extracts, concentrating by rotary evaporation, precisely removing 25mL of alkaloid obtained by dissolving dichloromethane, taking 5mL of alkaloid in a test tube, precisely adding 10mL of dichloromethane, adding 4mL of bromocresol green solution, transferring into a separating funnel, shaking up and down for 60 times, standing for 30min, separating a water layer and a dichloromethane layer, and measuring absorbance at 415.5 nm.
Influence of feed liquid ratio on the alkaloid content of sheep horn cotton: weighing 1g of sheep horn cotton powder, adding 80% ethanol solution with feed liquid ratios of 1:10, 1:15, 1:20, 1:25 and 1:30g/mL respectively, carrying out ultrasonic treatment for 1h, extracting sheep horn cotton alkaloids at 30 ℃, measuring the absorbance of the sample solution according to the absorbance of the sample solution, calculating the extraction amount of the alkaloids, and repeating for three times. As shown in FIG. 2, the yield of the sheep horn cotton alkaloid is higher when the feed liquid ratio is 1:15g/mL, 1:20g/mL and 1:25g/mL, and the optimal feed liquid ratio is 1:25 g/mL.
Influence of extraction temperature on the alkaloid content of sheep horn cotton: weighing 1g of sheep horn cotton powder, adding 80% ethanol solution with the feed-liquid ratio of 1:25g/mL, carrying out ultrasonic treatment for 1h, extracting sheep horn cotton alkaloids at 30, 40, 50, 60 and 70 ℃ respectively, measuring the absorbance at different feed-liquid ratios according to the measurement of the absorbance of the sample solution, further calculating the alkaloid extraction amount, and repeating for three times. As shown in figure 3, the extraction yield of the sheep horn cotton alkaloid is higher at 30, 40 and 50 ℃, and the optimal temperature is 40 ℃.
Influence of ethanol concentration on the alkaloid content of sheep horn cotton: weighing 1g of sheep horn cotton powder, adding ethanol solutions with the feed liquid ratio of 1:25g/mL and the concentration of 50, 60, 70, 80, 90 and 100 percent respectively, carrying out ultrasonic treatment for 1h, extracting sheep horn cotton alkaloids at 30 ℃, measuring the absorbance of different feed liquid ratios according to the absorbance measurement of the sample solution, further calculating the alkaloid extraction amount, and repeating for three times. As shown in figure 4, the extraction yield of the sheep horn cotton alkaloid is higher when the ethanol concentration is 70%, 80 and 90%, and the optimal concentration is 80%.
Influence of extraction time on the alkaloid content of sheep horn cotton: weighing 1g of sheep horn cotton powder, adding 80% ethanol solution with the feed liquid ratio of 1:25g/mL, respectively carrying out ultrasonic treatment for 20, 30, 40, 50 and 60min, extracting sheep horn cotton alkaloids at 30 ℃, respectively measuring the absorbance at different feed liquid ratios according to the measurement of the absorbance of the sample solution, further calculating the alkaloid extraction amount, and repeating for three times. As shown in fig. 5, the increase of the extraction amount of the sheep horn cotton alkaloid with time was not remarkable, and when the extraction time was 50min, the extraction amount reached the highest value, and therefore, the optimal extraction time was 50min.
Example 3 (response surface optimization experiment:)
Factor level selection and response surface design: on the basis of a single factor test, a response surface test is designed, 3 levels are selected by taking a feed liquid ratio A, an extraction temperature B and an ethanol concentration C as independent variables, the content of alkaloid is taken as a response value Y, a mathematical model is established, a model is established by using Design-Expert8 software, an ultrasonic-assisted sheep horn cotton alkaloid extraction process is optimized, test Design factors and levels are shown in table 1, and Design combinations and sheep horn cotton alkaloid content are shown in table 2.
Table 1 design of experimental factor level
Regression model creation and analysis
Polynomial fitting regression is carried out on experimental results by using Design-Expert8 software, a mathematical model is established by taking the feed liquid ratio A, the extraction temperature B and the ethanol concentration C and the content of alkaloid as a response value Y, and a regression equation is obtained: y=199.75+14.43a-3.75b+28.58c-1.83ab+2.27ac-13.19 BC-2.95A 2 -4.84B 2 -5.91C 2 The method comprises the steps of carrying out a first treatment on the surface of the The analysis of variance results are shown in Table 3.
TABLE 3 response surface regression model analysis of variance table
Note that: p <0.01 is extremely significant (x); p <0.05 is significant (; n indicates that the effect is insignificant.
As can be seen from Table 3, the F value was 6.63, indicating that the model was significant. P=0.0104, indicating significant differences in the established response surface models; the mismatch term p= 0.1274>0.05, the model fitting degree is good, and the model is established. Model correlation coefficient 0.8949, adjustment coefficient R2 adj The regression model is 0.7599, which shows that the actual value and the predicted value have better fitting degree and smaller test error, and the fitted regression model can be used for analyzing and predicting the optimal extraction parameters of the sheep horn cotton alkaloid.
The degree of influence of the independent variable on the extraction yield of alkaloid can be determined by the magnitude of the F value in Table 3, and the larger the F value, the larger the influence of the independent variable on the extraction yield is. From Table 3, it can be seen that F A ＝10.61，F B ＝0.72，F C The degree of influence of each factor on the extraction rate of the sheep horn cotton alkaloid is thus obtained as follows: ethanol concentration>Feed-to-liquid ratio>Extraction temperature.
And drawing a response surface diagram according to a regression equation, wherein the response surface diagram belongs to a three-dimensional space curved surface interacted between a response value and each factor, and mainly uses three factors of analysis feed-liquid ratio, extraction temperature and ethanol concentration to fix one factor, and the influence of the other two factors and interaction thereof on alkaloid content. The degree of interaction between the two factors can be determined based on the shape of the steepness of the response surface. As shown in fig. 6, the steepness of the response curve of the feed-liquid ratio is greater than the extraction temperature, which indicates that the effect of the feed-liquid ratio on the alkaloid extraction rate of the sheep horn cotton is greater than the extraction temperature; as shown in fig. 7, the steepness of the response curve of the ethanol concentration is greater than the feed-liquid ratio, which indicates that the effect of the ethanol concentration on the alkaloid extraction rate of the sheep horn cotton is greater than the feed-liquid ratio; as shown in fig. 8, the steepness of the response curve of the ethanol concentration is greater than the extraction temperature, which means that the effect of the ethanol concentration on the extraction rate of the sheep horn cotton alkaloid is greater than the extraction temperature, so we can also obtain that the effect of each factor on the extraction rate of the sheep horn cotton alkaloid is as follows: ethanol concentration > feed to liquid ratio > extraction temperature, which corresponds exactly to the result obtained by analysis of variance.
Solving the fitting equation to obtain the optimal alkaloid extraction condition: the feed liquid ratio of A is 23.41g/mL, the extraction temperature of B is 30.16 ℃, the concentration of C ethanol is 89.77%, and under the condition, the predicted alkaloid content is 244.892 mug/mL. Considering the feasibility of experiments and practical operations, the adjusted alkaloid extraction conditions are as follows: the feed liquid ratio of A is 23.5g/mL, the extraction temperature of B is 30.0 ℃, and the concentration of C ethanol is 90.0%. Under the condition, a verification experiment is carried out, the extraction content of the alkaloid is 246.3 mug/mL, the relative error of the same model predicted value is only 0.578%, and the production process parameters obtained by optimizing the technological conditions of the sheep horn cotton alkaloid extraction by the response surface method are accurate and reliable and have certain application value.
Example 4 (Process optimization of macroporous resin purification of sheep's horn cotton alkaloid)
Preparation of test solution: taking 100g of sheep horn cotton powder, ultrasonically extracting for 50min at the optimal extraction process A feed liquid ratio of 23.5g/mL and the extraction temperature of 30.0 ℃ and the concentration of C ethanol of 90.0%, and evaporating to dryness to obtain the sheep horn cotton total alkali. 200mg of sheep horn cotton total alkali is weighed and added into 400mL of 2% hydrochloric acid aqueous solution for dissolution, and the mixture is used as a sample solution.
Measurement of absorbance: transferring the acid aqueous solution obtained by dissolving hydrochloric acid into a 50mL centrifuge tube, centrifuging, alkalizing the supernatant by NaOH solution to a pH value of 10-11, extracting 3 times by using dichloromethane (25 mL each time), combining dichloromethane extracts, performing rotary evaporation concentration, precisely transferring 25mL of dichloromethane to dissolve the obtained alkaloid, taking 5mL of alkaloid in a test tube, precisely adding 10mL of dichloromethane, further adding 4mL of bromocresol green solution, transferring to a separating funnel, shaking up and down for 60 times, standing for 30min, separating a water layer and a dichloromethane layer, and measuring absorbance at 415.5 nm.
Screening of macroporous adsorbent resins
Accurately weighing 1g of wet resin of D101, X-5, LSA-21, AB-8, DPD-100 and NKA, placing into a 50mL centrifuge tube, adding 20mL of the sample solution, placing into a shaking table, vibrating and adsorbing for 24h at a shaking speed of 225 times/min, carrying out suction filtration on the total alkali extract of the adsorbed sheep horn cotton, measuring the absorbance of the filtrate according to the absorbance measuring method, further calculating the amount of the non-adsorbed sheep horn cotton alkaloids, and subtracting the amount of the non-adsorbed sheep horn cotton alkaloids from the amount of the added total sheep horn cotton alkaloids to obtain the amount of the sheep horn cotton alkaloids adsorbed by the macroporous adsorption resin. Pouring the wet resin after suction filtration into a 50mL centrifuge tube, adding 95% ethanol, vibrating and analyzing for 24h, suction filtering, rotationally evaporating the obtained ethanol, dissolving with 25mL hydrochloric acid aqueous solution, measuring the absorbance according to the absorbance measuring method, and further calculating the analyzed sheep horn cotton alkaloid amount.
The adsorption and analysis rates for D101, X-5, LSA-21, AB-8, DPD-100, NKA resins were calculated according to the following formula:
K suction pipe ＝(K Suction head -K After suction )/K Suction head ×100％；
K Solution ＝K After solution /(K Suction head -K After suction )×100％；
In the formula, K Suction pipe For adsorption rate, K Solution To resolution, K Suction head K is the initial concentration of adsorption liquid After suction K is the concentration of the solution after adsorption After solution The concentration of the solution after the analysis was determined.
As shown in the following Table 4, the adsorption rate of the six macroporous resins used in the experiment on the sheep horn cotton alkaloids is 98.20% -100%, the resolution rate is 62.56% -81.85%, and the adsorption rate and the resolution rate of the macroporous resin X-5 on the sheep horn cotton alkaloids are the highest, so that the macroporous resin of the X-5 model is selected for the experiment.
TABLE 4 adsorption and resolution of six macroporous resins to sheep's horn cotton alkaloids
Determination of optimal adsorption pH: 500mg of wet macroporous resin is placed in a 50mL centrifuge tube, 25mL of total alkali crude extract with the same total alkali content and pH of 2, 3, 4, 5 and 6 is added, a conical flask with a plug is placed in a shaking table, vibration absorption is carried out for 24 hours at the shaking speed of 225 times/min, suction filtration is carried out, the absorbance of the filtrate is measured according to the absorbance measuring method, the amount of unadsorbed sheep horn cotton alkaloids is calculated, when the pH of a sample is continuously increased, the content of the alkaloids in the solution after absorption is continuously reduced, and when the pH=5-6, the content of the alkaloids in the absorption solution is reduced, and the solubility of the alkaloids is reduced due to the increase of the pH, so that the pH=5 is selected as the optimal pH of the loading solution.
Determination of the ethanol concentration of the optimal eluent: weighing 6 parts of macroporous resin with adsorption saturation of 400mg, placing the macroporous resin into a 50mL centrifuge tube, precisely transferring 20mL of ethanol with concentration of 50%, 60%, 70%, 80% and 90% into a conical flask with a plug, vibrating and analyzing for 24 hours, filtering, evaporating filtrate, measuring the absorbance of the filtrate according to the absorbance measuring method, and further calculating the amount of desorbed alkaloids. As shown in fig. 10, the resolution ratio of the alkaloid increases and decreases with increasing concentration of the ethanol solution, and when the concentration of the ethanol is 70%, the content of the alkaloid in the resolution solution is the highest, so that 70% ethanol is selected as the optimal eluent of the sheep horn cotton alkaloid.
Determination of optimal loading: weighing 5g of X-5 macroporous resin, loading into a glass chromatographic column with the concentration of 1.5cm multiplied by 30cm, loading a goat horn cotton alkaloid solution with the concentration of 0.5mg/mL into the column at the flow rate of 1.0mL/min, collecting alkaloid effluent, measuring the absorbance value of the effluent, and stopping loading when the absorbance value of the effluent reaches 1/10 of the absorbance value of loading liquid. As shown in FIG. 11, the X-5 resin has better adsorption effect on the sheep horn cotton alkaloid, when the loading liquid reaches 630mL, the absorbance of the flowing alkaloid reaches 1/10 of the absorbance value of the alkaloid in the original solution, and the loading amount is confirmed to be 315mg so as to achieve the optimal adsorption.
Determination of optimal elution amount: weighing 5g of X-5 macroporous resin, loading into a 1.5cm multiplied by 30cm glass chromatographic column, loading a 0.5mg/mL wool-type biological alkali solution into the column at a flow rate of 1.0mL/min, washing with distilled water with a volume of 3 times of the column at the same speed, eluting with 70% ethanol, collecting effluent, measuring the absorbance value of the effluent, and drawing a biological alkaline analysis curve. As shown in fig. 12, the alkaloid content in the eluent was increased and then decreased with increasing elution volume, and when the amount of the eluent reached 220mL, the alkaloid concentration in the eluent was substantially 0, indicating that the alkaloid had been substantially eluted, confirming that 220mL was the optimal elution amount.
Verification of the optimal Process for macroporous resin purification
Weighing 5g of X-5 macroporous resin, loading into a 1.5cm multiplied by 30cm glass chromatographic column, loading a 0.5mg/mL wool alkaloid solution with a PH=5 concentration into the column at a flow rate of 1.0mL/min for adsorption, washing impurities with distilled water after complete adsorption, eluting with the determined optimal 70% ethanol, and calculating the purity of the alkaloid. As can be seen from the following Table 5, the purity of the sheep horn cotton alkaloid is increased from 71.56% to 89.14% after the sheep horn cotton alkaloid is purified by the X-5 macroporous resin, which shows that the process has better purifying effect on the sheep horn cotton alkaloid.
TABLE 5 calculation of purity of sheep's horn cotton alkaloids
Example 5 (detection of alkaloid Activity in sheep Horn cotton before and after purification)
Using HCT-15 cells in logarithmic phase, using 0.25% trypsin-EDTA solution to digest the cells for about 6min, using complete culture medium to stop digestion, centrifuging at 1000rpm for 5min, re-suspending cell sediment, counting by using a cell counter, adjusting cell density to 80000/mL, inoculating into 96-well plate at 8000, culturing for 24h to adhere to wall. After adherence, the culture medium was changed to 8 gradient concentration groups with total alkali final concentrations of 0, 5, 10, 20, 40, 60, 80 and 100. Mu.g/mL, and cultured for 48 hours. After the completion of the culture, the medium was replaced with a complete medium containing 10% of CCK-8, and the culture was continued for 45min, absorbance was measured at 450nm, and IC was calculated 50 Values. As shown in FIG. 13, the purified total alkali can increase sensitivity to HCT-15 cells, IC 50 The value was reduced from 51.80 + -1.40 μg/mL to 39.73 + -1.35 μg/mL.
It is to be understood that the invention is not limited to the precise arrangements and instrumentalities shown in the drawings, which have been described above, and that various modifications and changes may be effected without departing from the scope thereof. The scope of the invention is limited only by the appended claims.
Claims (10)
1. A method for extracting and purifying total alkaloids from sheep horn cotton, the method comprising:
s1, grinding the sheep horn cotton branches and leaves by using a grinder to obtain sheep horn cotton powder;
s2, selecting ethanol as an extracting agent, carrying out ultrasonic extraction on the sheep horn cotton powder in the step S1, and determining the optimal values of the concentration, the feed-liquid ratio, the extraction temperature and the extraction time of the extracting agent through a single-factor experiment;
s3, optimizing an ultrasonic-assisted goat horn cotton alkaloid extraction method by adopting a response surface method, and establishing a mathematical model by using Design-Expert8 software according to a single factor experimental result by taking a feed-liquid ratio A, an extraction temperature B and an ethanol concentration C as independent variables and the content of alkaloid as a response value Y to obtain a regression equation;
s4, calculating a regression equation in the step S3 to obtain the optimal feed-liquid ratio, extraction temperature and ethanol concentration;
s5, carrying out ultrasonic extraction on the wool powder under the conditions of the optimal feed-liquid ratio, the extraction temperature and the ethanol concentration in the S4 to obtain a wool alkaloid crude extract;
s6, evaporating the coarse extract of the goat horn cotton alkaloids in the S5 to obtain coarse goat horn cotton alkaloids, carrying out acid extraction, and then carrying out adsorption purification on the coarse extract by adopting macroporous resin, and determining the type of the macroporous adsorption resin, the pH value of the sample loading liquid and the optimal value of the sample loading quantity by single factor experiments; analyzing macroporous resin for adsorbing the sheep horn cotton alkaloid, and determining the optimal value of the ethanol concentration and the elution amount of the eluent through a single factor experiment;
and S7, alkalizing the parsed goat horn cotton alkaloid by using a NaOH solution, leading the pH value of the alkalized solution to be 10-11, and then extracting by using dichloromethane and evaporating to obtain the pure goat horn cotton alkaloid.
2. The method according to claim 1, wherein the size of the particle size ground by the pulverizer in the step S1 is 80 to 100 mesh.
3. The method according to claim 1, wherein the concentration of the extractant is 80%, the ratio of the liquid to the solid is 1:25g/mL, the extraction temperature is 40℃and the extraction time is 50min.
4. The method according to claim 1, wherein in the step S3, the regression equation is: y=199.75+14.43a-3.75b+28.58c-1.83ab+2.27ac-13.19 BC-2.95A 2 -4.84B 2 -5.91C 2 。
5. The method according to claim 1, wherein in the step S4, the optimal feed-liquid ratio of 23.41g/mL, the extraction temperature of 30.16 ℃ and the ethanol concentration of 89.77% are obtained by calculation.
6. The method according to claim 1, wherein in the step S5, the conditions for the acid extraction are: acid extraction is carried out by adopting hydrochloric acid solution with the mass fraction of 1-3%; preferably, the mass fraction of the hydrochloric acid is 2%; the volume ratio of the crude goat horn cotton alkaloid to the hydrochloric acid solution is 1mg: 1.34-4 mL; preferably 1mg:2mL.
7. The method according to claim 1, wherein in the step S6, the macroporous adsorbent resin is a macroporous resin of model X-5;
or, the pH of the loading solution is optimally ph=5.
8. The method of claim 1, wherein the loading is expressed in terms of a mass ratio of crude sheep's horn alkaloid to macroporous resin, and the loading has an optimum value of 0.063:1.
9. The method according to claim 1, wherein in the step S6, the optimal value of the ethanol concentration of the eluent is 70%.
10. The method according to claim 1, wherein in the step S6, the elution amount is expressed as a mass ratio of the volume of the eluent to the macroporous resin, and the optimal value of the elution amount is 44mL:1g.
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