CN113694128A - Response surface method optimized extraction process of strychnine-containing alkaloids in carex meyeriana - Google Patents
Response surface method optimized extraction process of strychnine-containing alkaloids in carex meyeriana Download PDFInfo
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- CN113694128A CN113694128A CN202110848038.4A CN202110848038A CN113694128A CN 113694128 A CN113694128 A CN 113694128A CN 202110848038 A CN202110848038 A CN 202110848038A CN 113694128 A CN113694128 A CN 113694128A
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- alkaloid
- strychnine
- extraction
- extraction process
- concentration
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- APUPEJJSWDHEBO-UHFFFAOYSA-P ammonium molybdate Chemical compound [NH4+].[NH4+].[O-][Mo]([O-])(=O)=O APUPEJJSWDHEBO-UHFFFAOYSA-P 0.000 description 2
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Abstract
And determining the optimal extraction process for extracting the strychnine-containing alkaloids from the carex meyeriana by an acid-water method by adopting a response surface analysis method, and identifying the optimal extraction process. Selecting three factors of extraction temperature, material-liquid ratio and hydrochloric acid concentration, and carrying out Box-Benhnke test design by taking the extraction rate of the Meyer sedge total alkaloid as a response value. The optimal extraction process comprises the following steps: the extraction time is 2h, the concentration of hydrochloric acid is 0.18 mol/L, the extraction temperature is 80 ℃, the liquid-material ratio is 30 mL/g, and the extraction rate of alkaloid can reach 2.74%. Purifying and separating alkaloid by silica gel column chromatography, identifying physicochemical properties, and identifying by ultraviolet spectrophotometry, thin layer chromatography and high performance liquid chromatography to prove that strychnine exists in Meyer sedge alkaloid.
Description
Technical Field
The invention relates to an extraction method for optimizing alkaloid and an identification method for strychnine in alkaloid.
Background
Carex meyeriana is an ancient plant, contains abundant bioactive components, and is a precious plant resource. Widely distributed in northeast, Sichuan, inner Mongolia and east Russia of China, and has wide development prospect in the fields of medicine, health care and the like. The carex meyeriana has peculiar warm-keeping function and functions of ventilation, cold prevention, bacteriostasis and the like. At present, the research on the wula sedge mainly focuses on the physical properties such as fiber and the like, and the research on the chemical components of the extracting solution is fresh. The grass extract contains alkaloids, and most of the alkaloids have anticancer, antitumor, antiinflammatory, antiviral, platelet aggregation inhibiting, arrhythmia resisting, and hypertension resisting effects.
Literature reports that the existing extraction methods of alkaloids in natural products include water extraction, acidic aqueous solution extraction, alkaline aqueous solution extraction, organic solvent extraction, cold leaching extraction, reflux extraction, Soxhlet extraction, ultrasonic extraction, membrane extraction and supercritical extraction. The extraction rate and chemical composition of alkaloid are influenced by many factors, and an orthogonal experiment method or a response surface analysis method is usually adopted to optimize the extraction process and determine the optimal extraction conditions. Selecting three factors of hydrochloric acid concentration, feed liquid volume and extraction temperature by Zhao Shengman, etc., taking yield of total alkaloids as response value, and performing response surface test design to obtain Chelidonium majus alkaloid with optimum extraction conditions of hydrochloric acid concentration 0.42%, feed liquid volume 97.59ml, extraction temperature 50.12 deg.C, and alkaloid obtained under the conditions of 2.7090mg g－1. Orthogonal experiments are adopted by Margil et al to select extraction time, material-liquid ratio, pH value and temperature to investigate the influence on the extraction rate of the total alkaloids of the red cyclopentadienyl grass, the optimal condition for extracting the total alkaloids of the red cyclopentadienyl grass by acid water is that the temperature is 90 ℃, the extraction time is 5h, the material-liquid ratio is 1:40, the pH value is 1.5, the optimal condition for extracting the total alkaloids of the red cyclopentadienyl grass is obtained, and the extraction rate of isocorydine is 9.28 percent.
In order to obtain alkaloid with high purity, the alkaloid is purified and separated. The typical purification method of alkaloid comprises organic solvent extraction, salting-out method, precipitation method, crystallization method, silica gel column chromatography and macroporous adsorbent resin method. For example, Panyan et al adopt solvent extraction, silica gel column chromatography and other methods to purify and separate the alkaloid components of the fermented semen Strychni product, dichloromethane-methanol elution is carried out, gradient increase is carried out according to 1% methanol, elution is carried out sequentially, each gradient is 5L, a thin layer plate is inspected under an ultraviolet lamp, the color development of a bismuth potassium iodide reagent is improved, after the same fractions are combined, the mixture is divided into 4 elution parts of 3-4% methanol, 6-7% methanol, 4-5% methanol and 7-15% methanol, and silica gel or Sephadex LH-20 column chromatography separation and recrystallization are repeatedly carried out to obtain 10 alkaloid components.
The monoterpene indole alkaloids of strychnine are effective components of semen Strychni, and are also toxic components, and the alkaloids are 70% of the total alkaloids. The results of the previous researches show that strychnine is a medium-strong alkali and is stable in an alkaline environment, and the strychnine is extracted by an acid solution and then subjected to alkaline extraction. The change of strychnine and strychnine in nux vomica preparation is measured by the neyan-fold-wai method using UV spectrophotometry; cao Ling et al qualitatively identify semen Strychni in the prescription by TLC; HuangyueChun et al used reverse phase high performance liquid chromatography with methanol-water (65: 35) (aqueous phase containing 0.029 mol. L-1Monopotassium phosphate and 0.037 md.L-1Sodium dodecyl sulfate) as mobile phase, measuring the content of strychnine in the Tongbiling tablets under the detection wavelength of 254nm to obtain the absorption peak of strychnine appearing in the solution of a test product and a reference product when the retention time is about 12min, and calculating the average content of strychnine in each tablet of Tongbiling sample to be 0.22mg by measuring the peak area.
So far, the research on the alkaloid in the carex meyeriana is less, and the optimization of the extraction process and the related report on the structure are not seen. The application patent firstly inspects the alkaloid in the carex meyeriana by using three factors of extraction temperature, material-liquid ratio and hydrochloric acid concentration by adopting a response surface design method, and performs chemical, spectral and chromatographic method identification through silica gel column chromatography purification and separation to determine that the alkaloid compounds contain strychnine.
Disclosure of Invention
The invention aims to provide a response surface method for optimizing the extraction method of total alkaloids in wula sedge and the identification of strychnine.
In order to solve the above problems, the present invention provides the following technical solutions.
(1) Drying the carex meyeriana, cutting into sections after constant weight.
(2) Extracting with acidic aqueous solution at a certain ratio for 2 hr, filtering with gauze, and concentrating under reduced pressure. Adjusting the pH value of all the wula sedge concentrated solution to be within the range of 8-10 by using 40% sodium hydroxide solution, extracting for 2 times by using dichloromethane with the volume of 1:1, combining extract liquor, washing by using water until alkaloid salt and alkaline water dissolved in the water are removed, collecting the extract liquor, concentrating and drying to obtain crude alkaloid. Selecting three factors of extraction temperature, liquid-material ratio and hydrochloric acid concentration, taking the extraction rate of the Meyer sedge total alkaloid as a response value, and carrying out Box-Benhnke test design, wherein the optimal extraction process is as follows: under the condition of extraction time of 2h, the concentration of hydrochloric acid is 0.18 mol/L, the extraction temperature is 80 ℃, the liquid-material ratio is 30 mL/g, and the extraction rate of total alkaloids is 2.74 +/-1.0%.
(3) The obtained crude alkaloid is subjected to color reaction. The color reaction phenomena are that the iodine-potassium iodide reagent generates orange precipitate, the concentrated sulfuric acid reagent generates yellow brown, the concentrated hydrochloric acid reagent does not generate color, and the ammonium molybdate-concentrated sulfuric acid solution (Frohde reagent) generates brown green respectively, which indicates that the alkaloid exists in the carex.
(4) The Meyer sedge total alkaloids are obtained after silica gel column chromatography purification and separation. Purifying and separating alkaloid by silica gel column chromatography, loading by a dry method, wherein the mass ratio of silica gel to a sample is 20:1, and the mass ratio of petroleum ether: dichloromethane: anhydrous ethanol: cyclohexane (50: 10:5: 10) is used as eluent to elute three column volumes, and impurities with smaller polarity are removed; purification exchange eluent was dichloromethane: anhydrous ethanol: cyclohexane (30: 10: 10) eluted three column volumes eluting purified alkaloid containing strychnine; finally, using methanol: dichloromethane (40: 10) elutes other highly polar alkaloids, and the elution sample is detected to be colorless by using an alkaloid precipitation reagent.
(5) Identifying strychnine by a chemical method: concentrating the Meyer sedge alkaloid acid-water extract, extracting with ethanol, purifying and separating the sample with AB-8 column, eluting with distilled water, then with 70% ethanol, eluting with 5 parts of alkaloid sample. Fraction 1 was washed with water to give a volume of 50ml, and fractions 2, 3, 4, 5 were washed with alcohol to give 50ml each. And performing color identification reaction on each alkaloid sample and the strychnine control solution by using 1% ammonium vanadate-concentrated sulfuric acid solution. The reaction of the part No. 3 with ammonium vanadate-concentrated sulfuric acid solution firstly shows purple color and then turns into red color, and the color change of the part No. 3 is the same as that of the strychnine reference substance.
(6) Ultraviolet, thin layer and high performance liquid chromatography to determine whether the alkaloid compound strychnine is contained therein. Dissolving purified alkaloid with methanol, and scanning at 254nm with ultraviolet full wavelength to obtain absorption peak identical to that of strychnine control; mixing with dichloromethane: cyclohexane: ethanol (3: 1: 1) is used as developing solvent to obtain spots which show the same color and shape with the strychnine reference substance on the same position of the TLC plate; using 0.4% phosphoric acid (pH 3.0 adjusted by triethylamine) -acetonitrile (85: 15) as mobile phase, detecting wavelength at 260nm, column temperature at 25 deg.C, and gradient eluting to obtain absorption peak identical to that of strychnine control at 3.47min, to obtain alkaloid sample containing strychnine.
Drawings
FIG. 1 is a graph showing the results of identification of a color reaction;
FIG. 2 is a graph of strychnine standard concentration versus absorbance standard;
FIG. 3 is a monogenic graph of different hydrochloric acid concentrations versus the concentration of Meyer sedge total alkaloids;
FIG. 4 is a monogenic graph of different extraction temperatures versus concentration of total alkaloids from Carex meyeriana;
FIG. 5 is a monogenic graph of the ratio of different feed solutions to the concentration of total alkaloids from carex meyeriana;
FIG. 6 is a response surface diagram showing the influence of solvent concentration (a), extraction temperature (b) and liquid-to-material ratio (c) on total alkaloid content;
FIG. 7 is a diagram of a response surface prediction model of residual scatter (d), prediction normal distribution (e), and actual normal distribution (f);
FIG. 8 is a graph showing the results of the ammonium vanadate-concentrated sulfuric acid solution assay;
FIG. 9 is a UV full wavelength scan of Merremia indica alkaloid separated strychnine;
FIG. 10 is a UV full wavelength scan of a strychnine control;
FIG. 11 is a thin layer chromatography identification chart (1 is alkaloid sample, 2 is strychnine control);
FIG. 12 is a high performance liquid chromatogram of an extract of Meyer sedge;
FIG. 13 is a high performance liquid chromatogram of a strychnine control.
Detailed Description
The invention is described in detail below with reference to the figures and the detailed description of the embodiments.
Example extraction of alkaloid as an active ingredient in Carex meyeriana
Air drying Carex meyeriana, weighing a certain amount of Carex meyeriana segments with an electronic balance, adding a certain proportion of solvent, placing in a 500 ml round bottom flask, heating under reflux in 80 deg.C constant temperature water bath for 2 hr, filtering with gauze, and concentrating under reduced pressure. Adjusting the pH value of all the wula sedge alkaloid concentrated solution to be within the range of 8-10 by using 40% sodium hydroxide solution, extracting for 2 times by using dichloromethane with the volume of 1:1, combining extract liquor, washing by using water until alkaloid salt and alkaline water dissolved in the water are removed, collecting the extract liquor, concentrating and drying.
Examples two chemical reaction identifications
Identification-color reaction:
in FIG. 1-a, from left to right, there are iodine-potassium iodide reagent group samples and their blank controls (1-a-1 and 1-a-2), concentrated sulfuric acid reagent group samples and their blank controls (1-a-3 and 1-a-4), concentrated hydrochloric acid reagent group samples and their blank controls (1-a-5 and 1-a-6), ammonium molybdate-concentrated sulfuric acid solution group samples and their blank controls (1-a-7 and 1-a-8). For clearer and more visual display, 1-b is an amplified iodine-potassium iodide reagent sample (1-a-1), 1-c is an amplified concentrated sulfuric acid reagent group (1-a-3 and 1-a-4), and 1-d is an amplified ammonium molybdate-concentrated sulfuric acid group (1-a-8 and 1-a-7).
(1) Iodine-potassium iodide reagent group: adding 1-2 ml of Meyer sedge extracting solution and 1-2 drops of iodine-potassium iodide alkaloid precipitation reagent into a test tube, and fully shaking. It is seen from FIGS. 1-a-1, 1-a-2 and 1-b that the iodine-potassium iodide reagent is precipitated orange.
(2) Concentrated sulfuric acid reagent group: colchicine appears yellow, codeine appears light blue, berberine appears green, atropine, cocaine, morphine and strychnine do not appear color. It is seen from FIGS. 1-a-3, 1-a-4 and 1-c that the concentrated sulfuric acid reagent is yellowish brown in color.
(3) Concentrated hydrochloric acid reagent group: veratrine appears red, and most other alkaloids do not appear. It can be seen from FIGS. 1-a-5 and 1-a-6 that the concentrated HCl reagent did not develop color.
(4) Ammonium molybdate-concentrated sulfuric acid solution reagent set: (Frohde reagent) is 1% ammonium molybdate concentrated sulfuric acid solution, 1g of ammonium molybdate is weighed and added into a beaker, and 100 mL of concentrated sulfuric acid (98%) is added for dissolution. Aconitine is yellowish brown, morphine is purple to brown, codeine is dark green to light yellow, berberine is brownish green, colchicine is yellow, atropine and strychnine are not colored. As shown in FIGS. 1-a-7, 1-a-8 and 1-d, the ammonium molybdate-concentrated sulfuric acid solution is brownish green and may contain berberine.
The 4 color reactions indicate that alkaloid exists in the carex meyeriana.
Example measurement of alkaloid content of Trifolium Linderae
Drawing of strychnine standard curve
Accurately weighing 1.0 mg of strychnine standard substance with an analytical balance, adding methanol solution to reach volume of 10 mL, shaking up, and preparing into 0.1 mg/mL strychnine standard substance solution. Diluting with methanol to obtain strychnine standard solution with concentration gradient of 0.004, 0.006, 0.008, 0.012 mg/mL. The absorbance A of strychnine was measured at a wavelength of 254 nm. Standard curves were plotted with strychnine concentration (mg/mL) as the abscissa and absorbance value (A) as the ordinate. Performing linear regression to obtain a regression equation: y = 73.7X-0.0754, R2=0.9994, as in fig. 2. The result shows that the concentration and the absorbance of the strychnine in 0.04-0.012 mg/L form a good linear relationship.
Content determination of sample
A sample is taken and added with 10 mL of methanol for ultrasonic dissolution. Transferring 1 mL of sample solution with a pipette, adding into a test tube, adding 3 mL of methanol, shaking, measuring absorbance A at 254nm wavelength, and measuring crude alkaloids content (mg/mL) of Meyer sedge by introducing the absorbance into strychnine standard curve. Calculating the concentration of the Meyer sedge crude alkaloid according to a formula, and calculating the extraction rate of the Meyer sedge total alkaloid according to the following formula:
c: the concentration of the alkaloid; v: volume of Meyer sedge alkaloid extract; n: dilution times; m: wula grass quality.
Example four Single factor experiments
(1) Influence of hydrochloric acid concentration on Meyer sedge total alkaloid content
The fixed extraction time is 2h, the liquid-material ratio is 20mL/g, and the extraction temperature is not changed at 80 ℃. Precisely weighing 5g of Meyer sedge segments, respectively placing into a 200 mL flask, extracting under the condition that the concentration of 5 groups of hydrochloric acid is respectively 0.06 mol/L, 0.12 mol/L, 0.18 mol/L, 0.24 mol/L and the other steps are the same as the above.
As is clear from the results in FIG. 3, the concentration of total alkaloids increases with the increase of the hydrochloric acid concentration, so the extraction rate of the total alkaloids from carex meyeriana increases proportionally, and reaches the maximum when the hydrochloric acid concentration reaches 0.18 mol/L, and begins to decrease when the hydrochloric acid concentration exceeds 0.18 mol/L.
(2) Influence of extraction temperature on extraction rate of Meyer sedge total alkaloids
The fixed extraction time is 2h, the liquid-material ratio is 20mL/g, and the hydrochloric acid concentration is 0.18 mol/L. Precisely weighing 5g of Meyer sedge segments, respectively placing into 200 mL flasks, extracting at 60 deg.C, 70 deg.C, 80 deg.C, 90 deg.C, and 100 deg.C at 5 groups of extraction temperatures, and performing the same steps as above.
From the results in FIG. 4, it is understood that the total alkaloid concentration C increases with the increase of the temperature, and therefore the wula sedge alkaloid extraction rate increases proportionally, and when the temperature reaches 80 ℃, the extraction rate reaches the maximum, and when the temperature exceeds 80 ℃, the extraction rate begins to decrease.
(3) Influence of feed liquid ratio on extraction rate of Meyer sedge total alkaloids
The extraction time is fixed for 2h, the extraction temperature is 80 ℃, and the hydrochloric acid concentration is unchanged at 0.18 mol/L. Weighing five parts of 5g Meyer sedge segments, respectively putting the five parts into 100 mL, 200 mL, 250 mL, 300 mL and 500 mL round-bottom flasks, respectively extracting according to the liquid-material ratio of 10, 20, 30, 40 and 50 mL/g, and carrying out other steps as above.
From the results in FIG. 5, it can be seen that the concentration of the total alkaloids in Wula grass increases continuously with the time of extraction, and when the liquid-to-material ratio reaches 1:30 g/mL, the concentration of the alkaloids increases greatly, and after 30 mL/g, the concentration of the total alkaloids increases slowly with the increase of the liquid-to-material ratio, and from the aspect of saving components, the optimal concentration of 30 mL/g is selected.
Therefore, the single-factor result is determined that the extraction rate of the wula sedge total alkaloids in each factor is the best under the conditions that the extraction temperature is 80 ℃, the hydrochloric acid concentration is 0.18 mol/L and the liquid-material ratio is 30 mL/g when the extraction time is 2 hours.
Example design of optimization experiment of five response surfaces
On the basis of the single-factor experiment, according to a Box-Behnken center combined experiment design principle and a single-factor experiment result, response surface statistical analysis software is adopted to carry out response surface experiment design by taking solvent concentration (A), extraction temperature (B) and liquid-material ratio (C) as influence variables, the content of Meyer sedge alkaloid is calculated by using weight coefficients obtained by hierarchical analysis as corresponding indexes, and 3-factor 3-level 17 groups of experiments are established, and are shown in tables 1 and 2.
TABLE 1 response surface test factor level design
TABLE 2 response surface analysis Experimental protocols and results
The extraction process of the Meyer sedge total alkaloid is calculated and analyzed by utilizing a statistical analysis software Design-Expert V, and the result of variance analysis is shown in a table 3 according to multiple regression fitting. Taking the solvent concentration (A), the extraction temperature (B) and the liquid-material ratio (C) as independent variables and the content of the Meyer sedge total alkaloids as response values, obtaining a quadratic regression model equation of the prediction value of the Meyer sedge total alkaloids content to A, B, C coding values:
Y=2.74+0.17A-0.21B+0.19C-0.37AB-8.3100E-003AC-0.74BC-0.81A2-0.61B2-0.99C2
the F value of the model was 17.48, meaning that the model was significant. Due to noise, the probability of such a large "model F value" is only 0.05%. "P>An F "value less than 0.05 indicates that the model term is significant. In this case, AB, BC, A2, B2, C2Are important model terms. When in usePValue of>At 0.1, the representation model term is not significant.
TABLE 3 regression model analysis of variance results
The influence of the solvent concentration, the extraction temperature and the liquid material ratio on the content of the wula sedge total alkaloids can be more clearly seen through the 3D response surface diagram and the 2D contour diagram of fig. 6. Experimental results show that the solvent concentration is within the range of 0.15-0.24 mol/L, the response curve is curved, the influence relationship on the index components of the carex meyeriana is visual, the curve is relatively curved within the extraction temperature range of 75-90 ℃, and the influence on the content of the carex meyeriana alkaloids is large. When the concentration of the solvent is 0.15-0.24 mol/L and the extraction temperature is 75-85 ℃, the image gradient of the two factors is larger, which shows that in the experiment, the two factors are combined with each other, and the influence on the experiment is more obvious; the relationship between the solvent concentration and the liquid-material ratio can be seen, the influence of the solvent concentration on the solvent concentration is obvious, and the liquid-material ratio tends to be gentle, so that the influence of the liquid-material ratio is small under the influence of the solvent concentration and the liquid-material ratio; the inclination between the two factors of the extraction temperature and the liquid-material ratio shows that when the temperature is 74-85 ℃, the liquid-material ratio is inclined and tends to be gentle, and the result shows that the liquid-material ratio plays an auxiliary role and the influence effect is not obvious.
As can be seen from the prediction model diagram of fig. 7, the experimental data are uniformly distributed in the linear range and close to the straight line, and there is no abnormal point, which shows that the linear relationship of the experiment is obvious. And secondly, analyzing the residual error and the equation predicted value, wherein the result shows that the distribution of each experimental point is uneven and irregular, so that the experiment is reasonable and reliable, has small error and is suitable for optimizing the extraction process of the wula sedge total alkaloids.
Example purification and separation of strychnine, an active ingredient in Linearia Nipponica
And (3) purification and separation: pretreatment: selecting a chromatographic column with proper length (2.6 cm multiplied by 40 cm), cleaning the chromatographic column by using distilled water, and performing ultrasonic treatment for 5 min to prevent the slow flow rate of the column blockage. And after the flow rate of the chromatographic column is ensured to be normal, naturally drying the chromatographic column for later use. Weighing 20 g of silica gel for column chromatography, mixing with a proper amount (5-10 mL) of dichloromethane, and uniformly stirring with a glass rod to discharge bubbles. Placing a proper amount of absorbent cotton at the bottom of the dried chromatographic column, adding a certain dichloromethane reagent to infiltrate the column, and quickly and completely pouring the mixture of the silica gel and the dichloromethane into the chromatographic column. Standing for 10 min to allow the silica gel to naturally settle, and opening the valve at the bottom of the column. Then, 50mL of dichloromethane was slowly poured into the column, and the column was repeatedly washed three times.
Loading: keeping the cylindrical surface flat, and reducing errors by adopting dry loading. And (3) ultrasonically dissolving 1g of a dried sample by using a small amount of dichloromethane, adding silica gel powder in the process of evaporating to dryness at low temperature until the silica gel is completely dried, weighing and loading.
And (3) purification: petroleum ether: dichloromethane: anhydrous ethanol: cyclohexane (50: 10:5: 10) elutes three column volumes, eluting the less polar impurities.
Separation: changing dichloromethane: anhydrous ethanol: cyclohexane (30: 10: 10) eluted three column volumes eluting purified alkaloid containing strychnine; finally, using methanol: dichloromethane (40: 10) elutes other highly polar alkaloids, and the elution sample is detected to be colorless by using an alkaloid precipitation reagent.
Example identification of strychnine as an active ingredient in Helianthus tuberosus
1. Chemical method identification of ammonium vanadate-concentrated sulfuric acid solution
1000 mL of the alkaloid/acid/water extract was concentrated to 150 mL, and the pH was adjusted with ammonia water to = 7. Concentrating the above solution to 60mL, adding 42mL anhydrous ethanol for extraction, centrifuging to obtain supernatant, and concentrating under reduced pressure to remove ethanol. And (3) obtaining a sample solution, purifying and separating the sample solution by an AB-8 column, eluting with distilled water, eluting with 70% ethanol, and eluting 5 parts of alkaloid sample. Fraction 1 was washed with water to give a volume of 50ml, and fractions 2, 3, 4, 5 were washed with alcohol to give 50ml each. And performing color identification reaction on each alkaloid sample and the strychnine control solution by using 1% ammonium vanadate-concentrated sulfuric acid solution. As can be seen from FIG. 8, 5 samples showed different colors, and strychnine reacted with 1% ammonium vanadate-concentrated sulfuric acid solution to turn purple first and then red, wherein the color change of part No. 3 was the same as that of strychnine control, indicating that strychnine, an indole alkaloid, may be contained in Ura.
2. Detecting the maximum absorption wavelength of ultraviolet
Taking the alkaloid sample and strychnine reference substance purified and separated by silica gel column chromatography, respectively metering to a 10 mL volumetric flask (with the concentration of 0.1 mg/mL) with methanol, performing full-wavelength scanning, and determining the maximum ultraviolet absorption wavelength. The results showed a distinct UV absorption peak at 254nm, consistent with the absorption peak of strychnine, as shown in FIGS. 9 and 10.
3. Thin layer chromatography identification method
Extracting 100 mL of alkaloid-acid water extract with dichloromethane at a ratio of 1:1 for 3 times. The extracts are combined and concentrated to about 5 mL by rotation for standby. A control solution was prepared and 1 mg of strychnine control was accurately weighed and added with dichloromethane to a 10 mL volumetric flask (0.1 mg/mL). Respectively pointing on the same silica gel G thin-layer plate, and preparing dichloromethane: cyclohexane: developing with ethanol (3: 1: 1) developer, taking out, pre-saturating in developing cylinder for 5 min, developing, and taking out. Spraying diluted potassium bismuth iodide solution for color development. It can be seen from FIG. 11 that the Ursoline alkaloid sample and the strychnine control showed spots of the same color and shape on the same position of the TLC plate.
4. High performance liquid chromatography identification method
Shim-pack VP C18(150 nm 4.6nm 5 um) and mobile phase of 0.4% phosphoric acid (prepared with triethylamine p)H3.0) -acetonitrile (85: 15), flow rate of 1.0mL/min, detection wavelength of 260nm, column temperature of 25 ℃, gradient elution, sample size of 20 μ L.
According to the above chromatographic conditions, sample solution purified and separated by silica gel chromatography is used as 20 μ L sample injection for sample solution and strychnine control, the sample and strychnine control have absorption peak at 3.47min, and the strychnine peak is well separated from adjacent peak, and the results are shown in FIGS. 12 and 13.
In conclusion, it was determined that the alkaloid extracted from wula sedge contains strychnine.
The invention adopts a response surface analysis method, selects extraction temperature, material-liquid ratio and solvent concentration as factors, inspects the optimal extraction condition of the Meyer sedge alkaloid, purifies and separates the alkaloid by silica gel column chromatography, identifies by a chemical method, and identifies the components of strychnine in the Meyer sedge alkaloid by an ultraviolet spectrophotometry, a thin layer chromatography and a high performance liquid chromatography.
Meyer sedge is taken as a raw material, acid water is used for reflux extraction of the total alkaloids, a response surface method is utilized to optimize an extraction process route of the total alkaloids, and strychnine is determined to be contained in the total alkaloids, so that certain technical support is provided for further development of the total alkaloids of the Meyer sedge. While the preferred embodiments of the present invention have been described, it is to be understood that the preferred embodiments described herein are for purposes of illustration and explanation only and are not intended to be limiting.
Claims (6)
1. A response surface method for optimizing the extraction process of strychnine-containing alkaloids in Carex meyeriana comprises the following steps:
(1) drying the carex meyeriana, cutting into sections after constant weight;
(2) extracting with acid water solution at a certain ratio for 2 hr, filtering with gauze, and concentrating under reduced pressure; adjusting the pH value of all the wula sedge concentrated solution to be within 8-10 range by using 40% sodium hydroxide solution, and then adding 1: extracting with 1 volume of dichloromethane for 2 times, combining the extract, washing with water until alkaloid salt and alkaline water dissolved in water are removed, collecting the extract, concentrating and drying to obtain crude alkaloid;
(3) carrying out color reaction on the obtained crude alkaloid;
(4) purifying and separating by silica gel column chromatography to obtain Meyer sedge total alkaloids, and identifying by ultraviolet, thin layer and high performance liquid chromatography to determine the alkaloid compound strychnine contained therein.
2. The extraction process according to claim 1, wherein the optimal extraction process optimized by the response surface analysis method is: the extraction time is 2h, the concentration of hydrochloric acid is 0.18 mol/L, the extraction temperature is 80 ℃, the liquid-material ratio is 30 mL/g, and the extraction rate of alkaloid in the carex meyeriana is 2.74 +/-1.0%.
3. The extraction process according to claim 1, wherein the crude alkaloid after extraction is subjected to a color reaction, wherein the color reaction phenomena are generation of orange precipitate of iodine-potassium iodide reagent, yellow-brown color development of concentrated sulfuric acid reagent, no color development of concentrated hydrochloric acid reagent and brown-green color development of ammonium molybdate-concentrated sulfuric acid solution (Frohde reagent), which indicates the presence of alkaloid in the carex.
4. The extraction process according to claim 1, wherein the alkaloid is purified and separated by silica gel column chromatography, and the mass ratio of silica gel to crude alkaloid sample is 20: 1; the method comprises the following steps of (1) mixing petroleum ether: dichloromethane: anhydrous ethanol: cyclohexane (50: 10:5: 10) is used as eluent to elute three column volumes, and impurities with smaller polarity are removed; purification exchange eluent was dichloromethane: anhydrous ethanol: cyclohexane (30: 10: 10) eluted three column volumes eluting purified alkaloid containing strychnine; finally, using methanol: dichloromethane (40: 10) elutes other highly polar alkaloids, and the elution sample is detected to be colorless by using an alkaloid precipitation reagent.
5. The extraction process according to claim 1, wherein the strychnine is identified by a chemical method of ammonium vanadate-concentrated sulfuric acid solution, the wula sedge alkaloid acid-acid aqueous extract is concentrated, the pH is adjusted to be neutral by ammonia water, the wula sedge alkaloid aqueous extract is extracted by absolute ethyl alcohol after being concentrated, the supernatant is obtained by centrifugation, and the sample solution is obtained by decompression and concentration; purifying and separating by using an AB-8 column, washing by using distilled water, then washing by using 70% ethanol, and eluting 5 flow parts of alkaloid samples; stream 1 was washed with water and streams 2, 3, 4, and 5 were washed with alcohol, with stream No. 3 changing color as the strychnine control.
6. The extraction process according to claim 1, wherein the purified alkaloid is dissolved in methanol, and the ultraviolet full wavelength scan has the same absorption peak at 254nm as the strychnine control; mixing with dichloromethane: cyclohexane: ethanol (3: 1: 1) is used as developing solvent to obtain spots which show the same color and shape with the strychnine reference substance on the same position of the TLC plate; using 0.4% phosphoric acid (pH 3.0 adjusted by triethylamine) -acetonitrile (85: 15) as mobile phase, detecting wavelength at 260nm, column temperature at 25 deg.C, and gradient eluting to obtain absorption peak identical to that of strychnine control at 3.47min, and concluding that the alkaloid sample contains strychnine.
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