CN113694128B

CN113694128B - Response surface method optimized extraction process of strychnine-containing alkaloids in carex meyeriana

Info

Publication number: CN113694128B
Application number: CN202110848038.4A
Authority: CN
Inventors: 周鸿立; 孙嘉祺; 赵爽
Original assignee: Jilin Institute of Chemical Technology
Current assignee: Jilin Yucao Workshop Technology Co.,Ltd.
Priority date: 2021-07-27
Filing date: 2021-07-27
Publication date: 2022-11-08
Anticipated expiration: 2041-07-27
Also published as: CN113694128A

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 0.18mol/L, the extraction temperature is 80 ℃, the liquid-material ratio is 30mL/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

Response surface method optimized extraction process of strychnine-containing alkaloids in carex meyeriana

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.

The prior extraction method of alkaloid in natural products reported in literature comprises water extraction method, acidic aqueous solution extraction method, alkaline aqueous solution extraction method, organic solvent extraction method, cold leaching method, and reflux extraction methodSoxhlet extraction, ultrasonic extraction, membrane extraction, and supercritical extraction. The extraction rate and chemical components 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. For example, zhao Shengnan, etc. by selecting three factors of hydrochloric acid concentration, feed liquid volume and extraction temperature, and using total alkaloid yield as response value, 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 2.7090 mg.g ^－1 . Ma Zhihong et al, by orthogonal experiments, examine the influence of extraction time, material-liquid ratio, pH value and temperature on the extraction rate of total alkaloids from Dicranopteris crenata, and obtain the optimal conditions of extracting total alkaloids from Dicranopteris crenata with acid water at 90 deg.C for 5h, pH value of 1.5 is the optimal conditions for extracting total alkaloids from Dicranopteris crenata, and obtain the extraction rate of isocorydine of 9.28%.

In order to obtain alkaloid with high purity, the alkaloid is purified and separated. Classical purification methods of alkaloids include organic solvent extraction, salting out, precipitation, crystallization, silica gel column chromatography, and macroporous adsorbent resin method. For example, pan Yang, etc. adopts solvent extraction, silica gel column chromatography, etc. to purify and separate alkaloid components of fermented semen Strychni product, dichloromethane-methanol elution is performed, gradient increase is performed according to 1% methanol, elution is performed sequentially, each gradient is 5L, thin layer plates are inspected under an ultraviolet lamp, improved bismuth potassium iodide reagent is developed, after 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, silica gel or Sephadex LH-20 column chromatography separation and recrystallization are repeatedly performed to obtain 10 alkaloid components.

The monoterpene indole alkaloids of strychnine are effective components of semen Strychni, and are also toxic components, and the strychnine and strychnine account for about 70% of 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. Nie Yanyan et al used UV spectrophotometry to measure different processing methods (frying, milk, baking, etc.),Licorice preparation method, sand scald method) to change strychnine and strychnine in semen Strychni; cao Ling et al qualitatively identifying semen Strychni in the prescription by TLC; huang Yuechun et al, by reverse phase high performance liquid chromatography, a column of methanol-water (65) (aqueous phase containing 0.029 mol. L. ^-1 Monopotassium phosphate and 0.037 md.L ^-1 Sodium 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 pH of all Meyer sedge concentrated solution to 8-10 with 40% sodium hydroxide solution, extracting with 1:1 volume of dichloromethane for 2 times, combining extract, washing with water until alkaloid salt and alkaline water dissolved in water are removed, collecting extract, 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 experimental design, wherein the optimal extraction process is as follows: under the condition of extraction time of 2h, the concentration of hydrochloric acid is 0.18mol/L, the extraction temperature is 80 ℃, the liquid-material ratio is 30mL/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 the alkaloid by silica gel column chromatography, loading by a dry method, wherein the mass ratio of silica gel to a sample is 20: dichloromethane: absolute ethanol: cyclohexane (50; purification exchange eluent was dichloromethane: anhydrous ethanol: cyclohexane (30; finally, methanol is used: dichloromethane (40.

(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 the raw materials in a ratio of dichloromethane: cyclohexane: ethanol (3; using 0.4% phosphoric acid (pH 3.0 adjusted by triethylamine) -acetonitrile (85) as a mobile phase, detecting the wavelength at 260nm, the column temperature at 25 ℃, and gradient elution to obtain an absorption peak which is the same as that of a strychnine control product at 3.47min, and obtain that the alkaloid sample contains 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 response surface prediction models of residual scatter (d), prediction normal distribution (e), and true normal distribution (f);

FIG. 8 is a diagram showing the result of identifying ammonium vanadate-concentrated sulfuric acid solution;

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 sections with an electronic balance, adding a certain proportion of solvent, placing in a 500 ml round-bottom flask, heating in 80 deg.C water bath under reflux for 2h, filtering with gauze, and concentrating under reduced pressure. Adjusting pH of the total Meyer sedge alkaloid concentrated solution to be within 8-10 by using 40% sodium hydroxide solution, extracting for 2 times by using 1:1 volume of dichloromethane, combining extract solutions, washing by using water until alkaloid salt and alkaline water dissolved in water are removed, collecting the extract solution, concentrating and drying.

Example two chemical reaction identification

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 enlarged iodine-potassium iodide reagent sample (1-a-1), 1-c is an enlarged concentrated sulfuric acid reagent group (1-a-3 and 1-a-4), and 1-d is an enlarged ammonium molybdate-concentrated sulfuric acid group (1-a-8 and 1-a-7).

(1) Iodine-potassium iodide reagent group: adding 1 to 2ml of Meyer sedge extracting solution and 1~2 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 ammonium molybdate is weighed and added into a beaker, and 100 mL concentrated sulfuric acid (98%) is added for dissolving. 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 showed a brownish green color 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 a strychnine standard substance 1.0 mg by using an analytical balance, adding a methanol solution to a constant volume of 10 mL, and shaking up to prepare a strychnine standard substance solution of 0.1 mg/mL. 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,R ² =0.9994, as in fig. 2. The result shows that the concentration and the absorbance of the strychnine are in a good linear relation within 0.04 to 0.012 mg/L.

Content determination of sample

A sample was taken and dissolved in 10 mL methanol by sonication. Transferring 1 mL sample solution with pipette, adding into test tube, adding 3 mL methanol, shaking, measuring absorbance A at 254nm wavelength, and bringing absorbance value into strychnine standard curve to determine crude alkaloid content (mg/mL) of Meyer sedge. 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 extraction time is 2h, the liquid-material ratio is 20mL/g, and the extraction temperature is 80 ℃. Precisely weighing 5g of Meyer sedge segments, respectively placing into a flask with 200 mL, and extracting under the conditions that the concentrations of 5 groups of hydrochloric acid are respectively 0.06, 0.12, 0.18, 0.24 and 0.30 mol/L, and the other steps are the same as the above steps.

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 0.18mol/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 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 a flask with 200 mL, and extracting at 60 deg.C, 70 deg.C, 80 deg.C, 90 deg.C and 100 deg.C at 5 groups of extraction temperatures, respectively, 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 2h, the extraction temperature is 80 ℃, and the hydrochloric acid concentration is 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-bottomed flasks, respectively extracting according to the liquid-material ratio of 10, 20, 30, 40 and 50 mL/g, and carrying out the other steps as above.

From the results in fig. 5, it can be seen that the concentration of the total alkaloids in wula sedge increases continuously with the extension of the extraction time, when the liquid-to-material ratio reaches 1 g/mL, the concentration of the alkaloids increases greatly, after 30mL/g, the concentration of the total alkaloids increases slowly and does not increase greatly with the increase of the liquid-to-material ratio, and from the perspective of saving components, the optimal value of 30mL/g is selected.

Therefore, the single factor result is determined that the extraction rate of the Meyer sedge total alkaloids is the best in each factor under the conditions that the extraction temperature is 80 ℃, the hydrochloric acid concentration is 0.18mol/L and the liquid-material ratio is 30mL/g when the extraction time is 2 h.

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 wula sedge total alkaloid content as a response value, obtaining a quadratic regression model equation of the prediction value of the wula sedge total alkaloid content to the A, B, C coding value:

Y=2.74+0.17A-0.21B+0.19C-0.37AB-8.3100E-003AC-0.74BC-0.81A ² -0.61B ² -0.99C ²

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, A ² , B ² , C ² Are 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 bent, the influence relationship on the index components of the carex, the curve is relatively bent within the range of 75-90 ℃, and the influence on the content of the carex 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 shows that the influence of the solvent concentration on the liquid-material ratio is obvious, and the liquid-material ratio tends to be smooth, 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 can be seen that when the temperature is between 74 and 85 ℃, the inclination is relatively inclined, the liquid-material ratio tends to be smooth, 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 silica gel for column chromatography, mixing with a proper amount (5 to 10 mL) of dichloromethane, and uniformly stirring with a glass rod to discharge air 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 dichloromethane was slowly poured into the column and the column was washed three times repeatedly.

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.

Separation: changing dichloromethane: anhydrous ethanol: cyclohexane (30; finally, using methanol: dichloromethane (40.

Example identification of strychnine as an active ingredient in Helianthus tuberosus

1. Chemical method identification of ammonium vanadate-concentrated sulfuric acid solution

The alkaloid acid water extract 1000 mL is concentrated to 150 mL, and the pH is adjusted with ammonia water =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. Color identification reaction was performed on each alkaloid sample and strychnine control solution with 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 10 mL volumetric flasks (with the concentration of 0.1 mg/mL) with methanol, performing full-wavelength scanning, and measuring the maximum ultraviolet absorption wavelength. The results show that there is a distinct uv absorption peak at 254nm, consistent with the strychnine absorption peak, as shown in figures 9, 10.

3. Thin layer chromatography identification method

The alkaloid acid water extract 100 mL is extracted with dichloromethane 1:1 for 3 times. Mixing extractive solutions, and rotary concentrating to about 5 mL. The control solution was prepared by accurately weighing the strychnine control, 1 mg, in dichloromethane to a 10 mL volumetric flask (0.1 mg/mL). Respectively pointing on the same silica gel G thin-layer plate, and configuring dichloromethane: cyclohexane: ethanol (3. 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 C ₁₈ (150nm 4.6nm 5um), mobile phase of 0.4% phosphoric acid (adjusted to pH3.0 with triethylamine) -acetonitrile (85).

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, the alkaloid extracted from the carex meyeriana is determined to contain 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

1. A response surface method for optimizing an extraction process of strychnine-containing alkaloids in carex meyeriana is characterized by comprising the following steps:

(1) Drying Carex meyeriana Kunth, cutting into segments;

(2) Extracting with acid water solution at a certain ratio for 2 hr, filtering with gauze, and concentrating under reduced pressure; regulating pH value of all Meyer sedge concentrated solution to be within 8-10 range by using 40% sodium hydroxide solution, extracting for 2 times by using 1:1 volume of dichloromethane, combining extract liquor, washing by using water until alkaloid salt and alkaline water dissolved in water are removed, collecting extract liquor, concentrating and drying to obtain crude alkaloid, and measuring alkaloid content by using strychnine as a standard curve;

(3) Selecting three factors of hydrochloric acid concentration, extraction temperature and material-liquid ratio to perform a single-factor experiment on the step (2), and determining the optimal levels of the hydrochloric acid concentration, the extraction temperature and the material-liquid ratio; obtaining an optimal extraction condition by taking a single-factor condition as an independent variable and taking the content of the total alkaloids as a response value;

(4) Performing response surface analysis on the multivariate quadratic equation established in the step (3), and taking the total alkaloid content as the maximum value to obtain the optimal extraction condition; in the step (3), the solvent concentration A, the extraction temperature B and the liquid-material ratio C are used as influence independent variables, the content of the Meyer sedge alkaloid is used as a response value, and the established multivariate quadratic equation is as follows: y =2.74+0.17A-0.21B +0.19C-0.37AB-8.3100E-003AC-0.74BC-0.81A ² -0.61B ² -0.99C ² (ii) a Wherein, in the step (3), the extraction time is 2h, the concentration of hydrochloric acid is 0.18mol/L, the extraction temperature is 80 ℃, the liquid-material ratio is 30mL/g, and the extraction rate of alkaloid in the carex meyeriana is 2.74 +/-1.0%; to the step(2) And purifying and separating the obtained crude alkaloid by silica gel column chromatography to obtain the Meyer sedge total alkaloid, purifying and separating the alkaloid by silica gel column chromatography, and loading the alkaloid by a dry method, wherein the mass ratio of silica gel to a sample is 20: dichloromethane: anhydrous ethanol: cyclohexane (50; purification exchange eluent was dichloromethane: absolute ethanol: cyclohexane (30; finally, using methanol: dichloromethane (40;

(5) And (3) checking the crude alkaloid obtained in the step (2) by using an alkaloid precipitation reagent to show a positive color reaction, purifying and separating by silica gel column chromatography to obtain the Meyer sedge total alkaloids, and identifying the Meyer sedge total alkaloids by ultraviolet, thin-layer and high performance liquid chromatography to determine that the Meyer sedge total alkaloids contain an alkaloid compound strychnine.