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

Abstract

Response surface methodology was used to determine the optimal extraction process of strychnine alkaloids from Ula grass by acid-water method and to identify them. Three factors, extraction temperature, material-to-liquid ratio and hydrochloric acid concentration were selected, and the extraction rate of total alkaloids from uran grass was the response value, and the Box-Benhnke experimental design was carried out. The optimal extraction process is as follows: extraction time is 2h, hydrochloric acid concentration is 0.18 mol/L, extraction temperature is 80℃, liquid-solid ratio is 30 mL/g, and the extraction rate of alkaloids can reach 2.74%. The alkaloids were purified and separated by silica gel column chromatography, identified by physicochemical properties, identified by UV spectrophotometry, identified by thin layer chromatography, and identified by high performance liquid chromatography to prove the presence of strychnine in urax alkaloids.

Description

A response surface methodology to optimize the extraction process of alkaloids containing strychnine from Ulagrass

technical field

The invention relates to an extraction method for optimizing alkaloids and a method for identifying strychnine in alkaloids.

Background technique

As an ancient plant, Ulagrass contains rich bioactive components and is a precious plant resource. Widely distributed in Northeast China, Sichuan, Inner Mongolia, Eastern Russia and other places, it has broad development prospects in the fields of medicine and health care. Ulagrass has a unique function of keeping warm, breathable, cold-proof, and antibacterial. At present, the research on Uralia ulata mainly focuses on the physical properties such as fiber, and there is little research on the chemical composition of its extract. Grass extracts often contain alkaloids, and most alkaloids have anti-cancer, anti-tumor, anti-inflammatory, anti-virus and anti-platelet aggregation, anti-arrhythmia and anti-hypertensive effects on cardiovascular diseases.

The literature reports that the current 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 method, supercritical extraction method. The extraction rate and chemical composition of alkaloids are affected by many factors. Orthogonal experiments or response surface analysis are often used to optimize the extraction process and determine the best extraction conditions. For example, Zhao Shengnan and others selected the three factors of hydrochloric acid concentration, feed liquid volume and extraction temperature, took the yield of total alkaloids as the response value, and carried out response surface experiment design, and concluded that the optimal extraction condition of celandine alkaloids is the concentration of hydrochloric acid 0.42%, the volume of feed liquid is 97.59ml, and the extraction temperature is 50.12℃. The alkaloid obtained under these conditions is 2.7090mg·g ^-1 . Ma Zhihong et al. used orthogonal experiments to select the extraction time, solid-liquid ratio, pH value and temperature to investigate the influence on the extraction rate of the total alkaloids of the red grass. ℃, extraction time 5h, solid-liquid ratio 1:40, pH value 1.5 are the best conditions for extracting the total alkaloids of C. chinensis, and the extraction rate of isochoryline is 9.28%.

In order to obtain alkaloids with higher purity, they are purified and separated. The classic purification methods of alkaloids include organic solvent extraction, salting out, precipitation, crystallization, silica gel column chromatography and macroporous adsorption resin method, etc. People such as Pan Yang use methods such as solvent extraction, silica gel column chromatography to purify and separate the alkaloid components of the fermented product of Nuxebra nuxe. Gradient 5L, inspect the TLC plate under ultraviolet light, improve the color development of bismuth potassium iodide reagent, combine the same fractions, divide them into 3%~4% methanol, 6%~7% methanol, 4%~5% methanol and 7% ~15% methanol for 4 elution sites, and then repeated silica gel or SephadexLH-20 column chromatography separation and recrystallization to obtain 10 alkaloid components.

Strychnine belongs to the class of monoterpene indole alkaloids, which is the active and toxic component of the traditional Chinese medicine Strychnine, accounting for about 70% of the alkaloids together with strychnine. Historical research results have shown that strychnine is a medium-strong base that is stable in an alkaline environment. The strychnine is extracted with an acid solution, and then the acid solution is subjected to an alkalized extraction method. Nie Yanyan and others used ultraviolet spectrophotometry to measure the changes of strychnine and strychnine in different processing methods (frying method, milk method, baking method, licorice method, sand scalding method); Cao Ling et al. used TLC method to qualitatively identify Nuxebra chinensis in the prescription; Huang Yuechun et al. used reversed-phase high-performance liquid chromatography, and used methanol-water (65:35) (the water phase contained 0.029mol L ^-1 diphosphate Potassium hydrogen and 0.037md L ^-1 sodium dodecylsulfonate) as the mobile phase, the content of strychnine in Tongbiling Tablets was measured at a detection wavelength of 254nm, and the test product was obtained when the retention time was about 12min Both the strychnine absorption peak and the reference substance solution appeared, and the average content of strychnine in each Tongbiling sample was calculated to be 0.22 mg by measuring the peak area.

So far, there are few studies on the alkaloids in Uralia sativa, and there are no related reports on the optimization of its extraction process and its structure. This patent application adopts the response surface design method to investigate the alkaloids in Uralia sativa for the first time with the three factors of extraction temperature, solid-liquid ratio and hydrochloric acid concentration, and then purifies and separates them through silica gel column chromatography, and conducts chemical, spectroscopic and chromatographic identification. , to determine the alkaloids containing strychnine.

Contents of the invention

The purpose of the present invention is to provide a response surface method to optimize the extraction method of total alkaloids in Ulagrass and the identification of strychnine.

In order to solve the above problems, the present invention provides the following technical solutions.

(1) Ulagrass is dried to a constant weight and then cut into sections.

(2) Extract with a certain proportion of acid aqueous solution for 2 hours, filter with gauze, and concentrate under reduced pressure. Use 40% sodium hydroxide solution to adjust the pH value in the range of 8 to 10, and then extract twice with 1:1 volume of dichloromethane. The combined extracts are washed with water until the water-soluble alkaloid salt and alkaline water, and the extract was collected, concentrated and dried to obtain crude alkaloid. Select the three factors of extraction temperature, liquid-to-solid ratio, and hydrochloric acid concentration, and use the response value of the total alkaloid extraction rate of A. /L, the extraction temperature was 80°C, the liquid-to-solid ratio was 30 mL/g, and the extraction rate of total alkaloids was 2.74±1.0%.

(3) Carrying out color reaction on the obtained crude alkaloid. The phenomenon of color reaction is that the iodine-potassium iodide reagent produces orange precipitate, the concentrated sulfuric acid reagent shows yellow-brown color, the concentrated hydrochloric acid reagent does not show color, and the ammonium molybdate-concentrated sulfuric acid solution (Frohde reagent) shows brown-green, indicating that there is biological alkali.

(4) After purification and separation by silica gel column chromatography, the total alkaloids of Uralia sativa were obtained. The alkaloids were purified and separated by silica gel column chromatography, and the sample was loaded by dry method. The mass ratio of silica gel to sample was 20:1, and petroleum ether: dichloromethane: absolute ethanol: cyclohexane (50:10:5:10) Elute three column volumes for the eluent to remove less polar impurities; purify and replace the eluent with dichloromethane: absolute ethanol: cyclohexane (30:10:10) to elute three column volumes, The purified alkaloids containing strychnine were eluted; finally, other alkaloids with high polarity were eluted with methanol:dichloromethane (40:10), and the eluted samples were detected to be colorless with an alkaloid precipitation reagent.

(5) Identification of strychnine by chemical method: after concentration and ethanol extraction of the alkaloid acid water extract, the sample was purified and separated by AB-8 column, first eluted with distilled water, and then eluted with 70% ethanol for a total of Five alkaloid samples were eluted. Fraction 1 was washed with water to obtain a volume of 50 ml, and fractions 2, 3, 4, and 5 were washed with alcohol to obtain a volume of 50 ml each. Use 1% ammonium vanadate-concentrated sulfuric acid solution to carry out color discrimination reaction on each alkaloid sample and strychnine control solution. Fraction No. 3 reacted with ammonium vanadate-concentrated sulfuric acid solution, and then turned purple first, and then turned red, and the color change was the same as that of the strychnine reference substance.

(6) Ultraviolet, thin-layer and high-performance liquid chromatography identification, it is determined that it contains the alkaloid compound strychnine. The purified alkaloids were dissolved in methanol, and the ultraviolet full-wavelength scanning had the same absorption peak as the strychnine reference substance at 254 nm; using dichloromethane:cyclohexane:ethanol (3:1:1) as the developing solvent to obtain Spots with the same color and shape as the strychnine reference substance were on the same position on the thin-layer chromatographic plate; 0.4% phosphoric acid (adjusted with triethylamine to pH 3.0)-acetonitrile (85:15) was used as the mobile phase, The detection wavelength is 260nm, the column temperature is 25°C, and the gradient elution results in the same absorption peak as the strychnine reference substance at 3.47min, indicating that the alkaloid sample contains strychnine.

Description of drawings

Figure 1 is a diagram of the identification results of the color reaction;

Fig. 2 is a standard curve figure of strychnine standard substance concentration and absorbance;

Fig. 3 is the univariate figure of different hydrochloric acid concentrations to the total alkaloid concentration of Uralia sativa;

Fig. 4 is a single factor diagram of different extraction temperatures on the total alkaloid concentration of Uralia sativa;

Fig. 5 is the univariate diagram of different solid-liquid ratios to the total alkaloid concentration of Uralia sativa;

Figure 6 is a response surface diagram of the influence of solvent concentration (a), extraction temperature (b), and liquid-solid ratio (c) on the total alkaloid content;

Fig. 7 is the response surface prediction model diagram of residual scatter point (d), predicted normal distribution (e), actual normal distribution (f);

Fig. 8 is the identification result figure of ammonium vanadate-concentrated sulfuric acid solution;

Fig. 9 is an ultraviolet full-wavelength scanning figure of strychnine separated from alkaloids of Uralia sativa;

Fig. 10 is strychnine reference substance ultraviolet full-wavelength scanning figure;

Figure 11 is a TLC identification diagram (1 is an alkaloid sample, and 2 is a strychnine reference substance);

Fig. 12 is the high-performance liquid chromatogram of Herba Ursa extract;

Fig. 13 is the high performance liquid chromatogram of strychnine reference substance.

Detailed ways

The present invention will be described in detail below in combination with diagrams and specific implementation operations.

Extraction of active ingredient alkaloids in embodiment 1 Ulagrass

Dried Uralia sativa, weighed a certain amount of Urassica ulata with an electronic balance, added a certain proportion of solvent, put it in a 500ml round bottom flask, heated it in a constant temperature water bath at 80 ℃ for 2 h, filtered it with gauze, and concentrated under reduced pressure. Use 40% sodium hydroxide solution to adjust the pH value in the range of 8 to 10, and then extract twice with 1:1 volume of dichloromethane, and wash the combined extracts with water until the dissolved The alkaloid salt of water and alkaline water, the extract is collected and concentrated to dryness.

Embodiment two chemical reaction identification

Identification - color reaction:

Figure 1-a, from left to right, is the iodine-potassium iodide reagent group samples and their blank controls (1-a-1 and 1-a-2), the 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 a clearer and more intuitive display, 1-b is the enlarged version of the iodine-potassium iodide reagent sample (1-a-1), 1-c is the enlarged version of the concentrated sulfuric acid reagent group (1-a-3 and 1-a-4), 1-d is an enlarged version of the ammonium molybdate-concentrated sulfuric acid group (1-a-8 and 1-a-7).

(1) Iodine-potassium iodide reagent group: Add 1~2 ml of Uralia ulata extract, 1~2 drops of iodine-potassium iodide alkaloid precipitation reagent into the test tube, and shake fully. It can be seen from Figure 1-a-1, 1-a-2 and 1-b that the iodine-potassium iodide reagent has an orange precipitate.

(2) Concentrated sulfuric acid reagent group: colchicine is yellow, codeine is light blue, berberine is green, atropine, cocaine, morphine and strychnine are not. It can be seen from Figure 1-a-3, 1-a-4 and 1-c that the concentrated sulfuric acid reagent is yellowish brown.

(3) Concentrated hydrochloric acid reagent group: Veratrine showed red color, but most of the other alkaloids showed no color. It can be seen from Figure 1-a-5 and 1-a-6 that the concentrated hydrochloric acid reagent does not develop color.

(4) Ammonium molybdate-concentrated sulfuric acid solution reagent group: (Frohde reagent) is a concentrated sulfuric acid solution of 1% ammonium molybdate, weigh 1 g of ammonium molybdate into a beaker, add 100 mL of concentrated sulfuric acid (98%) to dissolve Can. 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. It can be seen from Figures 1-a-7, 1-a-8 and 1-d that the ammonium molybdate-concentrated sulfuric acid solution is brownish green and may contain berberine.

The above 4 color reactions show that there are alkaloids in Uralia sativa.

The mensuration of the alkaloid content of embodiment three Ulagrass

Drawing of Strychnine Standard Curve

Accurately weigh 1.0 mg of standard strychnine with an analytical balance, add methanol solution to make up to 10 ml, shake well, and prepare a 0.1 mg/mL strychnine standard solution. Diluted with methanol to 0.004, 0.006, 0.008, 0.012 mg/mL serial concentration gradient strychnine standard solution. The absorbance A of strychnine was measured at a wavelength of 254 nm. Draw a standard curve with strychnine concentration (mg/mL) as the abscissa and absorbance value (A) as the ordinate. Perform linear regression to obtain the regression equation: Y=73.7X-0.0754, R ² =0.9994, as shown in Figure 2. The results showed that strychnine had a good linear relationship between its concentration and absorbance in the range of 0.04-0.012 mg/L.

Assay of samples

Take a sample and add 10 mL of methanol for ultrasonic dissolution. Use a pipette to pipette 1 mL of the sample solution into a test tube, add 3 mL of methanol, shake well, measure the absorbance A at a wavelength of 254 nm, and bring the absorbance value into the strychnine standard curve to determine the crude alkaloid Content (mg/mL). According to the formula to calculate the crude alkaloid concentration of Uralia sativa, the extraction rate of the total alkaloids of Urla sativa is calculated according to the following formula:

C: concentration of alkaloids; V: volume of alkaloid extract from A. ulata; n: dilution multiple; M: mass of A. ulaxa.

Embodiment four single factor experiment

(1) The effect of hydrochloric acid concentration on the total alkaloid content of Uralia sativa

The extraction time was fixed at 2 h, the liquid-to-solid ratio was 20 mL/g, and the extraction temperature was 80 °C. Precisely weigh 5g of Ulagrass segments into 200 mL flasks, select 5 groups of hydrochloric acid concentrations of 0.06, 0.12, 0.18, 0.24, and 0.30 mol/L for extraction, and the other steps are the same as above.

From the results in Figure 3, it can be seen that with the increase of the concentration of hydrochloric acid, the concentration of total alkaloids increases, so the extraction rate of total alkaloids of A. 0.18mol/L, the extraction rate began to decline.

(2) The effect of extraction temperature on the extraction rate of total alkaloids from Uralia sativa

The extraction time was fixed for 2 h, the ratio of liquid to solid was 20 mL/g, and the concentration of hydrochloric acid was 0.18 mol/L. Precisely weigh 5g of Ulagrass segments and put them into 200 mL flasks respectively. Select 5 sets of extraction temperatures, 60, 70, 80, 90, and 100 °C, and the other steps are the same as above.

From the results in Figure 4, it can be seen that with the increase of temperature, the concentration of total alkaloids C increases, so the extraction rate of alkaloids in Uralia sativa increases year-on-year. When the temperature reaches 80 °C, the extraction rate reaches the maximum, and when the temperature exceeds 80 °C, the extraction rate begins to decline. .

(3) The effect of solid-liquid ratio on the extraction rate of total alkaloids

The extraction time was fixed at 2 h, the extraction temperature was 80 °C, and the concentration of hydrochloric acid was 0.18 mol/L. Weigh 5 parts of 5g Uralia sativa, put them into 100 mL, 200 mL, 250 mL, 300 mL, 500 mL round bottom flasks respectively, and the corresponding liquid-to-solid ratios are 10, 20, 30, 40, 50 mL/g respectively Carry out extraction, other steps are the same as above.

It can be seen from the results in Figure 5 that with the prolongation of the extraction time, the concentration of total alkaloids in Uralia sativa increases continuously. When the liquid-to-solid ratio reaches 1:30 g/mL, the alkaloid concentration increases greatly. The concentration of total alkaloids increased slowly and did not increase much with the increase of ratio. From the perspective of saving ingredients, the best choice was 30 mL/g.

Therefore, the single-factor results determined that the extraction rate of total alkaloids in Uralia sativa was the highest in each factor under the conditions of extraction temperature of 80°C, hydrochloric acid concentration of 0.18 mol/L, and liquid-to-solid ratio of 30 mL/g when the extraction time was 2 h. good.

Embodiment 5 response surface optimization experiment design

On the basis of the above single factor experiments, according to the Box-Behnken central combination experiment design principle and the results of single factor experiments, the response surface statistical analysis software was used to set the solvent concentration (A), extraction temperature (B), and liquid-solid ratio (C) as Response surface experiment design was carried out on the influencing variables, and the weight coefficient obtained by AHP was used to calculate the alkaloid content of Uralia sativa as the corresponding index. A total of 17 experiments with 3 factors and 3 levels were established, as shown in Table 1 and Table 2.

Table 1 Response surface test factor level design

Table 2 Response surface analysis experiment scheme and results

The statistical analysis software Design-Expert V was used to calculate and analyze the extraction process of the total alkaloids of A. ulata. According to the multiple regression fitting, the variance analysis results are shown in Table 3. Taking solvent concentration (A), extraction temperature (B), liquid-to-solid ratio (C) as independent variables, and total alkaloid content of Uralia ulata as the response value, the predicted value of the total alkaloid content of Uralia ulata in relation to A, B, and C The quadratic regression model equation for coded values:

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 is 17.48, which means the model is significant. There is only a 0.05% chance of having such a large "model F-number" due to noise. A value of " P >F" 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 the P value > 0.1, it means that the model item is not significant.

Table 3 Regression Model Analysis of Variance Results

Through the 3D three-dimensional response surface diagram and 2D contour diagram in Figure 6, the influence of solvent concentration, extraction temperature and liquid-to-solid ratio on the total alkaloid content of Uralia sativa can be seen more clearly. The experimental results show that when the solvent concentration is in the range of 0.15 mol/L~0.24 mol/L, the response surface is curved, and the influence relationship on the index components of Uralia sativa is more intuitive. The alkali content has a greater influence. When the solvent concentration is 0.15 mol/L~0.24 mol/L, and the extraction temperature is 75~-85 ℃, the image slope of the two factors is relatively large, indicating that in the experiment, the combination of the two factors has a greater impact on the experiment. It can be seen that the solvent concentration and the relationship between the two factors of the liquid-to-solid ratio can be seen that the solvent concentration has a more significant impact on it, and the liquid-to-solid ratio tends to be gentle. It can be seen that under the influence of the solvent concentration and the liquid-to-solid ratio factors, The influence of liquid-solid ratio is small; and the gradient between the two factors of extraction temperature and liquid-solid ratio can be seen that when the temperature is between 74 and 85 ℃, it is relatively inclined, and the liquid-solid ratio tends to be flat. This result shows that the liquid-solid ratio Compared with the auxiliary effect, the effect is not significant.

It can be seen from the prediction model diagram in Figure 7 that the experimental data are evenly distributed within the linear range and close to the straight line, and there are no abnormal points, indicating that the linear relationship of the experiment is obvious. Secondly, by analyzing the residual and the predicted value of the equation, the results show that the distribution of each experimental point is uneven and irregular, which shows that the experiment is reasonable and reliable, with small errors, and is suitable for the optimization of the extraction process of the total alkaloids of Uralia sativa .

Purification and separation of active ingredient strychnine in embodiment six Ulagrass

Purification and separation: Pretreatment: Select a chromatographic column of appropriate length (2.6cm×40cm), wash it with distilled water, and ultrasonicate for 5 minutes to prevent the column from clogging and the flow rate is slow. After ensuring that the flow rate of the chromatographic column is normal, let it dry naturally for later use. Weigh 20 g of silica gel for column chromatography, mix with an appropriate amount (5-10 mL) of dichloromethane, and stir with a glass rod to evenly expel air bubbles. Place an appropriate amount of absorbent cotton at the bottom of the dry chromatography column, add some dichloromethane reagent to soak the column, and quickly pour the mixture of silica gel and dichloromethane into the chromatography column completely. After standing for 10 min to allow the silica gel to settle naturally, open the valve at the bottom of the column. Then slowly pour 50 mL of dichloromethane into the column, and wash it three times repeatedly.

Sample loading: keep the cylindrical surface flat, and use dry method to load samples to reduce errors. Take 1 g of the dried sample and dissolve it ultrasonically with a small amount of dichloromethane, then add silica gel powder during the process of evaporating to dryness at low temperature until it is completely dry, weigh it, and load the sample.

Purification: Petroleum ether: dichloromethane: absolute ethanol: cyclohexane (50:10:5:10) for three column volumes to elute less polar impurities.

Separation: Change dichloromethane: absolute ethanol: cyclohexane (30:10:10) to elute three column volumes to elute the purified alkaloid containing strychnine; finally use methanol: dichloromethane (40: 10) Elute other alkaloids with high polarity, and detect with alkaloid precipitation reagent until the eluted sample is colorless.

The identification of active ingredient strychnine in embodiment seven Ulagrass

1. Identification by chemical method: ammonium vanadate-concentrated sulfuric acid solution

Concentrate 1000 mL of alkaloid acid water extract to 150 mL, and adjust its pH to 7 with ammonia water. Continue to concentrate the above solution to 60 mL, add 42 mL of absolute ethanol for extraction, centrifuge to obtain the supernatant, concentrate under reduced pressure and evaporate the ethanol. The sample solution was obtained, and the sample solution was purified and separated through an AB-8 column, first eluted with distilled water, and then eluted with 70% ethanol, and a total of 5 alkaloid samples were eluted. Fraction 1 was washed with water to obtain a volume of 50 ml, and fractions 2, 3, 4, and 5 were washed with alcohol to obtain a volume of 50 ml each. Use 1% ammonium vanadate-concentrated sulfuric acid solution to carry out color discrimination reaction on each alkaloid sample and strychnine control solution. As can be seen from Figure 8, the five samples show different colors, and strychnine reacts with 1% ammonium vanadate-concentrated sulfuric acid solution to appear purple first, and then turn red, and the color change that occurs in No. 3 is the same as that of ± Strychnine is the same as the reference substance, indicating that strychnine may contain indole alkaloids in Uralia pratense.

2. Detection of UV maximum absorption wavelength

Take the alkaloid sample and strychnine reference substance purified and separated by silica gel column chromatography, and dilute to 10mL volumetric flask with methanol (concentration is 0.1 mg/mL), and perform full-wavelength scanning to determine the maximum ultraviolet absorption wavelength. The results show that there is an obvious ultraviolet absorption peak at 254 nm, which is consistent with the absorption peak of strychnine, as shown in Figures 9 and 10.

3. TLC identification method

Take 100 mL of alkaloid-acid water extract and extract 3 times with dichloromethane 1:1. The extracts were combined, concentrated by rotation to about 5 mL, and set aside. To prepare the reference substance solution, accurately weigh 1 mg of strychnine reference substance and add dichloromethane to a 10 mL volumetric flask (0.1 mg/mL). Spot on the same silica gel G thin-layer plate respectively, configure dichloromethane:cyclohexane:ethanol (3:1:1) developing agent, develop, take out the developing cylinder and pre-saturate for 5 min, develop and take out. Spray dilute potassium bismuth iodide test solution to develop color. It can be seen from Fig. 11 that the alkaloid sample and the strychnine reference substance are on the same position of the thin-layer chromatographic plate, showing spots of the same color and shape.

4. High performance liquid chromatography identification method

Shim-pack VP C ₁₈ (150nm*4.6nm*5um), the mobile phase is 0.4% phosphoric acid (adjust pH3.0 with triethylamine)-acetonitrile (85:15), the flow rate is 1.0mL/min, and the detection wavelength is 260nm , the column temperature was 25°C, gradient elution was performed, and the injection volume was 20 μL.

According to the above-mentioned chromatographic conditions, get the sample liquid after silica gel chromatography purification and separation respectively as need testing solution and 20 μ L sample injection of strychnine reference substance, sample and strychnine reference substance have absorption peak at 3.47min place, strychnine peak It is well separated from adjacent peaks, and the results are shown in Figures 12 and 13.

In summary, it is determined that the alkaloids extracted from Uralia sativa contain strychnine.

The present invention adopts the response surface analysis method, selects extraction temperature, material-liquid ratio and solvent concentration as factors, investigates the optimal extraction conditions of the alkaloids of A. , thin-layer chromatography, and high-performance liquid chromatography identified the components of strychnine in the alkaloids of Uralia sativa.

The total alkaloids were extracted by acid water reflux using Uralia sativa as raw material, and the extraction process was optimized by response surface methodology, and it was confirmed that strychnine was contained in it, which provided certain technical support for the further development of the total alkaloids . The preferred embodiments of the present invention are described, and it should be understood that the preferred embodiments described here are only used to illustrate and explain the present invention, and are not intended to limit the present invention.

Claims (1)

1. a response surface method optimizes the extraction process of the alkaloid that contains strychnine in Herba Uraldae, it is characterized in that comprising the steps: (1) Ulagrass is dried to a constant weight and then cut into sections; (2) Extract with a certain proportion of acid aqueous solution for 2 hours, filter with gauze, and concentrate under reduced pressure; use 40% sodium hydroxide solution to adjust the pH value within the range of 8 to 10, and then use 1:1 The volume of methylene chloride is extracted twice, the combined extracts are washed with water until the alkaloid salts and alkaline water soluble in water are removed, the extracts are collected, concentrated and dried to obtain crude alkaloids, and the alkaloid content is determined using strychnine as a standard curve; (3) Select the three factors of hydrochloric acid concentration, extraction temperature, and solid-liquid ratio to conduct a single-factor experiment on step (2) to determine the optimal level of hydrochloric acid concentration, extraction temperature, and solid-liquid ratio; The total alkaloid content is the response value, and the optimal extraction conditions are obtained; (4) Perform response surface analysis on the multivariate quadratic equation established in step (3), take the total alkaloid content as the maximum value, and obtain the optimal extraction conditions; wherein, in step (3), the solvent concentration A, Extraction temperature B and liquid-to-material ratio C are the independent variables, and the alkaloid content of Uralia sativa is the response value. The multivariate quadratic equation established is: 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 ; wherein, the extraction time in step (3) is 2 hours, the concentration of hydrochloric acid is 0.18mol/L, the extraction temperature is 80°C, and the liquid-to-solid ratio is 30mL/g , the extraction rate of alkaloids in Uralia sativa was 2.74±1.0%; the crude alkaloids obtained in step (2) were purified and separated by silica gel column chromatography to obtain the total alkaloids of Urami sativa, and the alkaloids were purified and separated by silica gel column chromatography , using the dry method to load the sample, the mass ratio of silica gel to sample is 20:1, and three columns are eluted with petroleum ether: dichloromethane: absolute ethanol: cyclohexane (50:10:5:10) as the eluent Volume, except for less polar impurities; Purify and replace the eluent with dichloromethane: absolute ethanol: cyclohexane (30:10:10) to elute three column volumes, and elute the purification containing strychnine Alkaloids; finally use methanol: dichloromethane (40:10) to elute other alkaloids with high polarity, and detect with alkaloid precipitation reagent until the eluted sample is colorless; (5) The crude alkaloids obtained in step (2) showed a positive color reaction with an alkaloid precipitation reagent. After purification and separation by silica gel column chromatography, the total alkaloids of Uralia sativa were obtained, which were subjected to ultraviolet, thin-layer and high-efficiency Liquid chromatographic identification determined that it contained the alkaloid compound strychnine.

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