CN114533771B - Extraction, separation, purification and detection method of Alangium chinense total alkaloids - Google Patents

Abstract

The invention relates to an extraction, separation, purification and detection method of Alangium total alkaloids, which adopts D101 type macroporous adsorption resin to separate and purify the Alangium total alkaloids, and the purity of the total alkaloids in the obtained Alangium extractum is improved from 21.61% to 63.03%; measuring total alkaloids in Alangium platanifolium with acid dye colorimetry, with precision RSD of 0.34%; the repetitive RSD is 1.40%; the sample recovery rate is 102.72 percent, the RSD is 2.66 percent, the method can truly, effectively and accurately determine the content of the veratrine poisoned by the alangium chinense harms, and the safety and the effectiveness of the medication are ensured.

Description

Extraction, separation, purification and detection method of Alangium chinense total alkaloids

Technical Field

The invention relates to the field of medicine invention, in particular to an extraction, separation, purification and detection method of alangium platanifolium total alkaloids.

Background

Alangium chinense (Alangium chinense), another name: huaguamu, white dragon beard, illicium verum and orange wood. Deciduous small trees or shrubs with a height of 4-5 m. The leaves are intergrown, have different shapes and are mainly distributed in wide areas in the south China; east and southeast asia. It is also common in mountain areas, bushes and miscellaneous forests, and around villages and roads. Warm in nature and pungent and bitter in flavor, and has the effects of dispelling pathogenic wind, removing dampness, relaxing muscles and tendons, activating collaterals, dissipating blood stasis and relieving pain.

The Chinese alangium has the functions of clearing away heat and toxic material, promoting blood circulation and dissipating blood stasis. The root and bark have the best efficacy, the root is white dragon beard, and the stem is white dragon strip. Can dispel wind and remove dampness, relax muscles and tendons, activate collaterals, remove blood stasis and relieve pain, and can be used for treating rheumatalgia, limbs anesthesia, and traumatic injury; the leaf can be used for treating traumatic fracture and hemorrhage due to external knife injury; the flower is used for treating headache and fullness and distention of chest and abdomen.

The alkaloids component of Alangium platanifolium has pharmacological activities of relieving pain, diminishing inflammation, resisting rheumatism, relaxing muscle, and treating central nerve and cardiovascular system, wherein the main pharmacological active component is Alangine.

At present, alkaloids in alangium platanifolium are mostly determined by HPLC:

1. HPLC (high performance liquid chromatography) is used for measuring the content of the alangium chinense harms in the alangium chinense, and the huyuxia and the yuzu honor and baili are obtained. Establishing a method for measuring the content of the alantoine in the prepared alangium platanifolium. The method comprises the steps of adopting Alltech Apollo C18 (4.6 mm multiplied by 250mm,5 mu m) as a chromatographic column, taking acetonitrile-buffer solution (0.2 g sodium heptane sulfonate and 3.5g potassium dihydrogen phosphate with constant volume of 1 000mL) (9: 91) as a mobile phase, setting the flow rate at 1.0 mL/min-1, detecting the wavelength at 259nm and keeping the column temperature at 30 ℃. As a result, the linear relationship between the content of the octagon seal gum base and the content of the star anise base was good at 0.024 95-0.499 g.L-1 (r =0.9999, n = 5), the average recovery rate was 98.73% (n = 6), and the RSD was 0.915%.

2. The content of the veratrine poisoned by the Guizhou nationality medicinal material Alangium chinense is determined by high performance liquid chromatography, and the Liu Yi, zhang Liyan, zhang Yongping Ying, wang Ying and Xuxiting are determined; guiyang college of traditional Chinese medicine. Discloses a method for measuring the content of veratrine poisoned by different producing areas and different medicinal parts (fibrous roots and branch roots) of Guizhou nationality medicinal material Alangium chinense. The method adopts chromatographic column SB-C18 (150 mm multiplied by 4.6mm,5 μm), uses methanol-0.1% triethylamine (8: 92, pH3.4 adjusted by phosphoric acid) as mobile phase, the flow rate is 1.0 mL.min-1; the detection wavelength is 259nm, and the column temperature is 25 ℃. The result shows that the taxinine is in good linear relation in the range of 0.086.500 mug, and r = 0.999; the average recovery rate was 98.22%, the RSD was 2.08% (n = 6), the content of veratrine poisoned by fibrous root of Alangium chinense of different origins was higher than that of taproot, 0.122% of fibrous root was 0.234%, and the percentage of taproot was 0.028 5% to 0.137% of each.

3. Analysis of adverse reaction reasons of Fengshiding capsules, namely Liu' an, piecewise yeabin, zhanyuan, qiqian and Zhang Wang Cheng; qinghai university affiliated hospital. Establishing a content determination method for alangium chinense medicinal material and chenopodium album poisoning of Fengshiding capsules, and simultaneously analyzing and comparing different medicine application parts of the alangium chinense (Lour) Harms medicinal material and the content change of the processed chenopodium album poisoning. The method comprises respectively measuring the content of taxine in fibrous root, branch root and main root of Alangium chinense DC and processed rhizoma et radix Dysosmatis by High Performance Liquid Chromatography (HPLC), simultaneously measuring the content of taxine in FENGSHIDING Capsule, using C18 and phosphate buffer-methanol (76: 24) as mobile phase, and detecting wavelength at 260nm. The result shows that the cause of the adverse reaction of the Fengshiding capsule is related to the use part and the content of the Chinese alangium medicinal material in the prescription, the distribution of the content of the chenopodine at different parts is fibrous root, branch root and main root respectively, the content of the chenopodine after water boiling is reduced by about 30 percent, the content of the chenopodine poisoning in the Fengshiding capsule exceeds more than 0.45mg per g, and the risk of the adverse reaction is increased.

4. High performance liquid chromatography is used for measuring the content of toxic veratrine in Fengding capsules, such as stone axe, yangshai and Likatong; institute of medicinal plants of Chinese academy of medical sciences. Establishing a method for measuring the content of toxic veratrine in Fengshiding capsules. The method comprises the steps of using a chromatographic column of Kromasil C18 (4.6 mm multiplied by 250mm,5 mu m), using a methanol-buffer solution (21: 79) as a mobile phase (buffer solution: 0.2g of sodium heptanesulfonate, 2.0g of potassium dihydrogen phosphate and 0.3mL of phosphoric acid for constant volume of 1000 mL), and using a detection wavelength of 260nm. The result shows that the toxic veratrine has a good linear relation with the peak area thereof within the range of 0.124-0.620 mu g, r =0.9999, the average recovery rate is 97.27%, and the RSD is 2.06%.

5. Application number CN201110020171.7 discloses a method for measuring content of octagonamine, which is used for measuring content of the octagonamine in Chinese herbal medicine alangium chinense. Comprises ultrasonic extracting Chinese medicinal material alangium chinense, and measuring its content with high performance liquid chromatography.

The technical problems of the technology are as follows: 1) The recovery rate is low by adopting high performance liquid chromatography for determination; 2) High performance liquid chromatography is adopted for determination, and the repeatability is poor; 3) Incomplete sample extraction results in misjudgment of the content of the veratrine, and drug poisoning is caused.

In order to solve the problems, the invention team adopts an acid dye colorimetric method to determine the content of total alkaloids in the alangium platanifolium through a large amount of experimental researches. Investigating the extraction, separation and purification effects of the aniseed maple total alkaloids by different types of macroporous adsorption resins through a static adsorption/desorption test; the method for measuring the total alkaloids in the alangium chinense by the acid dye colorimetric method is obtained by adopting a single-factor test to research the influence of the concentration, volume, pH value and diameter height ratio of a sample loading solution on the adsorption rate of resin and the influence of the concentration and volume of an eluent on the elution rate, wherein the precision RSD of the method is 0.34%; the repetitive RSD is 1.40%; the sample recovery rate is 102.72 percent, the RSD is 2.66 percent, the method can truly, effectively and accurately determine the content of the veratrine poisoned by the alangium chinense harms, and the safety and the effectiveness of the medication are ensured.

Disclosure of Invention

The invention aims to provide a method for extracting, separating and purifying alangium total alkaloids.

The invention also aims to provide a detection method of the alangium chinense total alkaloids.

The extraction, separation and purification method of the alangium platanifolium total alkaloids comprises the following steps:

1) Preparation of a crude extract: precisely weighing 150-250 g of alangium chinense medicinal material powder, placing the powder into a round-bottom flask, adding 8-12 times of 60-90% ethanol, performing reflux extraction for 1-3 times for 1-3 hours each time, filtering, and combining crude extract for later use;

2) Preparing a sample loading liquid: recovering ethanol from the crude extract by using a rotary evaporator, concentrating to 400-500 mL, adding a proper amount of 80% ethanol for dilution to prepare a solution with the concentration of 2.00-6.00 mg/mL, and adjusting the pH of the solution to 2-8 by using sodium hydroxide and hydrochloric acid to obtain a sample solution;

3) Separation and purification: loading macroporous adsorption resin into a column by a wet method, taking 5-45 mL of sample loading liquid, loading the sample at a flow rate of 1mL/min, collecting effluent liquid, and standing for 1 h; eluting with distilled water with the flow rate of 2mL/min, wherein the dosage is 20-40 mL; then eluting with 60-90% ethanol at a flow rate of 2mL/min, wherein the volume of the elution is 100-150 mL; collecting eluate to obtain purified total alkaloid solution of Alangium platanifolium.

Preferably, the first and second liquid crystal materials are,

the extraction, separation and purification method of the alangium platanifolium total alkaloids comprises the following steps:

1) Preparation of a crude extract: precisely weighing 200g of alangium chinense medicinal material powder, placing in a round-bottom flask, adding 10 times of 80% ethanol, reflux-extracting for 2 times (2 h each time), filtering, and mixing the crude extractive solutions;

2) Preparing a sample loading liquid: recovering ethanol from the crude extract by using a rotary evaporator, concentrating to 450-500 mL, adding a proper amount of 80% ethanol for dilution to prepare a solution with the concentration of 3.02mg/mL, and adjusting the pH of the solution to 6 by using sodium hydroxide and hydrochloric acid to obtain a sample solution;

3) Separation and purification: loading macroporous adsorbent resin into a column by a wet method, taking 15mL of sample loading liquid, loading the sample at a flow rate of 1mL/min, collecting effluent liquid, and standing for 1 h; eluting with distilled water at flow rate of 2mL/min, with the amount of 30mL; eluting with 90% ethanol at a flow rate of 2mL/min, wherein the volume of the eluate is 120mL; collecting eluate to obtain purified total alkaloid solution of Alangium platanifolium.

Preferably, the first and second liquid crystal materials are,

the model of the macroporous adsorption resin is D101.

The invention relates to a wet method for filling columns, which has the diameter-height ratio of 1:1 to 4.

Preferably, the first and second liquid crystal materials are,

the invention relates to a wet column packing method, wherein the diameter-height ratio is 1:3.

the detection method of the Alangium total alkaloids is an acid dye colorimetric method, and comprises the following detection steps:

1) Preparation of control solutions: taking a proper amount of a chenopodium album reference substance, adding methanol to prepare a stock solution with the concentration of 4.0mg/mL, and diluting with methanol to 0.4mg/mL for later use;

2) Preparing a test solution: precisely weighing 1-3 g of alangium chinense powder, adding 20mL of 70-90% ethanol solution, performing reflux extraction for 1-3 times for 1-3 h each time, combining filtrates, adding 70-90% ethanol to 50mL, and obtaining a test solution;

3) The determination method comprises the following steps: precisely measuring 1mL of each of a reference solution and a sample solution, respectively adding 5-15 mL of disodium hydrogen phosphate-citric acid buffer solution with the pH value of 3.5-5.5 and 5-15 mL of bromocresol green solution, respectively extracting by 1-3 times with 20-30 mL of chloroform, shaking vigorously for 1-5 minutes, standing for 20-40 min, fixing the volume to 25mL, performing 0401 ultraviolet-visible spectrophotometry under the condition of illumination on the volume of the volume to be 25mL, measuring at the wavelength of 415nm, and calculating to obtain the final product.

Preferably, the first and second liquid crystal materials are,

the preparation of the test solution of the invention is as follows: precisely weighing 2g of alangium chinense powder, adding 20mL of 80% ethanol solution, reflux-extracting for 2 times, each time for 2h, mixing filtrates, adding 80% ethanol, and dissolving to 50mL to obtain test solution.

In a preferred embodiment of the method of the invention,

the assay of the invention is: precisely measuring 1mL of each of a reference solution and a sample solution, respectively adding 12.5mL of disodium hydrogen phosphate-citric acid buffer solution with the pH value of 5.0 and 5mL of bromocresol green solution, extracting with 25mL of chloroform for 2 times, shaking vigorously for 2 minutes, standing for 30 minutes, fixing the volume to 25mL, performing ultraviolet-visible spectrophotometry with an irradiation mode 0401, measuring at a wavelength of 415nm, and calculating to obtain the test solution.

The pH value of the disodium hydrogen phosphate-citric acid buffer solution is 3.5-5.5.

In a preferred embodiment of the method of the invention,

the pH value of the disodium hydrogen phosphate-citric acid buffer solution is 5.

Advantageous effects

1. The extraction process of the total alkaloids in the alangium platanifolium is optimized, the content of the total alkaloids in the alangium platanifolium is determined by adopting an acid dye colorimetric method, the content of the total alkaloids in the alangium platanifolium is taken as an evaluation index, the influence of an extraction method, an extraction solvent, the dosage of the solvent, the extraction times and the extraction time on the extraction of the total alkaloids in the alangium platanifolium is investigated by single-factor tests, and the extraction process of the total alkaloids in the alangium platanifolium is optimized by adopting a Box-Benhnken response surface method test according to the single-factor test result. The result shows that the extraction effect of the total alkaloids of the alangium platanifolium is the best when 10 times of 80% ethanol is adopted for reflux extraction for 2 times and each time is 2 hours, the extraction content reaches 87.60mg/g, and the method proves that the extraction process of the total alkaloids of the alangium platanifolium, which is optimized by adopting a Box-Benhnken response surface method, is stable and feasible, and can provide a theoretical basis for the full utilization of the resource of the alangium platanifolium.

2. The process conditions and parameters of the total alkaloids in the alangium chinense are optimized, the basis is provided for the industrial production of the total alkaloids, and the content of the total alkaloids in the alangium chinense is determined by an acid dye colorimetric method. The purification effect of different types of macroporous adsorption resin on the alangium chinense total alkaloids is inspected through a static adsorption/desorption test; a single-factor test is adopted to research the influence of the concentration, volume, pH value and diameter height ratio of the sample loading liquid on the resin adsorption rate and the influence of the concentration and volume of the elution liquid on the elution rate. And as a result, the D101 macroporous adsorption resin is adopted to separate and purify the total alkaloids of the alangium chinense, and the optimal purification process parameters are as follows: the concentration of the sample loading solution is 3.02mg/mL, the volume is 45mL, the pH value is 6, the diameter-height ratio is 1. The purity of the total alkaloids in the obtained alangium chinense extract is improved from 21.61% to 63.03%. And (4) conclusion: the established process for separating and purifying the alangium platanifolium total alkaloids is scientific, reasonable, simple and feasible, and can provide experimental basis for industrial production of the alangium platanifolium total alkaloids.

3. The quantitative analysis method of the present invention is optimized by using the taxifoline as a standard substance and adopting a single factor test, and methodology investigation is performed. As a result: the acid dye is bromocresol green, the volume ratio of a buffer solution with pH =5.0, the bromocresol green to a test solution is 12.5. The veratrine is taken as a reference substance, the linear relation is good in the range of 0.1-0.8 mg/mL, the regression equation is Y =1.0572X +0.0089 (R2 = 0.9946), and the precision RSD is 0.34%; the repeatability RSD is 1.40 percent, and the stability is 0.86 percent; the sample recovery rate was 102.72% and the RSD was 2.66%. And (4) conclusion: the established method for measuring the total alkaloids in the alangium platanifolium by the acid dye colorimetric method has the advantages of simple operation, good precision, repeatability and recovery rate and higher sensitivity, so the method can be used for measuring the content of the total alkaloids in the alangium platanifolium.

Drawings

FIG. 1 blank control wavelength scan;

FIG. 2 standard wavelength scan;

FIG. 3 is a wavelength scan of a test article;

FIG. 4 is a taxine standard curve;

FIG. 5 is a graph comparing sonication and reflux;

FIG. 6 is a graph comparing acid addition with no acid addition;

FIG. 7 alcohol concentration investigation;

FIG. 8 extraction time review;

FIG. 9 extraction times review;

FIG. 10 alcohol volume study;

FIG. 11 three-dimensional response surface;

FIG. 12 static adsorption kinetics curves of D101 type macroporous resin;

FIG. 13 adsorption leak curves;

FIG. 14 is a graph showing the amount of water used for removing impurities from a cured product;

FIG. 15 alcohol elution profile.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1 extraction separation purification

(1) Preparation of a crude extract: precisely weighing 200g of alangium chinense medicinal material powder, placing in a round-bottom flask, adding 10 times of 80% ethanol, reflux-extracting for 2 times (2 h each time), filtering, and mixing the crude extractive solutions;

(2) Preparing a sample solution: recovering ethanol from the crude extractive solution with rotary evaporator, concentrating to 500mL, diluting with appropriate amount of 80% ethanol to obtain solution with concentration of 3.02mg/mL, and adjusting pH to 6 with sodium hydroxide and hydrochloric acid to obtain sample solution;

(3) Separation and purification: taking D101 type macroporous adsorption resin, and mixing the components in a ratio of diameter to height of 1:3, filling the column by a wet method, taking 15mL of sample loading liquid, loading the sample at the flow rate of 1mL/min, collecting effluent liquid, and standing for 1 h; eluting with distilled water at flow rate of 2mL/min, with the amount of 30mL; eluting with 90% ethanol at a flow rate of 2mL/min, wherein the volume of the eluate is 120mL; collecting the eluent to obtain the purified solution of the total alkaloids of Alangium chinense.

Example 2 extraction separation purification

(1) Preparation of a crude extract: precisely weighing 150g of alangium chinense medicinal material powder, placing the powder into a round-bottom flask, adding 8 times of 60% ethanol, performing reflux extraction for 1 time for 1 hour each time, filtering, and combining the crude extract for later use;

(2) Preparing a sample solution: recovering ethanol from the crude extractive solution with rotary evaporator, concentrating to 400mL, diluting with appropriate amount of 60% ethanol to obtain solution with concentration of 2.00mg/mL, and adjusting pH to 2 with sodium hydroxide and hydrochloric acid to obtain sample solution;

(3) Separation and purification: taking D101 type macroporous adsorption resin, and mixing the components in a ratio of diameter to height of 1:1, filling the column by a wet method, taking 5mL of sample loading liquid, loading the sample at the flow rate of 1mL/min, collecting effluent liquid, and standing for 1 h; eluting with distilled water at flow rate of 2mL/min, with the amount of 20mL; eluting with 60% ethanol at a flow rate of 2mL/min, wherein the volume of the eluate is 100mL; collecting the eluent to obtain the purified solution of the total alkaloids of Alangium chinense.

Example 3 extraction, separation and purification

(1) Preparation of a crude extract: precisely weighing 250g of alangium chinense medicinal material powder, placing the 250g of alangium chinense medicinal material powder into a round-bottom flask, adding 90% ethanol in an amount which is 12 times that of the powder, performing reflux extraction for 3 times, 3 hours each time, filtering, and combining crude extract for later use;

(2) Preparing a sample loading liquid: recovering ethanol from the crude extractive solution with rotary evaporator, concentrating to 450mL, diluting with appropriate amount of 90% ethanol to obtain solution with concentration of 6.00mg/mL, and adjusting pH to 8 with sodium hydroxide and hydrochloric acid to obtain sample solution;

(3) Separation and purification: taking D101 type macroporous adsorption resin, and mixing the components in a ratio of diameter to height of 1:4, filling the column by a wet method, taking 45mL of sample loading solution, loading the sample at the flow rate of 1mL/min, collecting effluent, and standing for 1 h; eluting with distilled water at flow rate of 2mL/min, with the amount of 40mL; eluting with 90% ethanol at flow rate of 2mL/min, with an elution volume of 150mL; collecting eluate to obtain purified total alkaloid solution of Alangium platanifolium.

Example 4 detection method

(1) Preparation of control solutions: taking a proper amount of a chenopodium album reference substance, adding methanol to prepare a stock solution with the concentration of 4.0mg/mL, and diluting with methanol to 0.4mg/mL for later use;

(2) Preparation of a test solution: precisely weighing 2g of alangium chinense powder, adding 20mL of 80% ethanol solution, reflux-extracting for 2 times, each for 2h, mixing filtrates, adding 80% ethanol to 50mL to obtain a test solution;

(3) The determination method comprises the following steps: precisely measuring 1mL of each of a reference solution and a test solution, respectively adding 12.5mL of disodium hydrogen phosphate-citric acid buffer solution with the pH value of 5.0 and 5mL of bromocresol green solution, respectively extracting with 25mL of chloroform for 2 times, shaking vigorously for 2 minutes, standing for 30 minutes, fixing the volume to 25mL, measuring at the wavelength of 415nm by an ultraviolet-visible spectrophotometry method under the condition of irradiation of 0401, and calculating to obtain the test solution.

Example 5 detection method

(1) Preparation of control solutions: taking a proper amount of a chenopodium album reference substance, adding methanol to prepare a stock solution with the concentration of 4.0mg/mL, and diluting with methanol to 0.4mg/mL for later use;

(2) Preparation of a test solution: precisely weighing 1g of alangium chinense powder, adding 20mL of 70% ethanol solution, reflux-extracting for 1 time for 1h each time, mixing filtrates, adding 70% ethanol, and dissolving to 50mL to obtain a test solution;

(3) The determination method comprises the following steps: precisely measuring 1mL of each of a reference solution and a sample solution, respectively adding 5mL of disodium hydrogen phosphate-citric acid buffer solution with the pH value of 3.5 and 5mL of bromocresol green solution, extracting with 20mL of chloroform for 1 time, shaking violently for 1 minute, standing for 20 minutes, fixing the volume to 25mL, performing ultraviolet-visible spectrophotometry with irradiation standard 0401, measuring at the wavelength of 415nm, and calculating to obtain the final product.

Example 6 detection method

(1) Preparation of control solutions: taking a proper amount of a chenopodium album reference substance, adding methanol to prepare a stock solution with the concentration of 4.0mg/mL, and diluting with methanol to 0.4mg/mL for later use;

(2) Preparing a test solution: precisely weighing 3g of alangium chinense powder, adding 20mL of 90% ethanol solution, reflux-extracting for 3 times, 3h each time, mixing filtrates, adding 90% ethanol, and dissolving to 50mL to obtain a test solution;

(3) The determination method comprises the following steps: precisely measuring 1mL of each of a reference solution and a test solution, respectively adding 15mL of disodium hydrogen phosphate-citric acid buffer solution with the pH value of 5.5 and 15mL of bromocresol green solution, extracting by using 30mL of chloroform for 3 times, shaking vigorously for 5 minutes, standing for 40 minutes, fixing the volume to 25mL, measuring at the wavelength of 415nm by using a 0401 ultraviolet-visible spectrophotometry method according to general rules, and calculating to obtain the test solution.

To further verify the effectiveness of the present invention, the inventors performed a series of verification tests, specifically as follows:

1. research on quantitative analysis method of total alkaloids in alangium platanifolium

1 Source and identification of medicinal materials

The alangium chinense medicinal material used in the experiment is purchased from orchard medicinal material markets. The purchased Alangium chinense medicinal material is identified as the dry root and rhizome of Alangium chinense (lour.) Harms by the crude drug laboratory king skilful teacher of the university of traditional Chinese medicine in Guizhou.

2 instruments and reagents

2.1 Instrument

One ten thousand analytical balance (METTLER TOLEDO ME204E Mettler-Tolliduo instruments (Shanghai) Co., ltd.), multifunctional disintegrator (LDP-150 Zhejiang Yongkang Red Sun electromechanical Co., ltd.), electronic balance (FA 2204N Shanghai Cyanine instruments Co., ltd.), ultraviolet spectrophotometer (UV-9000S Shanghai Yuan instruments Co., ltd.), quartz cuvette (10 mm (melt) JH), digital thermostat water bath (HH-4 Shanghai Wei Bangbi instruments Co., ltd.), qualitative filter paper (phi 10cm Hangzhou specialty paper industry Co., ltd.), medical sieve, round bottom flask, filter membrane, syringe, mortar, stoppered flask, volumetric flask, funnel, beaker, glass rod, and evaporating dish.

2.2 reagents

Distilled water (self-made); the following reagents were all analytically pure: acetic acid (20161227) is produced by Chongqing Chuanjiang chemical industries (group) Ltd; absolute ethanol (20190122), methanol (20190805), chloroform (20180418), disodium hydrogen phosphate dodecahydrate (20170227), citric acid (20190815) are all manufactured by national institute of chemical and pharmacy, ltd; bromophenol blue (20190613), bromocresol green (20190617) and bromopyrogallol blue (20181212) are produced by Tianjin, kemiou Chemicals Co., ltd.

3 methods and results

3.1 preparation of reagents

3.1.1 preparation of control solutions

Taking appropriate amount of taxinine as reference substance, adding methanol to obtain stock solution with concentration of 4.0mg/mL, and diluting with methanol to 0.4mg/mL for use.

3.1.2 preparation of test solutions

Precisely weighing about 2g of alangium chinense powder, adding 20mL of 80% ethanol solution, reflux-extracting for 2 times, each for 2h, mixing filtrates, and adding 80% ethanol to 50mL to obtain test solution.

3.2 optimization of Total alkaloid content determination method

3.2.1 examination of acid dyes with buffer solutions

Respectively and precisely measuring 3 parts of disodium hydrogen phosphate-citric acid buffer solutions with pH values of 3.5, 4.0, 4.5, 5.0 and 5.5 in volume of 12.5mL, respectively adding 7.5mL of 3 acid dye solutions of bromocresol green, bromothymol blue and bromophenol blue, respectively, adding 1.0mL of a reference solution, performing 2-time extraction by using 25mL of chloroform, carrying out volume determination to 25mL, and measuring at the wavelength of 415nm by using an ultraviolet-visible spectrophotometry method (generally 0401). As a result, bromocresol green was selected for dyeing, and when the pH of the buffer solution was 5.0, the absorbance was the best, and the results are shown in Table 1, so this system was selected for measurement.

Table 1 examination of stains and buffer solutions

3.2.2 examination of the amount of buffer solution

Precisely measuring 5.0mL, 7.5mL, 10.0mL, 12.5mL and 15mL of disodium hydrogen phosphate-citric acid buffer solution with the pH value of 5.0 respectively, adding 7.5mL of bromocresol green solution and 1.0mL of standard solution, extracting by using 25mL of chloroform for 2 times, fixing the volume to 25mL, and measuring at the wavelength of 415nm by an ultraviolet-visible spectrophotometry method (general instruction 0401). As a result, when the amount of the buffer solution was 12.5mL, the absorbance was the best, and the results are shown in Table 2, so that the amount of the buffer solution was determined to be 12.5mL.

TABLE 2 examination of the amount of buffer solution

3.2.3 examination of the amount of dye used

Precisely measuring 12.5mL of buffer solution, adding 5.0mL, 7.5mL, 10.0mL, 12.5mL and 15.0mL of bromocresol green solution and 1.0mL of reference solution, respectively, extracting with 25mL of chloroform for 2 times, diluting to 25mL, and measuring at 415nm wavelength by ultraviolet-visible spectrophotometry (general rule 0401). As a result, when the amount of bromocresol green solvent was 5.0mL, the absorbance value was the best, and the results are shown in Table 3, so that the amount of the coloring agent was determined to be 5.0mL.

Table 3 stain usage study

3.2.4 investigation of the measurement wavelength

Precisely measuring 12.5mL of disodium hydrogen phosphate-citric acid buffer solution with the pH value of 5.0, adding 5mL of bromocresol green solution, adding 1mL of reference solution under the condition of '3.1.1', transferring the solution into a separating funnel, extracting for 2 times by using 25mL of chloroform, and fixing the volume to 25mL to be used as the reference solution to prepare a test solution and a blank reference solution by the same method. Full wavelength scanning by ultraviolet-visible spectrophotometry (irradiation with light 0401). The result shows that the quinobases reference substance and the test solution have maximum absorption at 415nm, the blank solvent has no interference, and 415nm is selected as the measurement wavelength as shown in figure 1, figure 2 and figure 3.

3.2.5 determination of assay methods

Precisely measuring 1mL of a sample solution, respectively adding 12.5mL of disodium hydrogen phosphate-citric acid buffer solution with pH value of 5.0 and 5mL of bromocresol green solution, performing extraction with 25mL of chloroform for 2 times (shaking vigorously for 2 minutes, standing for 30 min), fixing the volume to 25mL, and measuring at 415nm by ultraviolet-visible spectrophotometry (general rule 0401).

3.3 examination of the establishment of the colorimetric method for acid dyes

3.3.1 examination of the Linear relationship

Accurately measuring 12.5mL buffer solution (pH = 5.0) and 5.0mL bromocresol green solution, respectively, adding into a separating funnel, and adding 0.40mg/mL taxine standard with volume of 0.25mL, 0.5mL, 1.0mL, 1.5mL, 2.0mLThe absorbance of the sample solution was measured by the method under "3.2.5". And (3) drawing a standard curve by taking the content (mg) of the taxinine as an abscissa and the absorbance as the abscissa. The regression equation is Y =1.0572X +0.0089 (R) ² = 0.9946) shows that the standard solution has a good correlation in the range of 0.1-0.8 mg, and the results are shown in fig. 4.

3.3.2 stability Studies

Collecting the sample solution under the item "3.1.2", treating and developing color according to the method under the item "3.2.5", collecting chloroform layer, standing at room temperature for 0, 20, 40, 60, 80, 100, 120min, and measuring absorbance at 415nm. The results show that the test solution is stable at room temperature for 120min, with an average absorbance of 0.359 and rsd =0.79% (n = 6), as shown in table 4.

TABLE 4 stability study

3.3.3 precision investigation

The sample solution under the item "3.1.2" was treated to develop color according to the method under the item "3.2.5", and then continuously measured for 6 times. The results showed that the method was accurate with an average absorbance of 0.442 and rsd of 0.34% (n = 6), as shown in table 5.

TABLE 5 examination of precision

3.3.4 repeatability tests

The test solution was treated under the item "3.1.2" for color development according to the method under the item "3.2.5", and then the absorbance was measured at a wavelength of 415nm. As a result: the average content of total alkaloids in the alangium chinense is 89.05mg/g, the RSD is 1.40% (n = 6), and the results are shown in Table 6, which indicates that the method has good repeatability.

TABLE 6 repeatability tests

3.3.5 sample application recovery test

6 parts of alangium chinense powder, about 1g of each part, 44mg of a veratrine standard substance is respectively added, a test solution is tested according to the item 3.1.2, the mixture is processed according to the item 3.2.5 to develop color, the absorbance is measured at the position of 415nm wavelength, the sample adding recovery rate is calculated, the average recovery rate is 102.72 percent, and the RSD is 2.66 percent (n = 6), and the results are shown in table 7.

(recovery = (a-b)/c × 100%; a true measurement value, b measured substance amount contained in sample, c standard substance amount)

TABLE 7 sample recovery rate test results table

4 summary and discussion

4.1 summary

The alkaloids component of Alangium platanifolium has pharmacological activities of relieving pain, diminishing inflammation, resisting rheumatism, relaxing muscle, and treating central nerve and cardiovascular system, wherein the main pharmacological active component is Alangine. In the experiment, factors (the pH value and the dosage of a buffer solution and the type and the dosage of an acid dye) influencing the determination of the content of total alkaloids in the alangium chinense are investigated, so that an analysis method for determining the total alkaloids in the alangium chinense by using a UV method is established, an acid dye colorimetric method is adopted, a chenopodium quinoa reference substance and bromocresol green are used as coloring agents, and the volume dosage ratio of the buffer solution with the pH =5.0 is 1.0:5.0:12.5, the test wavelength is 415nm.

4.2 discussion

The acid dye colorimetric method needs to be operated strictly according to the experimental steps, so that human errors are reduced, and more accurate experimental data are obtained. Since chloroform was used in large amounts in the experiments, it was handled in a fume hood and personal protection was applied. Most importantly, in the extraction link, the separating funnel needs to be fully oscillated to ensure that the acid dye mixed layer of the extracting solution is fully contacted with the chloroform layer, and the layering time needs to be kept long enough. The complex formed by alkaloid and acid dye is easy to decompose by light, and is protected from light and stored by using a brown volumetric flask. The stain and buffer solution are also formulated in situ, which would otherwise cause a bias in the experimental data.

2. Research on extraction process of Alangium chinense total alkaloids

1 Source and identification of medicinal materials

The alangium chinense medicinal material used in the test is purchased from a orchard medicinal material market. The purchased Alangium chinense medicinal material is identified as the dry root and rhizome of Alangium chinense (lour.) Harms by the crude drug laboratory wangshiki teacher of the Guizhou traditional Chinese medicine university.

2 instruments and reagents

2.1 instruments

The instruments used in this experiment are shown in Table 8.

TABLE 8 Instrument name and manufacturer

2.2 drugs and reagents

The drugs and reagents used in this experiment are shown in Table 9.

TABLE 9 drugs and reagents

3 methods and results

3.1 preparation of reagents

3.1.1 preparation of control solutions

Accurately weighing a proper amount of an octagon (toxigenin) reference substance, and adding methanol to prepare a standard substance solution with the concentration of 0.40mg/mL for later use.

3.1.2 preparation of bromocresol Green

Precisely weighing 0.02g of sodium hydroxide in a 10mL volumetric flask, adding water to prepare 0.05mol/L sodium hydroxide solution, weighing 2.8mL, precisely weighing 0.10g of bromocresol green, respectively placing in a mortar, grinding until the bromocresol green is dissolved, and adding 200mL of distilled water for dilution to obtain the sodium bromide.

3.1.3 preparation of buffer

Weighing 2.10g of 0.1mol/L citric acid, adding 100mL of distilled water for dissolving, 10.74g of 0.2mol/L disodium hydrogen phosphate dodecahydrate for dissolving, adding 150mL of distilled water for dissolving, taking 97mL of citric acid solution and 103mL of disodium hydrogen phosphate dodecahydrate solution, preparing a buffer solution with the pH value =5.0, and filling the buffer solution into a conical flask with a plug for storage.

3.2 acid dye colorimetric method for determining Total alkaloid content

Precisely measuring 1mL of a sample solution, respectively adding 12.5mL of disodium hydrogen phosphate-citric acid buffer solution with pH value of 5.0 and 5mL of bromocresol green solution, performing extraction with 25mL of chloroform for 2 times (shaking vigorously for 2 minutes, standing for 30 min), fixing the volume to 25mL, and measuring at a wavelength of 415nm by ultraviolet-visible spectrophotometry (general rule 0401).

3.3 Single factor test

3.3.1 examination of the extraction method

The test takes the alangium platanifolium total alkaloids as an index to respectively examine the influence of the ultrasonic extraction and reflux extraction method on the total alkaloid extraction content.

Precisely weighing about 2g of medicinal powder (sieving with a fourth sieve), placing in a round bottom flask, adding 10 times (20 mL) of 75% ethanol, weighing, respectively treating with ultrasound and reflux for 1h, cooling after extraction, complementing weight loss with 75% ethanol, and filtering with microporous membrane. The assay was performed as under "3.2" and two replicates were tested, the results are shown in FIG. 5.

As a result: the absorbance of the extracting solution obtained by different extracting methods is compared, so that the reflux extraction effect is better than that of the ultrasonic extraction.

3.3.2 investigation of auxiliary extraction solvents

Precisely weighing about 2g of alangium chinense powder (sieved by a sieve IV), placing the alangium chinense powder into a round-bottom flask, respectively adding 10 times (20 mL) of 75% ethanol and 75% ethanol containing 1% tartaric acid, weighing, carrying out reflux extraction for 1h, cooling after the extraction is finished, respectively adopting 75% ethanol and 75% ethanol containing 1% tartaric acid to complement the weight loss, and filtering by a microporous membrane. The assay was performed as under "3.2" and two replicates were tested, the results are shown in FIG. 6.

As a result: by comparing the data in fig. 6, it is found that the difference between the acid addition and the acid addition is not great, and the acid addition is selected to perform extraction research on the alangium chinense total alkaloids for considering a plurality of reasons such as simple operation, solvent saving and the like.

3.3.3 examination of extraction solvent

Precisely weighing about 2g of 4 parts of alangium chinense powder (screened by a No. four sieve), respectively placing the powder into a round-bottom flask, respectively adding 20mL of 60%, 70%, 80% and 90% ethanol solution, weighing, carrying out reflux extraction for 1h, cooling after the extraction is finished, complementing the weight loss by adopting ethanol with various concentrations, filtering by a microporous membrane, and measuring according to a method of 3.2, wherein the result is shown in figure 7.

As a result: as shown in FIG. 7, when the ethanol concentration is 80%, the absorbance is the maximum, and the total alkaloid extraction content is the maximum. Therefore 80% ethanol was chosen as the extraction solvent.

3.3.4 extraction time inspection

Precisely weighing about 2g of anise 4 parts of maple powder (screened by a No. four sieve), placing the anise 4 parts of maple powder into a round-bottom flask, adding 10 times of 80% ethanol solution, weighing, respectively performing reflux extraction for 0.5h, 1.0h, 1.5h and 2.0h, cooling after extraction is finished, adopting 80% ethanol to complement weight loss, filtering by a microporous membrane, and determining according to a method under the item '3.2', wherein the result is shown in figure 8.

The result shows that the extraction content of the alangium chinense total alkaloids is improved along with the increase of the extraction time and reaches the maximum value at 2h, so the extraction time is selected to be 2h.

3.3.5 number of extractions

Precisely weighing about 2g of 3 parts of alangium powder (screened by a No. four sieve), placing the powder into a round-bottom flask, adding 10 times of 80% ethanol solution, weighing, respectively carrying out reflux extraction for 1, 2 and 3 times (2 h/time), merging filtrates, placing the filtrates into a 100mL volumetric flask, and carrying out volume fixing by using 80% ethanol. 5mL of the extract was stained and extracted, and the measurement was carried out according to the method under "3.2", and the results are shown in FIG. 9.

As a result: the total alkaloid extraction content is improved along with the increase of extraction times, the absorbances of 2 times and 3 times of extraction are only slightly different, and the extraction times are selected to be 2 times for saving time.

3.3.6 volume examination of extraction solvent

Precisely weighing about 2g of 4 parts of alangium chinense powder (sieved by a No. four sieve), placing the powder into a round-bottom flask, respectively adding 12mL, 16mL, 20mL and 24mL of 80% ethanol solution, weighing, carrying out reflux extraction for 2 times, 2 hours each time, cooling after the extraction is finished, combining filtrates, placing the filtrates into a 50mL volumetric flask, and fixing the volume by 80% ethanol. The results of the measurement were as shown in FIG. 10, according to the method under "3.2".

As a result: as can be seen from the data in FIG. 10, the total alkaloid content is maximally absorbed when the extraction solvent is 20mL. Meanwhile, in order to save the solvent, 10 times of the amount of the extraction solvent is selected.

3.3.7 results

A single-factor test shows that the optimal extraction process of the alangium chinense is to adopt 10 times of 80 percent ethanol for reflux extraction for 2 times, and each time is 2 hours.

3.4 Box-Behnken response surface method optimization test

3.4.1 Box-Behnken response surface method optimization experiment design

In order to further optimize and research the extraction process of the total alkaloids in the alangium chinense, a Box-Behnken response surface method is applied based on a single-factor test, 3 main factors (ethanol concentration, ethanol volume and extraction time) are selected as independent variables, 3 levels are designed for each factor, and the response value is the content of the total alkaloids in the alangium chinense. (final volume of the extract was 50mL and the amount was measured) is shown in Table 10.

TABLE 10 design factors and levels

3.4.2 BBD and response surface test results

On the basis of the results of the single-factor test and the BBD principle design, a 3-factor (alcohol concentration, alcohol volume and extraction time) 3-level response surface analysis method is adopted to study the influence of the interaction among the 3 factors on the extraction content of the total alkaloids of the alangium chinense. The design and results are shown in table 11.

The result of the test is calculated and analyzed based on Design-expert8.0.6 software, and a quadratic polynomial regression model of the content of the total alkaloids of the alangium platanifolium to the above 3 factors is obtained as follows:

Y＝88.55～0.34A+0.52B+0.52C～0.28AB～1.52AC～0.90BC～4.00A ² ～2.41B ² ～1.58C ² in the formula, Y represents the content, A represents the alcohol concentration, B represents the alcohol volume, and C represents the extraction time. The results are shown in tables 11 and 12.

TABLE 11 BBD test conditions and response surface design tests and results

TABLE 12 response surface quadratic simulation analysis of variance

From Table 12, it can be seen that P of the extraction model of Alangium chinense total alkaloids is less than or equal to 0.05, and the factor affecting the extraction content of Alangium chinense total alkaloids can be deduced from the value F, that B is smaller than C and is smaller than A. Therefore, the model has significance and rationality in the optimization research of the extraction process of the total alkaloids of the Acer rubrum. The optimal extraction process comprises extracting with 80% ethanol for 2 times, each time for 2 hr, the volume of the extraction solvent is 20mL, and the total alkaloid content is 89.60mg/g.

3.4.3 response surface analysis

The steepness of the response surface may be used to reflect the significance of the interaction between the two factors, with the greater the interaction, the steeper the graph presented by the response surface. Fig. 11 is a response curved surface of the interaction among 3 factors of alcohol concentration, alcohol volume and extraction time on the alangium platanifolium total alkaloids, wherein the interaction curved surface of AC is steeper, which shows that the interaction of alcohol concentration and extraction time has a more significant effect on the extraction content of the alangium platanifolium total alkaloids.

3.4.4 best extraction Process verification test

Optimal extraction process parameters are obtained through calculation and analysis based on Design-Expert8.0.6 software, and the maximum extraction content of the total alkaloids of Chinese alangium is the target. Three verification tests are carried out under the conditions that the ethanol concentration of an extraction solvent is 80%, the extraction time is 2.0h, the alcohol volume is 20mL, and 2 times of extraction are carried out, so that the result shows that the average content of three times of extraction of the alangium chinense total alkaloids is 87.67mg/g, which is close to the theoretical predicted value of 88.63mg/g, and the optimization process of the method for extracting the alangium chinense total alkaloids is reasonable.

4 conclusion discussion

4.1 conclusion

The research investigates 6 influencing factors of the total alkaloids in the alangium chinense. On the basis, design-Expert8.0.6 software is used for carrying out 3-factor 3-level response surface Design test on alcohol concentration, alcohol volume and extraction time, the result shows that the alcohol concentration is 79.20%, the alcohol volume is 20.31mL, the extraction time is 2.09h and the extraction times are 2 times, and the extraction content of the alangium platanifolium total alkaloids is predicted to be 88.6mg/g. Finally, the optimal process conditions are determined by taking parameters of response surface optimization as guidance, wherein the alcohol concentration is 80%, the alcohol volume is 20mL (10 times), the extraction time is 2.0h, and the extraction is carried out for 2 times. The average extraction content of the Alangium total alkaloids is 87.67mg/g. Research shows that the alcohol reflux extraction has a good dissolution effect on the Alangium total alkaloids, can further effectively improve the extraction content of the total alkaloids, and can be applied to the extraction of the alkaloids. The reflux extraction of the total alkaloids in the alangium chinense has the characteristics of simple operation, material saving, environmental protection and the like, and provides a certain technical support and theoretical basis for the extraction, development and utilization of the alangium chinense.

4.2 discussion

The experiment adopts an ethanol reflux extraction method to extract the total alkaloids of the Alangium chinense, adopts an acid dye colorimetric method to dye the total alkaloids of the Alangium chinense, and adopts a Box-Behnken response surface method to optimize and research the extraction process of the total alkaloids of the Alangium chinense. In order to reduce human errors and obtain accurate experimental data in the experimental process, the operation is strictly carried out according to the experimental steps, and the volumetric flask is dried. When chloroform is used in the extraction link, personal protection and safety need to be kept, and due to the toxicity of chloroform, the chloroform needs to be operated in a fume hood, needs to be applied for use, and needs to be handed over to a teacher for storage after use. Most importantly, in the extraction step, in order to fully extract the total alkaloids in the Chinese alangium by the chloroform, the separating funnel needs to be fully shaken and layered for enough time, so that the extract dyed by the acid dye can be better contacted with the chloroform. To reduce errors, the buffer and stain need to be prepared on site. The complex formed by the acid dye and the alkaloid is unstable in light, so the dyed alkaloid needs to be stored in a brown bottle to play a role in avoiding light. Meanwhile, the same investigation factor needs to be carried out at the same time, the error is increased by the alternate-day investigation, and the measurement needs to be carried out after the system is stable in the ultraviolet measurement process.

3. Research on separation and purification process of Alangium chinense total alkaloids

1 instruments and reagents

1.1 apparatus, see table 13.

TABLE 13 Experimental instruments, models, and manufacturers

1.2 drugs and reagents, see Table 14.

TABLE 14 Experimental drugs and reagents

1.3 herbs

The alangium chinense medicinal material used in the experiment is purchased from orchard medicinal material markets. The purchased Alangium chinense medicinal material is identified as the dry root and rhizome of Alangium chinense Harms (Lour.) by Wangshi of the crude drug laboratory of the university of traditional Chinese medicine of Guizhou.

2 methods and results

2.1 preparation of test solutions

Accurately weighing 200g of medicinal powder, placing the medicinal powder in a 4000mL round-bottom flask, adding 2000mL of 80% ethanol solution, performing reflux extraction for 2 times, performing 2 hours each time, filtering, combining crude extract, recovering ethanol by using a rotary evaporator, concentrating to 450-500 mL, adding a proper amount of 80% ethanol for dilution, and preparing a sample solution with a proper concentration for later use.

2.2 preparation of control solutions

Precisely weighing a proper amount of a taxine reference substance, adding ethanol to prepare a standard substance solution with the concentration of 4.0mg/mL, and diluting the standard substance solution with the concentration of 4.0mg/mL to 0.4mg/mL to obtain a reference substance solution with the mass concentration of 0.4mg/mL for later use.

2.3 determination of Alangium total alkaloids

Precisely measuring 1mL of a sample solution, respectively adding 12.5mL of disodium hydrogen phosphate-citric acid buffer solution with pH value of 5.0 and 5mL of bromocresol green solution, performing extraction with 25mL of chloroform for 2 times (shaking vigorously for 2 minutes, standing for 30 min), fixing the volume to 25mL, and measuring at a wavelength of 415nm by ultraviolet-visible spectrophotometry (general rule 0401).

2.4 pretreatment of macroporous resins

Soaking the macroporous resin with 95% ethanol for 24h to fully expand the macroporous resin, removing floating resin impurities and fragments, filling the macroporous resin into a column by a wet method, washing the resin with 95% ethanol solution until no white turbidity exists (an effluent can be observed by distilled water = 1mL. Before use, the column is washed by a proper amount of 95% ethanol, and then the ethanol is washed by deionized water, dried in vacuum and reserved for use.

2.5 screening of resin types

And (3) screening the model of the resin by adopting a static adsorption method and taking the comprehensive scores of the adsorption rate and the desorption rate as indexes. The alternative resins include macroporous resins AB-8, D-101, HPD-200, NKA-9, HPD-600. Weighing 5 processed resins, 15g each, placing in a 50mL conical flask with a plug, adding 10mL of sample solution, placing in a constant temperature (25 ℃) oscillator for oscillation for 12h, after full adsorption, taking a proper amount of supernatant, and measuring the absorbance of the alkaloid before and after adsorption according to the method under the item '2.3'. And (3) sucking water on the surfaces of different types of resins, adding 10mL of 80% ethanol, sealing, placing in a constant-temperature (25 ℃) oscillator for oscillating for 12 hours, and taking a little supernatant. The absorbance was measured, and the saturated adsorption amount, adsorption rate and desorption rate were calculated, and the results are shown in Table 15. The calculation formula is as follows:

table 15 results of static adsorption experiments (n = 3)

The results of the adsorption capacities and desorption capacities of different types of macroporous resins are shown in Table 15, and the results show that the adsorption capacity HPD600 is greater than NKA-9 >. And D101 type macroporous adsorption resin is selected as the resin for separating and purifying the alangium chinense total alkaloids according to the comprehensive score.

2.6 Alangium total alkaloids macroporous adsorbent resin purification technology investigation

2.6.1 static adsorption kinetics feature investigation

Precisely weighing 15g of the pretreated D101 macroporous resin, adding 30mL of sample solution into a 50mL conical flask with a plug, sealing, placing in a constant-temperature (25 ℃) oscillator, oscillating, and filtering for 5min, 10min, 0.5h, 1h, 2h and 3h respectively, wherein 2mL of filtrate is taken each time. The absorbance was measured at 415nm according to the method under "2.3". And (4) calculating the static adsorption rate of the total alkaloids, and drawing a static adsorption kinetic curve. The results are shown in FIG. 12.

The results in FIG. 12 show that the adsorption rate increased with the increase in adsorption time, but the resin saturated and the adsorption rate increased slowly after reaching a certain time. The adsorption rates of the resins are increased rapidly within 30min, the later increase is slow, and the static adsorption rate is not increased obviously within the adsorption time of 1-2 h. In view of time cost, the static adsorption time of the sample is finally selected to be 1h.

2.6.2 investigation of the concentration of the sample liquid

And (4) wet-process column packing, namely weighing 15g of 4 parts of pretreated D101 type macroporous resin. Sequentially loading 15mL of total alkaloids with concentrations of 6.040, 4.030, 3.020 and 2.010mg/mL, loading at a flow rate of 1.0mL/min, collecting effluent, and diluting to a volume of 15mL. After standing for 1h, the mixture was eluted with distilled water at a flow rate of 2mL/min in an amount of 10mL. Eluting with 90% ethanol at a flow rate of 2mL/min, collecting eluate, and diluting to 15mL. Absorbance was measured at 415nm by the method of "2.3" to calculate the adsorption and desorption rates of total alkaloids, and the results are shown in Table 16.

Table 16 comparison of different concentrations of the sample solutions (n = 3)

As is clear from Table 16, when the concentration of the sample solution was 3.02mg/mL, the desorption rate and the adsorption rate were not the best, but the concentration of the sample solution was selected to be 3.02mg/mL because the concentration was the highest on a comprehensive scale when the concentration was 3.02mg/mL.

2.6.3 maximum sample size investigation

And (3) wet-process column packing, namely weighing 1 part of the treated D101 type macroporous resin, 15g, continuously adding column loading liquid with the concentration of 3.02mg/mL into the top end, loading at the flow rate of 1mL/min, and collecting 1 part of the column loading liquid every 5mL. The absorbance was measured at 415nm and the leakage curve was plotted according to the method under "2.3", see FIG. 13.

As can be seen from FIG. 13, the mass concentration of total alkaloids in every 5mL of effluent liquid gradually increases with the increase of the volume of the loading liquid, which indicates that the adsorption capacity of the resin gradually reaches a saturation state, and the adsorption effect is reduced or even disappeared. When the volume of the sample solution is 15mL, obvious leakage occurs, so that the volume of the sample solution is selected to be 15mL to reduce loss and improve the recovery rate of the total alkaloids.

2.6.4 pH investigation of the Loading solution

And (4) wet column packing, namely weighing 15g of each of 4 parts of pretreated D101 type macroporous resin. Taking 4 parts of sample solution with the concentration of 3.02mg/mL, wherein each part is 15mL; adjusting the pH of the sample liquid to 2, 4, 6 and 8 respectively by using sodium hydroxide and hydrochloric acid, carrying out sample loading at a flow rate of 1mL/min, collecting effluent liquid respectively, and fixing the volume to 15mL. Standing for 1h, eluting with distilled water at flow rate of 2mL/min, with the dosage of 10mL. Then 90% ethanol is taken to elute at the flow rate of 2mL/min, the dosage is 15mL, and the eluent is collected and the volume is determined to be 15mL. Absorbance was measured at 415nm according to the method under item "2.3", and adsorption and desorption rates of total alkaloids were calculated, and the results are shown in Table 17.

TABLE 17 comparison of pH values of the samples (n = 3)

As shown in table 17, the pH of the sample solution was selected to be 6 because the purification effect was good and the adsorption rate and desorption rate were highest when the pH of the sample solution = 6.

2.6.5 adsorption column diameter-height ratio investigation

Loading a column (column diameter: diameter height =3 cm); after standing for 1h, the mixture was eluted with distilled water at a flow rate of 2mL/min in an amount of 10mL. Then 15mL of 90% ethanol is taken to elute at the flow rate of 2mL/min, and the eluent is collected and is metered to 15mL. The absorbance at 415nm was measured according to the method under "2.3", and the adsorption rate and elution rate were calculated, and the results are shown in Table 18.

TABLE 18 comparison of column aspect ratios (n = 3)

From the results in Table 18, it is shown that the aspect ratio is 1:3, the elution capacity is optimal, and the optimal diameter-height ratio is determined to be 1.

2.6.6 Water consumption investigation for impurity removal

And (3) taking 45g of the treated D101 type macroporous resin with the diameter-height ratio of 1, adding 15mL of total alkaloid solution with the mass concentration of 3.02mg/mL and the pH value of 6.0, loading at the flow rate of 1mL/min, and collecting effluent. Standing for 60min, eluting with 2mL/min distilled water, collecting one part per 5mL eluate, volatilizing solvent, weighing the solidified substance in the eluate, and drawing a curve of the solidified substance-water consumption for impurity removal with water consumption for impurity removal as abscissa and the solidified substance as ordinate, wherein the result is shown in FIG. 14.

2.6.7 alcohol eluent concentration investigation

And (3) loading the column by a wet method (the diameter-height ratio is 1. After standing for 1h, the mixture was eluted with distilled water at a flow rate of 2mL/min in an amount of 30mL. Eluting at flow rate of 2mL/min, sequentially loading 60mL of 60%, 70%, 80%, and 90% ethanol, and collecting eluates. The absorbance at 415nm was measured according to the method under "2.3", and the adsorption rate and elution rate were calculated, and the results are shown in Table 19.

Table 19 different concentrations of alcohol eluents (n = 3)

As can be seen from table 19, the elution ability gradually increased with the increase in alcohol concentration, and the elution ability was higher when the alcohol concentration was 90%, so that the optimum alcohol eluent concentration was selected to be 90%.

2.6.8 alcohol eluent dosage Observation

Loading the column by a wet method (the diameter-height ratio is 1; after standing for 1h, the mixture was eluted with distilled water at a flow rate of 2mL/min in an amount of 30mL. Eluting with 90% ethanol at flow rate of 2mL/min, collecting eluate, and collecting one tube per 10mL. The absorbance at 415nm was measured according to the method under "2.3", and the elution rate of each fraction was calculated to prepare a curve, which is shown in FIG. 15.

As can be seen from FIG. 15, the concentration of total alkaloids increased with the increase of the elution volume, and then gradually decreased. When the volume dosage is 120mL, the alkaloid in the eluent can not be detected, indicating that the elution is complete, so the elution volume is determined to be 120mL.

The optimal purification process of the total alkaloids in the star anise is obtained according to the experimental results, and comprises the following steps: taking D101 type macroporous adsorption resin, and filling the resin into a column by a wet method (the diameter-height ratio is 1. Preparing a sample solution with pH =6 and a concentration of 3.02mg/mL, measuring 15mL of the sample solution, dynamically loading at a flow rate of 1.0mL/min, and collecting an effluent. Standing for 1h, and eluting with 30mL of distilled water at a flow rate of 2 mL/min; eluting with 90% ethanol at a flow rate of 2mL/min, with an elution volume of 120mL, and collecting eluate to obtain final product.

3 validation test of optimal purification Process

According to the optimal purification process, experiments are carried out 3 times in parallel, the absorbance is measured at 415nm according to the method under the item 2.3, and the adsorption rate and the elution rate are calculated. The results are shown in table 20, and the purification process conditions of the alangium chinense total alkaloids are stable and reliable. Under the optimal condition, the content of the Chinese alangium total alkaloids is increased from 21.61% to 63.03%. (crude extract M (mg) of alangium platanifolium total alkaloids and analytic solution M (mg) obtained after macroporous resin purification are concentrated by an evaporation dish, then are dried in vacuum to constant weight, a little dry powder is weighed, and is dissolved by 80% ethanol to be constant volume to 10 mL) according to the calculation formula:

table 20 repeatability test data (n = 3)

4 summary and discussion

4.1 summary

The macroporous adsorption resin is used for researching the separation and purification effect of the total alkaloids of the alangium chinense, and the separation and purification conditions are optimized. Static adsorption and desorption tests confirm that the D101 type nonpolar resin is more suitable for separating and purifying the alangium platanifolium total alkaloids; the optimal process parameters are determined through a dynamic adsorption test and a desorption test as follows: static kinetic profile: adsorbing for 1h to reach an equilibrium state, and measuring the concentration of a sample loading solution: 3.02mg/mL, maximum loading: 15mL, pH value of a sample loading solution is 6, and the diameter-height ratio is as follows: 1; parameters of the eluent were: 30mL of water for impurity removal, 90% of alcohol eluent and 120mL of alcohol eluent are used, under the condition, the alangium chinense total alkaloids are separated and purified by D101 type macroporous resin, and the content of the alkaloids is increased from 21.61% to 63.03%. The optimization process is stable and feasible, and provides a scientific reference basis for the production of the alangium chinense total alkaloids.

Although the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that various modifications and improvements can be made thereto without departing from the spirit of the invention.

Claims (5)

1. An extraction, separation and purification method of alangium platanifolium total alkaloids is characterized by comprising the following steps:

1) Preparation of a crude extract: precisely weighing 150-250 g of alangium chinense medicinal material powder, placing the powder into a round-bottom flask, adding 8-12 times of 60-90% ethanol, performing reflux extraction for 1-3 times for 1-3 hours each time, filtering, and combining crude extract for later use;

2) Preparing a sample solution: recovering ethanol from the crude extract by using a rotary evaporator, concentrating to 400-500 mL, adding a proper amount of 60-90% ethanol for dilution to prepare a solution with the concentration of 2.00-6.00 mg/mL, and adjusting the pH of the solution to 2-8 by using sodium hydroxide and hydrochloric acid to obtain a sample solution;

3) Separation and purification: loading macroporous adsorption resin into a column by a wet method, taking 5-45 mL of sample loading liquid, loading the sample at a flow rate of 1mL/min, collecting effluent liquid, and standing for 1 h; eluting with distilled water with the flow rate of 2mL/min, wherein the dosage is 20-40 mL; then eluting with 60-90% ethanol at a flow rate of 2mL/min, wherein the volume of the elution is 100-150 mL; collecting eluate to obtain purified total alkaloid solution of Alangium platanifolium.

2. The extraction separation and purification method according to claim 1, wherein the extraction separation and purification method comprises the following steps:

1) Preparation of a crude extract: precisely weighing 200g of alangium chinense medicinal material powder, placing in a round-bottom flask, adding 10 times of 80% ethanol, reflux-extracting for 2 times (2 h each time), filtering, and mixing the crude extractive solutions;

2) Preparing a sample solution: recovering ethanol from the crude extract by using a rotary evaporator, concentrating to 450-500 mL, adding a proper amount of 80% ethanol for dilution to prepare a solution with the concentration of 3.02mg/mL, and adjusting the pH value of the solution to 6 by using sodium hydroxide and hydrochloric acid to obtain a sample solution;

3) Separation and purification: loading macroporous adsorbent resin into a column by a wet method, taking 15mL of sample loading liquid, loading the sample at a flow rate of 1mL/min, collecting effluent liquid, and standing for 1 h; eluting with distilled water at flow rate of 2mL/min, with the amount of 30mL; eluting with 90% ethanol at a flow rate of 2mL/min, wherein the volume of the eluate is 120mL; collecting the eluent to obtain the purified solution of the total alkaloids of Alangium chinense.

3. The extraction, separation and purification method according to any one of claims 1 to 2, wherein the macroporous adsorbent resin in the separation and purification of step 3) is D101 type.

4. The extraction, separation and purification method according to any one of claims 1 to 2, wherein the wet column packing in the separation and purification of step 3) has a diameter-height ratio of 1:1 to 4.

5. The extraction, separation and purification method according to claim 4, wherein the wet column packing in the separation and purification of step (3) has a diameter-height ratio of 1:3.

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