CN100445726C - A kind of determination method of artemisinin content in Artemisia annua - Google Patents

Abstract

The invention relates to a method for determining the content of artemisinin in Artemisia annua. It is a kind of incomplete extraction of artemisinin in Artemisia annua to be tested. The content data M (mg/g) of artemisinin is measured by ultraviolet spectrophotometric analysis, and the liquid phase The linear model established by the corresponding relationship of the artemisinin content data measured by chromatography can be calculated to obtain the actual content X (mg/g) of artemisinin in the measured Artemisia annua. The invention simplifies the operation procedure, shortens the extraction time, and can complete the content determination of artemisinin within 1.5 hours. The required instrument is simple, easy to operate, saves reagents, and reduces pollution; due to the establishment of a related linear model , to ensure the reliability of the actual artemisinin content data obtained through the calculation process.

Description

A kind of determination method of artemisinin content in Artemisia annua

technical field

The invention relates to a method for determining the content of artemisinin in Artemisia annua, in particular to a method for determining the content of artemisinin in Artemisia annua by using ultraviolet spectrophotometry.

Background technique

Artemisinin and artemisinin-based antimalarial drugs such as artemether, dihydroartemisinin, and artesunate synthesized from artemisinin have been recommended by the World Health Organization as the best possible substitute for quinine in the treatment of malaria. Safe and effective medicines. Since the amount of artemisinin contained in Artemisia annua medicinal materials produced in different regions varies greatly (2‰～12‰), which has obvious ecological regional characteristics, it is necessary to carry out quality control on medicinal materials, especially when purchasing medicinal materials Therefore, both the supplier and the buyer need a method to quickly evaluate the quality of Artemisia annua, that is, a method for rapid determination of artemisinin content is urgently needed.

At present, there are many methods for the determination of artemisinin content at home and abroad. Such as high-performance liquid chromatography, thin-layer chromatography, supercritical fluid chromatography, and photometric enzyme-linked immunoassay. These methods are mainly used for the determination of artemisinin with high content and high purity, but require high equipment, The operation is complicated and time-consuming, which is not suitable for rapid on-site quality evaluation; in addition, there is ultraviolet spectrophotometry, which is mainly for the detection of artemisinin with high purity. Although the operation is simple, it takes a long time and is not suitable for direct on-site rapid quality evaluation. evaluate.

Contents of the invention

The purpose of the present invention is to provide a simple and quick method for detecting the content of Artemisia annua in Artemisia annua.

The object of the present invention is achieved in this way: a method for assaying the content of artemisinin in Artemisia annua, characterized in that: the artemisinin in Artemisia annua to be measured is not completely extracted, and the artemisinin is measured by ultraviolet spectrophotometric analysis The content data M (mg/g) is substituted into the linear model established by the corresponding relationship with the artemisinin content data measured by liquid chromatography after the complete extraction of artemisinin, and the measured artemisinin The actual content of artemisinin X (mg/g).

The incomplete extraction of artemisinin is extracted by ultrasonic extraction; the complete extraction of artemisinin is extracted by conventional Soxhlet extraction.

The linear model established by the corresponding relationship of the above artemisinin content data is X=(M+0.3995)/0.748.

The steps for incomplete extraction of artemisinin are as follows: Weigh 0.5±0.01g, 60-80 mesh samples of Artemisia annua powder to be tested, add 15ml of petroleum ether, and ultrasonicate for 12-17min at a temperature of 30-60°C. Record the volume V of petroleum ether; accurately measure 0.3ml of petroleum ether solution that has been ultrasonically treated in a 10ml graduated test tube, put it in boiling water until the petroleum ether is completely volatilized; then derivatize the remaining residue: add 3ml of 95% ethanol To dissolve, dilute to 10ml with 0.2% NaOH solution, mix well, and bathe in water at 50±1°C for 30min to obtain the sample solution to be tested.

The above-mentioned ultraviolet spectrophotometric analysis method measures the artemisinin content data Mmg/g to take the blank solution through derivatization as a reference, and at the ultraviolet spectrophotometer detection wavelength 292nm place, measure the artemisinin standard solution through derivatization and The absorbance of the sample solution to be tested, the measured absorbance is A _standard and A _sample respectively.

Calculate the content of artemisinin with the standard control method, and the calculation formula is:

M=(A _sample ×V×2)/(A _standard ×3)

Among them, A _sample is the measured absorbance value of the sample to be tested; V is the volume of petroleum ether in the sample after ultrasound, in ml; A _standard is the absorbance value of the 0.1 mg/ml artemisinin standard.

Derivatization of the above blank solution: dilute 3ml of 95% ethanol solution to 10ml with 0.2% NaOH solution, mix well, and then bathe in water at 50±1°C for 30min; derivation of the artemisinin standard solution: Add 2ml of 95% ethanol solution to 1ml of artemisinin standard 0.1mg/mL solution, dilute to 10ml with 0.2% NaOH solution, mix well, then put in water bath at 50±1℃ for 30min, then cool to room temperature, and use 0.22 obtained by filtration through a μm filter membrane.

The present invention replaces the traditional long-time reflux (usually 15 to 30 hours) method of extracting Artemisia annua with ultrasonic quick incomplete extraction, simplifies the operation procedure, shortens the extraction time, and directly measures the crude The determination of artemisinin in the extract can be completed within 1.5 hours. The required instrument is simple, easy to operate, saves reagents, and reduces pollution; The correlation linear model of Soxhlet (complete) extraction-high performance liquid chromatography (HPLC) detection data of artemisinin has guaranteed the reliability of the artemisinin content data that calculation process obtains, and therefore, the present invention can really be realized in Artemisia annua Rapid detection of artemisinin content is carried out at the site of medicinal material procurement.

Description of drawings

Fig. 1: The linear regression graph of the artemisinin content data detected by the incomplete extraction-ultraviolet spectrophotometric analysis method and the Soxhlet (complete) extraction-high performance liquid chromatography method of the present invention.

Figure 2: Variation curve of extraction time and artemisinin content in incomplete extraction.

Detailed ways

Embodiment 1: select 22 kinds of Artemisia annua medicinal materials with different artemisinin content and divide them into duplicates, one part adopts the method of incomplete extraction and ultraviolet spectrophotometric analysis of the present invention to measure the artemisinin content data M, and the other part adopts index The artemisinin content data Ms was measured by the liquid phase detection method, and then the two sets of data were subjected to linear regression analysis to obtain a related linear model.

1. Implementation of incomplete extraction and UV spectrophotometry

1.1 Instruments and reagents

Balance (0.01g), KQ2200B type ultrasonic cleaner, 150ml conical flask with stopper, 10ml graduated test tube with stopper, 25ml measuring cylinder, electric furnace, constant temperature water bath, 752 ultraviolet spectrophotometer, 0.22μm water-based filter, petroleum ether (30 ~60°C), 95% ethanol, 0.2% sodium hydroxide (NaOH) solution.

1.2 Test steps

1.2.1 Solution preparation:

a. Artemisinin standard solution: Accurately weigh 10mg of artemisinin standard substance into a 10ml volumetric flask, add 95% ethanol to dissolve and dilute, and dilute to 10ml to obtain a 1mg/ml reference substance stock solution, and store in a refrigerator at 4°C;

b. 0.2% NaOH solution: Accurately weigh 1g of NaOH into a 500ml reagent bottle, add 500ml of distilled water to dissolve, mix well, and store at room temperature;

1.2.2 Preparation of blank and standard solutions:

a. Preparation of blank solution: Take 3ml of 95% ethanol solution in a 10ml graduated test tube and dilute to 10ml with 0.2% NaOH solution, mix well to obtain blank solution;

b. Preparation of standard solution: draw 1ml of the stock solution into a 10ml graduated test tube and mix well to obtain a 0.1mg/ml standard solution.

1.2.3 Preparation of Artemisia annua sample solution.

a. Take Artemisia annua and put it into a pulverizer to pulverize, pass through 80 and 60 mesh sieves respectively, and obtain 60-80 mesh Artemisia annua powder;

b. Weigh 0.5-40.01g of Artemisia annua sample (60-80 mesh), add 15ml of petroleum ether (30-60°C) into 150ml with a stopper cone, and sonicate for 15min;

c. After ultrasonication, pour the petroleum ether solution into the graduated cylinder, and record the volume V of petroleum ether;

d. Precisely measure 0.3ml of the petroleum ether solution that has been ultrasonically treated in a 10ml graduated test tube, put it into boiling water that is no longer heated, and evaporate to dryness.

1.2.4 Derivation of Artemisinin

a. Add 3ml of 95% ethanol to the evaporated test tube in 1.2.3 and d to dissolve the residue, dilute to 10ml with 0.2% NaOH solution, mix well, and bathe in water at 50±1℃ for 30min;

b. Derivation of the blank solution: Take the blank solution and put it in a water bath at 50±1°C for 30 minutes;

c. Standard solution derivation: Take 1ml of 0.1mg/ml standard solution in a 10ml test tube, add 2ml of 95% ethanol solution, 0.2% NaOH solution to 10ml, mix well, and bathe in 50±1℃ water bath for 30min.

After derivatization, cool to room temperature with cold water, filter through a 0.22 μm filter membrane, and use the filtrate to measure the absorbance with an ultraviolet spectrophotometer.

1.2.5 Detection of Artemisinin

Using the blank solution as a reference, measure the absorbance of the artemisinin standard solution and the Artemisia annua sample solution at a detection wavelength of 292nm, and the measured absorbance is A _standard and A _sample , respectively.

1.3 Calculation of artemisinin content

Calculate the content of artemisinin with the standard control method, and the calculation formula is:

M(mg/g)＝(A _sample ×V×2)/(A _standard ×3)

In the above formula: A _sample is the measured value of the Artemisia annua sample solution; V is the volume of petroleum ether of the Artemisia annua sample after ultrasound, in ml, and the A _standard is the absorbance of the 0.1 mg/ml artemisinin standard solution.

The artemisinin content M data of 22 different Artemisia annua medicinal materials measured by the above detection method are shown in Table 1.

The measurement result of table 1 sample

Sample serial number Soxhlet liquid phase content Ms(mg/g) Ultrasonic UV content M (mg/g) 1 9.29 6.77 2 7.83 5.94 3 7.8 6.01 4 7.2 4.7 5 10.17 7.52 6 4.77 3.27 7 6.62 4.53 8 10.77 7.81 9 7.61 4.65 10 6.54 4.6 11 8.19 5.29 12 6.04 4.27 13 5.5 3.68 14 5.37 3.89 15 7.22 4.71 16 7.64 4.81 17 7.5 4.75 18 5.54 3.75 19 7.38 5.37 20 5.14 3.65 twenty one 7.49 5.07 twenty two 8.15 5.54

2. Implementation of Soxhlet liquid phase detection method

2.1 Instruments and reagents

Soxhlet extractor, American Agilent 1100 high performance liquid chromatograph, anhydrous sodium acetate, glacial acetic acid, acetonitrile, double distilled water.

2.2 Test steps

2.2.1 Soxhlet extraction of Artemisia annua: Take 1g of Artemisia annua sample and wrap it with filter paper. As for the Soxhlet extractor, add 70ml of petroleum ether (30-60℃) (a certain amount of petroleum ether can be added as appropriate during the water bath) , Soxhlet extraction in a water bath at 65±1°C for 30 hours. After the extraction, measure the volume V of the sample solution, take 0.5ml of the sample solution, and evaporate it to dryness in unheated boiling water.

2.2.2 Derivatization of artemisinin: Add 3ml of 95% ethanol to the evaporated test tube to dissolve the residue, dilute to 10ml with 0.2% NaOH, mix well, and bathe in water at 50±1°C for 30min.

2.2.3 Detection of artemisinin: after derivatization, cool to room temperature with cold water, take quantitative derivatization solution and mix equal volumes with 0.08mol/l acetic acid solution, inject 50 μl of sample, and perform HPLC detection.

Chromatographic conditions: Chromatographic column: Agilent Eclipse XDB C8 column (5μm, 4.6×150mm) mobile phase acetonitrile: acetate buffer (adjust the pH value to 5.8) = 23:77 Temperature: 30°C Flow rate: 1ml/min Wavelength: 260nm

2.3 Processing of test results:

Calculation formula of artemisinin content:

Ms(mg/g)=(A _sample ×V×2)/(A _standard ×5)

Among them, the A _sample is the peak area value of the Artemisia annua sample, V is the volume of the Artemisia annua sample, and the A _standard is the peak area value of the 0.1 mg/ml artemisinin standard product.

The artemisinin content Ms data of 22 different Artemisia annua medicinal materials measured by the above detection method are shown in Table 1 above.

3. The establishment of a linear model for the artemisinin content data measured by incomplete extraction, ultraviolet spectrophotometric analysis and Soxhlet liquid phase detection.

The two groups of data in Table 1 were subjected to linear regression analysis to obtain a related linear model, that is, the formula for calculating the true content of artemisinin in Artemisia annua is X=(M+0.3995)/0.748.

In the formula: X is the actual content of artemisinin in Artemisia annua, the unit is mg/g; M is the content of incompletely extracted artemisinin, the unit is mg/g.

Example 2: In this example, except that the time for the incomplete extraction of the same Artemisia annua medicinal material is 5, 12, 15, 20, 25, and 30 minutes, the other methods are the same as those in Example 1 for the incomplete extraction and ultraviolet spectroscopy. The detection method of the photometric analysis method is the same. The measured artemisinin content data of the same Artemisia annua medicinal material at different extraction time conditions are shown in Table 2, and the change curve of extraction time and artemisinin content for incomplete extraction is shown in Figure 2. It can be seen from Figure 2 that the measured artemisinin content data is basically stable within the extraction time range of 12 to 17 minutes.

Table 2

Extraction time min 5 12 15 20 25 30 Artemisinin content mg/g 0.94 1.7 1.74 1.75 1.86 2.0

Claims (2)

1, the assay method of artemislnin content in a kind of sweet wormwood, it is characterized in that: not exclusively extract the qinghaosu in the sweet wormwood to be measured, adopt the ultraviolet spectro-photometric analysis method to measure artemislnin content data M mg/g, substitution by with qinghaosu is extracted fully the linear model that the corresponding relation that adopts the artemislnin content data that liquid phase chromatography measures in the back is set up, calculate the actual content Xmg/g of the qinghaosu in the sweet wormwood of surveying;

Described qinghaosu not exclusively extracts and adopts the ultrasonic extraction method to extract; Described qinghaosu extracts fully and adopts soxhlet extraction to extract;

The linear model that the corresponding relation of described artemislnin content data is set up is X=(M+0.3995)/0.748;

The incomplete extraction step of described qinghaosu is as follows: weighing 0.54 ± 0.01g, and 60～80 orders sweet wormwood powder to be measured sample adds the 15ml sherwood oil therein, is under 30～60 ℃ of conditions in temperature, ultrasonic 15min, record sherwood oil volume V; Precision is measured the petroleum ether solution 0.3ml that crosses through sonicated in 10ml scale test tube, puts into boiling water and volatilizees fully until sherwood oil; The residue of retaining is derived: 95% ethanol that adds 3ml dissolves again, and uses 0.2%NaOH solution constant volume to 10ml, mixing, and water-bath 30min under 50 ± 1 ℃ of temperature gets testing sample solution;

It is being reference through the blank solution of derivation process that described ultraviolet spectro-photometric analysis method measures artemislnin content data M mg/g, at UV spectrophotometer measuring wavelength 292nm place, measure qinghaosu standard solution and testing sample solution absorbance, record absorbance and be respectively A through derivation process _StandardAnd A _Sample

Calculate artemislnin content with criterion keying method, computing formula is:

M=(A _Sample* V * 2)/(A _Standard* 3)

Wherein, A _SampleAbsorbance for the testing sample that records; V is the volume of ultrasonic back sample sherwood oil, in ml; A _StandardAbsorbance for 0.1mg/ml qinghaosu standard items.

2, the assay method of artemislnin content in the sweet wormwood as claimed in claim 1, it is characterized in that: the derivation process of described blank solution: with the 3ml95% ethanolic solution, with 0.2%NaOH solution constant volume to 10ml, mixing, water-bath 30min under 50 ± 1 ℃ of temperature again; Deriving of described qinghaosu standard solution: will contain and add the 2ml95% ethanolic solution in the 1ml solution of qinghaosu standard items 0.1mg/ml, with 0.2%NaOH solution constant volume to 10ml, mixing, water-bath 30min under 50 ± 1 ℃ of temperature again, be cooled to room temperature then, get with 0.22 μ m membrane filtration.

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