CN110967416B - Method for measuring asaricin in serum-nourishing brain water extract - Google Patents

Abstract

The invention relates to a method for measuring asaricin in a brain nourishing water extract, which comprises the following steps: 1) Preparation of control solutions: weighing a proper amount of asaricin reference substance, and adding methanol to dissolve to obtain a reference substance solution containing asaricin; 2) Preparation of a test solution: taking the blood-nourishing and brain-clearing water extract, adding 40-60% (v/v) methanol, ultrasonically dissolving, passing through a solid phase extraction column, washing with 50-60% methanol, discarding, eluting with pure methanol solution, collecting eluate, and adding methanol to scale to obtain sample solution; 3) And (3) detection: and respectively taking the reference solution and the test solution, injecting the reference solution and the test solution into an ultra-high performance liquid chromatograph, recording a chromatogram, calculating the content of the asarone in the test solution by using an external standard method according to the peak area, and converting to obtain the content of the asarone in the blood-nourishing brain-clearing water extract.

Description

Method for measuring asaricin in serum-nourishing brain water extract

Technical Field

The invention relates to a method for detecting and analyzing medicinal components, in particular to a method for determining asaricin in a brain nourishing water extract.

Background

The blood-nourishing and brain-refreshing granules and the blood-nourishing and brain-refreshing pills are Chinese patent medicines developed by the Tianshili group and are respectively recorded on pages 1281-1282 and 1280-1281 of 2015 part of Chinese pharmacopoeia. During preparation of the blood-nourishing brain-refreshing granules and the blood-nourishing brain-refreshing pills, traditional Chinese medicinal materials such as prepared rehmannia root, uncaria, suberect spatholobus stem, common selfheal fruit-spike, nacre, asarum and the like need to be extracted to prepare a traditional Chinese medicine extract, the traditional Chinese medicine extract is one of various preparation intermediates of the blood-nourishing brain-refreshing granules and the blood-nourishing brain-refreshing pills, and the preparation method comprises the following steps: decocting radix rehmanniae Preparata, ramulus Uncariae cum uncis, caulis Spatholobi, prunellae Spica, concha Margarit and herba asari with water twice, filtering, concentrating the filtrate to fluid extract, adding appropriate amount of ethanol, standing, recovering ethanol, and concentrating to appropriate amount.

The blood nourishing and brain refreshing granule and pill comprise herba asari which is dried root and rhizome of Aristolochiaceae plant such as Asarum heterotropoides, asarum sieboldii Hancheng or Asarum sieboldii. Collected in the fruit ripening period or early autumn in summer, the overground part and silt are removed, and the soil is dried in the shade to obtain the fertilizer. Pungent and warm in nature. It enters heart, lung and kidney meridians. Has the effects of relieving exterior syndrome, dispelling cold, dispelling pathogenic wind, relieving pain, inducing resuscitation, warming lung, and eliminating fluid retention. Can be used for treating wind-cold type common cold, headache, toothache, nasal obstruction, watery nasal discharge, nasosinusitis, rheumatalgia, phlegm retention, asthma, and cough.

The main components of herba asari include volatile oil, lignanoids, amino acids, etc. Wherein the volatile oil and lignans are main effective components. The lignanoid component of herba asari has antiviral and anti-tubercle bacillus effects, and is typically asarinin. Asarone is also an index for detection in pharmacopoeia (the first part of the 2015 edition, page 230-231 of the Chinese pharmacopoeia).

Asarinin (abbreviated as ASA), also known as (-) -asarinin, CAS:133-04-0, is lignanoid extracted from herba asari, and can be used as synergist for killing parasite and resisting virus and tubercle bacillus.

The extraction method of asarin is disclosed in Guo Zengjun, etc. and Zhang Shuixin, which uses 80% ethanol to heat and reflux for 120min, then uses ethyl acetate to make ultrasonic vibration extraction for 10min (Guo Zengjun, etc., HPLC method is used to measure the content of asarin and sesamolin in different varieties of commercial asarum, the Chinese medicinal material, no. 24, no. 4, no. 2001, 04: 273-274), or uses ethyl acetate and petroleum ether to make recrystallization (Zhang Shui Xin, the process research for separating asarin from asarum, TCM academy of traditional Chinese medicine, vol 31, no. 213, no. 2, no. 2016-257)

Methods for the detection of asaricin are also disclosed in Zhang Lei et al, which discloses a method for the simultaneous determination of L-asaricin, L-sesamin and carcumyl alcohol in asarum sieboldii, wherein the mobile phase in chromatographic conditions is acetonitrile and water in the range of 50:50 percent of the components. Ultrasonic dissolving test sample with ethyl acetate (Zhang Lei, etc. by RP-HPLC methodA method for preparing L-asarone, L-sesamin and carcumyl alcohol from herba asari comprises the following steps of (1) Chinese herbal medicine, no. 7 of volume 39, no. 7 of 2008: 1098-1100). Wang Guifang discloses a method for measuring the content of sesamin and asarone in asarum by an HPLC method, and the chromatographic conditions are as follows: hypersil BDS C18 chromatographic column, mobile phase acetonitrile and water (50), flow rate of 1.2mL/min, detection wavelength of 287, column temperature of 27-29 ℃; processing method of reference solution, wherein 4mg of ASA reference is added into 50mL of methanol; sample treatment: refluxing herba asari with ethyl acetate. (Wang Guifang, HPLC method for determining sesamin and asafetida content in Asarum heterotropoides, J.P.Analyzae, vol.19, no. 4, 1999: 251-253). Wu Guoqing et al disclose a method for determining contents of imperatorin and asaricin in liniment for relieving pain and removing blood stasis by using RP-HPLC method, wherein the method for determining the content of asaricin comprises the following steps: chromatographic conditions in Kromasil 100-5 C18 column, mobile phase in acetonitrile (B) and water (A), gradient conditions of 0-20min,50% B;20-26min,50-100% B, flow rate 1.0mL/min. In the preparation of the test solution, methanol is used for dissolving, centrifugation is carried out, the solvent is recovered under reduced pressure until the solution is dry, and then methanol is used for constant volume (Wu Guoqing and the like, and the RP-HPLC method is used for measuring the contents of imperatorin and asafetida in the pain-relieving and blood stasis-removing liniment, the journal of Chinese medicine, vol.32, no. 2 in 2014, 402-404). Zhang Shui Xin discloses a method for measuring the content of asafetida, wherein the chromatographic conditions are as follows: sunAire C ₁₈ Chromatography column (4.6 mm × 150mm,5 μm), mobile phase acetonitrile and water in a volume ratio of 55:45, the detection wavelength is 287nm, the column temperature is 30 ℃, and the flow rate is 1.0mL/min; sample preparation, taking asarum powder 0.5g, adding methanol 15mL, and performing ultrasonic treatment (power 500W, frequency 40 kHz) for 45min. (Zhang Shui Xin, research on the process of separating asarinin from Asarum sieboldii, reported in TCM, vol.31, no. 213, no. 2, 2016: 255-257).

However, in the detection method disclosed by the prior art, a target detection sample has single medicinal taste, the extraction and purification effects are poor, the chromatographic separation degree is low, the precision and the accuracy are not ideal, and the determination of the asaricin in the blood-nourishing and brain-refreshing water extract cannot be realized. The ultra-high performance liquid chromatography is adopted as a content determination method, the separation efficiency is high, and the analysis speed is high.

Disclosure of Invention

The invention relates to a blood-nourishing and brain-refreshing water extract, which is an intermediate of a preparation of blood-nourishing and brain-refreshing granules and blood-nourishing and brain-refreshing pills and is an extract of traditional Chinese medicinal materials such as prepared rehmannia root, uncaria, suberect spatholobus stem, common selfheal fruit-spike, nacre, asarum and the like.

The invention establishes a method for measuring the content of the asarone in the brain-nourishing water extract for further improving the quality control level of the product and comprehensively mastering the product quality.

The invention provides a method for measuring asaricin in a brain nourishing water extract, which comprises the following steps:

1) Chromatographic conditions are as follows: using a UPLC system, using an octadecyl bonded silica gel chromatographic column, with water: acetonitrile =50:50 is a mobile phase, and the flow rate is 0.3-0.5mL/min; detection wavelength: 280-295nm;

2) Preparation of control solutions: weighing a proper amount of asaricin reference substance, and adding methanol to dissolve to obtain a reference substance solution containing asaricin;

3) Preparation of a test solution: taking the blood-nourishing and brain-clearing water extract, adding 40-60% (v/v) methanol, ultrasonically dissolving, passing through a solid phase extraction column, washing with 50-60% methanol, discarding, eluting with pure methanol solution, collecting eluate, and adding methanol to scale to obtain sample solution;

4) And (3) detection: and respectively taking the reference solution and the test solution, injecting the reference solution and the test solution into an ultra-high performance liquid chromatograph, recording a chromatogram, calculating the content of the asarone in the test solution by using an external standard method according to the peak area, and converting to obtain the content of the asarone in the blood-nourishing brain-clearing water extract.

Wherein:

in the preparation of the test solution:

the solid phase extraction column is ProElut C18-U or Sep-Pak Vac C18;

the dosage of the solid phase extraction column is 0.5-1.5 times of the weight of the blood-nourishing brain-clearing water extract;

the methanol concentration for sample dissolution was 50%, and the methanol concentration for washing was 60%.

Preferably, the detection method of the present invention comprises the following steps:

1) Chromatographic conditions are as follows: using a UPLC system, using an octadecyl bonded silica gel chromatographic column, with water: acetonitrile =50:50 is a mobile phase, and the flow rate is 0.3-0.5mL/min; detection wavelength: 280-295nm;

2) Preparation of control solutions: accurately weighing appropriate amount of asaricin control, respectively, adding methanol to dissolve, and making into solution containing 0.01mg of asaricin per ml to obtain;

3) Preparation of a test solution: taking about 0.5g of blood-nourishing and brain-refreshing water extract, adding 10mL of 50% methanol, ultrasonically dissolving, shaking up, loading to a solid phase extraction column, washing with 10mL of 50-60% methanol, discarding, eluting with 5mL of pure methanol solution, collecting eluent, adding methanol to scale, and shaking up to obtain the final product;

4) And (3) detection: and respectively taking the reference solution and the test solution, injecting the reference solution and the test solution into an ultra-high performance liquid chromatograph, recording a chromatogram, calculating the content of the asarone in the test solution by using an external standard method according to the peak area, and converting to obtain the content of the asarone in the blood-nourishing brain-clearing water extract.

The chromatographic conditions were as follows:

the octadecyl bonded silica gel chromatography column is preferably a Waters HSS T3C 18 (2.1 x 100mm);

the flow rate is 0.4mL/min;

the detection wavelength was 287nm.

The preferred experimental method, chromatographic conditions and sample pretreatment method of the invention are obtained by screening. The chromatographic condition adopts ultra-high performance liquid chromatography, the mobile phase is eluted at equal degrees, the method is simple, and the analysis efficiency is improved.

The screening process is as follows:

(first) screening of sample pretreatment method

The sample pretreatment method adopts a solid phase extraction technology, can accurately and efficiently remove interfering components by screening solid phase extraction materials, and can effectively enrich the asaricin, thereby embodying the creativity of the application.

1. Solid phase extraction material and specification screening:

the blood-nourishing brain-refreshing water extract is prepared from medicinal materials such as prepared rehmannia root, uncaria, suberect spatholobus stem, common selfheal fruit-spike, nacre mother of pearl, manchurian wildginger and the like, has very complex components, and contains a large amount of components which interfere the determination of asarum lipid, such as saccharides, organic acid, alkaloid, saponin and the like. The method cannot be used for detection by adopting the existing literature and published measuring methods, so that the sample must be separated and purified in a targeted manner to realize accurate measurement of the asaricin.

Solid phase extraction columns which respectively adopt silica gel bonding and high molecular polymers as adsorbents are used for separating and purifying blood-nourishing and brain-refreshing water extract, and commercial columns with different specifications and models are compared, wherein the specific information of various SPE columns is as follows:

table 1: comparison of trade bars of different specifications and models

Test number	SPE adsorbent	SPE species	SPE model	Is commonly used in	SPE Specification
						1	Silica gel bonding	Adsorption	ProElut C18	Extraction of non-polar and weakly polar moderately polar compounds	500mg/6mL
2	Silica gel bonding	Adsorption	ProElut C18-U	Polar and non-polar compound extraction	500mg/6mL
						3	Silica gel bonding	Adsorption	Cleanert SC-18	Extraction of non-polar and weakly polar compounds	500mg/6mL
4	Silica gel bonding	Adsorption	Cleanert SC-18-N	Polar and non-polar compound extraction	500mg/6mL
						5	Silica gel bonding	Adsorption	Sep-Pak Vac tC18	Extraction of non-polar and weakly polar compounds	500mg/6mL
6	High molecular polymer	Adsorption	ProElut PLS	Extraction of non-polar and polar compounds	500mg/6mL
						7	Silica gel bonding	Adsorption	ProElut NH2	Weak anion and polar compound extraction	500mg/6mL
8	High molecular polymer	Ion exchange	ProElut PXA	Extraction of anionic and non-polar compounds	500mg/6mL
						9	High molecular polymer	Ion exchange	ProElut PXC	Weak cation and polar compound extraction	500mg/6mL
10	High molecular polymer	Ion exchange	ProElut PWA	Extraction of anionic and non-polar compounds	500mg/6mL
						11	Silica gel bonding	Adsorption	Sep-Pak Vac C18	Polar and non-polar compound extraction	500mg/6mL

1.1 solid phase extraction Material screening chromatography conditions

An ACQUITY UPLC HSS T3C 18 (2.1 x 100mm) column was used, and a UPLC chromatography system was used. Performing gradient elution according to the table 2 by using 0.5% formic acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B; detection wavelength: 287nm, 254nm and 320nm.

Table 2: gradient elution chart

1.2 sample treatment:

taking about 0.2g of the blood-nourishing and brain-refreshing water extract, placing the extract in a beaker, adding 10ml of water, ultrasonically dissolving, shaking up, and respectively loading to a solid phase extraction column. Gradient elution is carried out by adopting methanol with different concentrations, eluent is collected and is injected respectively, and the separation condition of components is examined. 1.3 screening results

According to the chromatogram map, the separation conditions of SPE columns with different specifications are compared, and the result (shown in figure 1) shows that: proElut C18-U type and Sep-Pak Vac C18 type SPE columns can effectively purify samples, and a asaricin chromatographic peak is separated from other chromatographic peaks, but the other types of SPE columns cannot be separated.

2. Solid phase extraction parameter optimization

The solid phase extraction separation and purification parameters are optimized respectively aiming at the method for measuring the asaricin, the strength of the elution solvent, the separation and purification cut point and the volume of the elution solvent are respectively considered, and the optimal separation and purification parameters are determined.

2.1 solid phase extraction parameters optimization of chromatographic conditions

An ACQUITY UPLC HST 3C 18 (2.1 x 100mm) column was used, with a UPLC chromatography system. Performing gradient elution according to the table 2 by using 0.5% formic acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B; detection wavelength: 287nm, 254nm and 320nm.

2.2 sample treatment:

taking about 0.2g of the blood-nourishing and brain-refreshing water extract, placing the extract in a beaker, adding 10ml of water, ultrasonically dissolving, shaking uniformly, and respectively feeding the extract to a ProElut C18-U solid phase extraction column. Eluting according to the solvent gradient elution conditions in Table 3, collecting the eluates, injecting sample, and examining the separation condition of the components. And determining the optimal SPE separation and purification parameters through chromatogram comparison.

Table 3: gradient elution conditions of different solid phase extraction

2.3 screening results

Comparing chromatograms of different solid phase extraction gradient elution conditions, wherein the result is shown in fig. 2, and finally determining the solid phase extraction gradient elution conditions as follows by integrating purification effects of different gradient elution conditions: dissolving sample with 50% methanol (to remove interference components such as saccharide and organic acid), washing with 50-60% (preferably 60%) methanol (to remove interference components such as alkaloid and saponin), eluting with methanol solution, and analyzing by sample injection.

(II) screening of chromatographic conditions

1. The sample pretreatment method comprises the following steps:

precisely weighing about 0.5g of blood-nourishing and brain-refreshing water extract, adding about 10ml of 50% methanol, ultrasonically dissolving, shaking uniformly, and feeding to a ProElut C18-U solid phase extraction column. Washed with 5ml of 60% methanol and discarded. Eluting with 5mL of methanol solution, collecting eluent to a 5mL volumetric flask, adding methanol to the mark, and shaking up to obtain the product.

2. Chromatographic column screening

The determination of the asaricin in the extract of the water extracts of blood-nourishing and brain-clearing is carried out by respectively adopting Waters HSS T3C 18, waters BEH C18 and Waters CSH C18 chromatographic columns, and the result is shown in a way of optimization (shown in a figure 3): the separation efficiency of the Waters HSS T3C 18 (2.1 x 100mm) column was the best.

3. Detection wavelength selection

From the full wavelength scan of asaricin, the maximum detection wavelength was 287nm. According to the ultraviolet absorption characteristics of main interference components, sample separation degrees are examined at 203nm, 254nm, 287nm and 334nm respectively, and chromatogram analysis shows that the absorption degree of the asaricin is high at 287nm, the separation degree meets the requirement, and the absorption degree of the asaricin is very low under other wavelength conditions. Therefore, 287nm was chosen as the optimal detection wavelength.

4. Mobile phase selection

The method is characterized in that a water phase is a mobile phase A, acetonitrile is used as a mobile phase B, organic acids with different concentrations are respectively added into the phase A to optimize the peak shape, the result shows that the peak shape of a pure water phase is good, the peak shape is not obviously improved by adding the organic acids, and therefore water and the acetonitrile are selected as the mobile phases.

5. Mobile phase elution condition optimization

In order to ensure chromatographic peak separation degree and improve separation efficiency, the mobile phase elution conditions are optimized, 3 gradient and isocratic elution conditions are respectively tested (see tables 4-1, 4-2 and 4-3), the results are shown in a result (figure 4), and the condition 3 can improve separation efficiency and ensure chromatographic peak separation degree, and is isocratic elution, so that the method is simpler, and the final gradient elution method is determined.

Table 4-1: gradient elution conditions 1

Tables 4-2: gradient elution Condition 2

Tables 4 to 3: isocratic elution conditions 3

(III) methodological validation

1. Linearity and range

Precisely weighing appropriate amount of asaricin reference substance, adding methanol to obtain reference solution containing asaricin 0.4mg per ml, adding mixed reference solutions of 0.05, 0.1, 0.2, 0.4, and 1ml, placing into 10ml measuring flask, adding methanol to scale, mixing, making into series solutions, injecting into liquid chromatograph, and measuring to obtain the result shown in Table 5. The measured peak areas were subjected to linear regression with concentration (see fig. 5), to find a regression equation, a linear relationship determination criterion: the correlation coefficient should be more than or equal to 0.999 (see Table 6 for details).

Table 5: different concentrations of asaricin and the measured peak areas

Concentration (mg/ml)	0.002116	0.004232	0.01058	0.02116	0.04232	0.2116
							Peak area	18220	25043	78113	154496	270242	1421701

Table 6: regression equation, correlation coefficient and linear range

And (4) conclusion: in the method, the peak area and the concentration of the asaricin are in a good linear relation in the concentration range. 2. Precision of sample introduction

Sampling, preparing a test sample solution according to the method, repeatedly injecting samples for 6 times, recording the area of the peak of the asaricin, and calculating the relative standard deviation.

Table 7: peak area obtained by repeated sample introduction of same batch of samples

Serial number	1	2	3	4	5	6	RSD％
								Peak area	34454	34600	34621	34623	34585	34950	0.5％

And (3) judging standard: the peak area RSD of 6 times of sample injection is less than or equal to 2.0 percent.

And (4) conclusion: the precision of sample introduction meets the verification requirement.

3. Repeatability examination

Taking samples, preparing 6 parts of test sample solution according to the method, respectively measuring the content of the asaricin, and calculating the relative standard deviation.

Table 8: repeatedly sampling the same sample to obtain the content of the asaricin

Serial number	1	2	3	4	5	6	Average	RSD％
									Content mg/g	0.0655	0.0638	0.0646	0.0674	0.0659	0.0654	0.0655	1.8％

And (3) judging standard: the RSD of the content determination result of 6 samples is less than or equal to 2.0 percent.

And (4) conclusion: the repeatability of the determination method meets the verification requirement.

4. Intermediate precision

Two detection personnel prepare 2 test sample solutions in parallel at different time, sample introduction and measurement are respectively carried out on two different instruments, 2 times of measurement results are analyzed, and the RSD value of measurement is calculated.

Table 9: detecting samples by different detectors at different times

And (4) judging standard: the RSD of the two measurement results is less than or equal to 2.0 percent.

And (4) conclusion: the RSD of the two measurement results is less than 2.0 percent, and the measurement method meets the requirement of intermediate precision.

5. Recovery rate

Precisely weighing 6 parts of sample, each part is about 0.4g, precisely adding 1.5ml of control solution (containing 0.01058mg of asaricin per ml) and about 8ml of 40% methanol respectively, treating according to the method under the content determination term from the ultrasonic dissolution to serve as test solution, taking 2ul of the test solution, injecting into a chromatograph, and respectively calculating the recovery rate of asaricin.

Table 10: experimental results of recovery

Sample recovery rate verification and judgment standard: the average sample recovery rate should be 95% -105%, and the RSD should be less than or equal to 3.0%.

And (4) conclusion: the average sample recovery rate of the asaricin is 96.1 percent, the RSD is less than or equal to 3.0 percent within 95 to 105 percent, and the method meets the verification requirement.

6. Sample stability

Sampling the test solution for 0, 2, 4, 8, 12 and 18h respectively, recording the peak area of the asaricin, and calculating the relative standard deviation.

Table 11: results of sample stability experiments

Time

0h

2h

4h

8h

12h

18h

RSD％

Peak area

51505

50779

50784

51924

50895

51297

0.9％

And (3) judging standard: when the peak area RSD of the sample solution is less than or equal to 2.0 percent, the solution is stable.

And (4) conclusion: the peak area RSD of the sample solution in 18 hours is less than or equal to 2.0 percent, which shows that the sample solution has good stability in 18 hours.

7. Durability of chromatographic conditions

The column durability test was carried out by the above-mentioned measuring method using the above-mentioned ACQUITY BEH C18 (2.1X 100mm) and ACQUITY CSH C18 (2.1X 100mm), respectively, and the results of 2 measurements were analyzed to calculate the RSD value measured.

Table 12: durability test results of chromatographic conditions

And (3) judging standard: the RSD of the three chromatographic column measuring results is less than or equal to 2.0 percent.

And (4) conclusion: the RSD of the three chromatographic column measuring results is less than 2.0 percent, and the RSD can be used for the measurement of the method.

8. Method specificity

Preparing blood-nourishing brain-refreshing negative water extract of default asarum medicinal materials, and measuring according to a asarin measuring method. And examining the specificity of chromatographic peaks. The chromatogram is shown in FIG. 6:

the results show that: the negative control sample has no corresponding chromatographic peak at the position corresponding to the asaricin, which indicates that the chromatographic peak is asaricin.

9. Verification conclusion

The verification result shows that the measuring method has good linear relation, durability and recovery rate, the precision meets the requirement, and the sample has good stability within 18 hours. The determination method meets the requirement of content determination, and can be used for determining the content of the asarone in the water extract for nourishing blood and refreshing brain.

The method of the invention has the following beneficial effects:

1. the blood-nourishing and brain-refreshing water extract is prepared by extracting six traditional Chinese medicines, and has very complex components. Meanwhile, the asarum accounts for a small proportion in the prescription due to toxic and side effects, and the content of asarinin in the medicinal materials is low. The asaricin is a fat-soluble component, and is extracted with other components by water (the extraction rate of aristolochic acid toxic substances is reduced by adopting water extraction), so that the content of the asaricin in the blood-nourishing and brain-refreshing water extract is extremely low (about 0.01 percent), and great difficulty is brought to content measurement. In addition, because the traditional Chinese medicine components are complex, the chromatographic baseline is raised, and the chromatographic behavior of low-content components is covered, the detection difficulty is increased. In conclusion, the difficulty of the method for detecting asarum extract alone increases compared to the method for detecting asarum extract alone.

2. Most of the existing literatures are used for measuring asarin in asarum medicinal materials, the content of asarin is dozens of times higher than that of a detection sample, and the purification difficulty is greatly reduced.

The blood-nourishing brain-refreshing water extract is prepared from radix rehmanniae Preparata, ramulus Uncariae cum uncis, caulis Spatholobi, prunellae Spica, concha Margaritifera, herba asari, etc., and contains a large amount of interfering components such as saccharides, organic acids, alkaloids, saponins, etc. The sample must be separated and purified to realize the accurate determination of the asaricin. The greatest difficulty of the determination method lies in the purification process of the sample, and the interference on the determination of the asaricin is reduced to the greatest extent. The purification difficulty is small.

According to the invention, through a large number of experimental researches, the most suitable solid phase extraction and purification material is screened out, and the asaricin component can be effectively purified and enriched. And through a large amount of experiments and data analysis, the optimal solid phase extraction elution parameters are optimized, and the interference components in the determination of the asaricin are effectively removed. The interfering components such as saccharides and organic acids are removed by 50% methanol sample solution, and the interfering components such as alkaloids and saponins are removed by 60% methanol washing. Finally, the optimal separation and purification effect of the asaricin is realized.

3. The method in the prior art is not suitable for measuring the blood-nourishing and brain-refreshing aqueous extract, has low content and is interfered by other substances, so that the detection result of other methods cannot be conjectured.

The method adopts the reverse phase silica gel solid phase extraction method as the pretreatment method, and is more convenient to operate compared with other pretreatment methods such as a solvent extraction method, the extraction material is more abundant in selection, and the parameter adjustment is more flexible. Can effectively separate and purify the water extract for nourishing blood and refreshing brain, and can enrich the asaricin, thereby reducing the interference of other components to the determination to the maximum extent. Meanwhile, the method only uses methanol as a reagent, thereby effectively reducing the use of organic reagents. Meanwhile, the operation is simpler, the solid phase extraction column is a commercial product, and the uniformity is better.

Compared with the high performance liquid chromatography, the ultra-high performance liquid chromatography is higher in separation efficiency and higher in analysis speed, and can save detection cost and analysis time.

4. The method adopts ultra-high performance liquid chromatography for measurement, can complete the measurement of the asaricin within 6 minutes by optimizing the method, and has the lower detection limit of about 0.7 ten thousandth.

5. The high-efficiency separation can improve the detection efficiency, reduce the detection cost and embody the advancement of the invention.

Drawings

FIG. 1: the separation effect graphs of SPE columns of different models are shown, wherein 1 is ProElut C18,2 is ProElut C18-U,3 is Cleanert SC-18,4 is Cleanert SC-18-N,5 is Sep-Pak Vac tC18,6 is ProElut PLS,7 is ProElut NH2,8 is ProElut PXA,9 is ProElut PXC,10 is ProElut PWA, and 11 is Sep-Pak Vac 18;

FIG. 2: the purification effect graphs of different solid phase extraction gradient elution conditions, the experiment numbers are consistent with the numbers in the table 3, and the gradient elution conditions are shown in the table 3;

FIG. 3: chromatogram obtained by chromatographic column selection in chromatographic condition optimization, wherein a No. 1 graph adopts a Waters CSH C18 chromatographic column, a No. 2 graph adopts a Waters BEH C18 chromatographic column, and a No. 3 graph adopts a Waters HSS T3C 18 chromatographic column;

FIG. 4 is a schematic view of: chromatograms obtained by selecting gradient elution conditions in chromatographic condition optimization, wherein the upper graph, the middle graph and the lower graph are chromatograms obtained by the gradient elution conditions in tables 4-1, 4-2 and 4-3 respectively;

FIG. 5 is a schematic view of: a linear relationship;

FIG. 6: determining a method-specific map;

FIG. 7: chromatogram of water extract for nourishing blood and refreshing brain;

FIG. 8: a chromatogram of a asaricin standard.

Detailed Description

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

Referring to the disclosure of blood-nourishing and brain-refreshing granules and blood-nourishing and brain-refreshing pills on 1280-1282 pages of 2015 version of Chinese pharmacopoeia, the prepared rehmannia root, uncaria, caulis spatholobi, selfheal, mother-of-pearl, asarum and other six traditional Chinese medicines are decocted with water twice, the first time is 2 hours, the second time is 1 hour, and then the filtrate is filtered and concentrated into clear paste with the relative density of 1.06.1.10 (80 ℃).

Example 1: method for measuring asaricin in blood-nourishing brain-clearing water extract

Chromatographic conditions are as follows: with UPLC system, with Waters HSS T3C 18 (2.1 × 100mm), water: the volume ratio of acetonitrile is 50:50 is a mobile phase, and the flow rate is 0.4mL/min; detection wavelength: 287nm.

Preparation of control solutions: accurately weighing appropriate amount of asaricin control, respectively, adding methanol to dissolve, and making into solution containing 0.01mg of asaricin per ml.

Preparation of a test solution: taking about 0.5g of the extract of the blood-nourishing and brain-clearing water extract, placing the extract in a beaker, adding 10ml of 50% methanol, ultrasonically dissolving, shaking up, and loading to a ProElut C18-U solid phase extraction column (500 mg/6 ml) or a Sep-Pak Vac C18 solid phase extraction column (500 mg/6 ml). Washed with 10ml of 60% methanol and discarded. Eluting with 5mL of methanol solution, collecting the eluent to a 5mL volumetric flask, adding methanol to the scale, and shaking up to obtain the product.

The determination method comprises the following steps: respectively taking 2ul of each of the reference solution and the test solution, injecting into an ultra-high performance liquid chromatograph, recording a chromatogram, calculating the content of the asarone in the test solution according to the peak area by using an external standard method, and converting to obtain the content of the asarone in the blood-nourishing and brain-refreshing water extract.

The 6 batches of the blood-nourishing and brain-refreshing water extracts were selected for determination, and the results are shown in the following table 13, fig. 7 (chromatogram of the blood-nourishing and brain-refreshing water extracts), and fig. 8 (chromatogram of the asarone standard):

TABLE 13 determination results of blood-nourishing and brain-refreshing water extract asaricin

Numbering	Asarone
		1	0.0752
2	0.0835
		3	0.0642
4	0.0655
		5	0.0646
6	0.07499

Comparative example 1:

the reference literature (Zhang Shui Xin, the research on the process of separating asarinin from asarum, reported in TCM, vol.31, no. 213, no. 2/2016: 255-257) includes the following specific steps:

the chromatographic conditions are as follows: sunAire C ₁₈ Chromatography column (4.6 mm × 150mm,5 μm), mobile phase acetonitrile and water in a volume ratio of 55:45, the detection wavelength is 287nm, the column temperature is 30 ℃, and the flow rate is 1.0mL/min;

sample preparation, taking asarum powder 0.5g, adding methanol 15mL, and performing ultrasonic treatment (power 500W, frequency 40 kHz) for 45min.

The control example has no purification process and cannot be used for measuring the asaricin in the blood-nourishing and brain-refreshing aqueous extract.

Comparative example 2:

the reference (Zhang Shui Xin, research on the process of separating asarinin from asarum, reported in TCM, vol.31, no. 213, no. 2/2016: 255-257) is a method, which is characterized in that the mobile phase is acetonitrile and water in a volume ratio of 50:50.

the control example has no purification process and cannot be used for measuring the asaricin in the blood-nourishing and brain-refreshing aqueous extract.

Claims (6)

1. A method for measuring asaricin in a brain nourishing water extract is characterized by comprising the following steps:

1) Chromatographic conditions are as follows: using a UPLC system, using an octadecyl bonded silica gel chromatographic column, with water: acetonitrile =50:50 is a mobile phase, and the flow rate is 0.3-0.5mL/min; detection wavelength: 280-295nm;

2) Preparation of control solutions: weighing a proper amount of asaricin reference substance, and adding methanol to dissolve to obtain a reference substance solution containing asaricin;

3) Preparing a test solution: taking the blood-nourishing and brain-clearing water extract, adding 40-60% (v/v) methanol, ultrasonically dissolving, passing through a solid phase extraction column, washing with 50-60% methanol, discarding, eluting with pure methanol solution, collecting eluate, and adding methanol to scale to obtain sample solution; wherein, the solid phase extraction column is ProElut C18-U or Sep-Pak Vac C18;

4) And (3) detection: and respectively taking the reference solution and the test solution, injecting the reference solution and the test solution into an ultra-high performance liquid chromatograph, recording a chromatogram, calculating the content of the asarone in the test solution by using an external standard method according to the peak area, and converting to obtain the content of the asarone in the blood-nourishing brain-clearing water extract.

2. The method for measuring according to claim 1, wherein the amount of the solid phase extraction column is 0.5-1.5 times of the weight of the extract.

3. The assay method according to claim 1, wherein in the preparation of the test solution: the methanol concentration for washing was 60%.

4. The assay method according to claim 1, characterized in that the method comprises the steps of:

1) Chromatographic conditions are as follows: using a UPLC system, using an octadecyl bonded silica gel chromatographic column, with water: acetonitrile =50:50 is a mobile phase, and the flow rate is 0.3-0.5mL/min; detection wavelength: 280-295nm;

2) Preparation of control solutions: accurately weighing appropriate amount of asaricin control, respectively, adding methanol to dissolve, and making into solution containing 0.01mg of asaricin per ml to obtain;

3) Preparing a test solution: taking about 0.5g of blood-nourishing and brain-clearing water extract, adding 10mL of 50% methanol, ultrasonically dissolving, shaking up, loading to a solid phase extraction column, washing with 10mL of 50-60% methanol, discarding, eluting with 5mL of pure methanol solution, collecting eluent, adding methanol to scale, and shaking up to obtain the final product;

4) And (3) detection: and respectively taking the reference solution and the test solution, injecting the reference solution and the test solution into an ultra-high performance liquid chromatograph, recording a chromatogram, calculating the content of the asarone in the test solution by using an external standard method according to the peak area, and converting to obtain the content of the asarone in the blood-nourishing brain-clearing water extract.

5. The assay of claim 1 wherein the chromatographic conditions are 2.1 x 100mm Waters HSS T3 c18 for an octadecyl bonded silica chromatographic column.

6. The assay according to claim 1, wherein, in the chromatographic conditions, the flow rate is 0.4mL/min; the detection wavelength was 287nm.

Images