CN104569200A - Measuring method for stephania tetrandra s. moore fingerprint spectrum - Google Patents

Abstract

The invention relates to a measuring method for stephania tetrandra s. moore fingerprint spectrum. The measuring method comprises the following steps: (1) taking a medical material of stephania tetrandra s. moore, pulverizing into medium powder, precisely weighing, adding ethyl alcohol, performing ultrasound dissolution, filtering, drying by distillation, dissolving residue through methanol, determining volume, filtering through a filtration film, and taking a filtrate, so as to obtain a test solution; (2) preparing a reference solution; (3) precisely absorbing the test solution and the reference solution respectively, and injecting into a high performance liquid chromatograph to be tested so as to obtain the stephania tetrandra s. moore fingerprint spectrum. The measuring method has an active function on effective feeding guide of raw medicines in clinical medication and production process and guarantee of reliable quality, and can be operated conveniently and quickly, and when the quality of the traditional Chinese medicine of the stephania tetrandra s. moore is evaluated according to the obtained similarity result, the conclusion is relatively objective and accurate.

Description

A method for measuring the fingerprint of Fangji

technical field

The invention belongs to the field of traditional Chinese medicine fingerprints, in particular to a method for measuring the fingerprints of Fangji.

Background technique

Fangji comes from the dry root of Fangjiaceae plant powder Fangji Stephania tetrandra S.Moore. Fangjigen mainly contains alkaloids, most of which are bisbenzylisoquinoline alkaloids. There are Tetrandrine (Tetrandrine), that is, HanfangchinA (Fanchinin); Fangchinoline), that is, Hanfangchin B (HanfangchinB), also known as Demethyltetrandrine (Demethyltetrandrine); Wood tetrandrine B) and Cyclanoline. It also contains Berbamine (Berba-mine), Oxofangchirine, Stephanthrine and so on. In addition, Fangjigen also contains flavonoid glycosides, phenols, organic acids, volatile oils, sugars and so on. The Chinese Pharmacopoeia 2010 edition records the content of tetrandrine and tetrandrine by liquid chromatography under the item of Fangji.

In the second issue of Chinese herbal medicine in 2007, Cui Guowen et al. used a liquid chromatography reversed-phase column, using acetonitrile-phosphoric acid solution as the mobile phase, and taking Fangchinoline as a reference, found 8 common peaks and determined 2 of them. . However, most of the subjects collected in this paper were produced in Jiangxi. The main producing areas of Fangji are Zhejiang, Anhui, Jiangxi, Fujian, Guangdong, Guangxi, Xianning, Huanggang and other places. In recent years, the research on fingerprints can not only measure the content of components in substances, but also monitor the ratio of components in traditional Chinese medicines containing multiple components according to the presence or absence of common peaks and the area or peak height of chromatographic peaks. It has a positive effect on the quality, safety and stability of traditional Chinese medicine preparations.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for measuring the fingerprint of Fangji, which can effectively monitor the quality of Fangji medicinal material and ensure its effectiveness, safety and stability in clinical or production application.

A kind of assay method of Fangji fingerprint spectrum of the present invention comprises:

(1) Take Fangji medicinal material and pulverize it into medium powder, accurately weigh it, add ethanol, dissolve it with 40KHz ultrasound for 40min, filter and evaporate to dryness, dissolve the residue with methanol and constant volume, filter through a filter membrane, and take the subsequent filtrate to obtain the test solution ;

(2) Get magnolialine reference substance, fangchinoline reference substance, and tetrandrine reference substance, accurately weigh, add methanol respectively to make reference substance solution;

(3) Accurately draw the test solution and the reference solution respectively, and inject them into a high-performance liquid chromatograph for measurement to obtain the Fangji fingerprint spectrum; wherein, the chromatographic conditions are: chromatographic column: Kromasil 100-5C ₁₈ chromatographic column; column temperature: 30 ℃; detection wavelength: 284nm; injection volume: 10μl; flow rate: 1ml·min ^-1 ; mobile phase: mobile phase A is 0.3% formic acid, mobile phase B is acetonitrile; gradient elution program: 0～20min: 15% B ～19% B, 20～30min: 19%B～30%B, 30～40min: 30%B～60%B.

The ratio of Fangji Zhongfen and ethanol in the step (1) is 0.1g:20ml.

The pore diameter of the microporous membrane in the step (1) is 0.22 μm.

The volume percentage of ethanol in the step (1) is 70%, and the volume percentage of methanol is 15%.

The volume percentage of the methanol in the step (2) is 15%.

The reference substance solution in the step (2) is specifically: each 1ml contains a mixed solution of magnolanine 0.080mg, tetrandrine 0.170mg, and tetrandrine 0.060mg.

The Kromasil 100-5C18 chromatographic column specification in the described step (3) is 4.6mm * 250mm, and the particle size of filler is 5 μm.

4 chromatographic peaks are identified as total chromatographic peaks in the fingerprint of Fangji in the described step (3), by comparing with the retention time of the reference substance, it is determined that peak 1 is Magnolin, peak 2 is Fangchinoline, and peak 3 is Tetrandrine.

Beneficial effect

In the present invention, the fingerprints of medicinal materials have been studied, the determination conditions of liquid phase fingerprints have been established, and methodological investigations have been carried out. According to many batches of large-scale production samples, the standards for liquid phase fingerprints of Fangji have been formulated, which can be effectively used in the production process. Guide the feeding and standardize the production operation, which truly ensure the safety, effectiveness and reliability of clinical medication. The identification of the measured fingerprints adopts the similarity evaluation system of traditional Chinese medicine chromatographic fingerprints provided by the National Pharmacopoeia Committee, which is convenient and fast to operate. The obtained similarity results are used to evaluate the fingerprints of medicinal materials, and the conclusions are more objective and accurate.

Description of drawings

Fig. 1 is the fingerprint of reference substance; Wherein, 1 is magnolialine; 2 is fangchinoline; 3 is tetrandrine;

Fig. 2 is 10 batches of Fangji fingerprints;

Fig. 3 is the standard fingerprint of Fangji; wherein, 1 is magnolialine; 2 is Fangchinoline; and 3 is tetrandrine.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1

(1) Preparation of the test solution: Precisely take 0.1000 g of Fangji powder (Fangji selected from decoction pieces), add 20 ml of 70% ethanol and ultrasonically (40KHz, 250W) for 40 minutes, evaporate to dryness, dissolve the residue with 15% methanol, and set the volume to 10ml, pass through a 0.22μm microporous membrane.

(2) Preparation of reference substance solution: Take appropriate amounts of magnolialine reference substance, fangchinoline reference substance, and powder tetrandrine reference substance respectively, accurately weigh them, and use methanol to prepare 0.080 mg of magnolialine and fangchinoline in each 1 ml. A mixed solution of 0.170 mg of lindine and 0.060 mg of tetrandrine is ready.

(3) Chromatographic conditions: chromatographic column is Kromasil 100-5C ₁₈ chromatographic column (4.6mm×250mm, 5μm); column temperature: 30°C; detection wavelength: 284nm; mobile phase is 0.3% formic acid (A)-acetonitrile (B) , Gradient elution program: 0-20min: 15%B-19%B, 20-30min: 19%B-30%B, 30-40min: 30%B-60%B.

(4) Determination method: according to above-mentioned testing data, obtain the HPLC chromatogram of Fangji, establish HPLC peak retention time and peak area value, then calculate the mean value of the relative retention time of each characteristic peak and the average value of relative peak area Value and standard deviation are divided by the average retention time and the average peak area of the reference peak respectively, as the relative peak retention time and relative peak area value of the standard fingerprint, derive the calculation results, formulate the standard fingerprint data, and draw the standard fingerprint; The fingerprint spectrum contains at least 3 characteristic peaks and 1 reference peak, and the fluctuation range of the relative retention time of each characteristic peak relative to the reference peak (S) complies with the regulations in the table below.

Table 1 Fluctuation range of relative retention time of standard fingerprint of Fangji

Fangji is the dry root of Fangji family plant powder Fangji Stephania tetrandra S. Moore. The HPLC fingerprints of the Fangji medicinal material were established, and the fingerprints of 10 batches of Fangji were tested according to the method provided by the present invention, and the obtained spectra had a relatively high similarity.

(1) Methodological investigation:

Choice of detection wavelength

Scanning the ultraviolet full wavelength of 3 kinds of reference substances (magnoline, fangchinoline, and tetrandrine), the maximum absorption wavelength of magnoline is 268nm, the maximum absorption wavelength of fangchinoline is 284nm, and the maximum absorption wavelength of tetrandrine is 268nm. The wavelength is 280nm, so the detection wavelength of the Fangji fingerprint spectrum should be selected within 268nm-284nm, and the fingerprints obtained by comparing the UV absorption scans of each reference substance are compared at two wavelengths (268nm, 284nm) respectively. The absorption wavelength is determined to be 284nm.

precision test

Get the same production place batch of Fangji medicinal material test solution, continuous sample injection 6 times, investigate the relative retention time of the chromatographic peak, the consistency of the relative peak area ratio, thereby investigate the precision of the instrument, the results of Table 2 and Table 3 show that, The RSD values of the relative retention time and relative peak area ratio of each common chromatographic peak were all within 2%, indicating that the precision of the instrument was good.

Table 2 The results of the precision test of the Fangji fingerprint methodological investigation (relative retention time)

serial number 1 2 3 4 5 6 RSD(%) peak 1 0.308 0.307 0.307 0.307 0.307 0.307 0.123 peak 2 0.525 0.524 0.525 0.525 0.524 0.525 0.058

peak 3 0.772 0.772 0.772 0.772 0.771 0.771 0.052 peak 4 1.000 1.000 1.000 1.000 1.000 1.000 0.000

Table 3 Results of the precision test (relative peak area) of the methodological investigation of Fangji fingerprint

serial number 1 2 3 4 5 6 RSD(%) peak 1 0.063 0.061 0.061 0.061 0.061 0.061 1.651 peak 2 0.365 0.365 0.366 0.366 0.366 0.366 0.100 peak 3 0.566 0.565 0.566 0.566 0.567 0.566 0.091 peak 4 1.000 1.000 1.000 1.000 1.000 1.000 0.000

Stability study

Get the need testing solution of the antichristine medicinal material of the same place of origin batch, detect respectively at 0h, 4h, 8h, 12h, 16h, 24h, to investigate the stability of the sample, table 4, table 5 results show that each common chromatographic peak The RSD values of relative retention time and relative peak area ratio were both less than 2%, indicating that the sample solution was basically stable within 24 hours.

Table 4 Investigation of Relative Retention Time Stability of Fangji Fingerprint Spectrum

serial number 1 2 3 4 5 6 RSD(%) peak 1 0.307 0.307 0.308 0.308 0.308 0.308 0.128 peak 2 0.524 0.525 0.526 0.526 0.526 0.525 0.178 peak 3 0.770 0.773 0.775 0.773 0.774 0.772 0.197 peak 4 1.000 1.000 1.000 1.000 1.000 1.000 0.000

Table 5 Investigation on the Relative Peak Area Stability of Fangji Fingerprint

serial number 1 2 3 4 5 6 RSD(%) peak 1 0.063 0.063 0.063 0.062 0.062 0.062 0.424 peak 2 0.369 0.370 0.370 0.370 0.370 0.370 0.121 peak 3 0.562 0.563 0.563 0.563 0.563 0.562 0.103 peak 4 1.000 1.000 1.000 1.000 1.000 1.000 0.000

repeatability test

Get 6 parts of Fangji medicinal materials of the same place of origin batch, prepare test solution respectively and detect, as can be seen from Table 6 and Table 7, the relative retention time and relative peak area RSD value of each common chromatographic peak are all less than 2%, show that Good repeatability.

Table 6 Investigation on the Reproducibility of Relative Retention Time of Fangji Fingerprint Spectrum

serial number 1 2 3 4 5 6 RSD(%)

peak 1 0.308 0.308 0.307 0.308 0.308 0.308 0.082 peak 2 0.525 0.526 0.525 0.526 0.526 0.525 0.122 peak 3 0.771 0.775 0.774 0.775 0.774 0.772 0.180 peak 4 1.000 1.000 1.000 1.000 1.000 1.000 0.000

Table 7 Investigation on the Reproducibility of Relative Peak Area of Fangji Fingerprint

serial number 1 2 3 4 5 6 RSD(%) peak 1 0.064 0.062 0.065 0.065 0.063 0.062 1.897 peak 2 0.381 0.371 0.373 0.373 0.363 0.362 1.916 peak 3 0.584 0.567 0.568 0.567 0.566 0.566 1.215 peak 4 1.000 1.000 1.000 1.000 1.000 1.000 0.000

Similarity evaluation of fingerprints of Fangji medicinal materials:

The similarity calculation of the HPLC fingerprint of Fangji was carried out using the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint (version 2012), and the results are shown in Table 8.

Table 8 Calculation results of fingerprint similarity of 10 batches of Fangji medicinal materials

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 0.947 0.988 0.982 0.986 0.974 0.991 0.840 0.968 0.970 0.983

Claims (8)

1. A method for measuring the fingerprint of Fangji, comprising: (1) Take Fangji medicinal material and pulverize it into medium powder, accurately weigh it, add ethanol, dissolve it with 40KHz ultrasound for 40min, filter and evaporate to dryness, dissolve the residue with methanol and constant volume, filter through a filter membrane, and take the subsequent filtrate to obtain the test solution ; (2) Get magnolialine reference substance, fangchinoline reference substance, tetrandrine reference substance, accurately weigh, add methanol respectively to make reference substance solution; (3) Accurately draw the test solution and the reference solution respectively, and inject them into a high-performance liquid chromatograph for measurement to obtain the Fangji fingerprint spectrum; wherein, the chromatographic conditions are: chromatographic column: Kromasil 100-5C ₁₈ chromatographic column; column temperature: 30 ℃; detection wavelength: 284nm; injection volume: 10μl; flow rate: 1ml·min ^-1 ; mobile phase: mobile phase A is 0.3% formic acid, mobile phase B is acetonitrile; gradient elution program: 0～20min: 15% B ～19% B, 20～30min: 19%B～30%B, 30～40min: 30%B～60%B. 2. the assay method of a kind of Fangji fingerprint spectrum according to claim 1, is characterized in that: the ratio of Fangji powder in the described step (1) and ethanol is 0.1g:20ml. 3. The assay method of a kind of Fangji fingerprint according to claim 1, characterized in that: the aperture of the microporous membrane in the step (1) is 0.22 μm. 4. the assay method of a kind of Fangji fingerprint spectrum according to claim 1, is characterized in that: the volume percentage of the ethanol in the described step (1) is 70%, and the volume percentage of methyl alcohol is 15%. 5. the assay method of a kind of Fangji fingerprint spectrum according to claim 1, is characterized in that: the volume percentage of the methyl alcohol in the described step (2) is 15%. 6. the assay method of a kind of Fangjiu fingerprint spectrum according to claim 1, is characterized in that: the reference substance solution in the described step (2) is specially: every 1ml respectively contains Magnolin 0.080mg, Fangchinoline 0.170mg, tetrandrine 0.060mg mixed solution. 7. the assay method of a kind of Fangji fingerprint according to claim 1, is characterized in that: the Kromasil 100-5C18 chromatographic column specification in the described step (3) is 4.6mm * 250mm, and the particle size of filler is 5 μ m. 8. the assay method of a kind of Fangji fingerprint collection according to claim 1, it is characterized in that: 4 chromatographic peaks are determined as total chromatographic peak in the Fangji fingerprint collection in the described step (3), by retaining with reference substance The comparison of time determined that peak 1 was magnolialine, peak 2 was fangchinoline, and peak 3 was tetrandrine.