Construction method and application of fingerprint spectrum of Hubei radix ophiopogonis medicinal material

Abstract

The invention relates to the technical field of detection, and particularly provides a construction method and application of a fingerprint spectrum of a Hubei radix ophiopogonis medicinal material. According to the present invention, the Hubei ophiopogon japonicus medicinal material is detected and analyzed by using the specific HPLC chromatographic condition, the Hubei ophiopogon japonicus medicinal material fingerprint containing a plurality of characteristic peaks is constructed, and the method has advantages of simpleness, reliability, strong objectivity, good precision, good repeatability, good specificity and good stability. Furthermore, the obtained fingerprint spectrum of the Hubei radix ophiopogonis medicinal material is analyzed, 10 common peaks are extracted in total, liriope saponin B and liriope saponin C are identified as index components, and an overall quality control basis is provided for the quality standard of the Hubei radix ophiopogonis genuine medicinal material. Besides, by optimizing the extraction time and the extraction method, the construction method of the fingerprint spectrum of the radix ophiopogonis in Hubei has high specificity and can distinguish liriope muscari, radix ophiopogonis in Zhejiang and radix ophiopogonis in Sichuan. The method has good reproducibility and stability, is high in accuracy, and can quickly and efficiently control the quality of the Hubei radix ophiopogonis medicinal material.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to a construction method and application of a Hubei Radix Ophiopogonis medicinal material fingerprint. Background Art

[0002] Hubei Ophiopogon comes from the dried rhizome of Liriopespicata (Thunb.) Lour.var.prolifera Y.T.Ma, a plant of the genus Liriopespicata in the Liliaceae family. Hubei Ophiopogon is one of the original plants under the item of "Mountain Ophiopogon" included in the 2020 edition of the "Chinese Pharmacopoeia" (Part I). It is mainly produced in Xiangfan, Hubei. Its dried tubers have the effects of nourishing yin, promoting body fluid, moistening the lungs and clearing the heart. Modern pharmacology shows that Hubei Ophiopogon has pharmacological activities such as treating cardiovascular diseases, regulating immunity and lowering blood sugar. Mountain Ophiopogon is used as a medicine equivalent to Ophiopogon japonicus in clinical practice, among which Hubei Ophiopogon is one of the mainstream varieties in the current domestic market. At present, the 2020 edition of the "Chinese Pharmacopoeia" only has moisture, ash content, thin layer identification and other items for the quality control of Hubei Ophiopogon, and there are no fingerprints and active ingredient content determination items. Therefore, Hubei Ophiopogon medicinal materials lack a complete set of scientific quality control methods and cannot truly control their quality.

[0003] The Chinese medicine fingerprint is based on the chromatogram of the characteristic components of Chinese medicinal materials or Chinese medicine preparations. With the help of the Chinese medicine chromatographic fingerprint similarity evaluation system, it reflects the data and variables of the intrinsic chemical composition information of Chinese medicine and its preparations, and realizes the understanding of the overall effect of the Chinese medicine substance group. Because of its strong specificity, good reproducibility and accurate analysis results, it has become an internationally recognized means of quality control for Chinese medicine.

[0004] Through literature search, a comparative study on the HPLC fingerprints of Ophiopogon japonicus and Radix Ophiopogonis japonicus was found (Dong Zhengqi et al., Yanbian University), and Sichuan Ophiopogon japonicus, Zhejiang Ophiopogon japonicus and Radix Ophiopogonis japonicus were distinguished by the overall appearance of the fingerprint and the individual component indicators; a method for identifying Ophiopogon japonicus and Radix Ophiopogonis japonicus medicinal materials (CN115306964A), constructed UPLC characteristic spectra of Ophiopogon japonicus from different origins and / or Radix Ophiopogonis japonicus medicinal materials from different origins, and Ophiopogon japonicus from Sichuan and Zhejiang can be distinguished by Ophiopogon japonicus saponin D, Ophiopogon japonicus saponin-3-O-α-L-rhamnopyranosyl-(1→2)-β-D-pyranoglucoside can distinguish Ophiopogon japonicus and Radix Ophiopogonis japonicus, and Ophiopogon japonicus borneol can distinguish Ophiopogon japonicus from Hubei and Radix Ophiopogonis japonicus from short-stemmed Radix Ophiopogonis.

[0005] At present, the legal standards have set standards for the origin, properties, microscopy, thin layer, and inspection items of Radix Ophiopogonis and Radix Ophiopogonis japonicus respectively. However, the identification of properties requires the appraiser to have a certain theoretical foundation and practical experience, which is difficult to promote. In addition, the properties cannot be used for identification after preparation. The above studies have some defects for Radix Ophiopogonis japonicus from Hubei Province. (1) The similarity of fingerprint spectra can be used to identify Radix Ophiopogonis japonicus from Zhejiang Province, but no similarity is specified for Radix Ophiopogonis japonicus from Hubei Province, so it is still impossible to distinguish them; (2) When the above methods were established, the samples collected were mainly for Radix Ophiopogonis japonicus. The established methods did not involve the specific components of Radix Ophiopogonis japonicus from Hubei Province, and are not suitable for the identification of Radix Ophiopogonis japonicus from Hubei Province; (3) The above detection methods require the additional configuration of an electric aerosol detector, which has a high application threshold. Summary of the invention

[0006] In view of this, the present invention provides a method for constructing a fingerprint of Hubei Ophiopogon japonicus medicinal materials. The method has the advantages of strong specificity, high stability, good repeatability, etc., solves the problems existing in the prior art, achieves the purpose of effectively identifying Hubei Ophiopogon japonicus medicinal materials, and helps to improve the quality standards of Hubei Ophiopogon japonicus medicinal materials.

[0007] The technical solution of the present invention is implemented as follows: The present invention provides a method for constructing a fingerprint spectrum of Hubei Radix Ophiopogonis, which is implemented by the following steps:

[0008] (1) Preparation of reference solution: Weigh an appropriate amount of ophiopogonin B and ophiopogonin C reference substances, accurately weigh them, and add 80% methanol to prepare reference solutions with concentrations of 5-20 μg / ml and 45-55 μg / ml, respectively;

[0009] (2) Preparation of test solution: Take 2-4 g of Hubei Radix Ophiopogonis powder that has passed through a No. 2 sieve, place it in a stoppered conical flask, accurately add 25-75 ml of 80% methanol, stopper it, weigh it, heat it in a water bath at 80°C for 30-90 min, let it stand at room temperature, add 80% methanol to make up for the weight loss, filter it, take 20-30 ml of the filtrate and evaporate it to dryness, add 25 ml of water to dissolve the residue, shake and extract it with water-saturated n-butanol for 3 times, 20 ml each time, combine the n-butanol solutions, evaporate to dryness, add an appropriate amount of 30% methanol to dissolve the residue, transfer it to a 5 ml volumetric flask, add 30% methanol to the mark, shake well, and filter it through a 0.22 μm microporous filter membrane to obtain the test solution;

[0010] (3) The test solution and reference solution of Hubei Ophiopogon japonicus were detected by high performance liquid chromatography, respectively, to construct the fingerprint of Hubei Ophiopogon japonicus.

[0011] In some embodiments, the chromatographic conditions are: the chromatographic column is a C18 chromatographic column, 250mm×4.6mm, 5μm; the column temperature is 25-35°C; acetonitrile-water solution is used for gradient elution: 0-5min, 20% acetonitrile, 5-50min, 20%-70% acetonitrile, 50-55min, 70%-90% acetonitrile; the flow rate is 0.8-1.2 / min; the detection wavelength is 203nm; the injection volume is 5-20μl.

[0012] In some embodiments, the chromatographic peaks of the Hubei Ophiopogon japonicus fingerprint map contain 10 characteristic peaks; among which the two identified common peaks include: Peak 7 is Ophiopogonin C, and Peak 10 is Ophiopogonin B.

[0013] In the second aspect, the present invention also provides a fingerprint of Hubei Ophiopogon japonicus constructed by the above-mentioned construction method, which contains 10 characteristic peaks, of which 2 common peaks identified include: Peak 7 is Ophiopogonin C, and Peak 10 is Ophiopogonin B.

[0014] In a third aspect, the present invention also provides an application of the above construction method in distinguishing the medicinal materials of Ophiopogon japonicus, Ophiopogon japonicus and Ophiopogon japonicus.

[0015] In a fourth aspect, the present invention also provides an application of the above-mentioned construction method, which is used to detect the content of ophiopogonin C and ophiopogonin B in the Ophiopogon japonicus medicinal material to be tested, and to control the quality of the medicinal material through the content of each component.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The Hubei Radix Ophiopogonis fingerprint established by the present invention can be used to identify and distinguish the medicinal materials of Zhejiang Radix Ophiopogonis, Sichuan Radix Ophiopogonis and Brachypogon japonicus. The method has strong specificity and is simple and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 It is the fingerprint superposition diagram of 30 batches of Hubei Radix Ophiopogonis in Example 1;

[0020] Figure 2 The HPLC reference fingerprint of Hubei Radix Ophiopogonis in Example 1, wherein S1 is the Hubei Radix Ophiopogonis with index number S1;

[0021] Figure 3This is the chromatographic peak identification diagram of the HPLC fingerprint of Hubei Radix Ophiopogonis in Example 1, wherein the upper curve is the reference peak, wherein No. 7 is Ophiopogonin C, and No. 10 is Ophiopogonin B; the lower curve is the HPLC reference fingerprint of Hubei Radix Ophiopogonis samples;

[0022] Figure 4 It is an overlay diagram of the precision test spectrum in Example 1;

[0023] Figure 5 It is an overlapping diagram of the repetitive test spectrum in Example 1;

[0024] Figure 6 It is an overlay of the stability test spectra in Example 1;

[0025] Figure 7 It is an overlay of fingerprint characteristic spectra of 10 batches of Ophiopogon japonicus in Example 1;

[0026] Figure 8 It is an overlay of fingerprint characteristic spectra of 10 batches of Radix Ophiopogonis in Example 1;

[0027] Fig. 9 It is an overlay of fingerprint characteristic spectra of 10 batches of Radix Ophiopogonis japonicus in Example 1;

[0028] Fig.10 It is the specific property investigation map in Example 1, wherein S1 is the reference fingerprint map of Hubei Radix Ophiopogonis, S2 is the reference fingerprint map of Zhejiang Radix Ophiopogonis, S3 is the reference fingerprint map of Sichuan Radix Ophiopogonis, and S4 is the reference fingerprint map of Brachypogon thunbergii.

[0029] Fig.11 The liquid chromatograms of the extraction solvents for the test sample in Example 2 are examined, wherein 1 is water extraction without ammonia washing, 2 is 30% methanol extraction without ammonia washing, 3 is 50% methanol extraction without ammonia washing, 4 is 80% methanol extraction without ammonia washing, 5 is methanol extraction without ammonia washing, 6 is water extraction and ammonia washing, 7 is 30% methanol extraction and ammonia washing, 8 is 50% methanol extraction and ammonia washing, 9 is 80% methanol extraction and ammonia washing, and 10 is methanol extraction and ammonia washing;

[0030] Fig.12 Liquid chromatograms for the sample extraction method in Example 2, where A is reflux extraction and B is ultrasonic extraction;

[0031] Fig.13 The liquid chromatograms for the extraction time of the test sample in Example 2, where A is 15 min, B is 30 min, and C is 45 min;

[0032] Fig.14Liquid chromatograms for different chromatographic column chromatographic conditions in Example 2, wherein A is a Diamonsil Plus C18 chromatographic column, B is a Kromaisil 100-S-C18 chromatographic column, and C is a YMC Triart C18 chromatographic column;

[0033] Fig.15 It is the DAD full-wavelength three-dimensional spectrum of the Hubei Ophiopogon japonicus test sample in Example 2;

[0034] Fig.16 This is a liquid chromatogram of the investigation of the separation effect of different column temperatures on the Hubei Ophiopogon japonicus test solution in Example 2, wherein A is 25°C, B is 30°C, and C is 35°C. DETAILED DESCRIPTION

[0035] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the embodiments of the present invention belong. If the definitions set forth in this section are contrary to or otherwise inconsistent with the definitions set forth in the patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions listed in this section take precedence over the definitions incorporated herein by reference.

[0037] Unless otherwise specified, the methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents and instruments used are all conventional materials, reagents and instruments in the art, and can be obtained by those skilled in the art through commercial channels.

[0038] When an amount, concentration or other value or parameter is expressed as a range, a preferred range or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including the range "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range. In the present specification and claims, range definitions can be combined and / or interchanged, and if not otherwise stated, these ranges include all subranges contained therein.

[0039] Example 1

[0040] This embodiment provides a method for constructing a fingerprint of Hubei Radix Ophiopogonis, which is as follows:

[0041] 1 Experimental Materials

[0042] The 60 batches of experimental samples came from major production areas and authentic production areas across the country. Both Ophiopogon japonicus and Radix Ophiopogonis japonicus meet the requirements of the 2020 edition of the "Chinese Pharmacopoeia". The medicinal material information is shown in Table 1.

[0043] Table 1 Origin information of Radix Ophiopogonis and Radix Ophiopogonis from different origins

[0044]

[0045]

[0046] 2 Methods

[0047] 2.1 Preparation of reference stock solution

[0048] Take appropriate amount of ophiopogonin B and ophiopogonin C reference substances, weigh accurately, add 80% methanol to make reference substance solutions with concentrations of 50 μg / ml and 10 μg / ml respectively for qualitative analysis.

[0049] 2.2 Preparation of test solution

[0050] Take about 3g of the powder of Hubei Ophiopogon japonicus to be tested, crush it through a No. 2 sieve, weigh it accurately, put it in a stoppered conical flask, accurately add 50ml of 80% methanol, stopper it, weigh it, heat and reflux for 30 minutes, cool it, weigh it again, make up the lost weight with 80% methanol, shake it well, filter it, take 25ml of the filtrate and evaporate it to dryness, add 25ml of water to the residue to dissolve it, shake it with water-saturated n-butanol to extract it 3 times, 20ml each time, combine the n-butanol liquid, evaporate it to dryness, add an appropriate amount of 30% methanol to dissolve the residue, transfer it to a 5ml volumetric flask, add 30% methanol to the scale, shake it well, take the filtrate to obtain the test solution.

[0051] 2.3 Chromatographic conditions

[0052] Octadecylsilane bonded silica gel is used as filler; acetonitrile (A)-water solution (B) is used for gradient elution according to the provisions in the following table; the detection wavelength is 203nm, the column temperature is 30℃; the flow rate is 1ml / min. The theoretical plate number calculated based on Ophiopogonin B should not be less than 5000.

[0053]

[0054] Accurately pipette 20 μl of reference solution and test solution respectively, inject into liquid chromatograph, measure, and record the chromatogram.

[0055] 2.4 Data Processing

[0056] The spectra obtained in the experiment were processed and exported by the Thermo workstation; the exported sample spectra were analyzed by the traditional Chinese medicine chromatographic fingerprint similarity evaluation system to calculate the sample similarity.

[0057] 3 Establishment of fingerprint of Hubei Ophiopogon japonicus

[0058] 3.1 Sample detection and similarity investigation

[0059] Take 30 batches of Hubei Ophiopogon japonicus samples, prepare them according to the test solution preparation method proposed in 2.2 above, inject and analyze them according to the chromatographic conditions proposed in 2.3 above, import the obtained chromatograms into the similarity evaluation system, and obtain the HPLC and fingerprint overlap diagrams of 30 batches of Hubei Ophiopogon japonicus samples, as shown in the figure. Figure 1 As shown. Using the S1 sample chromatogram as the reference spectrum, according to the median method, setting the time window width to 1, after multi-point calibration of chromatographic peaks 7 and 10, and matching the Mark peaks, a control fingerprint spectrum was generated, see Figure 2 By calculating the similarity, it can be seen that the similarity between the 30 batches of samples and the control fingerprint is greater than 0.85, indicating that although the quality of Hubei Ophiopogon japonicus medicinal materials in various places is relatively stable, there are certain differences in the types of chemical components they contain.

[0060] 3.2 Identification of common peaks in fingerprints

[0061] According to the fingerprints of 30 batches of samples, 10 common peaks were marked. The above-mentioned reference solution was detected by high performance liquid chromatography to obtain the reference spectrum, and the common peaks of the fingerprint spectrum of Hubei Ophiopogon japonicus were identified by comparing the retention time of the reference. Figure 3 Finally, two common peaks were identified: Peak 7 was ophiopogonin C with a retention time of 16.898 min; Peak 10 was ophiopogonin B with a retention time of 26.536 min.

[0062] 3.3 Results Analysis

[0063] This example conducts a HPLC fingerprint study on Hubei Ophiopogon japonicus medicinal materials. By comparing with the reference fingerprint, it can be seen that the similarity between each sample and the reference fingerprint is greater than 0.85, which can provide an overall quality control basis for the quality standard of Hubei Ophiopogon japonicus authentic medicinal materials.

[0064] 4 Methodological investigation

[0065] 4.1 Precision

[0066] Take the Hubei Ophiopogon japonicus sample of batch S1, prepare it according to the test solution preparation method described in 1.2 above, set the chromatographic conditions according to 1.3 above, take the filtrate and inject it continuously for 6 times, import the spectrum into the similarity evaluation system to calculate the similarity, such as Figure 4 As shown, the results showed that the similarity of each spectrum was greater than 1; at the same time, the RSDs of the peak areas of ophiopogonin C and ophiopogonin B were measured to be 0.87% and 0.23%, respectively, indicating that the instrument has good precision.

[0067] 4.2 Repeatability

[0068] Take Hubei Ophiopogon japonicus sample of batch S1, prepare 6 samples according to the test solution preparation method described in 2.2 above, inject and analyze them respectively according to the chromatographic conditions proposed in 2.3 above, record their respective fingerprint chromatograms, and import the chromatograms into the similarity evaluation system to calculate the similarity, such as Figure 5 As shown, the results showed that the similarity of each spectrum was greater than 0.99, and the RSDs of the peak areas of ophiopogonin C and ophiopogonin B were 1.64% and 0.86%, respectively, indicating that the method had good repeatability.

[0069] 4.3 Stability

[0070] Take the Hubei Ophiopogon japonicus sample of batch S1, prepare it according to the test solution preparation method proposed in item 2.2 above, and according to the chromatographic conditions proposed in item 2.3 above, inject and analyze at 0h, 3h, 6h, 9h, 12h, and 24h respectively, and import the spectrum into the similarity evaluation system to calculate the similarity, such as Figure 6 As shown, the results showed that the similarity of each spectrum was greater than 0.99; at the same time, the RSDs of the peak areas of ophiopogonin C and ophiopogonin B were measured to be 1.31% and 0.42%, respectively. The results showed that the above two components in the test solution had good stability within 24 hours, and the method had good stability.

[0071] 4.4 Exclusivity

[0072] Take batches of S31-S40 Zhejiang Radix Ophiopogonis powder, S41-S50 Sichuan Radix Ophiopogonis powder, S51-S60 short-tending Radix Ophiopogonis powder, prepare according to the test solution preparation method proposed in 2.2 above, and analyze according to the chromatographic conditions proposed in 1.3 above. The fingerprints of 10 batches of Zhejiang Radix Ophiopogonis are superimposed. Figure 7 , 10 batches of Sichuan Radix Ophiopogonis medicinal materials fingerprint superposition Figure 8 , 10 batches of Radix Ophiopogonis short-spined herb fingerprint superposition Fig. 9 , the obtained spectrum is imported into the similarity analysis software to calculate the similarity between the sample and the control fingerprint spectrum, such as Fig.10 As shown, the results show similarities of 0.34, 0.65 and 0.68, indicating that the method has good specificity.

[0073] Example 2

[0074] This example provides a condition investigation of the method for constructing a fingerprint spectrum of Hubei Radix Ophiopogonis, which is as follows:

[0075] 1 Investigation of the preparation method of the test solution

[0076] 1.1 Investigation of extraction solvent

[0077] The experiment compared water, 30% methanol, 50% methanol, 80% methanol, and methanol to prepare the test samples, as follows:

[0078] Take an appropriate amount of this product, crush it, take about 6.0g respectively, weigh it accurately, put it in a stoppered conical flask, and accurately add water, 30% methanol, 50% methanol, 80% methanol, and 100ml of methanol respectively, heat and reflux for 30 minutes, take it out, let it cool, filter it, take 50ml of the filtrate and evaporate it to dryness, add 50ml of water to the residue to dissolve it, shake and extract it with water-saturated n-butanol for 3 times, 40ml each time, combine the n-butanol liquid, take 50ml of the n-butanol liquid and evaporate it directly, take another 50ml and wash it with ammonia test solution twice, 5ml each time, discard the ammonia test solution, and evaporate the n-butanol liquid. Add an appropriate amount of 30% methanol to the residue to dissolve it, transfer it to a 5ml volumetric flask, add 30% methanol to the scale, shake well, filter it, take the filtrate as the test solution, take 20μL respectively, inject it into the high performance liquid chromatograph, and record the chromatogram, as shown Fig.11 As shown, peak areas greater than 5 and characteristic component peaks were taken as representatives, and the ratio of peak area to sampling amount (g) was taken as the evaluation index. The results are shown in Tables 2 and 3.

[0079] Table 2 Ratio of peak area to sampling amount of different extraction solvents without ammonia washing

[0080]

[0081]

[0082] Table 3 Ratio of peak area to sampling volume of different extraction solvents and ammonia washing

[0083] Characteristic peak water 30% methanol 50% methanol 80% Methanol Methanol Peak 1(Unknown) 8.0862 6.9591 5.5895 5.7438 6.2448 Peak 2 (Unknown) 7.0362 5.3126 5.1885 5.5086 5.7638 Peak 4 (Unknown) 6.0663 4.5095 4.4084 5.5985 6.9062 Peak 6 (Unknown) 3.6543 9.8816 11.5560 12.7157 14.4050 Peak 7 (Ophiopogon japonicus saponin C) 0.7779 3.1731 3.6030 3.8174 4.2465 Peak 10 (Ophiopogon japonicus saponin B) 2.9634 14.3231 17.0159 18.7902 19.0919 Total area / g 4.7848 7.3181 7.8484 8.6944 9.4096

[0084] It can be seen from the above table that the total integrated area of ​​the chromatographic peaks of the main components of Hubei Ophiopogon japonicus extracted with 80% methanol solvent without ammonia washing is the largest, and the extracted liquid chromatogram has a large number of peaks, the chromatographic peaks of each main component are clearly visible, the peak position and peak shape are good, and the separation degree is high. Therefore, 80% methanol was selected as the extraction solvent for the test sample in the fingerprint study of Hubei Ophiopogon japonicus medicinal materials.

[0085] 1.2 Investigation of extraction methods

[0086] Take 2 portions of Hubei Ophiopogon japonicus medicinal materials from batch S1, 3g each, add 50mL of 80% methanol respectively, weigh accurately, extract one portion under reflux for 30min, and ultrasonicate the other for 30min, cool, and use 80% methanol to make up the weight loss of the two extracts, filter, take 25ml of the filtrate and evaporate to dryness, add 25ml of water to the residue to dissolve, shake and extract 3 times with water-saturated n-butanol, 20ml each time, combine the n-butanol solution, wash twice with ammonia test solution, 5ml each time, discard the ammonia test solution, and evaporate the n-butanol solution. Add an appropriate amount of 30% methanol to the residue to dissolve, transfer to a 5ml volumetric flask, add 30% methanol to the scale, shake well, filter, take the filtrate as the test solution, and inject 20μL into the high performance liquid chromatograph, record the chromatogram, and the results are as follows Fig.12 As shown, peak areas greater than 5 and characteristic component peaks were taken as representatives, and the ratio of peak area to sampling amount (g) was taken as the evaluation index. The results are shown in Table 4 below.

[0087] Table 4 Ratio of peak area to sampling volume of different extraction methods

[0088]

[0089]

[0090] From the chromatogram, we can see that the HPLC spectra of the two extraction methods are basically the same, but from the ratio of the peak area of ​​the main peak to the sampling amount, we can see that the efficiency of reflux extraction is higher than that of ultrasonic extraction. Therefore, reflux was selected as the method for extracting the effective ingredients of the test sample in the fingerprint study of Hubei Ophiopogon japonicus.

[0091] 1.3 Extraction time investigation

[0092] Take 3 batches of Hubei Ophiopogon japonicus medicinal materials of S1, 3g each, add 50mL of 80% methanol respectively, weigh accurately, reflux extract for 30min, 60min, 90min respectively, cool, use 80% methanol to make up the weight lost of the two extracts, filter, take 25ml of the filtrate and evaporate to dryness, add 25ml of water to the residue to dissolve, shake and extract 3 times with water-saturated n-butanol, 20ml each time, combine the n-butanol solution, wash twice with ammonia test solution, 5ml each time, discard the ammonia test solution, and evaporate the n-butanol solution. Add an appropriate amount of 30% methanol to the residue to dissolve, transfer to a 5ml volumetric flask, add 30% methanol to the scale, shake well, filter, take the filtrate as the test solution, and take 20μL of each into the high performance liquid chromatograph, record the chromatogram, such as Fig.13 As shown, peak areas greater than 5 and characteristic component peaks were taken as representatives, and the ratio of peak area to sampling amount (g) was taken as the evaluation index. The results are shown in Table 5 below.

[0093] Table 5 Ratio of peak area to sampling volume at different extraction times

[0094]

[0095] From the chromatogram, we can see that the HPLC chromatograms of different extraction times are the same, and from the table above, as the extraction time increases to 30 minutes, the peak area of ​​the main component increases accordingly. When the extraction time is further extended, the peak area of ​​the main component does not increase significantly. Therefore, 30 minutes was selected as the optimal extraction time for the test sample in the fingerprint study of Hubei Ophiopogon japonicus.

[0096] 1.4 Optimal test sample preparation method

[0097] Based on the above results, the best test sample preparation method was determined as follows: about 3 g of Hubei Radix Ophiopogonis powder was crushed and passed through a No. 2 sieve, accurately weighed, placed in a stoppered conical flask, 50 ml of 80% methanol was accurately added, the flask was sealed, the weight was determined, the flask was heated to reflux for 30 minutes, cooled, and the weight was determined again. 80% methanol was used to make up the lost weight, shaken, filtered, 25 ml of the filtrate was evaporated to dryness, 25 ml of water was added to the residue to dissolve it, and it was shaken and extracted 3 times with water-saturated n-butanol, 20 ml each time, the n-butanol liquids were combined, evaporated to dryness, an appropriate amount of 30% methanol was added to the residue to dissolve it, and the residue was transferred to a 5 ml volumetric flask, 30% methanol was added to the scale, shaken, and the filtrate was taken to obtain the test sample solution.

[0098] 2 Comparison and optimization of chromatographic conditions

[0099] 2.1 Selection of chromatographic columns

[0100] Referring to the construction method in Example 1, keeping other conditions unchanged, the mobile phase is acetonitrile (A)-water (B); different models of C18 chromatographic columns (5 μm, 4.6 mm×250 mm), including Diamonsil Plus C18 chromatographic column, Kromaisil100-S-C18 chromatographic column and YMC Triart C18 chromatographic column, were investigated in turn. The results are as follows Fig.14 shown.

[0101] From the results in the figure, we can see that different types of C18 columns have different separation effects under the same chromatographic conditions. The Diamonsil Plus C18 column has a good separation effect, a stable baseline, good separation and symmetry of the main component chromatographic peaks, and good stability. Therefore, the Diamonsil Plus C18 column was selected as the chromatographic column for the fingerprint study of Hubei Ophiopogon japonicus.

[0102] 2.2 Selection of detection wavelength

[0103] According to Fig.15 The three-dimensional spectrum detected by DAD shown in the figure selects 203nm, which can detect all the substances in the whole spectrum, as the detection wavelength for the fingerprint spectrum research of Hubei Ophiopogon japonicus.

[0104] 2.3 Investigation of column temperature

[0105] Referring to the construction method in Example 1, keeping other conditions unchanged, using Diamonsil Plus C18 chromatographic column, acetonitrile-water as mobile phase, setting column temperature to 25 ° C, 30 ° C, 35 ° C, respectively, to investigate the effect of different column temperatures on the separation of Hubei Ophiopogon japonicus samples. The results are as follows Fig.16 As shown in the figure, different column temperatures have a significant effect on the chromatographic peaks. When the column temperature is 30℃, there is a better separation effect, and the separation and symmetry of the main component chromatographic peaks are good. Therefore, 30℃ was selected as the column temperature for the fingerprint study of Hubei Ophiopogon japonicus.

[0106] Example 3

[0107] This embodiment provides a method for detecting the content of multiple index components of Hubei Ophiopogon japonicus, that is, simultaneously determining the contents of two saponin components in Hubei Ophiopogon japonicus medicinal materials by HPLC.

[0108] 1 Method

[0109] 1.1 Chromatographic conditions

[0110] Octadecylsilane bonded silica gel is used as filler; acetonitrile (A)-water solution (B) is used for gradient elution according to the provisions in the following table; the detection wavelength is 203nm, the column temperature is 30℃; the flow rate is 1ml / min. The theoretical plate number calculated based on Ophiopogonin B should not be less than 5000.

[0111]

[0112] Accurately pipette 20 μl of reference solution and test solution respectively, inject into liquid chromatograph, measure, and record the chromatogram.

[0113] 1.2 Preparation of reference solution

[0114] Take appropriate amount of ophiopogonin B and ophiopogonin C reference substances, weigh accurately, add 80% methanol to prepare reference substance solutions with concentrations of 10 μg / ml and 50 μg / ml respectively for qualitative analysis.

[0115] 1.3 Preparation of test solution

[0116] Take about 3g of the powder of Hubei Ophiopogon japonicus to be tested, crush it through a No. 2 sieve, weigh it accurately, put it in a stoppered conical flask, accurately add 50ml of 80% methanol, stopper it, weigh it, heat and reflux for 30 minutes, cool it, weigh it again, make up the lost weight with 80% methanol, shake it well, filter it, take 25ml of the filtrate and evaporate it to dryness, add 25ml of water to the residue to dissolve it, shake it with water-saturated n-butanol to extract it 3 times, 20ml each time, combine the n-butanol liquid, evaporate it to dryness, add an appropriate amount of 30% methanol to dissolve the residue, transfer it to a 5ml volumetric flask, add 30% methanol to the scale, shake it well, take the filtrate to obtain the test solution.

[0117] 2 Methodological investigation

[0118] 2.1 Repeatability study

[0119] Take sample S1, make 6 parallel portions, accurately weigh them, and prepare test solutions in parallel according to the above method. Inject 20 μL of each sample for detection and analysis. The results showed that the peak area RSDs of ophiopogonin C and ophiopogonin B were 1.54% and 1.13%, respectively, indicating that the method has good repeatability and meets the requirements.

[0120] 2.2 Precision investigation

[0121] 20 μL of the test solution of Hubei Ophiopogon japonicus sample S1 was accurately aspirated and sampled 6 times continuously for detection and analysis. The RSDs of the peak areas of ophiopogonin C and ophiopogonin B were measured to be 0.57% and 0.08%, respectively, indicating that the instrument has good precision and meets the requirements.

[0122] 2.3 Stability investigation

[0123] The test solution of sample S1 was taken and placed at room temperature. 20 μL was injected at 0, 3, 6, 9, 12, and 24 h for detection and analysis. The results showed that the RSDs of the peak areas of ophiopogonin C and ophiopogonin B were 1.31% and 1.98%, respectively, indicating that the above two components in the test solution were stable within 24 h. This method has good stability and meets the requirements.

[0124] 2.4 Investigation of linear relationship

[0125] Accurately weigh 5.413 mg of reference substance ophiopogonin C and 8.430 mg of reference substance ophiopogonin B into a 25 mL volumetric flask, add 80% methanol solution to dissolve and prepare a stock solution, accurately pipette 1.0 mL of stock solution No. 1 into 5 mL, 10 mL, 25 mL, 50 mL, and 100 mL volumetric flasks, add 80% methanol solution to the scale, and inject 20 μL of each sample for determination under the chromatographic conditions described in 1.1 above. Draw a standard curve with peak area Y as the ordinate and reference substance concentration X (mg / L) as the abscissa. The results are shown in Table 6 below, indicating that ophiopogonin C and ophiopogonin B have a good linear relationship between concentration and peak area within their respective linear ranges, and the linear correlation coefficient R 2 Not less than 0.99, with a good linear relationship.

[0126] Table 6 Linear relationship of reference substances

[0127]

[0128] 3. Investigation of sample recovery rate

[0129] Take 6 portions of S1 sample with known determination, take about 1.5g of each portion, accurately weigh, and place them in conical flasks respectively. Add appropriate amount of reference stock solution of precision ophiopogonin C and ophiopogonin B, prepare the test solution according to the method described in 1.3 above, and inject 20μL of each sample for determination under the chromatographic conditions described in 1.1 above. Calculate the recovery rate. The results are shown in Table 7 below.

[0130] Table 7 Sample recovery test results

[0131]

[0132]

[0133] 4. Sample testing

[0134] The test solution was prepared according to the method described in 1.3 above. Two samples were prepared in parallel for each batch. The samples were injected and analyzed according to the chromatographic conditions described in 1.1 above, and the content was calculated. The results are shown in Table 8 below.

[0135] Table 8 Hubei Radix Ophiopogonis medicinal material content test results

[0136]

[0137] In summary, this application has established the fingerprint of Hubei Ophiopogon japonicus medicinal materials by measuring 30 batches of Hubei Ophiopogon japonicus medicinal materials through chromatographic conditions and methodology investigation, extracted 10 common peaks, and identified 2 of them by using the retention time of the reference substance, namely, Ophiopogonin C and Ophiopogonin B. The similarity evaluation shows that the similarity between the fingerprints of medicinal materials from different origins and the reference spectra is greater than 0.9, and the established fingerprint is stable and reliable, which can be used to evaluate the quality of Hubei Ophiopogon japonicus.

[0138] Furthermore, HPLC was used to simultaneously determine the two saponin components of Hubei Ophiopogon japonicus, and the quality of 30 Hubei Ophiopogon japonicus samples collected from different production areas was studied. The comprehensive evaluation of the quality of Hubei Ophiopogon japonicus medicinal materials from different production areas based on the content of Ophiopogonin C and Ophiopogonin B is of great significance for accurately controlling the quality of Hubei Ophiopogon japonicus.

[0139] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for constructing a fingerprint of Hubei Radix Ophiopogonis, characterized in that: include: (1) Extracting the Hubei Radix Ophiopogonis with an extraction solvent to prepare a test solution; (2) Take ophiopogonin C and ophiopogonin B to prepare a reference solution; (3) HPLC detection of the test solution and the reference solution, matching characteristic peaks, identifying common peaks, and establishing the fingerprint of Hubei Ophiopogon japonicus; The HPLC conditions are as follows: the chromatographic column is filled with octadecylsilane bonded silica gel; the mobile phase is acetonitrile (A)-water solution (B), gradient elution; the detection wavelength is 200-280nm, the column temperature is 25-35°C; the flow rate is 0.7-1.3ml / min.

2. The method for constructing the fingerprint of Hubei Radix Ophiopogonis as claimed in claim 2, characterized in that: The chromatographic column is Diamonsil Plus C18, and the gradient elution conditions are: 0-5 min, 20% A; 5-50 min, 20-70% A; 40-45 min, 70-90% A; the detection wavelength is 203 nm; the column temperature is 30° C.; and the flow rate is 1 ml / min.

3. The method for constructing the fingerprint of Hubei Radix Ophiopogonis as claimed in claim 1, characterized in that: The chromatographic peaks of the Hubei Radix Ophiopogonis fingerprint spectrum include 10 characteristic peaks; The two common peaks identified include: Peak 7 is ophiopogonin C, and Peak 10 is ophiopogonin B.

4. The method for constructing the fingerprint of Hubei Radix Ophiopogonis as claimed in claim 1, characterized in that: The preparation method of the test solution is as follows: take 2-4g of Hubei Ophiopogon japonicus powder that has passed through No. 2 sieve, place it in a stoppered conical flask, accurately add 25-75ml of 80% methanol, seal it, weigh it, heat it in a water bath at 80℃ and reflux it for 30-90min, then place it at room temperature, make up the weight loss with 80% methanol, filter it, take 20-30ml of the filtrate and evaporate it to dryness, add 25ml of water to the residue to dissolve it, shake and extract it 3 times with water-saturated n-butanol, 20ml each time, combine the n-butanol liquid, evaporate it to dryness, add an appropriate amount of 30% methanol to dissolve the residue, transfer it to a 5ml volumetric flask, add 30% methanol to the scale, shake it well, and filter it through a 0.22μm microporous filter membrane to obtain the test solution.

5. The method for constructing the fingerprint of Hubei Radix Ophiopogonis as claimed in claim 1, characterized in that: The preparation method of the reference solution is as follows: weigh appropriate amounts of ophiopogonin B and ophiopogonin C reference substances, accurately weigh them, and add 80% methanol to prepare reference solutions with concentrations of 5-20 μg / ml and 45-55 μg / ml, respectively.

6. A Hubei Radix Ophiopogonis medicinal material fingerprint, characterized in that: The Hubei Ophiopogon medicinal material fingerprint is constructed by detection according to the Hubei Ophiopogon medicinal material fingerprint construction method described in any one of claims 1-5.

7. The Hubei Radix Ophiopogonis medicinal material fingerprint as claimed in claim 6, characterized in that: The fingerprint spectrum of Hubei Ophiopogon japonicus contains 10 characteristic peaks, of which 2 common peaks are identified: Peak 7 is Ophiopogonin C, and Peak 10 is Ophiopogonin B.

8. Application of the method for constructing the Hubei Radix Ophiopogonis medicinal material fingerprint as described in any one of claims 1 to 5 in distinguishing the Zhejiang Radix Ophiopogonis, Sichuan Radix Ophiopogonis and Brachypogon japonicus medicinal materials.

9. The application of the method for constructing the Hubei Radix Ophiopogonis medicinal material fingerprint spectrum as described in any one of claims 1 to 5, characterized in that: Used to detect the content of ophiopogonin C and ophiopogonin B in the Ophiopogon japonicus medicinal material to be tested.

Images