CN105572258B - Orobanche pycnostachya Hance fingerprint - Google Patents

Abstract

The present invention relates to a method for establishing the fingerprint of Chrysanthemum chrysalis, comprising: 1) preparation of the test solution: take chrysanthemum chrysogenum as medicinal material powder, add 70% methanol, weigh, soak, ultrasonically treat, use solvent to make up the lost Weight, filter, take the subsequent filtrate and filter it with a 0.45 μm microporous membrane; 2) inject the test solution into a high-performance liquid chromatograph to obtain a fingerprint, wherein, the chromatographic column: octadecylsilane bonded silica gel Chromatographic column; mobile phase: acetonitrile (A)-1‰ phosphoric acid aqueous solution (B), gradient elution 89% B (0min) ~ 85% B (13min) ~ 81% B (22min) ~ 78% B (33min) ~ 76% B(50)min; flow rate: 1ml/min; detection wavelength: 230-400nm; column temperature: 20-30°C. The invention also provides a method for evaluating the quality of Huanghualiedang medicinal material by using fingerprint spectrum. The method for analyzing the chrysanthemum chrysalis fingerprint established according to the invention has high sensitivity and good reproducibility; the obtained standard fingerprint can comprehensively reflect the types and quantities of the chemical components contained in the chrysanthemum chrysalis medicinal material.

Description

Method for establishing fingerprint of Liedang chrysanthemum

technical field

The invention relates to the field of drug analysis, in particular to a method for establishing fingerprints of Chinese medicinal materials and a method for evaluating the quality of medicinal materials.

Background technique

Liedang chrysanthemum, also known as one-root grass. It is the dry whole herb of Orobanche pycnostachya Hance. Distributed in Northeast China, North China, Shanxi, Shaanxi and other places. It has the functions of invigorating the kidney and yang, and strengthening the bones and muscles. It is used to treat cold pain in the waist and knees due to kidney deficiency, impotence and spermatorrhea, neurosis, and diarrhea in children. Folks are also used as a substitute for the precious medicinal material "desert ginseng" meat congrong. Plants of the Lidanaceae generally contain phenylethanol glycosides and are important resource plants for phenylethanoid glycosides. Modern pharmacological studies have shown that this type of compound has good medical value, such as: antibacterial, anti-inflammatory, antiviral, antitumor, antioxidative, scavenging free radicals, immune regulation, enhancing memory, protecting liver, strengthening the heart and so on.

Fingerprint of traditional Chinese medicine is recognized at home and abroad as one of the effective means to comprehensively reflect the information of medicinal materials, identify medicinal materials and evaluate the quality of traditional Chinese medicine. or image.

At present, there is no comprehensive report on the quality control of Liedang medicinal materials, and its quality standards are not included in the "Chinese Pharmacopoeia". Therefore, it is very necessary and meaningful to establish a method that can reflect the quality information of Liedang medicinal materials more comprehensively, to guide the use of Liedang medicinal materials in medicine, and to increase the limit of development and utilization of Liedang medicinal materials.

Contents of the invention

The present invention proposes a method for establishing a fingerprint of chrysanthemum japonica, comprising the following steps:

1) Preparation of the test solution: take chrysanthemum as medicinal material powder, add 70% methanol, weigh, soak, sonicate, make up the lost weight with solvent, filter, and take the subsequent filtrate to filter through 0.45 μm microporous Membrane filtration, standby;

2) Inject the test solution into a high-performance liquid chromatograph to obtain fingerprints. The chromatographic conditions adopted are: chromatographic column: octadecylsilane bonded silica gel chromatographic column; mobile phase: acetonitrile (A)-1‰ phosphoric acid Aqueous solution (B), gradient elution 89% B (0min) ~ 85% B (13min) ~ 81% B (22min) ~ 78% B (33min) ~ 76% B (50)min; flow rate: 1ml/min; The detection wavelength is 230-400nm; the column temperature is 20-30°C; the injection volume is 5-15μl.

Preferably, the chromatographic column adopts a Shimadzu Inertsil ODS-SP HPLC chromatographic column or a YMC-Pack ODS-A chromatographic column with a specification of 250 mm×4.6 mm and 5 μm.

More preferably, in step 1), take 0.15 g of chrysanthemum as medicinal material powder, add 25 ml of 70% methanol, weigh it, soak for 1 hour, ultrasonically treat for 40 minutes, make up the lost weight with solvent, filter, and take the subsequent filtrate Filter through a 0.45 μm microporous membrane.

More preferably, the flow rate is 1 ml/min; the detection wavelength is 295 nm; the column temperature is 25° C.; the injection volume is 10 μl.

The present invention also provides a method for using fingerprints to evaluate the quality of Huanghualiedang medicinal materials, comprising the following steps: Step 1: establishing a standard fingerprint, including: a) preparing a test solution from more than 10 batches of samples from different origins: taking As medicinal material powder, add 70% methanol, weigh, soak, sonicate, use solvent to make up for the lost weight, filter, take the subsequent filtrate and filter it with a 0.45 μm microporous membrane, and set aside; b) Reference substance Solution preparation: Weigh 1) α-L-rhamnose (1→3)[β-D-glucose (1→6)]-O-(4-O-caffeoyl)-D-glucose caffeoyl tri Glycosides, 2) sinapinic acid-4-O-β-D glucoside, 3) 7-hydroxy verbascoside, 4) 7-methoxy verbascoside, 5) verbascoside, 6) crestinate, 7) 3'-methoxyverbacoside), 8) 2'-acetyl verbascoside, and 9) isosteridoside, a total of 9 kinds of reference substances were dissolved in methanol to make a mixed solution of a certain concentration, and the microporous filter Filter through a membrane of 0.45 μm and set aside; c) Inject each test solution and reference solution into a high-performance liquid chromatograph successively to obtain fingerprints respectively. The chromatographic conditions are: chromatographic column: octadecylsilane bonded silica gel chromatography Column; mobile phase: acetonitrile (A)-1‰ phosphoric acid aqueous solution (B), gradient elution 89% B (0min) ~ 85% B (13min) ~ 81% B (22min) ~ 78% B (33min) ~ 76 %B(50)min; flow rate: 1ml/min; detection wavelength 230-400nm; column temperature 20-30°C; A standard fingerprint. Step 2: Obtain a fingerprint of a sample to be evaluated in the same manner as above; Step 3: Use the aforementioned similarity evaluation software to evaluate the similarity between the fingerprint of the sample to be evaluated and the standard fingerprint, and determine if the similarity is greater than 0.9 The quality of the sample to be evaluated meets the requirements.

Further, the standard fingerprint contains the following 15 characteristic peaks, wherein peak No. 10 is a reference peak, its relative retention time and relative peak area are 1.00, and the relative retention times of each peak are respectively: 8.07, 12.22, 20.11, 20.74 , 25.17, 26.56, 27.00, 28.32, 32.58, 35.76, 38.46, 40.35, 41.06.

Furthermore, the relative peak areas of the 15 characteristic peaks of the standard fingerprint are 0.0035, 0.0053, 0.0038, 0.0099, 0.0114, 0.0086, 0.0073, 0.0041, 0.0042, 1.000, 0.1995, 0.0263, 0.0546, 0.0081, 0.0037

Furthermore, the parameters of the peaks corresponding to the 9 reference substances in the standard fingerprint were used to evaluate the similarity with the peak parameters corresponding to the sample to be tested, and when the similarity was greater than 0.90, it was determined that the medicinal material compounding requirement.

According to the preparation method of the test product solution of the present invention, the operation solution has high precision, stability, and reproducibility, and has strong system applicability, and can be applied to the production of medicinal materials of this genus. The established fingerprint analysis method of Huanghua Liedang has high sensitivity and good reproducibility; the obtained standard fingerprint is typical and representative, and can comprehensively reflect the types and quantities of chemical components contained in the medicinal material of Huanghua Liedang. Then provide a more comprehensive basis for the overall evaluation and description of medicinal materials. The high-efficiency liquid phase fingerprinting technique for Liedang medicinal material established in the present invention makes up for the blank quality control method of Liedang medicinal material at present, and makes the quality control technology of Liedang medicinal material more comprehensive.

Description of drawings

Fig. 1 is the identification map of the reference substance obtained according to the method of the present invention.

Fig. 2 is a standard fingerprint spectrum obtained according to the method of the present invention.

Figure 3 is an overlay of HPLC fingerprints of 17 batches of Huanghua Liedang medicinal materials.

detailed description

Preparation of the test solution

In this link, take Huanghualiu as medicinal material powder, add a calculated amount of methanol solution, and perform ultrasonic treatment after soaking, use solvent to make up for the lost weight, filter, take the subsequent filtrate, filter through a 0.45 μm microporous membrane, and set aside .

Among the present invention, use methanol aqueous solution as need testing sample solvent, the concentration range of methanol solution can be between 50-90%, preferably between 60-80%, most preferably 70% methanol solution.

In the present invention, it is preferred to grind the yellowing column to a size below 40 mesh, and pass through a 40 mesh sieve during use. The extraction method can be reflux or ultrasonic treatment, preferably soaking for a certain period of time before treatment, preferably soaking for 1 hour. Ultrasonic extraction is preferred, and the extraction time may be 20-80 min, preferably 40 min.

In the present invention, the amount of sampling can be in the range of 0.1-0.3g. In the case of adopting the above-mentioned most preferred method, preferably sampling 0.15g, adding 25ml of 70% methanol, weighed, soaked for 1h, ultrasonically treated for 40min, placed in After room temperature, weigh again, make up the lost weight with a solvent, filter, take the subsequent filtrate and filter it with a 0.45 μm microporous membrane to obtain the test solution.

Therefore, the preparation method of the most preferred Huang Hua Lie as the test solution of the present invention is: take about 0.15g of the medicinal material powder, accurately weigh it, put it in a 100mL conical flask with a stopper, add 25mL of 70% methanol accurately, and soak for 1h , Ultrasonic extraction for 40min, put it at room temperature, weigh again, use solvent to make up the lost weight, filter, take the filtrate and filter it with a 0.45μm microporous membrane to obtain the product.

Preparation of reference solution

Take an appropriate amount of each reference substance, number them separately, dissolve them in methanol to a constant volume, make a solution of a certain concentration, filter through a microporous membrane (0.45 μm), and set aside. The reference substance used has the following 9:

α-L-rhamnose(1→3)[β-D-glucose(1→6)]-O-(4-O-caffeoyl)-D-glucose

Sinapoyl-4-O-β-D-glucopyranoside

7-hydroxyverbacoside (campneosideⅡ)

7-methoxy verbascoside (campneosideⅠ, also known as sarcoside)

3'-Methoxyverbacoside (leucosceptosideA)

2'-acetylacteoside (2'-acetylacteoside)

Isocrenatoside

Chromatographic conditions

The chromatographic conditions used in the present invention are preferably: the chromatographic column is an octadecylsilane bonded silica gel chromatographic column; the mobile phase is acetonitrile (A)-1‰ phosphoric acid aqueous solution (B), and adopts gradient elution 89% B (0min) ～85%B (13min)～81%B(22min)～78%B(33min)～76%B(50)min; detection wavelength 230～400nm; column temperature 20～30℃; injection volume 5～15μl.

In the best embodiment of the present invention, it is determined that the optimal chromatographic condition of Huanghua Liedang HPLC fingerprint analysis is: chromatographic column: Shimadzu Inertsil ODS-SP HPLC chromatographic column or YMC-Pack ODS-A chromatographic column (250mm * 4.6 mm, 5μm); mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution, gradient elution: 89% B (0min) ~ 85% B (13min) ~ 81% B (22min) ~ 78% B (33min) ~ 76% B (50)min; flow rate: 1.0ml/min; detection wavelength 295nm; column temperature 25°C; injection volume 10μl.

Establishment of Standard Fingerprints

Using the above-mentioned chromatographic conditions, 17 batches of Huanghualiedang samples from different origins and different collection times were measured, 17 fingerprints were obtained, 15 common peaks were determined, and 9 of them were identified with the aforementioned 9 compounds as controls, and evaluated by similarity The software evaluates the fingerprints of 17 samples, and selects a fingerprint with the highest similarity—the fingerprint of No. S9 medicinal material as the standard fingerprint. The comparison between the identification map of the reference substance (obtained from the aforementioned reference substance) and the map of No. S9 medicinal material is shown in Fig. 1 and Fig. 2 .

Using the aforementioned most preferred parameters and chromatographic conditions, the standard fingerprint contains 15 common peaks, the retention times and relative peak areas are shown in Table 1.

Table 1 The relative retention time and relative peak area of the common peaks in the standard fingerprint of Huanghua Liedang

In this standard fingerprint, the 9 peaks whose structure is determined correspond to the peak numbers in the standard fingerprint as follows:

No. 2: -L-rhamnose (1→3)[β-D-glucose (1→6)]-O-(4-O-caffeoyl)-D-glucose

No. 4: Sinapoyl-4-O-β-D-glucopyranoside

No. 6: 7-Hydroxy verbascoside (campneosideⅡ)

No. 7: 7-methoxy verbascoside (campneosideⅠ)

No. 10: Actascoside

No. 11: crenatoside

No. 12: 3'methoxy verbascoside (leucosceptosideA)

No. 13: 2-acetylacteoside (2'-acetylacteoside)

No. 14: isocrenatoside

In the embodiment of the present invention, standard fingerprints were established from 17 samples of Rhizoma japonica from the following sources.

Table 2. Information on 17 batches of Huanghua Liedang medicinal materials

Quality Evaluation of Huanghua Liedang Medicinal Material

According to the present invention, the fingerprint of the sample to be evaluated can be obtained using the conditions established by the above-mentioned fingerprint, and then compared with the aforementioned standard fingerprint, evaluated by similarity evaluation software, and the similarity is investigated, and the similarity is 0.90-1.00 Then it is determined that the sample meets the requirements. The evaluation software that adopts in the typical embodiment of the present invention is the Chinese medicine chromatographic fingerprint spectrum similarity evaluation system (2004A version) recommended by the National Pharmacopoeia Commission. In another embodiment, only the similarity evaluation is performed on the peak parameters of the aforementioned reference product and the corresponding peak parameters of the sample to be evaluated. If the similarity is greater than 0.90, it is determined that the product meets the requirements. In another embodiment, the first two methods can be combined, and when the similarity obtained twice is greater than 0.90, the analysis result is more reliable and accurate.

Methodology Validation

Inspection of precision

According to the above-mentioned most preferred method for preparing the test solution, the sample is prepared, and according to the above-mentioned most preferred fingerprint chromatographic conditions, continuous sampling analysis is carried out 6 times. The obtained data is input into the similarity evaluation system of the chromatographic fingerprint of traditional Chinese medicine, and the precision matching data and the similarity data of the precision matching are calculated, and the relative retention time of the common peak and the ratio of the relative peak area are calculated at the same time. Results The RSD values of relative retention time and relative peak area ratio of each common peak after HPLC analysis of Huanghualiedang test solution were between 0.01%～0.08% and 0.75%～3.87%, indicating the precision of the whole instrument detection system. good degree. The results are shown in Table 3 and Table 4.

Table 3. The precision matching data of Huanghua Liedang and the RSD values of relative retention time and relative peak area

Table 4. Similarity data for precision matching

Repeated inspection

Prepare 6 parts of No. S11 sample in parallel according to the above-mentioned most preferred method for preparing the test solution, and continuously inject and analyze according to the above-mentioned most preferred fingerprint chromatographic conditions. The obtained data is input into the similarity evaluation system of chromatographic fingerprints of traditional Chinese medicine, and the repetitive matching data and the similarity data of the repetitive matching are calculated. At the same time, the ratio of relative retention time and relative peak area of common peaks is calculated. The results are shown in Tables 5 and 6. The results showed that the RSD values of 6 samples of Huanghua Liedang tested by HPLC were between 0.01%-0.14% and 0.87%-4.40%, indicating that the repeatability of the method was good.

Table 5. Repeatability matching data and RSD values of relative retention time and relative peak area

Table 6. Similarity data for repetitive matches

Stability study

Take the same aliquot of the test solution and inject 10 μL for analysis at 0, 2, 4, 8, and 12 hours respectively. The obtained data is input into the similarity evaluation system of the chromatographic fingerprint of traditional Chinese medicine, and the similarity data and the stability matching data of the stability matching are calculated. At the same time, the ratio of relative retention time and relative peak area of common peaks is calculated. The results are shown in Tables 7 and 8. Results The RSD values of relative retention time and relative peak area ratio were between 0.01%-0.10% and 1.16%-4.77% respectively within 12h, indicating that the test solution had good stability within 12h.

Table 7. Huanghua Liedang stability matching data and relative retention time, RSD value of relative peak area

Table 8 Similarity data of stability matching

The above methodological investigation shows that the fingerprint operation method established at present is feasible and can be used for the establishment and analysis of the fingerprint of Huanghua Liedang.

Similarity Evaluation of 17 Batches of Huanghua Liedang Samples

A sample is respectively taken from the aforementioned 17 batches of Huang Hua Lie as medicinal materials, and the HPLC fingerprints of each batch of medicinal materials are obtained by the same method as described above. The common peaks were analyzed and evaluated, and the similarity was calculated by the average method to obtain the similarity and similarity matching data of 17 batches of medicinal materials (Table 9, 10). The overlay of HPLC fingerprints of each batch of samples is shown in Figure 3.

Claims (6)

1. A method for setting up the fingerprint of Huanghua Liedang medicinal material, comprising the following steps: Establish a standard fingerprint, including: a) Prepare the test solution from more than 10 batches of samples from different origins: take Huanghua Lie as medicinal powder, add 70% methanol, weigh, soak, sonicate, and make up for the loss with a solvent weight, filtered, take the filtrate and filter it with a 0.45μm microporous membrane, and set aside; b) Preparation of the reference solution: Weigh 1) α-L-rhamnose (1→3)[β-D- Glucose (1→6)]-O-(4-O-caffeoyl)-D-glucocaffeoyl triglucoside, 2) sinapinic acid-4-O-β-D glucoside, 3) 7-hydroxyverbacoside, 4) 7-methoxyverbacoside, 5) verbascoside, 6) crestoside, 7) 3'-methoxyverbacoside), 8) 2'-acetyl verbascoside, 9) isoscarnate A total of 9 kinds of reference substances of glycosides were dissolved in methanol to constant volume to make a mixed solution of a certain concentration, filtered through a microporous membrane of 0.45 μm, and set aside; c) each test solution and reference solution were injected into the high-efficiency liquid phase In the chromatograph, the fingerprints are obtained respectively, and the chromatographic conditions are: chromatographic column: octadecylsilane bonded silica gel chromatographic column; mobile phase: acetonitrile (A)-1‰ phosphoric acid aqueous solution (B), gradient elution 89% B ( 0min)～85%B(13min)～81%B(22min)～78%B(33min)～76%B(50)min; flow rate: 1ml/min; detection wavelength 230～400nm; column temperature 20～30℃ ; The injection volume is 5-15 μl; d) Evaluate the fingerprints of 17 samples with the similarity evaluation software, and determine a standard fingerprint from them. 2. The method according to claim 1, wherein the chromatographic column adopts a Shimadzu Inertsil ODS-SP HPLC chromatographic column or a YMC-Pack ODS-A chromatographic column whose specification is 250mm×4.6mm and 5 μm. 3. The method as claimed in claim 1, wherein, in step a), take 0.15g of chrysanthemum as medicinal material powder, add 25ml of 70% methanol, weigh it, soak for 1h, sonicate for 40min, and make up the lost amount with solvent Weight, filter, take the subsequent filtrate and filter it with a 0.45 μm microporous membrane, and set it aside. 4. The method according to claim 3, wherein the flow rate is 1 ml/min; the detection wavelength is 295 nm; the column temperature is 25° C.; the injection volume is 10 μl. 5. method as claimed in claim 4, wherein, described standard fingerprint spectrum comprises following 15 characteristic peaks, wherein No. 10 peak is reference peak, and its relative retention time and relative peak area are 1.00, and the retention time of each peak is respectively For: 8.07, 8.34, 11.02, 12.22, 20.11, 20.74, 25.17, 26.56, 27.00, 28.32, 32.58, 35.76, 38.46, 40.35, 41.06. 6. The method according to claim 5, wherein, the relative peak areas of 15 characteristic peaks of the standard fingerprint are respectively 0.0035, 0.0053, 0.0038, 0.0099, 0.0114, 0.0086, 0.0073, 0.0041, 0.0042, 1.000, 0.1995 , 0.0363, 0.0546, 0.0086, 0.003711.