CN111257456A - Fingerprint detection method for overground part of ligusticum wallichii - Google Patents
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Abstract
The invention discloses a fingerprint detection method for the overground part of ligusticum wallichii. The fingerprint detection method of the overground part (stem leaf) of the ligusticum wallichii fills the blank of the quality detection of the overground part of the ligusticum wallichii, and establishes a standard quality control system for the overground part of the ligusticum wallichii. The fingerprint of the overground part of the ligusticum wallichii is marked with 20 common peaks, and two components of ligustilide and butenyl phthalide are identified. Compared with the reference map, the similarity of the fingerprint maps of the overground parts of the ligusticum wallichii of different batches is 0.942-0.990, and the similarity is good, so that the chemical components of the overground parts of the ligusticum wallichii of different producing areas have good consistency, and the method can be used for quality control of the overground parts of the ligusticum wallichii.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine fingerprint spectrums, in particular to a fingerprint spectrum detection method for the overground part of ligusticum wallichii.
Background
Rhizoma Ligustici Chuanxiong is dried rhizome of Umbelliferae plant rhizoma Ligustici Chuanxiong Lingusticicum chuanxiong Hort, and has effects of promoting blood circulation, activating qi-flowing, removing blood stasis and relieving pain. It mainly contains volatile oil, alkaloid, phenolic acid, lactone, terpene, phthalide derivative, polysaccharide and inorganic element. At present, the research and application of the ligusticum wallichii are concentrated on underground rhizomes, and the research and the utilization of the overground parts of the ligusticum wallichii are very little. The overground part of the ligusticum wallichii is rich in resources, accounts for 75 percent of the fresh weight of the whole plant, but is discarded more, so that the resource is greatly wasted. The overground part of the rhizoma ligustici wallichii can dispel wind, calm the liver and dispel wind-cold in the brain. Can be used for treating dizziness, infantile convulsion, lacrimation due to wind-induced eye, headache due to wind-induced headache, and excessive nasal discharge. The record of Shen nong Ben Cao Jing records that the Chinese herbal medicine is effective in treating cough, arresting convulsion, repelling evil and evil, removing poison and poison, promoting recovery from ghost, removing three insects, and promoting the recovery of spirit after long-term administration. The famous physicians' book records that it is "the wind dizziness due to the main body attacking the middle-aged wind and the long-term wind in the head". Modern researches show that the water decoction of the overground part of the ligusticum wallichii obviously prolongs the bleeding and blood coagulation time of mice, prolongs the cough incubation period of the mice caused by ammonia water, reduces the cough frequency of the mice caused by the ammonia water, and has certain effects of promoting blood circulation and relieving cough.
At present, research reports on one or more chemical components of the overground part of the ligusticum wallichii are reported and used as a method for evaluating the quality of the overground part of the ligusticum wallichii, but the content level of the single component cannot achieve the aim of controlling and evaluating the quality of the overground part of the ligusticum wallichii. The fingerprint spectrum as a quality control method of single Chinese medicine and compound full components and extracts thereof can more comprehensively reflect the full chemical spectrum of the Chinese medicine, and has become an internationally recognized effective means for controlling and evaluating the quality of the Chinese medicine and natural medicine. The aboveground parts of the ligusticum wallichii have great differences in chemical component properties, polarities and the like, the existing single component detection is difficult to reflect the overall appearance of the ligusticum wallichii, and the integral quality control of the aboveground parts of the ligusticum wallichii is difficult. However, no research report about the fingerprint of the overground part of the ligusticum wallichii exists at present.
Disclosure of Invention
In order to solve the problems, the invention provides a fingerprint detection method for the overground part of the ligusticum wallichii, which adopts high performance liquid chromatography for detection and comprises the following steps:
1) preparing a test solution: pulverizing aerial part of rhizoma Ligustici Chuanxiong, extracting with methanol, and filtering to obtain filtrate;
2) respectively sucking the test solution to be tested, injecting the test solution into a high performance liquid chromatograph, and recording a chromatogram; the chromatographic conditions were as follows:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; mobile phase: taking 0.05-0.2% phosphoric acid solution as a mobile phase A; acetonitrile is used as a mobile phase B; the gradient elution procedure was as follows:
further, the extraction in the step 1) is ultrasonic extraction, and the ultrasonic extraction time is 30 min.
Further, the mass volume ratio of the overground part of the ligusticum wallichii in the step 1) to the methanol is 1-10 g: 10ml, preferably 2 g: 10 ml.
Further, the chromatographic column in the chromatographic conditions of the step 2) is Kromasil C18, the specification is 4.6X 250mm, and the size is 5 μm; the concentration of the mobile phase A phosphoric acid solution is 0.1%.
Further, in the chromatographic conditions in the step 2), the flow rate is 1.0mL/min, the sample injection amount is 20 mu L, the column temperature is 35 ℃, and the detection wavelength is 254 nm.
Further, the detection method further comprises the following steps: dissolving ligustilide and butenyl phthalide in methanol to obtain reference solution, sucking the reference solution, injecting into high performance liquid chromatograph, and detecting according to the chromatographic conditions in step 2).
Furthermore, each 1mL of the control solution contains 0.1-0.5 mg/mL of ligustilide and 0.01-0.08 mg/mL of butenyl phthalide.
Further, the fingerprint map of the overground part of the ligusticum wallichii should have 20 common characteristic peaks, the peak corresponding to the ligustilide reference substance is a peak 16, the peak corresponding to the butenyl phthalide reference substance is a peak 17, the relative retention time of each characteristic peak and the peak 17 is calculated, and the relative retention time is within +/-5% of a specified value; the specified values are: peak 1: 0.2446, Peak 2: 0.2631, peak 3: 0.4306, Peak 4: 0.5600, peak 5: 0.6588, Peak 6: 0.6962, peak 7: 0.7567, Peak 8: 0.7703, peak 9: 0.8191, peak 10: 0.8226, peak 11: 0.8447, peak 12: 0.8877, peak 13: 0.9269, peak 14: 0.9657, peak 15: 0.9814, peak 16: 0.9921, peak 17: 1.0000, peak 18: 1.1425, peak 19: 1.1856, peak 20: 1.3283.
the fingerprint detection method of the overground part (stem leaf) of the ligusticum wallichii fills the blank of the quality detection of the overground part of the ligusticum wallichii, and establishes a standard quality control system for the overground part of the ligusticum wallichii. The fingerprint of the overground part of the ligusticum wallichii is marked with 20 common peaks, and two components of ligustilide and butenyl phthalide are identified. Compared with the reference map, the similarity of the fingerprints of the overground part medicinal materials of the ligusticum wallichii of different batches is 0.942-0.990, and the similarity is good, so that the chemical components of the overground parts of the ligusticum wallichii of different producing areas have good consistency, and the mass control method can be used for quality control of the overground parts of the ligusticum wallichii.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 HPLC control map of aboveground part of rhizoma Ligustici Chuanxiong
FIG. 2 HPLC finger print of aboveground part of Ligusticum wallichii
FIG. 3 cluster analysis of samples of aboveground part of Ligusticum chuanxiong
Detailed Description
The reagents, reagents and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products.
Example 1 fingerprint detection of overground part of Ligusticum chuanxiong Hort of the present invention
1) Preparation of a test solution: taking 2.0g of sample powder (sieved by a No. six sieve) of the overground part of the ligusticum wallichii, precisely weighing, placing the sample powder into a 50mL conical flask with a plug, precisely adding 10mL of methanol, weighing, ultrasonically treating for 30 minutes, cooling, complementing weight loss, shaking up, filtering by a 0.22 mu m microporous filter membrane, and taking the subsequent filtrate to obtain the ligusticum wallichii extract powder;
2) preparation of control solutions
Precisely weighing 10mg of ligustilide reference substance, dissolving in 10mL volumetric flask with methanol to scale, and shaking to obtain the reference ligustilide stock solution with concentration of 1.0780 mg/mL. Precisely measuring 1.0mL of stock solution, adding methanol into a 5mL volumetric flask, diluting to scale, and shaking to obtain a ligustilide reference substance with a concentration of 0.2156 mg/mL;
taking about 10mg of a butenyl phthalide reference substance, precisely weighing, adding methanol into a 25mL volumetric flask to dissolve to a scale, and shaking up to obtain a butenyl phthalide reference substance stock solution with the concentration of 0.4300 mg/mL. Precisely measuring 1.0mL of stock solution, adding methanol into a 10mL volumetric flask to dilute to a scale, and shaking up to obtain a butenyl phthalide reference substance with the concentration of 0.0430 mg/mL;
3) respectively sucking the test solution and the reference solution, injecting into a high performance liquid chromatograph, and recording the chromatogram; the chromatographic conditions were as follows:
a chromatographic column: kromasil C18 (250X 4.6mm,5 μm); detection wavelength: 254 nm; the sample injection volume is 20ul, the flow rate is 1.0ml/min, and the column temperature is 35 ℃; mobile phase: taking 0.1% phosphoric acid water solution as a mobile phase A and acetonitrile as a mobile phase B; the gradient elution procedure was as follows:
4) the fingerprint of the overground part of the ligusticum wallichii has 20 common characteristic peaks, the peak corresponding to the ligustilide reference substance is a peak 16, the peak corresponding to the butenyl phthalide reference substance is a peak 17, the relative retention time of each characteristic peak and the peak 17 is calculated, and the relative retention time is within +/-5% of a specified value; the specified values are: peak 1: 0.2446, Peak 2: 0.2631, peak 3: 0.4306, Peak 4: 0.5600, peak 5: 0.6588, Peak 6: 0.6962, peak 7: 0.7567, Peak 8: 0.7703, peak 9: 0.8191, peak 10: 0.8226, peak 11: 0.8447, peak 12: 0.8877, peak 13: 0.9269, peak 14: 0.9657, peak 15: 0.9814, peak 16: 0.9921, peak 17: 1.0000, peak 18: 1.1425, peak 19: 1.1856, peak 20: 1.3283.
the beneficial effects of the present invention are illustrated by the following experimental examples:
experimental example 1
1 Instrument and reagent
1.1 instruments
HPLC-DAD3000 type high performance liquid chromatograph (seimer feishell science ltd); chromatographic column Kromasil C18(250X 4.6mm,5 μm); an electronic balance of FA 2004N type (shanghai national bridge precision scientific instruments ltd); UPS-11-20L model ultrapure water machine (Sichuan Yopu ultrapure technology, Inc.), KQ5200E model ultrasonic cleaner (Kunshan ultrasonic instruments, Inc.).
1.2 reagents and drugs
Butenyl phthalide (BP0061, Kyopu scientific development Co., Ltd.); ligustilide (DDYJ0412-08, Chengdu Dingdang times pharmaceutical science and technology Co., Ltd.); acetonitrile (chromatographically pure, SIGMA-ALORICH); phosphoric acid (AR, metropolis chemicals ltd); methanol (chromatographically pure, SIGMA-ALORICH); methanol (AR, metropolis chemicals, ltd); ultrapure water. 11 batches of above-ground parts of Chuan Xiong from different producing areas in Sichuan are shown in Table 1 for sample information and numbers.
TABLE 1 sample information
2 methods and results
2.1 creation of fingerprint
2.1.1 chromatographic conditions
A chromatographic column Kromasil C18(250 x 4.6mm,5 μm) and a 0.1% phosphoric acid aqueous solution (A) -acetonitrile (B) are used as mobile phases, and gradient elution is carried out (0-1 min, 10% B; 1-9 min, 10% → 16% B; 9-10 min, 16% → 20% B; 10-21 min, 20% B; 21-27 min, 20% → 60% B; 27-39 min, 60% B; 39-40 min, 60% → 90% B; 40-50 min, 90% B; 50-51 min, 90% → 100% B; 51-60 min, 100% B; after 60min, 100% B is eluted for 30min, so that chromatographic conditions are not added, the flow rate is 1.0mL/min, the sample volume is 20 μ L, and the column temperature is 35 ℃ and 254nm detection is carried out.
2.1.2 preparation of control solutions
Precisely weighing about 10mg ligustilide reference substance, dissolving in 10mL volumetric flask with methanol to scale, and shaking to obtain reference ligustilide stock solution with concentration of 1.0780 mg/mL. Precisely measuring 1.0mL of stock solution, adding methanol to dilute to scale in a 5mL volumetric flask, and shaking up to obtain a ligustilide control product with a concentration of 0.2156 mg/mL.
Taking about 10mg of a butenyl phthalide reference substance, precisely weighing, adding methanol into a 25mL volumetric flask to dissolve to a scale, and shaking up to obtain a butenyl phthalide reference substance stock solution with the concentration of 0.4300 mg/mL. Precisely measuring 1.0mL of stock solution, adding methanol to dilute to a scale in a 10mL volumetric flask, and shaking up to obtain a butenyl phthalide reference substance with the concentration of 0.0430 mg/mL.
2.1.3 preparation of test solutions
Taking about 2.0g of sample powder (passing through a No. six sieve) of the overground part of the ligusticum wallichii, precisely weighing, placing the sample powder into a 50mL conical flask with a plug, precisely adding 10mL of methanol, weighing, ultrasonically treating for 30 minutes, cooling, complementing weight loss, shaking up, passing through a 0.22 mu m microporous filter membrane, and taking the subsequent filtrate to obtain the ligusticum wallichii extract.
2.1.4 precision investigation
Taking an S8 sample, preparing a sample according to the method under the item 2.1.3, continuously injecting samples for 6 times according to the chromatographic condition under the item 2.1.1, and recording the retention time and the peak area of each chromatographic peak. The results show that the RSD value of 20 common peaks relative to the retention time is between 0.01% and 0.07%, and the RSD value of the relative peak area is between 0.42% and 2.69%, which indicates that the precision of the instrument is good.
2.1.5 repeatability test
6 parts of S8 sample is taken to prepare a sample according to the method under the item 2.1.3, sample injection is carried out according to the chromatographic condition under the item 2.1.1, and the retention time and the peak area of each chromatographic peak are recorded. The results show that the RSD value of 20 common peaks relative to the retention time is between 0.00% and 0.12%, and the RSD value of the relative peak area is between 0.44% and 4.65%, which indicates that the method has good repeatability.
2.1.6 stability Studies
Taking an S8 sample to prepare a sample according to the method under the item 2.1.3, injecting samples according to the chromatographic condition under the item 2.1.1 at 0, 3, 6, 9, 12 and 24 hours after the preparation, and recording the retention time and the peak area of each chromatographic peak. The results show that the RSD value of 20 common peaks relative to the retention time is between 0.00% and 0.13%, and the RSD value of the relative peak area is between 0.47% and 4.58%, which indicates that the sample has good stability within 24 h.
2.1.7 determination of common Peak
Taking 11 batches of aboveground samples of rhizoma Ligustici Chuanxiong, preparing sample according to the method of '2.1.3 items', injecting sample according to the chromatographic condition of '2.1.1 items', and recording chromatogram. Introducing the chromatogram of the upper part samples of each region into software of a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition) after 5min, taking the chromatogram of S1 sample as a reference chromatogram, adopting a median method, setting the time width to be 0.1, calibrating 20 common peaks through chromatographic peak multipoint correction, automatically matching the chromatographic peaks of the fingerprints of the upper part samples of 11 batches of ligusticum wallichii to generate a reference chromatogram which is used as the fingerprint of the upper part of the ligusticum wallichii, and showing in figure 1 and a fingerprint superposition chart in figure 2. Comparison of the retention time and the ultraviolet spectrum of the sample with those of the control sample confirmed 2 common peaks, i.e., peaks 16 and 17, which are shown in fig. 3 as ligustilide and butenyl phthalide, respectively. According to the technical principle of fingerprint spectrum reference peak selection, in order to facilitate the identification of fingerprint spectrum, the No. 17-butenyl phthalide peak as the main active component is selected as the reference peak. The retention time and peak area of the reference peak are set as reference 1, the relative retention time and relative peak area of the common peak of 11 batches of aboveground samples of ligusticum wallichii are calculated respectively, and the peak area percentage of the single common peak is calculated by using a normalization method, which is shown in tables 2 and 3.
The results show that the RSD value of the common peak relative retention time of different batches of aboveground sample of the ligusticum wallichii is between 0.00 and 0.33 percent, and the RSD of the relative peak area is between 8.13 and 88.12 percent; the percentage of the No. 1 peak and the No. 4 peak in the common peaks is larger and is between 10 and 15 percent, the percentage of the No. 3 peak and the No. 16 and 17 peaks is between 5 and 10 percent, and the percentage of the rest peaks is less than 2 percent.
2.1.8 evaluation of similarity
The similarity calculation of fingerprints of 11 batches of ligusticum wallichii overground part samples is carried out by adopting software of a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition), and the result is shown in table 4. Compared with the contrast map, the similarity of 11 batches of aboveground samples of the ligusticum wallichii is between 0.942 and 0.990, and the similarity is good, which indicates that the chemical components of aboveground samples of the ligusticum wallichii in different producing areas have good consistency. Analysis of similarity between 11 batches of samples found: the similarity of the other 10 samples except the S4 sample is more than 0.9; the similarity of S4 to S2, S3, S5 and S8 is 0.843-0.866, the similarity is low, but the similarity to the rest of samples exceeds 0.9, possibly related to the production area, and further analysis is needed.
Table 411 batch Ligusticum chuanxiong Hort ground part fingerprint similarity
2.1.9 Cluster analysis
The SPSS 22.0 software is adopted, the peak areas of 20 common peaks are taken as variables, the interclass connection method and the Euclidean square distance are adopted to perform cluster analysis on 11 batches of aboveground sample of the ligusticum wallichii, and the result is shown in figure 3. 11 batches of samples of the overground part of the ligusticum wallichii can be divided into two types: class I is S4; the other samples were of class II. The similarity of the sample S4 with other 10 batches of samples is low, and is basically consistent with the conclusion drawn by the fingerprint.
3. Conclusion
The invention establishes a fingerprint chromatogram method of the overground part of the ligusticum wallichii for the first time on the basis of 11 batches of overground part samples of the ligusticum wallichii in different producing areas. The chromatogram of each sample is led into software of a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition), so that the fingerprint of the overground part of the ligusticum wallichii is established, 20 common peaks are calibrated, and two components of ligustilide and butenyl phthalide are identified. Compared with the reference map, the similarity of the fingerprints of the overground part medicinal materials of the ligusticum wallichii of different batches is 0.942-0.990, and the similarity is good, which shows that the chemical components of the overground parts of the ligusticum wallichii of different producing areas have good consistency. However, the RSD of the relative peak area of part of the common peaks is large, and may be related to the soil conditions, cultivation modes and fertilization techniques of different producing areas.
In conclusion, the fingerprint detection method for the aboveground part (stem leaf) of the ligusticum wallichii fills the blank of quality detection of the aboveground part system of the ligusticum wallichii, and establishes a standard quality control system for the aboveground part of the ligusticum wallichii. The fingerprint of the overground part of the ligusticum wallichii is marked with 20 common peaks, two components of ligustilide and butenyl phthalide are identified, and the similarity is good, so that the fingerprint provided by the invention can be used for quality control of the overground part of the ligusticum wallichii in different producing areas, and has practical popularization and application values.
Claims (8)
1. A fingerprint detection method for overground parts of ligusticum wallichii is characterized by comprising the following steps: the detection is carried out by adopting a high performance liquid chromatography, and the method comprises the following steps:
1) preparing a test solution: pulverizing aerial part of rhizoma Ligustici Chuanxiong, extracting with methanol, and filtering to obtain filtrate;
2) absorbing the test solution, injecting the test solution into a high performance liquid chromatograph, and recording a chromatogram; the chromatographic conditions were as follows:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; mobile phase: taking 0.05-0.2% phosphoric acid solution as a mobile phase A; acetonitrile is used as a mobile phase B; the gradient elution procedure was as follows:
2. the detection method according to claim 1, characterized in that: the extraction in the step 1) is ultrasonic extraction, and the ultrasonic extraction time is 30 min.
3. The detection method according to claim 1, characterized in that: step 1), the mass volume ratio of the overground part of the ligusticum wallichii to methanol is 1-10 g: 10ml, preferably 2 g: 10 ml.
4. The detection method according to claim 1, characterized in that: the chromatographic column in the chromatographic condition of the step 2) is Kromasil C18, the specification is 4.6 x 250mm, and the size is 5 μm; the concentration of the mobile phase A phosphoric acid solution is 0.1%.
5. The detection method according to claim 1, characterized in that: and 2) under the chromatographic condition, the flow rate is 1.0mL/min, the sample injection amount is 20 mu L, the column temperature is 35 ℃, and the detection wavelength is 254 nm.
6. The detection method according to claim 1, characterized in that: the detection method further comprises the following steps:
dissolving ligustilide and butenyl phthalide in methanol to obtain reference solution, sucking the reference solution, injecting into high performance liquid chromatograph, and detecting according to the chromatographic conditions in step 2).
7. The detection method according to claim 6, characterized in that: the contrast solution contains 0.1-0.5 mg/mL ligustilide and 0.01-0.08 mg/mL butenyl phthalide per 1 mL.
8. The detection method according to any one of claims 1 to 7, characterized in that: the fingerprint map of the overground part of the ligusticum wallichii is provided with 20 common characteristic peaks, the peak corresponding to the ligustilide reference substance is a peak 16, the peak corresponding to the butenyl phthalide reference substance is a peak 17, the relative retention time of each characteristic peak and the peak 17 is calculated, and the relative retention time is within +/-5% of a specified value; the specified values are: peak 1: 0.2446, Peak 2: 0.2631, peak 3: 0.4306, Peak 4: 0.5600, peak 5: 0.6588, Peak 6: 0.6962, peak 7: 0.7567, Peak 8: 0.7703, peak 9: 0.8191, peak 10: 0.8226, peak 11: 0.8447, peak 12: 0.8877, peak 13: 0.9269, peak 14: 0.9657, peak 15: 0.9814, peak 16: 0.9921, peak 17: 1.0000, peak 18: 1.1425, peak 19: 1.1856, peak 20: 1.3283.
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Cited By (2)
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|---|---|---|---|---|
| CN114813987A (en) * | 2022-02-14 | 2022-07-29 | 四川新绿色药业科技发展有限公司 | Compound composition containing ligusticum wallichii, borneol and artificial musk and method for constructing characteristic spectrum of medicinal preparation |
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