CN109030694B - Method for identifying authenticity of subprostrate sophora by using fingerprint spectrum - Google Patents
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- 239000000243 solution Substances 0.000 claims description 33
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- QZVQYTIOGPCCRU-JOCHJYFZSA-N sophoranone Natural products COc1c(O)c(OC)c(cc1CC=C(C)C)[C@]2(O)COc3cc(O)cc(O)c3C2=O QZVQYTIOGPCCRU-JOCHJYFZSA-N 0.000 claims description 2
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- 208000002454 Nasopharyngeal Carcinoma Diseases 0.000 description 1
- 206010061306 Nasopharyngeal cancer Diseases 0.000 description 1
- 206010068319 Oropharyngeal pain Diseases 0.000 description 1
- 201000007100 Pharyngitis Diseases 0.000 description 1
- AAGFPTSOPGCENQ-UHFFFAOYSA-N Sophocarpin I Natural products C1CCC2CN3C(=O)C=CCC3C3C2N1CCC3 AAGFPTSOPGCENQ-UHFFFAOYSA-N 0.000 description 1
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- 238000013441 quality evaluation Methods 0.000 description 1
- AAGFPTSOPGCENQ-JLNYLFASSA-N sophocarpine Chemical compound C1CC[C@H]2CN3C(=O)C=CC[C@@H]3[C@@H]3[C@H]2N1CCC3 AAGFPTSOPGCENQ-JLNYLFASSA-N 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
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- Engineering & Computer Science (AREA)
- Library & Information Science (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a method for identifying the authenticity of subprostrate sophora by a fingerprint, which judges the authenticity of a subprostrate sophora medicinal material sample by measuring a liquid phase map of the subprostrate sophora medicinal material sample. The identification method provided by the invention can comprehensively reflect the effective chemical components of the subprostrate sophora, integrally reflect the quality of the subprostrate sophora medicinal material sample, is accurate and reliable, is simple and easy to operate, low in cost and strong in practicability, is favorable for large-scale popularization, and is very suitable for detecting and monitoring the quality of the subprostrate sophora medicinal material and the product thereof.
Description
Technical Field
The invention relates to a method for identifying the authenticity of subprostrate sophora. More specifically, the invention relates to a method for identifying the authenticity of subprostrate sophora by using a fingerprint, belonging to the technical field of traditional Chinese medicine material identification methods.
Background
The subprostrate sophora is the dry root and rhizome of leguminous plant sophora tonkinensis, has the effects of clearing away heat and toxic material, reducing swelling and relieving sore throat, contains matrine, oxymatrine, sophocarpine, dauricine, genistein, β -sterol, flavone and the like, and the existing Chinese pharmacopoeia has simpler quality control method for the subprostrate sophora, only takes the content of the matrine and the oxymatrine as the index of the quality evaluation of medicinal materials, but the matrine and the oxymatrine are not the specific components of the subprostrate sophora root, and the index determination of the matrine and the oxymatrine is difficult to reflect the quality of the medicinal materials integrally.
The fingerprint spectrum technology is used as an effective quality control method of a multi-component chemical component sample, can integrally reflect the integral characteristics of a sample to be detected, and is widely applied to quality control of traditional Chinese medicinal materials at present. Therefore, in order to evaluate the quality of the subprostrate sophora medicinal material more accurately, a fingerprint capable of comprehensively reflecting the effective chemical components of the subprostrate sophora needs to be established.
Disclosure of Invention
The invention aims to provide a method for identifying the authenticity of subprostrate sophora by using a fingerprint, which can comprehensively reflect the effective chemical components in the subprostrate sophora and integrally reflect the quality of a subprostrate sophora medicinal material sample.
To achieve these objects and other advantages in accordance with the present invention, a method for identifying the authenticity of a sample of subprostrate sophora root by measuring the liquid phase spectrum of the sample is provided.
Preferably, 10 chromatographic peaks in a standard subprostrate sophora non-alkaloid fingerprint spectrum are used as characteristic peaks, and when the 10 characteristic peaks do not appear in a liquid chromatogram of a subprostrate sophora medicinal material sample at the same time, the subprostrate sophora medicinal material sample is a pseudo-inferior product; when the similarity between the relative retention time of 10 characteristic peaks in the liquid chromatogram of the radix sophorae tonkinensis medicinal material sample and the standard non-alkaloid fingerprint chromatogram of the radix sophorae tonkinensis is higher than 85%, the radix sophorae tonkinensis medicinal material sample is a high-quality product, wherein the third characteristic peak in the 10 characteristic peaks is used as a reference peak according to the peak appearance sequence, and the relative retention time of the other 9 characteristic peaks is respectively as follows: 0.403, 0.462, 1.077, 1.600, 1.883, 1.899, 1.948, 1.975, 2.081.
Preferably, the compounds corresponding to the first four characteristic peaks in the order of appearance are pratensein, isopratensein, formononetin and 2- (2',4' -dihydroxyphenyl) -5, 6-dioxymethylene benzofuran, the compound corresponding to the sixth characteristic peak is 3-methoxy-Korean-sophoricide, and the compound corresponding to the ninth characteristic peak is Vietnam-sophorol.
Preferably, the method comprises the steps of preparing a subprostrate sophora medicinal material sample into a test sample solution, and then determining a liquid phase map of the test sample solution, wherein the preparation process of the test sample solution specifically comprises the following steps: weighing tonka bean root medicinal material sample powder, adding an extracting solution which is 5-20 times the weight of the tonka bean root medicinal material sample powder, performing reflux extraction for 1-2 h, filtering, taking filtrate, performing reduced pressure rotary evaporation on the filtrate to recover the extracting solution to dryness to obtain a crude extract, adding water into the crude extract to dissolve, passing through macroporous resin, eluting with an ethanol solution with the volume fraction of 10-39%, eluting with an ethanol solution with the volume fraction of 40-95%, collecting an eluting part of the ethanol solution with the volume fraction of 40-95%, performing reduced pressure rotary evaporation to recover the ethanol solution to dryness to obtain a fine extract, adding methanol into the fine extract to dissolve, filtering with a 0.45 mu m microporous filter membrane, and fixing the volume.
Preferably, the extracting solution is 65-95% ethanol solution by volume fraction.
Preferably, the specific conditions for determining the liquid phase spectrum of the subprostrate sophora medicinal material sample comprise: a chromatographic column used for the high performance liquid chromatograph takes octadecylsilane chemically bonded silica as a filler, and the column temperature is 25-35 ℃; the wavelength is 200-320 nm during liquid chromatography detection, the sample injection amount is 5-10 mu l, and the flow rate is 0.8-1.2 ml/min; gradient elution is adopted in liquid chromatography detection, wherein acetonitrile is a mobile phase A, water is a mobile phase B, and the change of the volume ratio of the acetonitrile is specifically as follows: within 0-5 min, the volume ratio of acetonitrile is 25%, within 5-55 min, the volume ratio of acetonitrile is gradually increased from 25% to 50%, and within 55-80 min, the volume ratio of acetonitrile is gradually increased from 50% to 95%.
Preferably, the specific conditions for determining the liquid phase spectrum of the subprostrate sophora medicinal material sample comprise: the chromatographic column used by the high performance liquid chromatograph is a Waters Sunfire-C18 column, the inner diameter of the column is 4.6mm, the height of the column is 250mm, the particle size of silica gel is 5 μm, and the temperature of the column is 30 ℃; the liquid chromatography detection wavelength is 205nm, the sample injection amount is 10 μ l, and the flow rate is 1.0 ml/min.
The invention at least comprises the following beneficial effects: the invention can comprehensively reflect the effective chemical components of the subprostrate sophora root by measuring the liquid phase atlas of the subprostrate sophora root medicinal material sample and comparing with the standard subprostrate sophora root non-alkaloid fingerprint atlas, and takes 10 characteristic peaks as the evaluation index of the subprostrate sophora root quality, thereby reflecting the quality of the subprostrate sophora root medicinal material as a whole, accurately and reliably identifying the authenticity of the subprostrate sophora root medicinal material. The method is verified through a similarity test, a precision test, a stability test, a repeatability test and a characteristic peak identification test, and the result shows that the detection method provided by the invention is accurate and reliable, can reflect a plurality of effective chemical components in the subprostrate sophora, and reflects the quality of the subprostrate sophora medicinal material as a whole, so that a more accurate and reliable detection result is obtained. In addition, the identification method provided by the invention is simple and easy to operate, low in cost, strong in practicability, suitable for large-scale popularization and very suitable for detecting and monitoring the quality of the subprostrate sophora medicinal material and the product thereof.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a liquid phase spectrum of a 10 batch subprostrate sophora root product according to an embodiment of the present invention;
FIG. 2 is a standard non-alkaloid fingerprint of subprostrate sophora according to an embodiment of the present invention.
Detailed Description
The present invention is further described in detail below with reference to the drawings and examples so that those skilled in the art can practice the invention with reference to the description.
It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.
< example >
The method for identifying the authenticity of the subprostrate sophora by the fingerprint comprises the following steps:
the method comprises the following steps: weighing 2g of sample powder of the radix sophorae tonkinensis, adding ethanol solution with the weight being 20 times that of the sample powder of the radix sophorae tonkinensis and the volume fraction being 85%, performing reflux extraction for 2h, filtering, taking filtrate, performing reduced pressure rotary evaporation on the filtrate to recover the ethanol solution until the ethanol solution is evaporated to dryness to obtain a crude extract, adding 20ml of water into the crude extract to dissolve the crude extract, passing through macroporous resin, eluting with 39% of ethanol solution, eluting with 95% of ethanol solution, collecting 95% of ethanol solution eluate, performing reduced pressure rotary evaporation to recover the ethanol solution until the ethanol solution is evaporated to dryness to obtain a fine extract, adding methanol into the fine extract to dissolve the fine extract, filtering with a 0.45 mu m microporous filter membrane, and performing constant volume to 10ml volumetric flask to obtain a sample solution;
step two: determining a liquid phase map of the test solution, wherein the specific conditions for determining the liquid phase map comprise:
chromatograph: waters 2695 high performance liquid chromatograph (Waters corporation, usa);
chromatographic column Waters Sunfire-C18(4.6mm × 250mm, 5 μm) chromatographic column;
column temperature: 30 ℃;
detection wavelength: 205 nm;
sample introduction amount: 10 mu l of the mixture;
flow rate: 1.0 ml/min;
mobile phase: acetonitrile as mobile phase a and water as mobile phase B, gradient elution was performed as per table 1:
[ Table 1]
Step three: comparing the liquid phase chromatogram of the radix sophorae tonkinensis medicinal material sample with 10 characteristic peaks in a standard radix sophorae tonkinensis non-alkaloid fingerprint chromatogram, wherein when the 10 characteristic peaks do not appear in the liquid phase chromatogram of the radix sophorae tonkinensis medicinal material sample at the same time, the radix sophorae tonkinensis medicinal material sample is a pseudo-inferior product; and when the similarity between the relative retention time of 10 characteristic peaks in the liquid chromatogram of the radix sophorae tonkinensis medicinal material sample and the standard non-alkaloid fingerprint chromatogram of the radix sophorae tonkinensis is higher than 85%, the radix sophorae tonkinensis medicinal material sample is a high-quality product.
Wherein, 10 characteristic peaks are respectively marked as No. 1-10 peaks according to the peak appearance sequence, the No. 3 peak is taken as a reference peak, and the relative retention time of the other 9 characteristic peaks is respectively as follows: 0.403, 0.462, 1.077, 1.600, 1.883, 1.899, 1.948, 1.975, 2.081.
< similarity test >
Taking 10 batches of subprostrate sophora root genuine products (the sources are shown in table 2), preparing the subprostrate sophora root genuine products into genuine product solutions according to the preparation method of the test sample solutions in the examples, measuring the liquid phase maps of the 10 batches of subprostrate sophora root genuine product solutions by adopting the same liquid phase map measuring conditions as the examples, and recording the liquid phase maps of the 10 batches of subprostrate sophora root genuine products, wherein the result is shown in figure 1.
The liquid phase chromatogram separation degree and peak shape of the subprostrate sophora root genuine product No. 3 are both best, the liquid phase chromatogram separation degree and peak shape are used as standard subprostrate sophora root non-alkaloid fingerprint chromatogram, see figure 2, and 1-10 marked in figure 2 are selected 10 characteristic peaks and are used for comparing with the liquid phase chromatogram of the subprostrate sophora root medicinal material sample so as to identify the authenticity of the subprostrate sophora root medicinal material sample. The retention times of the 10 characteristic peaks are respectively: 15.291, 17.562, 37.976, 40.894, 60.753, 71.513, 72.119, 73.993, 75.003, 79.046 (units: min). Since formononetin appears in each batch of subprostrate sophora root genuine products and has good separation degree, formononetin (namely, peak 3) is selected as a reference peak (marked as S), and the relative retention time of the other 9 characteristic peaks is respectively as follows: 0.403, 0.462, 1.077, 1.600, 1.883, 1.899, 1.948, 1.975, 2.081.
Taking an formononetin standard sample, preparing a standard solution with the same concentration as the authentic product solution, measuring a liquid phase diagram of the formononetin standard solution by adopting the same liquid phase diagram measuring conditions of the embodiment to obtain the liquid phase diagram of the formononetin standard sample, and calculating the relative retention time (marked as S1-S10) of a reference peak (S) in the liquid phase diagrams of 10 batches of subprostrate sophora root authentic products by taking the retention time of the formononetin in the liquid phase diagram of the formononetin standard sample as a reference, wherein the result is shown in Table 3. As can be seen from Table 3, S1-S10 is between 0.893-0.981, which shows that the similarity between the retention time of formononetin peak in the liquid phase chromatogram of 10 batches of subprostrate sophora root true products and the retention time of formononetin standard sample is between 0.893-0.981, the similarity is very high, and the liquid phase fingerprint chromatogram is used for identifying the authenticity of the subprostrate sophora root medicinal material sample more accurately and reliably.
[ Table 2]
[ Table 3]
< precision test >
Taking a genuine product solution of the same subprostrate sophora root genuine product for continuous sample injection for 6 times, adopting the same liquid chromatogram determination conditions as the embodiments for determination, recording the liquid chromatogram after each determination, taking the No. 3 peak as a reference peak, calculating the relative retention time and the relative peak area of the other 9 characteristic peaks, and displaying the result: RSD (relative standard deviation) of relative retention time of each characteristic peak in 6 times of measurement is less than 0.12%, and RSD of relative peak area is less than 2.93%, which indicates that the method provided by the invention has good precision.
< stability test >
Taking a genuine product solution of the same subprostrate sophora, respectively measuring the liquid phase spectrum of the genuine product solution in 0, 2, 4, 8, 12 and 24 hours, wherein the measuring conditions of the liquid phase spectrum are the same as those in the embodiment, recording the liquid phase spectrum after each measurement, taking the No. 3 peak as a reference peak, calculating the relative retention time and the relative peak area of the other 9 characteristic peaks, and displaying the result: RSD of each characteristic peak relative to retention time was < 0.31% and RSD of the relative peak area was < 2.04% within 24h, indicating that the test solution was stable within 24 h.
< reproducibility test >
Taking 6 parts of the same subprostrate sophora root genuine product, preparing the subprostrate sophora root genuine product into genuine product solution according to the preparation method of the test solution in the embodiment, determining the liquid phase spectrum of the 6 parts of genuine product solution by adopting the same liquid phase spectrum determination conditions as the embodiment, recording the liquid phase spectrum after each determination, calculating the relative retention time and the relative peak area of the other 9 characteristic peaks by taking the No. 3 peak as a reference peak, and displaying the result: the RSD of each characteristic peak in 6 true product solutions relative to the retention time is less than 0.15%, and the RSD of the relative peak area is less than 2.94%, which indicates that the method provided by the invention has good repeatability.
< test for identifying characteristic peaks >
Through single comparison of various standard samples, compounds corresponding to 6 characteristic peaks in 10 characteristic peaks are identified as follows: the peak 1 is red pratensein, the peak 2 is isored pratensein, the peak 3 is formononetin, the peak 4 is 2- (2',4' -dihydroxyphenyl) -5, 6-dioxymethylene benzofuran, the peak 6 is 3-methoxy-Korean-sophoricoside, and the peak 9 is Vietnam-sophorol. The compounds in the 6 are all active ingredients of the subprostrate sophora for resisting nasopharyngeal carcinoma, which shows that the method provided by the invention can better reflect the effective chemical ingredients of the subprostrate sophora.
While embodiments of the invention have been disclosed above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in a variety of fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concepts defined by the claims and the equivalents thereof.
Claims (1)
1. The method for identifying the authenticity of the subprostrate sophora root by the fingerprint is characterized in that the authenticity of the subprostrate sophora root medicinal material sample is judged by measuring the liquid phase map of the subprostrate sophora root medicinal material sample;
taking 10 chromatographic peaks in a standard subprostrate sophora non-alkaloid fingerprint spectrum as characteristic peaks, and taking the subprostrate sophora medicinal material sample as a pseudo-inferior product when the 10 characteristic peaks do not appear in the liquid-phase spectrum of the subprostrate sophora medicinal material sample at the same time; when the similarity between the relative retention time of 10 characteristic peaks in the liquid chromatogram of the radix sophorae tonkinensis medicinal material sample and the standard non-alkaloid fingerprint chromatogram of the radix sophorae tonkinensis is higher than 85%, the radix sophorae tonkinensis medicinal material sample is a high-quality product, wherein the third characteristic peak in the 10 characteristic peaks is used as a reference peak according to the peak appearance sequence, and the relative retention time of the other 9 characteristic peaks is respectively as follows: 0.403, 0.462, 1.077, 1.600, 1.883, 1.899, 1.948, 1.975, 2.081;
according to the peak appearance sequence, the compounds corresponding to the first four characteristic peaks are respectively red pratensein, isored pratensein, formononetin and 2- (2',4' -dihydroxyphenyl) -5, 6-dioxymethylene benzofuran, the compound corresponding to the sixth characteristic peak is 3-methoxy-Korean-sophoricine, and the compound corresponding to the ninth characteristic peak is Vietnam-sophorol;
firstly, preparing a subprostrate sophora medicinal material sample into a test sample solution, and then determining a liquid phase map of the test sample solution, wherein the preparation process of the test sample solution specifically comprises the following steps: weighing tonka bean root medicinal material sample powder, adding an extracting solution which is 5-20 times the weight of the tonka bean root medicinal material sample powder, performing reflux extraction for 1-2 h, filtering, taking filtrate, performing reduced pressure rotary evaporation on the filtrate to recover the extracting solution to dryness to obtain a crude extract, adding water into the crude extract to dissolve, passing through macroporous resin, eluting with an ethanol solution with the volume fraction of 10-39%, eluting with an ethanol solution with the volume fraction of 40-95%, collecting the eluted part of the ethanol solution with the volume fraction of 40-95%, performing reduced pressure rotary evaporation to recover the ethanol solution to dryness to obtain a fine extract, adding methanol into the fine extract to dissolve, filtering with a 0.45 mu m microporous filter membrane, and fixing the volume;
the extracting solution is an ethanol solution with the volume fraction of 65-95%;
the specific conditions for determining the liquid phase map of the subprostrate sophora medicinal material sample comprise: the chromatographic column used by the high performance liquid chromatograph takes octadecylsilane chemically bonded silica as a filler, and the column temperature is 30 ℃; the wavelength is 205nm during liquid chromatography detection, the sample injection amount is 10 μ l, and the flow rate is 1.0 ml/min; gradient elution is adopted in liquid chromatography detection, wherein acetonitrile is a mobile phase A, water is a mobile phase B, and the change of the volume ratio of the acetonitrile is specifically as follows: within 0-5 min, the volume ratio of acetonitrile is 25%, within 5-55 min, the volume ratio of acetonitrile is gradually increased from 25% to 50%, and within 55-80 min, the volume ratio of acetonitrile is gradually increased from 50% to 95%;
the chromatographic column used by the high performance liquid chromatograph is a Waters Sunfire-C18 column, the inner diameter of the column is 4.6mm, the height of the column is 250mm, and the particle size of silica gel is 5 μm.
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Application publication date: 20181218 Assignee: Pubei County Hongwei Nut Planting Co.,Ltd. Assignor: GUANGXI BOTANICAL GARDEN OF MEDICINAL PLANTS Contract record no.: X2023980046052 Denomination of invention: A Method for Identifying the Authenticity of Shandou Root by Fingerprint Spectroscopy Granted publication date: 20200707 License type: Common License Record date: 20231108 |