CN111624294A - UPLC content detection method for lycium barbarum ethyl and mulberroside A in Xiebai powder particles - Google Patents
UPLC content detection method for lycium barbarum ethyl and mulberroside A in Xiebai powder particles Download PDFInfo
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Abstract
A UPLC content detection method of lycium barbarum ethyl and mulberroside A in Xiebai san particles adopts chromatographic conditions as follows: a chromatographic column: BEH Shield RP18 (2.1X 100mm, 1.7 μm), column temperature: 30 ℃; flow rate: 0.2 mL/min; mobile phase: the acetonitrile (A) -0.1 percent phosphoric acid water (B), the preparation of the test sample and the reference solution thereof, the detection by the UPLC content detection method, the reproducibility is good, the method is simple and feasible after the methodological verification, the operation is convenient, the acetonitrile (A) -0.1 percent phosphoric acid water (B) is applied to the quality evaluation of the Xiebai san particles, and a content detection method is provided for the quality standard research of the Xiebai san particles.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine analysis and detection, and particularly relates to a UPLC content detection method of lycium barbarum extract and mulberroside A in Xiebai powder particles.
Background
Xiebai san originated from Xiao Er Yao Zheng Zhi Jue (Children's medicine syndrome directly know what), originated by Song Dynasty physicians Qian second, listed in the ancient classical famous prescription catalog (first batch) by the State administration of traditional Chinese medicine, is composed of cortex Mori, cortex Lycii, Glycyrrhrizae radix and semen oryzae Sativae, has the effects of clearing lung heat, relieving asthma and cough, and is a common prescription for treating lung heat and cough and asthma. In the formula, the root bark of white mulberry is sweet and cold and enters lung, clears lung heat and purges heat, relieves asthma and cough, although purging lung without hurting lung, is a monarch drug; cortex lycii radicis is sweet, bland and cold in nature, clears away stagnated fire in the lung and has the effect of nourishing yin, and is used as a ministerial drug; the liquorice and the polished round-grained rice have the effects of nourishing the stomach and regulating the middle warmer, banking up earth and generating gold to strengthen lung qi, are sweet and moderate, can prevent cold from damaging the stomach, can coordinate the medicines, are used as adjuvant medicines together, and have the effects of purging lung heat, relieving cough and asthma by combining the medicines. It is mainly used for treating cough caused by stagnated fire in lung and lung heat. Modern researches have shown that the Xiebai powder has the main functions of relieving fever, resisting inflammation, resisting pathogenic microorganism, eliminating phlegm and relieving cough.
The invention patent application with the application number of '201910812642.4' provides an HPLC characteristic map determination method based on the Xiebai substance, and establishes a characteristic map based on the Xiebai substance. Although the method can comprehensively and accurately evaluate the overall and intrinsic quality of the reference effusion mass, no research is made on the detection method of the content of each reference substance, particularly the content of effective active ingredients.
The present inventors have searched for the existing research data on the Xiebai powder and the raw materials used therein, and found that the prior art has not established a method for detecting the content of active ingredients effective in Xiebai powder, and the applicant has not searched for in other patents or documents.
Disclosure of Invention
Brief summary of the invention:
the medicine is nontoxic, and has certain curative effect and quality controllability. The Xiebai powder is one of the ancient classical prescriptions which are supported and developed by the nation, and the practice of hundreds of years shows that the toxicity and the curative effect of the Xiebai powder are undoubted, and for the quality controllability, the key point is how to establish the quality standard based on the pharmacodynamic action and strictly control the quality condition in the preparation process, and for the traditional Chinese medicine prescription with complex components, the measurement of fingerprint and the content of multi-index components is an important means for evaluating the quality condition. The research takes the above as the starting point, develops the UPLC content detection method of the lycium barbarum extract and the mulberroside A in the traditional Chinese medicine classical famous prescription Xiebai san granules, is simple and feasible through methodology verification and convenient to operate, is applied to the quality evaluation of the Xiebai san granules, and provides a content detection method for the quality standard research of the Xiebai san granules.
The content detection items of the mulberroside A in the white mulberry root-bark and the lycine B in the cortex lycii radicis are mainly used for research, and the reason is that (1) the white mulberry root-bark is the dried root bark of mulberry of the family Moraceae. Belongs to a medicine for relieving cough and reducing sputum. Has effects in purging heat, relieving asthma, inducing diuresis, and relieving swelling, and can be used for treating cough and asthma due to lung heat, edema, oliguria, and edema of face and eyes. The mulberry bark is cold in nature and also has the effects of clearing liver and reducing blood pressure, and the mulberroside A is a main stilbene glucoside active ingredient in the mulberry bark, has strong cough relieving effect and certain asthma relieving effect, is matched with the main treatment function of the Xiebai powder and is a main active ingredient in the mulberry bark; (2) cortex Lycii is the dry root bark of Lycium barbarum or Lycium barbarum of Solanaceae, collected in the first part of the 2010 version of the Chinese pharmacopoeia, and has cold and sweet flavor and enters lung, liver and kidney meridians. Has effects of cooling blood, removing heat, clearing lung-heat and lowering fire, and is mainly used for treating yin deficiency hectic fever, bone steaming night sweat, lung heat cough, hemoptysis, epistaxis, internal heat and diabetes. Modern pharmacological research finds that the cortex lycii radicis also has the effects of reducing blood sugar and blood pressure and resisting bacteria. Kukoamine B is a main active ingredient in Chinese medicine kukoamine.
Detailed description of the invention
The invention discloses a UPLC content detection method of lycium barbarum B and mulberroside A in Xiebai powder particles, which takes the lycium barbarum B contained in the lycium barbarum in Xiebai powder formula and the mulberroside A contained in the cortex mori as detection objects and provides a content detection method.
In order to achieve the purpose, according to the national development and research requirements of the classical famous prescription, firstly, the Xiebai powder decoction is prepared into granules, the granules are used as detection samples, and the content of the mulberroside A and the lycium barbarum B is detected by a UPLC method.
The specific technical scheme of the invention is as follows:
1. the preparation method of the Xiebai powder granule comprises the following steps:
placing cortex Mori, cortex Lycii, Glycyrrhrizae radix and semen oryzae Sativae in an extractor, decocting with water twice, adding 10-12 times of water for the first time, decocting for 1-2 hr, adding 8-10 times of water for the second time, decocting for 0.5-1.5 hr, mixing the two extractive solutions, filtering, concentrating, drying, pulverizing, and granulating to obtain XIEBAISAN granule.
2. The mulberry bark glycoside A content detection method comprises the following steps:
2.1 chromatographic conditions and systematic testing: chromatographic column BEHShield RP18 (2.1X 100mm, 1.7 μm) using octadecylsilane chemically bonded silica as a filler; gradient elution is carried out by taking acetonitrile (A) -0.1 percent phosphoric acid water (B) as a mobile phase; gradient elution conditions: 0-1min, 5% A; 1-2min, 5-6% of A; 2-8min, 6% A; 8-9min, 6-8% of A; 9-16min, 8-9% of A; 16-17min, 9-35% A; 17-29min, 35-40% A; 29-30min, 40-5% A. Sample introduction amount: 1 μ L, detection wavelength: 280 nm. The number of theoretical plates is not less than 1000 calculated according to the mulberroside A; column temperature: 30 ℃; flow rate: 0.2 mL/min.
2.2 preparation of control solutions:
precisely measuring appropriate amount of mulberroside A reference substance, and adding methanol to obtain solutions with mass concentration of 0.2 mg/mL-1Shaking the reference solution to obtain the final product.
2.3 preparation of test solution:
dissolving 0.5g of the above Chinese medicinal powder in 15mL of water, placing 1mL of the solution in a centrifuge tube for 13000r min-1Centrifuging for 5min, collecting supernatant, and filtering with 0.22 μm microporous membrane.
2.4 content determination method:
precisely sucking 1 μ L of each of the reference solution and the sample solution, injecting into a liquid chromatograph, measuring according to chromatographic conditions, recording chromatogram, and calculating by substituting peak area into standard curve to obtain the content of mulberroside A in the XIEBAISAN granule.
The chromatogram of mulberroside A in the reference and Xiebai san particles are shown in FIG. 1 and FIG. 2.
3. Method for detecting lycium barbarum ethyl content
3.1 chromatographic conditions and systematic testing: BEH Shield RP18 (2.1X 100mm, 1.7 μm); column temperature: 30 ℃; flow rate: 0.2 mL/min; mobile phase: acetonitrile (a) -0.1% phosphoric acid water (B); gradient elution conditions: 0-1min, 5% A; 1-2min, 5-6% of A; 2-8min, 6% A; 8-9min, 6-8% of A. Sample introduction amount: 1 μ L. Detection wavelength: 280nm, and the number of theoretical plates is not less than 1000 calculated according to lycium barbarum B; column temperature: 30 ℃; flow rate: 0.2 mL/min.
3.2 preparation of test solution:
dissolving 0.5g of the above Chinese medicinal powder in 15mL of water, placing 1mL of the solution in a centrifuge tube for 13000r min-1Centrifuging for 5min, collecting supernatant, and filtering with 0.22 μm microporous membrane.
3.3 preparation of control solutions:
precisely measuring appropriate amount of wofberry bark ethyl reference substance, adding methanol to obtain extract with mass concentration of 0.25 mg/mL-1The control stock solution was shaken well.
3.4 content determination method:
precisely absorbing 1 μ L of each of the test solution and the reference solution, injecting into a liquid chromatograph, measuring according to chromatographic conditions, recording chromatogram, and substituting peak area into a standard curve for calculation to obtain cortex Lycii B content in the XIEBAISAN granule.
Chromatogram of wogonin B in wogonin B control and Xiebai san granule are shown in FIG. 3 and FIG. 4.
It should be noted that the minimum limit standard for detecting the active ingredients should not limit the actual protection scope of the present invention.
Advantageous effects
The UPLC content detection method for the kukoamine B and the mulberroside A in the Xiebai granules has the advantages that the UPLC content detection method is used for establishing quality standard requirements for developing and researching the national classic famous Xiebai powder, is simple and feasible through methodology verification and convenient to operate, is applied to quality evaluation of the Xiebai granules, and provides a content detection method for quality standard research of the Xiebai granules, so that the quality detection method has certain quality controllability.
Drawings
FIG. 1: the chromatogram of the sangusin A reference substance.
FIG. 2: chromatogram of mulberroside A in Xiebai san particles.
FIG. 3: chromatogram of cortex lycii radicis B reference substance.
FIG. 4: chromatogram map of cortex lycii radicis B in Xiebai san particles.
Detailed Description
The method for detecting the content of kukoamine B and mulberroside A in the Xiebai san particles, which is claimed by the invention, adopts a UPLC detection method, and necessary linear relations, reproducibility and stability of chromatographic conditions, system applicability tests, reference substance preparation, test substance preparation and the like are all within the concept of the invention, so that the technical scheme in the embodiment of the invention is clearly and completely described. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example one
Firstly, respectively carrying out mulberroside A content detection on 15 batches of Xiebai powder particles prepared from 15 batches of mulberry bark with different production places and different numbers.
1. Instrument for measuring the position of a moving object
Waters H-Class high performance liquid chromatograph;
analytical balance model ME204 (Metler-Torlodo, Switzerland);
KQ500D model digital control ultrasonic cleaner (kunshan ultrasonic instrument ltd);
2. reagent
Mulberroside A (94.8%, LOT: 5343, Shanghai Shidande Standard technology services, Inc.);
acetonitrile (chromatographically pure, Fisher, usa);
phosphoric acid, methanol, ethanol and phosphoric acid are all analytically pure; the water is ultrapure water.
3. The information of 15 batches of cortex mori radicis is shown in table 3:
table 3: 15 batches of cortex mori information tables in different producing areas
Batch number | Producing area | Batch number | Producing area |
191006 | Guangxi nanning | 191014 | Anhui Mazhou |
191007 | Guangxi nanning | 191015 | Anhui Mazhou |
191008 | Guangxi nanning | 191016 | Shanghai Shangqiu |
191009 | Guangxi nanning | 191017 | Shanghai Shangqiu |
1910010 | Guangxi nanning | 191018 | Shanghai Shangqiu |
191011 | Anhui Mazhou | 191019 | Shanghai Shangqiu |
191012 | Anhui Mazhou | 191020 | Shanghai Shangqiu |
191013 | Anhui Mazhou |
4. And (3) content detection:
4.1 preparation of test solution:
dissolving the above 15 batches of the XIEBAISAN granules respectively prepared from cortex Mori in 15mL of water, respectively, collecting 1mL of the solution, placing in a centrifuge tube, and processing at 13000 r.min-1Centrifuging for 5min, collecting supernatant, and filtering with 0.22 μm microporous membrane to obtain 15 sample solutions.
4.2 preparation of control solutions:
precisely measuring appropriate amount of mulberroside A reference substance, and adding methanol to obtain solutions with mass concentration of 1.0 mg/mL-1The control stock solution was shaken well.
4.3 chromatographic conditions
BEH Shield RP18 (2.1X 100mm, 1.7 μm); column temperature: 30 ℃; flow rate: 0.2 mL/min; mobile phase: acetonitrile (a) -0.1% phosphoric acid water (B); gradient elution conditions: 0-1min, 5% A; 1-2min, 5-6% of A; 2-8min, 6% A; 8-9min, 6-8% of A; 9-16min, 8-9% of A; 16-17min, 9-35% A; 17-29min, 35-40% A; 29-30min, 40-5% A. Sample introduction amount: 1 μ L. Detection wavelength: 280 nm.
4.4 content determination:
precisely sucking 1 μ L of each of the reference solution and 15 batches of test solutions, respectively, injecting into a liquid chromatograph, measuring according to chromatographic conditions, recording chromatogram, and substituting peak area into a standard curve for calculation to obtain the content of mulberroside A in 15 Xiebai san particles. The results are shown in Table 4.
Table 4: determination result of content of mulberroside A in Xiebai powder granules
Batch number | Mulberry bark glycoside A (mg/mL) | Batch number | Mulberry bark glycoside A (mg/mL) |
191006 | 0.404 | 191014 | 0.527 |
191007 | 0.413 | 191015 | 0.534 |
191008 | 0.455 | 191016 | 0.353 |
191009 | 0.411 | 191017 | 0.360 |
191010 | 0.424 | 191018 | 0.378 |
191011 | 0.482 | 191019 | 0.311 |
191012 | 0.521 | 191020 | 0.339 |
191013 | 0.481 |
And secondly, detecting the content of the lycium pigment B in 15 batches of the Xiebai powder particles prepared from 15 batches of the lycium pigment with different production places and different batches respectively.
1. Instrument for measuring the position of a moving object
Waters H-Class high performance liquid chromatograph;
analytical balance model ME204 (Metler-Torlodo, Switzerland);
KQ500D model digital control ultrasonic cleaner (kunshan ultrasonic instrument ltd);
2. reagent
Lycin B (99.17%, LOT: DST191118-105, Doudsite biotechnology, Inc.);
acetonitrile (chromatographically pure, Fisher, usa);
phosphoric acid, methanol, ethanol and phosphoric acid are all analytically pure; the water is ultrapure water.
3. The information for 15 batches of cortex lycii radicis is shown in table 1.
Table 115 batch-to-batch information table for different producing areas of cortex lycii radicis:
batch number | Producing area | Batch number | Producing area |
190306 | Gansu Zhuang Lang | 190314 | Yichuan Shaanxi |
190307 | Gansu Zhuang Lang | 190315 | Yichuan Shaanxi |
190308 | Gansu Zhuang Lang | 191016 | Shanxi fortune city |
190309 | Gansu Zhuang Lang | 190317 | Shanxi fortune city |
190310 | Gansu Zhuang Lang | 190318 | Shanxi fortune city |
190311 | Yichuan Shaanxi | 190319 | Shanxi fortune city |
190312 | Yichuan Shaanxi | 190320 | Shanxi fortune city |
190313 | Yichuan Shaanxi |
4. And (3) content detection:
4.1 preparation of test solution:
dissolving the above 15 batches of XIEBAISAN granule 0.5g respectively in 15mL of water, placing 1mL of the solution in a centrifuge tube, 13000r min-1Centrifuging for 5min, collecting supernatant, and filtering with 0.22 μm microporous membrane to obtain 15 test solutions.
4.2 preparation of control solutions:
precisely measuring a proper amount of lycium barbarum ethyl reference substance, adding methanol to prepare a reference substance stock solution with the mass concentration of 1.0 mg/mL-1, and shaking up.
4.3 chromatographic conditions and systematic testing:
a column chromatography using octadecylsilane bonded silica as a packing material (BEH Shield RP18 (2.1X 100mm, 1.7 μm)); gradient elution is carried out by taking acetonitrile (A) -0.1 percent phosphoric acid water (B) as a mobile phase; gradient elution conditions: 0-1min, 5% A; 1-2min, 5-6% of A; 2-8min, 6% A; 8-9min, 6-8% of A. Sample introduction amount: 1 μ L, detection wavelength: 280nm, column temperature: 30 ℃; flow rate: 0.2 mL/min.
4.4 measurement of content
Precisely sucking 1 μ L of each of the reference solution and 15 sample solutions, respectively, injecting into a liquid chromatograph, measuring according to chromatographic conditions, recording chromatogram, and substituting peak area into a standard curve for calculation to obtain cortex Lycii B content in the XIEBAISAN granule.
The results of the measurements are shown in Table 2,
table 2: determination result of lycium barbarum ethyl content in 15 batches of Xiebai san particles
Batch number | Cortex lycii radicis B element (mg/mL) | Batch number | Cortex lycii radicis B element (mg/mL) |
190306 | 0.251 | 190314 | 0.054 |
190307 | 0.240 | 190315 | 0.049 |
190308 | 0.321 | 191016 | 0.346 |
190309 | 0.157 | 190317 | 0.527 |
190310 | 0.231 | 190318 | 0.427 |
190311 | 0.077 | 190319 | 0.564 |
190312 | 0.067 | 190320 | 0.430 |
190313 | 0.057 |
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (3)
1. A UPLC content detection method for kukoamine B and mulberrin A in Xiebai powder particles is characterized by comprising a mulberrin A UPLC content detection method and a kukoamine B UPLC content detection method.
2. The UPLC content detection method of kukoamine B and mulberroside A in Xiebai san granules according to claim 1, wherein the UPLC content detection method of mulberroside A comprises the following steps:
2.1 preparation method of Xiebai san granule: placing cortex Mori, cortex Lycii, Glycyrrhrizae radix and semen oryzae Sativae in an extractor, decocting with water twice, adding 10-12 times of water for the first time, decocting for 1-2 hr, adding 8-10 times of water for the second time, decocting for 0.5-1.5 hr, mixing the two extractive solutions, filtering, concentrating the filtrate under reduced pressure to relative density of 55-60 deg.C of 1.25-1.30, drying, pulverizing, granulating to obtain XIEBAISAN granule,
2.2 the method for detecting the content of the mulberroside A comprises the following steps:
2.2.1 chromatographic conditions and systematic testing: chromatographic column BEHShield RP18 (2.1X 100mm, 1.7 μm) using octadecylsilane chemically bonded silica as a filler; gradient elution is carried out by taking acetonitrile (A) -0.1 percent phosphoric acid water (B) as a mobile phase; gradient elution conditions: 0-1min, 5% A; 1-2min, 5-6% of A; 2-8min, 6% A; 8-9min, 6-8% of A; 9-16min, 8-9% of A; 16-17min, 9-35% A; 17-29min, 35-40% A; 29-30min, 40-5% A. Sample introduction amount: 1 μ L, detection wavelength: 280 nm. The number of theoretical plates is not less than 1000 calculated according to the mulberroside A; column temperature: 30 ℃; flow rate: 0.2mL/min of the reaction solution,
2.2.2 preparation of control solutions: precisely measuring appropriate amount of mulberroside A reference substance, and adding methanol to obtain solutions with mass concentration of 0.2 mg/mL-1The reference substance solution is evenly shaken to obtain the compound,
2.2.3 preparation of test solutions: dissolving 0.5g of the above Chinese medicinal powder in 15mL of water, placing 1mL of the solution in a centrifuge tube for 13000r min-1Centrifuging for 5min, collecting supernatant, and filtering with 0.22 μm microporous membrane.
2.2.4 content determination method: precisely sucking 1 μ L of each of the reference solution and the sample solution, injecting into a liquid chromatograph, measuring according to chromatographic conditions, recording chromatogram, and calculating by substituting peak area into standard curve to obtain the content of mulberroside A in the XIEBAISAN granule.
3. The method for detecting UPLC content of kukoamine B and mulberroside A in Xiebai san granules according to claim 1, wherein the method for detecting UPLC content of kukoamine B comprises the following steps:
3.1, a method for detecting the content of lycium barbarum B:
3.1.1 chromatographic conditions and systematic testing: BEH Shield RP18 (2.1X 100mm, 1.7 μm); column temperature: 30 ℃; flow rate: 0.2 mL/min; mobile phase: acetonitrile (a) -0.1% phosphoric acid water (B); gradient elution conditions: 0-1min, 5% A; 1-2min, 5-6% of A; 2-8min, 6% A; 8-9min, 6-8% of A. Sample introduction amount: 1 μ L. Detection wavelength: 280nm, and the number of theoretical plates is not less than 1000 calculated according to lycium barbarum B; column temperature: 30 ℃; flow rate: 0.2mL/min of the reaction solution,
3.1.2 preparation of test solution: dissolving 0.5g of the Xiebai san granules of claim 2.1 in 15mL of water, placing 1mL of the solution in a centrifuge tube at 13000 r.min-1Centrifuging for 5min, collecting supernatant, filtering with 0.22 μm microporous membrane,
3.1.3 preparation of control solutions: precisely measuring appropriate amount of wofberry bark ethyl reference substance, adding methanol to obtain extract with mass concentration of 0.25 mg/mL-1The control stock solution was shaken well.
3.1.4 content determination method: precisely absorbing 1 μ L of each of the test solution and the reference solution, injecting into a liquid chromatograph, measuring according to chromatographic conditions, recording chromatogram, and substituting peak area into a standard curve for calculation to obtain cortex Lycii B content in the XIEBAISAN granule.
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CN112656869B (en) * | 2020-12-30 | 2022-03-08 | 辽宁上药好护士药业(集团)有限公司 | Composition for treating lung-heat cough and asthma, preparation method and application |
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CN118191140A (en) * | 2024-03-18 | 2024-06-14 | 湖南易能生物医药有限公司 | Quality control method of traditional Chinese medicine compound containing cortex lycii radicis and cortex mori radicis and application thereof |
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