CN115326974A - Quality double-label visual quality control technology of fructus aurantii medicinal material - Google Patents

Quality double-label visual quality control technology of fructus aurantii medicinal material Download PDF

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CN115326974A
CN115326974A CN202210997910.6A CN202210997910A CN115326974A CN 115326974 A CN115326974 A CN 115326974A CN 202210997910 A CN202210997910 A CN 202210997910A CN 115326974 A CN115326974 A CN 115326974A
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peak
medicinal material
fructus aurantii
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孟宪生
罗曦
包永睿
王帅
李天娇
孟莹
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Liaoning University of Traditional Chinese Medicine
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Abstract

The invention discloses a quality-quality dual-standard visual quality control technology of a bitter orange medicinal material, belonging to the technical field of quality control of traditional Chinese medicines. Establishing characteristic map of fructus Aurantii reference medicinal material by HPLC technique, and comparing with the test medicinal material map to identify the authenticity of medicinal material, i.e. "quality"; the internal standard substance is adopted to carry out relative quantification on the chemical components of the characteristic peak so as to distinguish the quality of the medicinal materials, namely the quantity; and (4) visualizing the quality-quantity dual-standard control method by using Visual Basic programming language design. The established method for analyzing the quality of the bitter orange meets the requirement of methodology investigation, and except for the samples of the bitter orange in Hubei filial piety, the similarity of the other samples and the reference medicinal material is more than 0.98; the lower limit of the relative content of the chemical components of the characteristic peak of the fructus aurantii is specified. The method has high utilization degree and good repeatability, can improve the quality control system of Chinese medicinal materials, and reduce economic burden of quality control departments of enterprises, so as to promote sustainable development of Chinese medicinal industry.

Description

Quality double-label visual quality control technology of fructus aurantii medicinal material
Technical Field
The invention belongs to the technical field of quality control of traditional Chinese medicines, and particularly relates to a method for comprehensively controlling the quality of medicinal materials by taking a fructus aurantii reference medicinal material as a reference substance, which is independent of a reference substance.
Background
The fructus Aurantii is Citrus aurantium of RutaceaeCitrus aurantium L. and its cultivars dried immature fruits mainly produced in Sichuan, jiangxi and Zhejiang, and also distributed in Hubei, hunan and Chongqing provinces. The original plants of the bitter orange are complex in history, and counterfeit products such as green tangerine peel, trifoliate orange and the like are used as the bitter orange to be used as medicaments, so that the clinical reasonable use of the traditional Chinese medicine bitter orange is influenced.
With the progress of modern traditional Chinese medicine analysis means, the quality control method of the bitter orange mainly comprises a single-index content measurement method, a multi-index content measurement method and a fingerprint and content measurement combined method, the methods have the advantages of high sensitivity and good repeatability, and can provide quantitative data for controlling the bitter orange medicinal material, but the defects that the single index cannot reflect the integral characteristics of the medicinal material, the multi-index content measurement depends on various reference substances, certain index components cannot reflect the drug effect, and the fingerprint can only fuzzily evaluate the similarity of the medicinal material and cannot clearly judge the authenticity and quality of a test sample exist.
Disclosure of Invention
Aiming at the problems, the invention provides a method for controlling the quality-quantity dual-standard of a bitter orange medicament. The method is characterized in that the bitter orange reference medicinal material is used as a qualitative and quantitative reference substance, and a high performance liquid chromatography and Q-TOF-MS technology are combined to establish a set of method which does not depend on a reference substance and can comprehensively and scientifically control the quality of the medicinal material.
In order to achieve the above object, the present invention provides the following technical solutions.
The invention provides a method for establishing quality control standard of fructus aurantii, which is characterized by comprising the following steps:
step 1, performing qualitative research on a fructus aurantii medicinal material based on a reference medicinal material;
step 2, relative quantitative research of chemical components of characteristic peaks based on internal standard substances;
step 3 methodology investigation.
Further, the specific steps of step 1 include:
(1) Preparation of the solution: precisely weighing fructus Aurantii reference material and test material powder, placing into a conical flask with a plug, precisely adding 50 times of 50% methanol, weighing, heating and refluxing to 1 h, cooling, supplementing weight loss, shaking, and filtering; volatilizing the filtrate, diluting with 50% methanol to desired volume, shaking, and filtering with 0.22 μm filter membrane to obtain the final product;
(2) Chromatographic conditions and system applicability test: agilent EC-C adopting octadecylsilane chemically bonded silica as filler 18 A chromatographic column with the specification of 3.0 multiplied by 100 mm and 2.7-Micron; gradient elution is carried out by taking 0.1% formic acid water as a mobile phase A and acetonitrile as a mobile phase B; the flow rate is 1.0 mL/min; the column temperature is 30 ℃; the DAD detector detects at a wavelength of 330 nm; the sample injection amount is 5 mu L;
(3) Establishing a characteristic spectrum: each sample is paralleled for 2 times, sample injection detection is carried out in sequence according to chromatographic conditions, and the HPLC superposed spectra of 10 batches of test medicines are obtained by leading out the spectrum data under the wavelength of 330 nm from an analytical detection instrument; automatically matching 10 batches of test medicines by using a median, performing multi-point correction to generate a common mode R, and comparing the common mode R with a reference medicine characteristic map;
(4) And (3) similarity evaluation: and determining the authenticity of the bitter orange medicinal material according to the similarity of the characteristic peaks. Except for Hubei filial piety bitter orange sample, the similarity of other samples and the reference medicinal material is more than 0.98;
(5) Analyzing the chemical components of the characteristic peaks: identifying 9 chemical components by analyzing retention time, first-order ion mass-to-charge ratio and second-order ion fragment information of the compound and matching with related literature report information by adopting a Q-TOF-MS technology;
(6) The visual quality-quality double-standard quality control method of the traditional Chinese medicinal materials (decoction pieces) comprises the following steps: a Visual Basic programming language is utilized to design a quality-quality dual-standard quality control program which comprises 1 OEL control and 4 Command controls and is suitable for visualization of fructus aurantii medicinal materials (decoction pieces).
Further, the 9 chemical components identified in (5) were: the eriocitrin is shown in the No. 3 peak, the eriocitrin in the No. 4 peak, the naringin in the No. 6 peak, the naringin in the No. 7 peak, the hesperidin in the No. 9 peak, the neohesperidin in the No. 10 peak, the poncirin in the No. 12 peak, the nobiletin in the No. 15 peak and the hesperetin in the No. 16 peak.
Further, the specific steps of step 2 include:
taking the 7 th peak naringin determined in the step 1 as an internal standard substance, accurately quantifying chemical components of the internal standard substance, calculating the relative content of the chemical components of the characteristic peaks of the rest 9 batches of test medicines except the Hubei filial piety bitter orange, and taking the median-standard deviation as the lower limit of the relative content of the chemical components of the characteristic peaks relative to the chemical components of the internal standard substance.
Furthermore, the characteristic peaks of the step 2 are all characteristic active chemical components of the fructus aurantii playing the efficacy and pharmacological action.
Further, the specific steps of step 3 include:
(1) And (3) precision experiment: precisely absorbing the same sample solution, measuring according to chromatographic conditions, continuously feeding samples for 6 times, measuring relative retention time and relative peak area of each chromatographic peak, and calculating relative standard deviation; the relative retention time of each chromatographic peak and the RSD value of the relative peak area are both less than 1.0 percent, which indicates that the precision of the instrument is good;
(2) And (3) stability test: precisely sucking the same sample solution, respectively injecting samples at 0, 2, 4, 8, 12 and 24 h for 6 times according to chromatographic conditions, measuring relative retention time and relative peak area of each chromatographic peak, and calculating relative standard deviation; the relative retention time of each chromatographic peak and the relative peak area RSD value are both less than 2.4 percent, which indicates that the sample solution is stable in 24 h;
(3) And (3) repeatability experiment: preparing 6 parts of test sample according to a test sample solution preparation method, respectively carrying out sample injection detection according to chromatographic conditions, measuring relative retention time and relative peak area of each chromatographic peak, and calculating relative standard deviation; the relative retention time of each chromatographic peak and the RSD value of the relative peak area are both less than 1.9 percent, which indicates that the method has good repeatability;
(4) And (3) linear relation investigation: preparing 6 solutions with mass concentration of naringin, respectively injecting samples and detecting according to the chromatographic condition under the item of 2.1.2, drawing a standard curve by taking the mass (X) as a horizontal ordinate and the peak area (Y) as a vertical coordinate, and performing linear regression to obtain a linear regression equation (Y =1327.2X + 87.58) and a correlation coefficient (r =0.999 1). The results show that the linear relation of naringin is good in the range of 0.6800-5.7800 mug.
Furthermore, the method can definitely identify the authenticity of the medicinal material by taking a characteristic peak in the characteristic spectrum of the bitter orange medicinal material as a qualitative standard of the bitter orange medicinal material.
Further, the method can distinguish the advantages and disadvantages of the bitter orange through the established lower limit of the relative content of each characteristic peak determined by the calculation method of the relative content of the characteristic peaks.
Further, the method is suitable for the fructus aurantii medicinal materials (decoction pieces) through a Visual quality-quality dual-standard quality control program designed by Visual Basic programming language.
Compared with the prior art, the invention has the beneficial effects.
(1) The invention discloses a method for controlling the quality of a bitter orange medicament for the first time, which can overcome the defects that the single index in the existing method for controlling the quality of the bitter orange medicament cannot reflect the integral characteristics of the medicament, the multi-index content measurement depends on various reference substances, certain index components cannot reflect the medicament effect, and the fingerprint can only fuzzily evaluate the similarity of the medicament and cannot clearly judge the authenticity and quality of a test product.
(2) The invention utilizes modern analysis and detection technology to indicate that the basic parent nucleus of the chemical component of the characteristic peak is 2-phenyl chromone. Wherein the narirutin has antioxidant and small intestine promoting effects; naringin has biological activity of resisting gastric ulcer; hesperidin can promote gastrointestinal motility; neohesperidin has gastrointestinal hormone regulating effect; poncirin can inhibit gastric cancer cell proliferation; nobiletin has pharmacological effects of resisting inflammation and regulating gastrointestinal motility; the hesperetin has a certain treatment effect on colon cancer. The chemical components are all fructus aurantii, and the fructus aurantii plays roles of regulating qi, relieving epigastric distention, activating stagnancy and relieving flatulence, and has anti-inflammation and antioxidation functions. The active chemical components with pharmacological action of resisting cancer can comprehensively and scientifically reflect the internal quality of the traditional Chinese medicine bitter orange.
Drawings
Fig. 1 is an overlay map of 10 batches of fructus aurantii test drug materials.
FIG. 2 is a reference drug characteristic spectrum and a test drug common mode.
FIG. 3 is the result of visualization of the Hubei filial piety bitter orange sample quality-quantity dual-standard quality control method.
FIG. 4 is the visualized result of the quality-quality control method of Yichun fructus Aurantii sample in Jiangxi.
Detailed Description
The following examples will help to understand the present invention, but they are only for illustrative purposes and the present invention is not limited to these contents.
Example 1.
Fructus Aurantii control drug (batch No. 120981-201605, china institute for testing and testing food and drug). The sample of fructus Aurantii (Jiangxi Yichun, jiangxi Jian, jiangxi camphor tree, zhejiang Quzhou, zhejiang Jinhua, sichuan Bazhong, hunan Yuan Jiang, chongqing Dabieshan mountain, hebei Baodin) was identified as Ruxiang plant Citrus aurantium by professor Xu Liang of Liaoning Chinese medicine universityCitrus aurantium L.And dried unripe fruits of cultivars thereof. According to the method under the item of ' content determination ' of fructus Aurantii in ' pharmacopoeia of the people's republic of China ' 2020 edition, 10 batches of test samples in production places are detectedIn the test, except the test sample from Hubei filial piety, other test samples can accord with the pharmacopoeia regulation (naringin content is not less than 4.0%, and neohesperidin content is not less than 3.0%).
1. And performing qualitative research on the fructus aurantii medicinal material based on the control medicinal material.
1.1 preparation of the solution.
Taking 0.5 g of reference medicinal material powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50% methanol 25 mL, weighing, heating and refluxing 1 h, cooling, complementing weight loss, shaking up, filtering, volatilizing filtrate, fixing the volume of 50% methanol to 5 mL volumetric flask, shaking up, and passing through a 0.22 μm filter membrane as reference solution; taking test medicinal material powder (passing through No. 4 sieve) 0.5 g, and making into test solution by the above method.
1.2 chromatographic conditions.
And (3) chromatographic column: agilent EC-C 18 A chromatography column (3.0X 100 mm, 2.7-Micron); mobile phase: 0.1% formic acid water (a) -acetonitrile (B) with gradient elution; gradient elution procedure: 0-10 min,5% → 15% B;10 to 15 min,15% → 18% by weight of B; 10-25 min,18% → 50% B; flow rate: 1.0 mL/min; column temperature: 30. DEG C; DAD detector wavelength: 330 nm; sample introduction amount: 5.μ L.
1.3 And establishing a characteristic map.
And (3) paralleling each sample of the reference substance solution and the test sample solution for 2 times, sequentially injecting samples according to chromatographic conditions for detection, and exporting the spectrum data under the wavelength of 330 nm from an analytical detection instrument. HPLC stacking maps of 10 test drugs are shown in figure 1; the 10 batches of test drugs are automatically matched by using median, and subjected to multipoint correction to generate a common mode R, and the common mode R is compared with a reference drug characteristic map, which is shown in figure 2.
1.4 And (5) evaluating the similarity.
Except for Hubei filial piety bitter orange sample, the similarity of other samples and the reference medicinal material is more than 0.98.
1.5 And (4) analyzing the chemical components of the characteristic peaks.
And (3) identifying 9 characteristic peaks in total by analyzing retention time, primary ion mass-to-charge ratio and secondary ion fragment information of the compound and matching with related literature report information by adopting a Q-TOF-MS technology.
The method for controlling the quality of the bitter orange medicinal material can be used for judging the authenticity of the bitter orange medicinal material by the obtained bitter orange medicinal material characteristic spectrum.
2. Relative quantitative studies of chemical composition based on characteristic peaks of internal standard substances.
The 7 peak naringin has good separation effect, large peak area and stable and centered retention time, so that the naringin is used as an internal standard substance to carry out relative quantitative research on characteristic peaks based on the internal standard substance. According to the detection method under the item of ' content determination ' of fructus Aurantii in ' pharmacopoeia of the people's republic of China ' 2020 edition, in 10 batches of production area test samples, except the test sample from filial piety in Hubei, the other test samples all accord with the regulation of pharmacopoeia. The relative content of the chemical components of the characteristic peaks of the rest 9 batches of test medicinal materials except the Hubei filial piety bitter orange is calculated through the accurate quantification of the chemical components of the internal standard substance. The "median-standard deviation" was taken as the lower limit of the relative content of the chemical components of the characteristic peak to the chemical components of the internal standard substance, and is shown in table 1.
TABLE 1 relative quantitation of characteristic peaks based on internal standard substances
Figure DEST_PATH_IMAGE001
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3. Methodology investigation.
3.1 And (5) testing the precision.
Precisely absorbing the same sample solution, measuring according to chromatographic conditions, continuously feeding sample for 6 times, measuring relative retention time and relative peak area of each chromatographic peak, and calculating relative standard deviation. The relative retention time of each chromatographic peak and the relative peak area RSD value are less than 1.0 percent, which indicates that the precision of the instrument is good.
3.2 And (5) stability test.
Precisely sucking the same sample solution, injecting samples at 0, 2, 4, 8, 12, 24 h for 6 times according to chromatographic conditions, measuring relative retention time and relative peak area of each chromatographic peak, and calculating relative standard deviation. The relative retention time of each chromatographic peak and the relative peak area RSD value are both less than 2.4 percent, which indicates that the test solution is stable in 24 h.
3.3 And (5) performing repeated experiments.
Preparing 6 parts of test sample according to the preparation method of the test sample solution, respectively injecting and detecting according to chromatographic conditions, measuring the relative retention time and the relative peak area of each chromatographic peak, and calculating the relative standard deviation. The relative retention time of each chromatographic peak and the RSD value of the relative peak area are both less than 1.9 percent, which indicates that the method has good repeatability.
3.4 And (5) observing a linear relation.
Preparing 6 solutions with mass concentration of naringin, respectively injecting samples for detection according to chromatographic conditions, drawing a standard curve by taking mass (X) as a horizontal ordinate and peak area (Y) as a vertical coordinate, and performing linear regression to obtain a linear regression equation (Y =1327.2X + 87.58) and a correlation coefficient (r =0.999 1). The results show that the linear relation of naringin is good in the range of 0.6800-5.7800 mug.
Example 2 identification of test samples of Hubei filial piety bitter orange.
The similarity between the Hubei filial piety bitter orange sample and the reference medicinal material is 0.776 which is less than 0.90. Through the visualized quality-quantity dual-standard quality control method program (version 01) of the traditional Chinese medicinal materials (decoction pieces), which is designed by the invention, the samples of the bitter orange in Hubei filial piety are visually judged to be counterfeit products, as shown in figure 3.
Example 3 identification of test samples of bitter orange in Yichun Jiangxi.
The similarity between the Hubei filial piety bitter orange sample and the reference medicinal material is 0.984. Through the visual 'quality-quantity' dual-standard quality control method program (version 01) of the traditional Chinese medicinal materials (decoction pieces) designed by the invention, the samples of the fructus aurantii immaturus in Hubei filial piety can be intuitively judged to be 'true' and 'excellent' in quality, which is shown in figure 4.

Claims (9)

1. A method for establishing quality control standards of fructus aurantii medicinal materials is characterized by comprising the following steps:
step 1, performing qualitative research on a fructus aurantii medicinal material based on a reference medicinal material;
step 2, relative quantitative research of chemical components of characteristic peaks based on internal standard substances;
step 3 methodology investigation.
2. The method for establishing the quality control standard of fructus aurantii according to claim 1, wherein the specific steps of the step 1 comprise:
(1) Preparation of the solution: taking fructus Aurantii reference medicinal material and test medicinal material powder, accurately weighing, placing in a conical flask with a plug, accurately adding 50 times of 50% methanol, weighing, heating and refluxing for 1 h, cooling, supplementing weight loss, shaking, and filtering; volatilizing the filtrate, diluting with 50% methanol to desired volume, shaking, and filtering with 0.22 μm filter membrane to obtain the final product;
(2) Chromatographic conditions and system applicability test: agilent EC-C adopting octadecylsilane chemically bonded silica as filler 18 A chromatographic column with the specification of 3.0 multiplied by 100 mm and 2.7-Micron; gradient elution is carried out by taking 0.1% formic acid water as a mobile phase A and acetonitrile as a mobile phase B; the flow rate is 1.0 mL/min; the column temperature is 30 ℃; the DAD detector detects at a wavelength of 330 nm; the sample injection amount is 5 mu L;
(3) Establishing a characteristic spectrum: each sample is paralleled for 2 times, sample injection detection is carried out in sequence according to chromatographic conditions, and the HPLC superposed spectra of 10 batches of test medicines are obtained by leading out the spectrum data under the wavelength of 330 nm from an analytical detection instrument; automatically matching 10 batches of test medicines by using a median, performing multi-point correction to generate a common mode R, and comparing the common mode R with a reference medicine characteristic map;
(4) And (3) similarity evaluation: determining the authenticity of the bitter orange medicinal material according to the similarity of the characteristic peaks; except for Hubei filial piety bitter orange sample, the similarity of other samples and the reference medicinal material is more than 0.98;
(5) Analyzing the chemical components of the characteristic peaks: identifying 9 chemical components by analyzing retention time, first-order ion mass-to-charge ratio and second-order ion fragment information of the compound and matching with related literature report information by adopting a Q-TOF-MS technology;
(6) The visual quality-quality double-standard quality control method of the traditional Chinese medicinal materials (decoction pieces) comprises the following steps: a Visual Basic programming language is utilized to design a quality-quantity dual-standard quality control program which comprises 1 OEL control and 4 Command controls and is suitable for visualizing bitter orange medicinal materials (decoction pieces).
3. The method of claim 2, wherein the 9 chemical components identified in (5) are: the eriocitrin is shown in the No. 3 peak, the eriocitrin in the No. 4 peak, the naringin in the No. 6 peak, the naringin in the No. 7 peak, the hesperidin in the No. 9 peak, the neohesperidin in the No. 10 peak, the poncirin in the No. 12 peak, the nobiletin in the No. 15 peak and the hesperetin in the No. 16 peak.
4. The method for establishing the quality control standard of fructus aurantii according to claim 1, wherein the specific steps of the step 2 comprise:
taking the 7 th peak naringin determined in the step 1 as an internal standard substance, accurately quantifying chemical components of the internal standard substance, calculating the relative content of the chemical components of the characteristic peaks of the rest 9 batches of test medicines except the Hubei filial piety bitter orange, and taking the median-standard deviation as the lower limit of the relative content of the chemical components of the characteristic peaks relative to the chemical components of the internal standard substance.
5. The method for controlling the quality of the fructus aurantii medicinal material according to claim 1, wherein the characteristic peaks in the step 2 are all characteristic active chemical components of the fructus aurantii playing the efficacy and pharmacological action thereof.
6. The method for establishing the quality control standard of fructus aurantii according to claim 1, wherein the specific steps of the step 3 comprise:
(1) And (3) precision experiment: precisely absorbing the same sample solution, determining according to chromatographic conditions, continuously feeding sample for 6 times, determining relative retention time and relative peak area of each chromatographic peak, and calculating relative standard deviation; the relative retention time of each chromatographic peak and the RSD value of the relative peak area are both less than 1.0 percent, which indicates that the precision of the instrument is good;
(2) And (3) stability test: precisely absorbing the same sample solution, injecting samples for 6 times at 0, 2, 4, 8, 12 and 24 h respectively according to chromatographic conditions, measuring relative retention time and relative peak area of each chromatographic peak, and calculating relative standard deviation; the relative retention time of each chromatographic peak and the relative peak area RSD value are both less than 2.4 percent, which indicates that the sample solution is stable in 24 h;
(3) And (3) repeatability experiment: preparing 6 parts of test sample according to a test sample solution preparation method, respectively carrying out sample injection detection according to chromatographic conditions, measuring relative retention time and relative peak area of each chromatographic peak, and calculating relative standard deviation; the relative retention time of each chromatographic peak and the RSD value of the relative peak area are both less than 1.9 percent, which indicates that the method has good repeatability;
(4) And (3) linear relation investigation: preparing 6 solutions with mass concentration of naringin, respectively injecting samples and detecting according to the chromatographic condition under the item of 2.1.2, drawing a standard curve by taking the mass (X) as a horizontal ordinate and the peak area (Y) as a vertical coordinate, and performing linear regression to obtain a linear regression equation (Y =1327.2X + 87.58) and a correlation coefficient (r =0.999 1); the results show that the linear relation of naringin is good in the range of 0.6800-5.7800 mug.
7. The method for controlling the quality of the fructus aurantii medicinal material according to claim 1, wherein characteristic peaks in a characteristic spectrum of the fructus aurantii medicinal material are used as a qualitative standard of the fructus aurantii medicinal material, so that the authenticity of the medicinal material can be definitely identified.
8. The method for controlling the quality of fructus aurantii according to claim 1, wherein the method can distinguish the quality of fructus aurantii by establishing a lower limit of the relative content of each characteristic peak determined by a calculation method of the relative content of the characteristic peaks.
9. The method for controlling the quality of fructus aurantii crude drug according to claim 1, wherein the method is suitable for the fructus aurantii crude drug (decoction pieces) through a Visual quality-to-quantity dual-standard quality control program designed by Visual Basic programming language.
CN202210997910.6A 2022-08-19 2022-08-19 Quality double-label visual quality control technology of fructus aurantii medicinal material Pending CN115326974A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116990420A (en) * 2023-09-27 2023-11-03 江西中医药大学 Feature-map-based honey bran fructus aurantii comprehensive quality evaluation method

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116990420A (en) * 2023-09-27 2023-11-03 江西中医药大学 Feature-map-based honey bran fructus aurantii comprehensive quality evaluation method

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