CN115326980A - Quality double-label visual quality control technology of Chinese angelica medicinal material - Google Patents

Quality double-label visual quality control technology of Chinese angelica medicinal material Download PDF

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CN115326980A
CN115326980A CN202210998280.4A CN202210998280A CN115326980A CN 115326980 A CN115326980 A CN 115326980A CN 202210998280 A CN202210998280 A CN 202210998280A CN 115326980 A CN115326980 A CN 115326980A
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medicinal material
angelica
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孟宪生
李晓晨
赵琳
王帅
包永睿
李天娇
郑莹
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 for a Chinese angelica medicinal material, and belongs to the technical field of quality control of traditional Chinese medicines. The invention adopts HPLC technology to establish the characteristic map of angelica sinensis reference medicinal material, and compares the characteristic map with the map of the test medicinal material to distinguish the authenticity of the medicinal material, namely '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 angelica sinensis medicine material-quantity analysis method meets the methodology investigation requirement, and the similarity of each test medicine and the reference medicine is more than 0.98; the lower limit of the relative content of the chemical components of the characteristic peak of the angelica medicinal material 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 Chinese angelica 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 a medicinal material by taking an angelica sinensis reference medicinal material as a reference substance, wherein the method is independent of a reference substance, has good stability and precision, can make up for the defects of quality control of the angelica sinensis medicinal material at present, and can provide a new idea for improving the quality of the angelica sinensis medicinal material.
Background
The radix Angelicae sinensis is derived from Angelica gigas of UmbelliferaeAngelic sinensis(Oliv.)DielsThe dried root is recorded in Shennong Bencao Jing, also named as gan Gui, ma taili Dang, ma taili Gui, yun Gui, shanmin Gui and the like, is one of the most commonly used Chinese medicines in China in clinic, has the effects of enriching blood, activating blood, regulating menstruation and relieving pain, is named as 'qi medicine in blood', 'essential medicine for enriching blood', 'good medicine for gynecology', and mainly contains various compounds such as volatile oil, organic acid, polysaccharide, nucleoside, amino acid and the like, and has long medicinal history and is used as medicine by a plurality of classical famous prescriptions. Gansu, as the local area of Dang Dan, has a long history, and its medical records, dang Gui, produced in Longxi (jin Gansu Longxi-Lanzhou), and Hei Shui (jin Gansu Ding)West), west (west of today's Sichuan). In recent years, with the increase of the market demand of angelica, the planting area is continuously enlarged, and with the global warming and the large environment with high temperature, the traditional main production area of angelica has certain changes and gradually migrates to the west, the seedling quality is good and uneven, and the healthy development of angelica medicinal material production is influenced; the angelica sinensis is long in cultivation duration, the collection and breeding work of high-quality germplasm is relatively lagged, and the variety degeneration is serious, which can be important factors influencing the quality of medicinal materials.
Disclosure of Invention
Aiming at the problems, the invention provides a Chinese angelica medicine material-quantity dual-standard control method. The angelica sinensis reference medicinal material is taken as a qualitative and quantitative reference substance, and a method which does not depend on a reference substance and can comprehensively and scientifically control the quality of the medicinal material is established by adopting high performance liquid chromatography and a Q-TOF-MS technology.
In order to achieve the above object, the present invention provides the following technical solutions.
The invention provides a method for establishing a quality control standard of a Chinese angelica medicinal material, which is characterized by comprising the following steps of:
step 1, carrying out qualitative research on angelica medicinal materials based on reference medicinal materials;
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 radix Angelicae sinensis reference material and test material powder, placing into conical flask with plug, precisely adding 70% methanol, weighing, ultrasonically extracting for 30 min (frequency: 40 KHZ), cooling, adding methanol to supplement loss, shaking, filtering, collecting filtrate, and filtering with 0.22 μm filter membrane to obtain the final product;
(2) Chromatographic conditions and system applicability test: agilent poroshell 120 SB-C adopting octadecylsilane chemically bonded silica as filler 18 The specification of the chromatographic column is 100 mm multiplied by 4.6 mm and 2.7 mu m; gradient elution is carried out by taking 0.1% formic acid water as a mobile phase A and acetonitrile as a mobile phase B; flow rate 0.6mL/min; the column temperature is 30 ℃; the DAD detector detects at a wavelength of 254 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 8 batches of tested medicinal materials are obtained by leading out the spectrum data under the wavelength of 254 nm from an analytical detection instrument; 8 batches of test medicines are automatically matched by using a median, and subjected to multipoint correction to generate a common mode R which is compared with a characteristic map of a reference medicine;
(4) And (3) similarity evaluation: and (4) determining the authenticity of the Chinese angelica medicinal material according to the similarity of the characteristic peaks. The similarity of the sample and the reference medicinal material is more than 0.98;
(5) Analyzing the chemical components of the characteristic peaks: by adopting a Q-TOF-MS technology, 6 chemical components are identified by analyzing retention time, a primary ion mass-to-charge ratio and secondary ion fragment information of a compound and matching with related literature report information;
(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 visualizing Chinese angelica medicinal materials (decoction pieces).
Further, the 6 chemical components identified in (5) were: (4) The peak is chlorogenic acid, the peak (6) is ferulic acid, the peak (7) is senkyunolide I, the peak (11) is imperatorin, the peak (14) is ligustilide and the peak (15) is angelicin.
Further, the specific steps of step 2 include:
taking the ferulic acid of the peak (6) determined in the step 1 as an internal standard substance, and accurately quantifying the chemical components of the internal standard substance to calculate the relative content of the chemical components of the characteristic peak of 8 batches of the test drugs; 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 angelica which exert the efficacy and pharmacological action of the angelica.
Further, the specific steps of step 3 include:
(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 relative peak area RSD value are less than 1.0 percent, which shows that the precision of the instrument is good;
(2) And (3) stability test: precisely absorbing the same test solution, respectively injecting samples for 6 times at 0, 2, 4, 8, 12 and 24 h 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 less than 2.4 percent, which indicates that the test solution is stable within 24 hours;
(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 relative peak area RSD value are less than 2.0 percent, which indicates that the method has good repeatability;
(4) Examination of linear relationship: preparing 6 mass concentration solutions of ferulic acid, carrying out sample injection detection respectively 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 carrying out linear regression to obtain a linear regression equation (Y = 0.7021X + 0.7703), a correlation coefficient (r = 0.9991) and a linear range (0.0510-0.2020 mg); the result shows that the sample amount range and the peak area have good linear relation.
Furthermore, the method can definitely identify the truth of the medicinal materials by taking the characteristic peaks in the characteristic spectrum of the Chinese angelica medicinal materials as the qualitative standard of the Chinese angelica medicinal materials.
Further, the method can distinguish the advantages and disadvantages of angelica 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 Chinese angelica 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 Chinese angelica medicine material-quantity dual-standard control method for the first time, which can overcome the defects that the single index in the traditional Chinese angelica medicine material quality control method can not reflect the integral characteristics of the medicine material, the multi-index content measurement depends on various reference substances, certain index components can not reflect the medicine effect, and the fingerprint spectrum can only fuzzily evaluate the similarity of the medicine material and can not clearly judge the authenticity and the quality of a test product.
(2) The invention utilizes modern analysis and detection technology to indicate that chlorogenic acid in the characteristic peak chemical components has wide pharmacological activities of protecting liver, benefiting gallbladder, resisting tumor, resisting virus and the like. The phthalide compounds comprise ligustilide, senkyunolide I, and levistilide, which are main components of radix Angelicae sinensis volatile oil, wherein ligustilide has the highest content, and the compounds have various pharmacological effects of resisting tumor, resisting oxidation, inhibiting angiogenesis, diminishing inflammation, relieving pain, etc. The coumarin compounds comprise imperatorin and the like, and have various pharmacological activities of resisting tumors, hypertension, myocardial ischemia, inflammation, pain and the like, which shows that the characteristic peak selected by the method can relatively scientifically reflect the drug effect of the Chinese angelica medicinal material and can comprehensively and scientifically reflect the internal quality of the Chinese angelica.
Drawings
FIG. 1 is a superimposed spectrum of the angelica reference drug and 8 batches of the angelica test drug.
FIG. 2 is a reference drug characteristic spectrum and a test drug common mode.
FIG. 3 is a visualization result of Gansu Min county sample quality-quantity double-standard quality control method.
Fig. 4 is a visualization of the anhuzhou-like quality-quantity dual-standard quality control method.
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.
Chinese angelica as reference material (batch number 120927-201617, china institute for testing and testing food and drugs). The Chinese angelica medicinal material test sample (Gansu Min county, gansu Dingxi, gansu Weiyuan county, gansu Wen county, anhuo Mazhou, hebei, yunnan and Ningxia) is identified as the Umbelliferae swertia plant by the professor xueliang in Liaoning traditional Chinese medicine university
Angelic sinensis(Oliv.)DielsDried root of (4). According to the detection method under the item of ' content determination ' of Chinese angelica in ' pharmacopoeia of the people's republic of China ' 2020 edition, the test products in 8 batches of production places can all accord with the regulation of the pharmacopoeia (containing ferulic acid is not less than 0.05%).
1. And (3) carrying out qualitative research on the angelica medicinal material based on the reference medicinal material.
1.1 preparation of the solution.
Precisely weighing 2.0 g of radix Angelicae sinensis control material, placing in a conical flask with a plug, precisely adding 25 mL of 70% methanol, weighing, ultrasonically extracting for 30 min (frequency: 40 KHZ), cooling, adding methanol to supplement loss mass, shaking, filtering, collecting filtrate, and filtering with 0.22 μm filter membrane to obtain reference solution; taking 2.0 g of test drug powder (screened by No. 4 sieve), and preparing the test solution according to the method.
1.2 chromatographic conditions.
A chromatographic column: agilent ponoshell 120 SB-C18, the specification of a chromatographic column is 100 mm multiplied by 4.6 mm,2.7 mu m; gradient elution is carried out by taking 0.1% formic acid water as a mobile phase A and acetonitrile as a mobile phase B; 0 to 15 min,5% → 40% by weight of B; 15-17 min,40% → 60% B;17 to 25 min,60% → 75% B,25 to 31 min,75% → 87% B;31 to 35 min,87% → 95% B; the flow rate is 0.6 mL/min; the column temperature is 30 ℃; DAD detector at wavelength: 254 nm; the sample size was 5. Mu.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 254 nm from an analysis detection instrument. HPLC stacking maps of 8 test medicinal materials are shown in figure 1;8 batches of test drugs are automatically matched by using median, and are 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.
The similarity between the test medicine and the reference medicine is greater than 0.98.
1.5 And (4) analyzing the chemical components of the characteristic peaks.
And (3) identifying 6 characteristic peaks in total by analyzing retention time, primary ion mass-to-charge ratio and secondary ion fragment information of the compound by adopting a Q-TOF-MS technology and matching with relevant literature report information.
According to the quality control method of the angelica sinensis medicinal material, the obtained angelica sinensis medicinal material characteristic spectrum can be used for judging the authenticity of the angelica sinensis medicinal material.
2. Relative quantitative studies of chemical composition based on characteristic peaks of internal standard substances.
The No. 6 peak ferulic acid has good separation effect, large peak area and stable and centered retention time, so that the ferulic acid is used as an internal standard substance to carry out relative quantitative research on a characteristic peak based on the internal standard substance. According to the method under the item of ' content determination ' of Chinese angelica in ' pharmacopoeia of the people's republic of China ' 2020 edition, 8 batches of test samples in production places all accord with the regulations of the pharmacopoeia. And accurately quantifying the chemical components of the internal standard substance, and calculating the relative content of the chemical components of the characteristic peaks of 8 batches of test medicaments. 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 internal standard-based characteristic peak relative quantification results of 8 batches of Chinese angelica medicinal materials
Figure DEST_PATH_IMAGE001
。
3 methodology investigation.
3.1 precision assay.
Precisely absorbing 5 mu L of the same sample solution, determining according to chromatographic conditions, continuously injecting samples 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 relative peak area RSD value are less than 1.0 percent, which indicates that the precision of the instrument is good.
3.2 stability test.
Precisely absorbing 5 mu L of the same test solution, injecting samples for 6 times at 0, 2, 4, 10, 16 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 test solution is stable within 24 hours.
3.3 repeatability 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 2.0 percent, which indicates that the method has good repeatability.
3.4 Linear relationship examination.
Preparing 6 mass concentration solutions of ferulic acid, carrying out sample injection detection respectively 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 carrying out linear regression to obtain a linear regression equation (Y = 0.7021X + 0.7703), a correlation coefficient (r = 0.9991) and a linear range (0.0510-0.2020 mg). The result shows that the sample amount range and the peak area have good linear relation.
Example 2 identification of the quality of the angelica medicinal material.
And calculating the relative content of the characteristic peaks of the test samples according to a visual program, comparing with the reference medicinal materials, grading, and drawing to judge the quality. Wherein the Bozhou medicinal material score is 37.29, and the quality is judged to be medium and lower.
Due to the increase of the market demand of angelica sinensis, the quality of seedlings is not uniform, and the fertilization, cultivation mode, altitude gradient, seedling raising mode and period can be important factors influencing the quality of medicinal materials. The characteristic peaks in the characteristic spectrum of the angelica medicinal material are used as the qualitative standard of the angelica medicinal material, so that the advantages and the disadvantages can be obviously distinguished, the established lower limit of the relative content of the characteristic peaks can distinguish the advantages and the disadvantages of the angelica, and the problem of poor clinical efficacy caused by the poor angelica can be solved. The invention can clearly judge the quality of the test sample, make up the deficiency of the quality control of the angelica medicinal material at present and achieve the purpose of comprehensively controlling the angelica medicinal material, which is shown in figure 4.

Claims (9)

1. A method for establishing a quality control standard of a Chinese angelica medicinal material is characterized by comprising the following steps:
step 1, carrying out qualitative research on angelica medicinal materials based on reference medicinal materials;
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 the angelica sinensis medicinal material according to claim 1, wherein the specific steps of the step 1 comprise:
(1) Preparation of the solution: precisely weighing radix Angelicae sinensis reference material and test material powder, placing into conical flask with plug, precisely adding 70% methanol, weighing, ultrasonically extracting for 30 min (frequency: 40 KHZ), cooling, adding methanol to supplement loss, shaking, filtering, collecting filtrate, and filtering with 0.22 μm filter membrane to obtain the final product;
(2) Chromatographic conditions and system applicability test: agilent ponoshell 120 SB-C using octadecylsilane chemically bonded silica as filler 18 The specification of the chromatographic column is 100 mm multiplied by 4.6 mm and 2.7 mu m; 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 0.6 mL/min; the column temperature is 30 ℃; the DAD detector detects at a wavelength of 254 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 8 batches of tested medicinal materials are obtained by leading out the spectrum data under the wavelength of 254 nm from an analytical detection instrument; carrying out automatic matching on 8 batches of test medicines by using a median, carrying out 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 truth of the angelica medicinal material according to the similarity of the characteristic peaks; the similarity of the sample and the reference medicinal material is more than 0.98;
(5) Analyzing the chemical components of the characteristic peaks: by adopting a Q-TOF-MS technology, 6 chemical components are identified by analyzing retention time, a primary ion mass-to-charge ratio and secondary ion fragment information of a compound and matching with related literature report information;
(6) The visual quality-quality dual-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 visualizing Chinese angelica medicinal materials (decoction pieces).
3. The method of claim 2, wherein the 6 chemical components identified in (5) are: (4) The peak is chlorogenic acid, (6) ferulic acid, (7) senkyunolide I, (11) imperatorin, (14) ligustilide and (15) angelicin.
4. The method for establishing the quality control standard of the angelica sinensis medicinal material according to claim 1, wherein the specific steps of the step 2 comprise:
taking the (6) peak ferulic acid determined in the step 1 as an internal standard substance, and accurately quantifying the chemical components of the internal standard substance to calculate the relative content of the chemical components of the characteristic peak of 8 batches of test drugs; 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 quality control method of angelica sinensis according to claim 1, wherein the characteristic peaks in the step 2 are all characteristic active chemical components of angelica sinensis for exerting the efficacy and pharmacological action thereof.
6. The method for establishing the quality control standard of the angelica sinensis medicinal material according to claim 1, wherein the specific steps of the step 3 comprise:
(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 absorbing the same test solution, respectively injecting samples for 6 times at 0, 2, 4, 8, 12 and 24 h 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 within 24 hours;
(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 2.0 percent, which indicates that the method has good repeatability;
(4) Examination of linear relationship: preparing 6 mass concentration solutions of ferulic acid, carrying out sample injection detection respectively 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 carrying out linear regression to obtain a linear regression equation (Y = 0.7021X + 0.7703), a correlation coefficient (r = 0.9991) and a linear range (0.0510-0.2020 mg); the result shows that the sample amount range and the peak area have good linear relation.
7. The method for establishing the quality control standard of the angelica sinensis medicinal material according to claim 1, wherein the method can definitely identify the authenticity of the medicinal material by taking a characteristic peak in a characteristic map of the angelica sinensis medicinal material as a qualitative standard of the angelica sinensis medicinal material.
8. The method for establishing the quality control standard of the angelica sinensis medicinal material as claimed in claim 1, wherein the method can distinguish the quality of the angelica sinensis by the lower limit of the relative content of each characteristic peak determined by the established calculation method of the relative content of the characteristic peaks.
9. The method for establishing the quality control standard of Chinese angelica root crude drugs according to claim 1, wherein the method is applied to Chinese angelica root crude drugs (decoction pieces) through a Visual quality-quality dual-standard quality control program designed by Visual Basic programming language.
CN202210998280.4A 2022-08-19 2022-08-19 Quality double-label visual quality control technology of Chinese angelica medicinal material Pending CN115326980A (en)

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