CN113156001B - Fingerprint construction method and application of Chinese herbal compound containing angelica sinensis - Google Patents
Fingerprint construction method and application of Chinese herbal compound containing angelica sinensis Download PDFInfo
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- CN113156001B CN113156001B CN202110351933.5A CN202110351933A CN113156001B CN 113156001 B CN113156001 B CN 113156001B CN 202110351933 A CN202110351933 A CN 202110351933A CN 113156001 B CN113156001 B CN 113156001B
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Abstract
The invention discloses a detection method of a Chinese herbal compound containing angelica, which is characterized by comprising the following stepsThe method comprises the following steps: detecting a traditional Chinese medicine compound test sample and a reference substance, wherein the traditional Chinese medicine compound comprises angelica sinensis, cassia twig, liquorice, white paeony root, ginger and Chinese date, the reference substance is gallic acid, paeoniflorin, ferulic acid, liquiritin, ammonium glycyrrhizate and 6-gingerol, and the chromatographic conditions of the detection are as follows: by C18The chromatographic column takes methanol or acetonitrile as a mobile phase A and acid water solution as a mobile phase B, and the gradient elution procedure is as follows: 0-20 min, 5% -12% A; 20-90 min, 12% -85% A; the flow rate is 0.8-1.2 mL/min; the column temperature is 25-40 ℃; the detection wavelength is 210-330 nm; and obtaining the component information of the Chinese herbal compound or the component and content information thereof according to the detection result. The invention adopts the high performance liquid chromatography technology to comprehensively and systematically analyze the chemical components of the Chinese herbal compound containing the angelica, and provides a theoretical basis for the deep research of quality control and pharmacodynamic substance basis.
Description
Technical Field
The application relates to the technical field of traditional Chinese medicine quality identification, in particular to a fingerprint construction method of a traditional Chinese medicine compound containing angelica sinensis and application thereof.
Background
With the rapid development of Chinese herbal medicine preparations in China, the quality management problem of the Chinese herbal medicine preparations is also widely concerned by people, and the quality of the Chinese herbal medicine preparations can directly influence the medical quality and the life safety of patients. The factors influencing the quality of the traditional Chinese medicine preparation mainly comprise: (1) the quality of raw medicinal materials and the quality of the Chinese medicinal materials are one of key reasons which directly influence the clinical curative effect of the Chinese medicinal preparation, and because the traditional Chinese medicinal materials are relatively complex in source and various in variety, a large amount of economic benefits are often obtained from the traditional Chinese medicinal materials in the market. In addition, the relative shortage of the wild medicinal materials in the market of Chinese medicinal materials is limited by the collection of wild natural resources by China, the quality of the medicament is seriously influenced by adopting the planted medicinal materials to replace the wild medicinal materials in many places, so that the quality of the medicinal materials is reduced, and particularly, the yield of the Chinese medicinal materials is greatly improved by excessively using chemical fertilizers, so that the smell of the Chinese medicinal materials and the wild medicinal materials is changed, and the curative effect of the Chinese medicament is influenced; (2) in the production process, in each link of the traditional Chinese medicine preparation, from feeding to preparation, feeding personnel needs to carefully check the taste and quantity of the fed medicines according to the prescription in the process, and if the feeding is less or more, the overall quality of the product is affected. In addition, the processes of crushing, extracting, concentrating, drying, refining and the like of the traditional Chinese medicine preparation are also required to be paid attention to, and the processes have a great influence on the content of the components of the finished traditional Chinese medicine. Therefore, comprehensive thinking is carried out according to the properties of the medicinal materials during process design, the optimal preparation conditions and the optimal process are selected, and quality supervision work in the working procedures is well done, so that the quality of the Chinese medicinal preparation is guaranteed; (3) process water, clean area environment and process sanitation are also an important step in preparation, and if the process sanitation and the clean area environment are unqualified, the breeding and pollution of microorganisms can be directly caused, so that the produced preparation is unqualified; (4) the packaging material and modern packaging technology, the packaging material of the present preparation is also the key that affects the quality of the preparation, for example, plastics have permeability and adsorptivity, it can not only ensure the solvent volatilization of liquid medicine, but also change the medicine performance of volatile components, accelerate the decomposition of the medicine, use inferior packaging bag, can also make harmful substance permeate into the preparation, in addition, because the Chinese medicinal preparation has the characteristics of many dosage forms, various and small production capacity, etc., therefore, the problems of liquid leakage, liquid mildew, reaction between liquid medicine and packaging, solid preparation deterioration due to moisture, etc. caused by the poor quality of packaging bag will appear, which will seriously affect the quality of the preparation.
The fingerprint of Chinese medicine (including single Chinese medicine and compound Chinese medicine) is a chromatogram or a spectrogram which can mark the chemical characteristics of some Chinese medicine or Chinese medicine preparation and is obtained by adopting a certain analysis means after the Chinese medicine or the Chinese medicine preparation is properly processed. The traditional Chinese medicine fingerprint spectrum is a comprehensive and quantifiable identification means, is established on the basis of the systematic research of the chemical components of the traditional Chinese medicine, and is mainly used for evaluating the authenticity, the excellence and the stability of the quality of the semi-finished products of the traditional Chinese medicine and the traditional Chinese medicine preparation. "integrity" and "fuzziness" are its distinguishing features.
The traditional Chinese medicine and the preparation thereof are all multi-component complex systems, so that the quality of the traditional Chinese medicine and the preparation thereof is evaluated by adopting a detection method which is adaptive to the traditional Chinese medicine and can provide rich identification information, but the existing methods such as microscopic identification, physicochemical identification, content measurement and the like are not enough to solve the problem, the establishment of the traditional Chinese medicine fingerprint spectrum can more comprehensively reflect the types and the quantities of chemical components contained in the traditional Chinese medicine and the preparation thereof, and further the overall description and evaluation of the quality of the medicine are carried out. This also corresponds to the holistic theory of traditional Chinese medicine. On the basis, if the research on the spectrum effect is further carried out, the quality of the traditional Chinese medicine and the drug effect thereof can be really combined, which is helpful for clarifying the action mechanism of the traditional Chinese medicine. In a word, the research and establishment of the traditional Chinese medicine fingerprint spectrum have important significance for improving the quality of the traditional Chinese medicine and promoting the modernization of the traditional Chinese medicine.
Angelica sinensis (Oliv.) Diels is dried root of Angelica sinensis (Oliv.) Diels, Umbelliferae. Mainly produced in southeast of Gansu province, has high yield and good quality due to Min county, and is cultivated in Yunnan province, Sichuan province, Shaanxi province, Hubei province and the like. Angelica has the effects of replenishing blood and activating blood, regulating menstruation and relieving pain, and loosening bowel to relieve constipation, and is commonly used for blood deficiency and chlorosis, dizziness and palpitation, irregular menstruation, amenorrhea and dysmenorrhea, asthenia cold and abdominal pain, rheumatic arthralgia, traumatic injury, superficial infection, pyocutaneous disease, constipation due to intestinal dryness, etc. The wine prepared with angelica can activate blood and dredge channels, and is used for treating amenorrhea, dysmenorrhea, rheumatalgia, traumatic injury, etc.
Cassia twig, dried tender skin of Cinnamomum cassia Cassia Presl of Lauraceae. Mainly produced in Guangxi, Guangdong and Yunnan provinces. Cutting off twig in spring and summer, drying in the sun or in the shade, and cutting into slices or small sections. Gui Zhi is pungent, sweet and warm in flavor, and enters heart, lung and bladder meridians. Ramulus Cinnamomi has effects of inducing sweat, expelling pathogenic factors from muscles, warming and dredging channels, supporting yang, regulating qi-flowing, and regulating qi-flowing. It is commonly used for wind-cold type common cold, abdominal cold pain, amenorrhea due to blood cold, arthralgia, phlegm and fluid retention, edema and palpitation.
Glycyrrhiza uralensis Fisch, Glycyrrhiza inflata Bat, or Glycyrrhiza glabra L, which is a dried root and rhizome of Glycyrrhiza uralensis Fisch. Distributed in northeast, northeast China, Shaanxi, Gansu, Qinghai, Xinjiang, Shandong, etc. The licorice has the effects of tonifying spleen and qi, clearing away heat and toxic materials, eliminating phlegm and stopping cough, relieving spasm and pain and harmonizing the drugs. It is commonly used for weakness of spleen and stomach, lassitude and hypodynamia, palpitation and shortness of breath, cough with profuse sputum, spasm and pain of abdomen and limbs, carbuncle and sore, and alleviating toxicity and intensity of drugs.
Radix Paeoniae alba is dried root of Paeonia lactiflora pall. Collected in summer and autumn, cleaned, removed head, tail and fine root, boiled in boiling water, peeled or boiled again, and dried in the sun. Radix Paeoniae alba has effects of nourishing blood, regulating menstruation, astringing yin, arresting sweating, softening liver, relieving pain, and suppressing liver yang. It is commonly used for blood deficiency, sallow complexion, irregular menstruation, spontaneous perspiration, night sweat, hypochondriac pain, abdominal pain, limb spasm pain and headache vertigo.
Rhizoma Zingiberis recens is fresh rhizome of Zingiber officinale Rosc of Zingiber of Zingiberaceae, and is also named as rhizoma Zingiberis recens root, BAIYANYUN, ramulus Uncariae cum uncis, DIXIN, YANLIANGXIAOZI, and fresh rhizoma Zingiberis recens. The rhizome (dried ginger), the bark (ginger peel) and the leaf (ginger leaf) of Zingiber officinale can be used as the raw materials. Ginger, rhizoma Zingiberis recens, with pungent flavor and slightly warm nature, enters lung, spleen and stomach meridians. Rhizoma Zingiberis recens has effects of relieving exterior syndrome, dispelling cold, warming spleen and stomach, relieving vomit, warming lung, relieving cough, and removing toxic substance, and can be used for treating wind-cold type common cold, spleen and stomach cold syndrome, stomach cold type emesis, lung cold type cough, fish and crab toxin, etc.
Fructus Jujubae is dried mature fruit of Ziziphus jujuba Mill of Ziziphus of Rhamnaceae, and contains protein, fat, saccharide, carotene, vitamin B group, vitamin C, vitamin P, and nutritional components such as calcium, phosphorus, iron and cyclic adenosine monophosphate. The content of vitamin C is listed as the top of the fruits, and the vitamin C is known as vitamin king. Fructus Jujubae is sweet in taste, warm in nature, and nontoxic, and can be used for relieving fatigue, relieving diarrhea, preventing blood transfusion reaction, reducing serum glutamic pyruvic transaminase, resisting tumor, resisting oxidation, lowering blood pressure, reducing cholesterol, protecting liver, improving immunity, preventing and treating cerebral ischemia, resisting allergy, preventing and treating cardiovascular disease, osteoporosis and anemia.
The Chinese herbal compound comprising angelica sinensis claimed in the application consists of angelica sinensis, cassia twig, liquorice, white paeony root, ginger and Chinese date. The high performance liquid chromatography has the characteristics of high resolution, high sensitivity, high selectivity and the like, and is widely applied to the research fields of traditional Chinese medicine effective substance basis, chemical component analysis and the like. The analysis of chemical components of traditional Chinese medicine is one of the key problems of the clarification of effective substances of traditional Chinese medicine and the quality control. The traditional Chinese medicine fingerprint refers to a chromatogram or a spectrogram which can mark chemical characteristics of certain traditional Chinese medicinal materials or traditional Chinese medicine preparations by adopting a certain analysis means after the traditional Chinese medicinal materials or the traditional Chinese medicine preparations are properly processed. The traditional Chinese medicine fingerprint spectrum is a comprehensive and quantifiable identification means, can relatively comprehensively reflect the types and the quantities of chemical components contained in the traditional Chinese medicine and the preparation thereof, and further performs overall description and evaluation on the quality of the medicine. Therefore, the traditional Chinese medicine fingerprint spectrum has become a more international advanced traditional Chinese medicine quality control means at present. At present, no literature report is available for comprehensively analyzing the chemical components of the compound and researching the fingerprint spectrum.
The united states Food and Drug Administration (FDA) plant drug product industry guidelines, WHO herbal medicine evaluation guidelines, indicate that consistency in product quality can be demonstrated by fingerprint (fingerprint) if the active ingredients of the herbal preparation cannot be identified. The European Community also pointed out in the notes of herbal quality guidelines that herbs and their preparations are used as a whole as active ingredients, and therefore, the quality stability of herbs is not sufficient only by measuring known active ingredients, and various ingredients contained therein should be indicated by finger-print. The Indian grass pharmacopoeia, British grass pharmacopoeia, German Association of medicinal plants, Canadian Association of medicinal and aromatic plants, etc. are also accepted for use in fingerprinting to ensure consistency in quality of herbal products with unknown active ingredients. At present, the national drug administration has required the traditional Chinese medicine injection to have relevant fingerprint, plays a key role in improving the quality of the traditional Chinese medicine injection, but has no mandatory requirement on the traditional Chinese medicine of other dosage forms.
The traditional Chinese medicine fingerprint comprises traditional Chinese medicine chemical fingerprint, protein fingerprint, DNA fingerprint, biological effect fingerprint, etc. The fingerprint is a chemical fingerprint obtained by properly processing Chinese medicinal materials, Chinese medicinal extracts or Chinese patent medicaments and adopting a certain analysis means to obtain a chromatogram or a spectrum capable of marking the characteristics of main chemical components of the Chinese medicinal materials.
The chemical fingerprints of Chinese medicines can be classified into Chinese medicine spectrum fingerprints such as infrared spectrum (IR), ultraviolet spectrum (UV), X-ray diffraction spectrum, etc.; chromatographic fingerprint such as Thin Layer Chromatography (TLC), Gas Chromatography (GC), High Performance Liquid Chromatography (HPLC), Capillary Electrophoresis (CE), etc.; spectral fingerprints, such as Mass Spectrometry (MS), nuclear magnetic resonance spectroscopy (NMR), and the like; DNA fingerprint spectrum; and multidimensional multi-information profiles (characteristic finger print of muhrdimension and muhrdata) obtained by using various modern analytical instruments, such as HPLC/MS, HPLC/Ms/MS, GC/MS, CE/MS and the like.
Although there are many traditional Chinese medicine fingerprint spectrum research methods, chromatographic methods are mainstream methods, especially TLC, HPLC and GC chromatographic techniques, which have become three generally accepted analytical methods, especially HPLC, which should be considered first in the prior art for researching traditional Chinese medicine fingerprint spectrum.
The traditional Chinese medicine fingerprint has two functions: firstly, the authenticity or the producing area of the traditional Chinese medicinal materials can be effectively identified through the characteristics of the fingerprint: secondly, the quality of the product can be effectively controlled by setting the area or the proportion of the main characteristic peak of the fingerprint, and the relative consistency of the product quality is ensured.
The preparation and application of the traditional Chinese medicine fingerprint usually comprise: (1) collecting representative Chinese medicinal samples (single Chinese medicinal material and Chinese medicinal compound), preparing test solution by appropriate method, and selecting appropriate standard substance (reference substance) for optimization of analysis method; (2) according to the determined analysis method, performing separation analysis on a certain batch of traditional Chinese medicine samples, and processing the obtained multiple batches of data to obtain a standard fingerprint; (3) after the sample is processed and separated according to the method same as the standard fingerprint spectrum, the obtained fingerprint spectrum is compared with the standard fingerprint spectrum in similarity, and the sample can be regarded as qualified when the similarity generally reaches more than 80 percent. The similarity calculation and evaluation method usually adopts a correlation coefficient and a coincidence coefficient or an included angle cosine method. Regarding similarity evaluation, a traditional Chinese medicine chromatography fingerprint similarity evaluation system recommended by the national pharmacopoeia committee is more applied.
Disclosure of Invention
In view of the above, the invention provides a Chinese herbal compound component detection method containing angelica and a fingerprint construction method thereof. The spectrum measured by the detection method can comprehensively reflect chemical components in the traditional Chinese medicine compound, the separation of various spectrum peaks is good, the base line is stable, the peak pattern is good, the repeatability is good, and the quality of a sample can be objectively evaluated by the obtained contrast fingerprint spectrum.
In order to achieve the above object, the present invention provides the following technical solutions:
according to one aspect of the invention, the detection method of the Chinese herbal compound containing the angelica is provided, and comprises the following steps:
detecting test sample and reference substance of Chinese medicinal composition, wherein the Chinese medicinal composition comprises radix Angelicae sinensis, ramulus Cinnamomi, Glycyrrhrizae radix, radix Paeoniae alba, rhizoma Zingiberis recens and fructus Jujubae, the reference substance is gallic acid, paeoniflorin, ferulic acid, liquiritin, ammonium glycyrrhizate and 6-gingerol,
the chromatographic conditions for the detection were: by C18The chromatographic column takes methanol or acetonitrile as a mobile phase A and acid water solution as a mobile phase B, and the gradient elution procedure is as follows: 0-20 min, 5% -12% A; 20-90 min, 12% -85% A; the flow rate is 0.8-1.2 mL/min; the column temperature is 25-40 ℃; the detection wavelength is 210-330 nm;
and obtaining the component information of the Chinese herbal compound or the component and content information thereof according to the detection result.
Further, the preparation process of the test article comprises the following steps: adding water or alcohol into the Chinese medicinal compound to obtain Chinese medicinal compound solution; and filtering the traditional Chinese medicine compound solution after ultrasonic extraction or reflux extraction to obtain the test sample.
Further, the preparation process of the reference substance comprises the following steps: weighing appropriate amount of gallic acid, paeoniflorin, ferulic acid, liquiritin, ammonium glycyrrhizate and 6-gingerol; and adding 30-70% methanol to obtain a reference substance with the concentration of each component of 0.02-0.2 mg/mL-1.
Further, the flow rate is 1.0-1.2 mL/min, and the column temperature is 30-40 ℃.
Further, the number of theoretical plates is not less than 8000 in terms of gallic acid.
Further, the C18The chromatographic column is Agilent Zorbax SB-C18Chromatographic column, Phenomenex Gemini C18Chromatography column, Thermo syncronis C18Chromatographic column, Agilent5TC-C18Chromatographic column, Kromasil 100-5C18Column, Waters symmetry C18Chromatographic column or Phenomenex Luna C18A chromatographic column.
Further, the acid aqueous solution is selected from one or more of weak acids and salts thereof, weak bases and salts thereof, and polybasic weak acids and salts thereof in different concentrations.
Further, the acid is formic acid, acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid.
Further, the acid aqueous solution is a 0.08% -0.3% phosphoric acid aqueous solution.
Further, the acid aqueous solution is a 0.08% -0.12% phosphoric acid aqueous solution.
Further, the acid aqueous solution was a 0.1% phosphoric acid aqueous solution.
Further, the traditional Chinese medicine compound is prepared from the following components in parts by weight: 1-10 parts of angelica, 1-10 parts of cassia twig, 1-10 parts of liquorice, 1-10 parts of white paeony root, 1-10 parts of ginger and 1-10 parts of Chinese date.
Further, the traditional Chinese medicine compound is prepared from the following components in parts by weight: 4 parts of angelica, 3 parts of cassia twig, 2 parts of liquorice, 6 parts of white paeony root, 3 parts of ginger and 4 parts of Chinese date.
Further, the licorice is prepared licorice.
According to another aspect of the invention, a fingerprint construction method of a Chinese herbal compound containing angelica is provided,
preparation of a test solution: adding water or alcohol into the Chinese medicinal compound to obtain Chinese medicinal compound solution; and the test solution is obtained by filtering after the ultrasonic extraction or reflux extraction of the traditional Chinese medicine compound solution, wherein the traditional Chinese medicine compound solution consists of angelica, cassia twig, liquorice, white paeony root, ginger and Chinese date;
preparation of control solutions: weighing gallic acid, paeoniflorin, ferulic acid, liquiritin, ammonium glycyrrhizate and 6-gingerol, adding 30% -70% methanol to obtain the final product with concentration of each component of 0.02-0.2 mg/mL-1The control solution of (a);
the chromatographic conditions of the high performance liquid detection are as follows: adopting a C18 chromatographic column, taking methanol or acetonitrile as a mobile phase A and acid water solution as a mobile phase B, and performing gradient elution procedure: 0-20 min, 5% -12% A; 20-90 min, 12% -85% A; the flow rate is 0.8-1.2 mL/min; the column temperature is 25-40 ℃; the detection wavelength is 210-330 nm;
and obtaining the fingerprint of the Chinese herbal compound according to the high performance liquid detection result.
Further, the flow rate of the high performance liquid detection is 1.0-1.2 mL/min, and the column temperature is 30-40 ℃.
Further, the number of theoretical plates is not less than 8000 in terms of gallic acid.
Further, the fingerprint comprises a peak 1-13, wherein the peak 1 is gallic acid as a reference peak, the peak 7 is paeoniflorin, the peak 8 is ferulic acid, the peak 9 is liquiritin, the peak 12 is ammonium glycyrrhizate, and the peak 13 is 6-gingerol, and the retention time is respectively 6.4-7.2 min, 28.4-29.2 min, 34.4-35.2 min, 36.4-37.2 min, 72.4-73.2 min, and 77.6-78.4 min.
Further, the traditional Chinese medicine compound is prepared from the following components in parts by weight: 1-10 parts of angelica, 1-10 parts of cassia twig, 1-10 parts of liquorice, 1-10 parts of white paeony root, 1-10 parts of ginger and 1-10 parts of Chinese date.
Further, the traditional Chinese medicine compound is prepared from the following components in parts by weight: 4 parts of angelica, 3 parts of cassia twig, 2 parts of liquorice, 6 parts of white paeony root, 3 parts of ginger and 4 parts of Chinese date.
Further, the licorice is prepared licorice.
Further, the acid aqueous solution is selected from one or more of weak acids and salts thereof, weak bases and salts thereof, and polybasic weak acids and salts thereof in different concentrations.
Further, the acid is formic acid, acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid.
Further, the acid aqueous solution is a 0.08% -0.3% phosphoric acid aqueous solution.
Further, the C18The chromatographic column is Agilent Zorbax SB-C18Chromatography column, Agilent5TC-C18(2)Column, Phenomenex Kinetex C18Chromatographic column, Ultimate XB C18Chromatographic column, Hypersil GOLD C18Column chromatography, Diamonsil C18Chromatographic column or Supersil ODS 2C18A chromatographic column.
Further, peak 8 is from angelica; peak 10 and peak 11 are from cinnamomi; peak 9 and peak 12 are from licorice; peak 1, peak 2, peak 6, and peak 7 are from white peony; no. 13 is derived from rhizoma Zingiberis recens; the peak 3 and the peak 5 are common peaks of angelica and white peony root; peak 4 is the common peak of licorice and white peony root.
Further, the retention time of peak 1-13 is 6.4-7.2 min, 7.6-8.4 min, 17.2-18.0 min, 19.6-20.4 min, 22.4-23.2 min, 26-26.8 min, 28.4-29.2 min, 34.4-35.2 min, 36.4-37.2 min, 46.4-47.2 min, 60-60.8 min, 72.4-73.2 min and 77.6-78.4 min respectively.
According to a further aspect of the present invention, there is provided a standard fingerprint of a reference composition, wherein the reference is gallic acid, paeoniflorin, ferulic acid, liquiritin, ammonium glycyrrhizate and 6-gingerol, and the standard fingerprint is a high performance liquid chromatography, wherein 6 chromatographic peaks are present in the standard fingerprint, wherein the peak 1 is a chromatographic peak with the same retention time as that of the gallic acid as the reference peak, and the relative retention times of the other 5 chromatographic peaks are respectively: 4.23 +/-10%, 5.12 +/-10%, 5.41 +/-10%, 10.71 +/-10% and 11.47 +/-10%.
According to another aspect of the present invention, there is provided a standard fingerprint of a reference composition, wherein the reference is gallic acid, paeoniflorin, ferulic acid, liquiritin, glycine and 6-gingerol, and the standard fingerprint is a high performance liquid chromatography with the following chromatographic conditions: adopting a C18 chromatographic column, taking methanol or acetonitrile as a mobile phase A and acid water solution as a mobile phase B, and performing gradient elution procedure: 0-20 min, 5% -12% A; 20-90 min, 12% -85% A; the flow rate is 0.8-1.2 mL/min; the column temperature is 25-40 ℃; the detection wavelength is 210-330 nm.
Further, after the standard fingerprint is established, the fingerprint of the Chinese herbal compound containing the angelica is established according to the detection method, and then the similarity is compared with the standard fingerprint and is not lower than 0.90.
Further, after the standard fingerprint is established, the fingerprint established by the establishing method is compared with the standard fingerprint, and the similarity is not lower than 0.90.
According to a further aspect of the invention, the application of the detection method in constructing the fingerprint of the Chinese herbal compound containing angelica is provided.
According to the detection method, the component information or the component and content information of the Chinese herbal compound containing the angelica can be accurately obtained. The obtained spectrum can comprehensively reflect chemical components in the Chinese herbal compound containing the angelica, the separation of various spectrum peaks is good, the base line is stable, the peak pattern is good, the repeatability is good, and the obtained fingerprint can objectively evaluate the quality of a sample. The present invention identified 13 compounds in total, of which 6 were validated against controls. The result lays a foundation for clarifying the drug effect substance basis of the Chinese herbal compound containing the angelica, and provides reference for quality control.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without exceeding the protection scope of the present application.
FIG. 1 shows the comparison fingerprint spectra generated from 26 representative samples of the above Chinese medicinal compound material standard corresponding substance (lyophilized powder). Wherein the batches referred from bottom to top are DGJZT-1-1, DGJZT-1-2, DGJZT-2-1, DGJZT-3-2, DGJZT-4-1, DGJZT-4-2, DGJZT-5-1, DGJZT-5-2, DGJZT-6-2, DGJZT-7-1, DGJZT-7-2 and DGJZT-8-1 respectively, DGJZT-9-1, DGJZT-9-2, DGJZT-11-1, DGJZT-11-2, DGJZT-10-1, DGJZT-12-2, DGJZT-13-1, DGJZT-13-2, DGJZT-14-2, DGJZT-15-1, DGJZT-15-2 and DGJZT-15-3.
FIG. 2 is an HPLC fingerprint of a Chinese herbal compound sample containing Angelica sinensis. Wherein the fingerprint comprises No. 1-13 peak, wherein No. 1 peak is gallic acid as reference peak, No. 7 peak is paeoniflorin, No. 8 peak is ferulic acid, No. 9 peak is liquiritin, No. 12 peak is ammonium glycyrrhizate, and No. 13 peak is 6-gingerol; wherein peak 8 is from Angelica sinensis; peak 10 and peak 11 are from cinnamomi; peak 9 and peak 12 are from licorice; peak 1, peak 2, peak 6, and peak 7 are from white peony; no. 13 is derived from rhizoma Zingiberis recens; the peak 3 and the peak 5 are common peaks of angelica and white peony root; peak 4 is the common peak of licorice and white peony root.
Detailed Description
The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are some, but not all, embodiments of the present application. 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 application.
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.
The present invention is described in further detail below with reference to specific examples, which are not to be construed as limiting the scope of the invention as claimed herein.
The invention discloses a detection method of a Chinese herbal compound containing angelica and a fingerprint construction method thereof, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention.
Except for special points, the medicines, reagents and instruments used in the technical scheme provided by the invention can be purchased from conventional channels or markets.
According to one aspect of the invention, the detection method of the Chinese herbal compound containing the angelica is provided, and comprises the following steps:
detecting test sample and reference substance of Chinese medicinal composition, wherein the Chinese medicinal composition comprises radix Angelicae sinensis, ramulus Cinnamomi, Glycyrrhrizae radix, radix Paeoniae alba, rhizoma Zingiberis recens and fructus Jujubae, the reference substance is gallic acid, paeoniflorin, ferulic acid, liquiritin, ammonium glycyrrhizate and 6-gingerol,
the chromatographic conditions for the detection were: by C18The chromatographic column takes methanol or acetonitrile as a mobile phase A and acid water solution as a mobile phase B, and the gradient elution procedure is as follows: 0-20 min, 5% -12% A; 20-90 min, 12% -85% A; the flow rate is 0.8-1.2 mL/min; the column temperature is 25-40 ℃; the detection wavelength is 210-330 nm;
and obtaining the component information of the Chinese herbal compound or the component and content information thereof according to the detection result.
In a preferred embodiment, the process for preparing the test article comprises: adding water or alcohol into the Chinese medicinal compound to obtain Chinese medicinal compound solution; and filtering the traditional Chinese medicine compound solution after ultrasonic extraction or reflux extraction to obtain the test sample.
In a preferred embodiment, the process for preparing the control comprises: weighing appropriate amount of gallic acid, paeoniflorin, ferulic acid, liquiritin, ammonium glycyrrhizate and 6-gingerol; and adding 30-70% methanol to obtain a mixture containing 0.02-0.2 mg/mL of each component-1The control of (1).
In order to obtain better chromatographic peak separation effect and lower column pressure, in a preferred embodiment, the flow rate is 1.0-1.2 mL/min, and the column temperature is 30-40 ℃.
In a preferred embodiment, the number of theoretical plates is not less than 8000, calculated as gallic acid.
In a preferred embodiment, the C18The chromatographic column is Agilent Zorbax SB-C18Chromatographic column, Agilent5TC-C18(2)Column, Phenomenex Kinetex C18Chromatographic column, Ultimate XB C18Chromatographic column, Hypersil GOLD C18Column chromatography, Diamonsil C18Chromatographic column or Supersil ODS 2C18A chromatographic column.
In a preferred embodiment, the aqueous acid solution is selected from one or more of weak acids and salts thereof, weak bases and salts thereof, and weak polybasic acids and salts thereof at different concentrations.
In a preferred embodiment, the acid is formic acid, acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid.
In order to achieve better separation of chromatographic peaks, appropriate retention time, maximization of fingerprint information, and better reproducibility and stability, in a preferred embodiment, the acid aqueous solution is 0.08% -0.3% phosphoric acid aqueous solution.
In a preferred embodiment, the aqueous acid solution is a 0.08% to 0.12% aqueous phosphoric acid solution.
In a preferred embodiment, the aqueous acid solution is a 0.1% aqueous phosphoric acid solution.
In a preferred embodiment, the traditional Chinese medicine compound is prepared from the following components in parts by weight: 1-10 parts of angelica, 1-10 parts of cassia twig, 1-10 parts of liquorice, 1-10 parts of white paeony root, 1-10 parts of ginger and 1-10 parts of Chinese date.
In a preferred embodiment, the traditional Chinese medicine compound is prepared from the following components in parts by weight: 4 parts of angelica, 3 parts of cassia twig, 2 parts of liquorice, 6 parts of white paeony root, 3 parts of ginger and 4 parts of Chinese date.
In a preferred embodiment, the licorice is honey-fried licorice.
According to another aspect of the invention, a fingerprint construction method of a Chinese herbal compound containing angelica is provided,
preparation of a test solution: adding water or alcohol into the Chinese medicinal compound to obtain Chinese medicinal compound solution; and the test solution is obtained by filtering after the ultrasonic extraction or reflux extraction of the traditional Chinese medicine compound solution, wherein the traditional Chinese medicine compound solution consists of angelica, cassia twig, liquorice, white paeony root, ginger and Chinese date;
preparation of control solutions: weighing gallic acid, paeoniflorin, ferulic acid, liquiritin, ammonium glycyrrhizate and 6-gingerol, adding 30% -70% methanol to obtain the final product with concentration of each component of 0.02-0.2 mg/mL-1The control solution of (a);
the chromatographic conditions of the high performance liquid detection are as follows: by C18The chromatographic column takes methanol or acetonitrile as a mobile phase A and acid water solution as a mobile phase B, and the gradient elution procedure is as follows: 0-20 min, 5% -12% A; 20-90 min, 12% -85% A; the flow rate is 0.8-1.2 mL/min; the column temperature is 25-40 ℃; the detection wavelength is 210-330 nm;
and obtaining the fingerprint of the Chinese herbal compound according to the high performance liquid detection result.
The fingerprint spectrum construction method has the beneficial effects that:
(1) the fingerprint of the Chinese herbal compound containing angelica built by the method provided by the invention can effectively represent the quality of the Chinese herbal compound and is beneficial to comprehensively monitoring the quality of medicinal materials;
(2) the fingerprint pays attention to the front-back sequence and the mutual relation of each fingerprint characteristic peak and the overall facial features, thereby not only avoiding determining the one-sidedness of the overall quality of the Chinese herbal compound due to the determination of individual chemical components, but also reducing the possibility of considering treatment for reaching the standard quality;
(3) the method has the advantages of simple and stable method, high precision, good reproducibility and the like;
(4) the method can rapidly and accurately identify the authenticity of the product.
In order to obtain better chromatographic peak separation effect and lower column pressure, in a preferred embodiment, the flow rate of the high performance liquid phase detection is 1.0-1.2 mL/min, and the column temperature is 30-40 ℃.
In a preferred embodiment, the number of theoretical plates is not less than 8000, calculated as gallic acid.
In a preferred embodiment, the fingerprint comprises peaks 1-13, wherein peak 1 is gallic acid as reference peak, peak 7 is paeoniflorin, peak 8 is ferulic acid, peak 9 is liquiritin, peak 12 is glycine, and peak 13 is 6-gingerol, and the retention times are respectively 6.4-7.2 min, 28.4-29.2 min, 34.4-35.2 min, 36.4-37.2 min, 72.4-73.2 min, and 77.6-78.4 min.
In a preferred embodiment, the traditional Chinese medicine compound is prepared from the following components in parts by weight: 1-10 parts of angelica, 1-10 parts of cassia twig, 1-10 parts of liquorice, 1-10 parts of white paeony root, 1-10 parts of ginger and 1-10 parts of Chinese date.
In a preferred embodiment, the traditional Chinese medicine compound is prepared from the following components in parts by weight: 4 parts of angelica, 3 parts of cassia twig, 2 parts of liquorice, 6 parts of white paeony root, 3 parts of ginger and 4 parts of Chinese date.
In a preferred embodiment, the licorice is honey-fried licorice.
In a preferred embodiment, the aqueous acid solution is selected from one or more of weak acids and salts thereof, weak bases and salts thereof, and weak polybasic acids and salts thereof at different concentrations.
In a preferred embodiment, the acid is formic acid, acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid.
In order to achieve better separation of chromatographic peaks, appropriate retention time, maximization of fingerprint information, and better reproducibility and stability, in a preferred embodiment, the acid aqueous solution is 0.08% -0.3% phosphoric acid aqueous solution.
In a preferred embodiment, the aqueous acid solution is a 0.08% to 0.12% aqueous phosphoric acid solution.
In a preferred embodiment, the aqueous acid solution is a 0.1% aqueous phosphoric acid solution.
In a preferred embodiment, the C18The chromatographic column is Agilent Zorbax SB-C18Chromatographic column, Phenomenex Gemini C18Chromatography column, Thermo syncronis C18Chromatography column, Agilent5TC-C18Chromatographic column, Kromasil 100-5C18Column, Waters symmetry C18Chromatographic column or Phenomenex Luna C18A chromatographic column.
In a preferred embodiment, peak 8 is from angelica; peak 10 and peak 11 are from cinnamomi; peak 9 and peak 12 are from licorice; peak 1, peak 2, peak 6, and peak 7 are from white peony; no. 13 is derived from rhizoma Zingiberis recens; the peak 3 and the peak 5 are common peaks of angelica and white peony root; peak 4 is the common peak of licorice and white peony root.
In a preferred embodiment, the retention time of peak 1-13 is 6.4-7.2 min, 7.6-8.4 min, 17.2-18.0 min, 19.6-20.4 min, 22.4-23.2 min, 26-26.8 min, 28.4-29.2 min, 34.4-35.2 min, 36.4-37.2 min, 46.4-47.2 min, 60-60.8 min, 72.4-73.2 min, 77.6-78.4 min.
The invention screens out the optimum analysis conditions of mobile phase composition, gradient elution mode, detection wavelength, chromatographic column, column temperature and the like through a large number of experiments, and multiple times of experimental verification show that the fingerprint spectrum detection method provided by the invention can establish a good fingerprint spectrum. The compound traditional Chinese medicine fingerprint detection method provided by the invention can be used for simultaneously detecting 13 active ingredients in the traditional Chinese medicine compound, so that the quality of the traditional Chinese medicine compound can be comprehensively, objectively and accurately detected and evaluated, and the method has important significance for ensuring the clinical curative effect.
According to a further aspect of the present invention, there is provided a standard fingerprint of a reference composition, wherein the reference is gallic acid, paeoniflorin, ferulic acid, liquiritin, ammonium glycyrrhizate and 6-gingerol, and the standard fingerprint is a high performance liquid chromatography, wherein 6 chromatographic peaks are present in the standard fingerprint, wherein the peak 1 is a chromatographic peak with the same retention time as that of the gallic acid as the reference peak, and the relative retention times of the other 5 chromatographic peaks are respectively: 4.23 +/-10%, 5.12 +/-10%, 5.41 +/-10%, 10.71 +/-10% and 11.47 +/-10%.
According to another aspect of the present invention, there is provided a standard fingerprint of a reference composition, wherein the reference is gallic acid, paeoniflorin, ferulic acid, liquiritin, glycine and 6-gingerol, and the standard fingerprint is a high performance liquid chromatography with the following chromatographic conditions: adopting a C18 chromatographic column, taking methanol or acetonitrile as a mobile phase A and acid water solution as a mobile phase B, and performing gradient elution procedure: 0-20 min, 5% -12% A; 20-90 min, 12% -85% A; the flow rate is 0.8-1.2 mL/min; the column temperature is 25-40 ℃; the detection wavelength is 210-330 nm.
In a preferred embodiment, after the standard fingerprint is established, the fingerprint of the Chinese herbal compound containing angelica is established according to the detection method, and then the similarity is compared with the standard fingerprint and is not lower than 0.90.
In a preferred embodiment, after the standard fingerprint is established, the similarity of the fingerprint established by the establishing method and the standard fingerprint is not lower than 0.90.
According to a further aspect of the invention, the application of the detection method in constructing the fingerprint of the Chinese herbal compound containing angelica is provided.
Examples
Method for preparing test solution
Taking 12g of angelica, 9g of cassia twig, 6g of honey-fried licorice root, 18g of white paeony root, 9g of ginger and 12g of Chinese date, adding 2000ml of water, soaking for 50 minutes, decocting for 150 minutes, filtering by 120-mesh double-layer filter cloth, reserving filtrate for later use, and freeze-drying to obtain about 19g of freeze-dried powder. Precisely weighing about 0.9g of the freeze-dried powder, placing the freeze-dried powder in a conical flask with a plug, precisely adding 25ml of water, weighing, ultrasonically treating for 15 minutes (the power is 250W40kHz), taking out, cooling, weighing, supplementing the lost weight with water, shaking up, filtering, and taking the subsequent filtrate to obtain the sample solution.
Preparation method of reference substance solution
Taking appropriate amount of ammonium glycyrrhizinate control, ferulic acid control, gallic acid control, liquiritin control, and paeoniflorin control, precisely weighing, and adding 50% methanol to obtain control stock solutions containing ammonium glycyrrhizinate 0.5mg, ferulic acid 0.2mg, gallic acid 0.5mg, liquiritin 2.0mg, and paeoniflorin 1.5mg per 1 ml. Taking another appropriate amount of 6-gingerol control, precisely weighing, and adding methanol to obtain control stock solution containing 6-gingerol 2mg per 1 ml. Respectively and precisely sucking 5ml of ammonium glycyrrhizinate reference stock solution, 5ml of ferulic acid reference stock solution, 10ml of gallic acid reference stock solution, 5ml of liquiritin reference stock solution, 10ml of paeoniflorin reference stock solution and 1ml of 6-gingerol reference, and preparing into a mixed solution containing 0.05mg of ammonium glycyrrhizinate, 0.02mg of ferulic acid, 0.1mg of gallic acid, 0.2mg of liquiritin, 0.3mg of paeoniflorin and 0.2mg of 6-gingerol per 1ml, namely a reference solution (wherein the weight of glycyrrhizic acid is equal to the weight of ammonium glycyrrhizinate/1.0207).
The invention provides an optimum condition screening process of a fingerprint spectrum detection method of a Chinese herbal compound containing angelica, which comprises the following steps:
1. instruments and reagents
An Agilent 1100 chromatographic system of a high performance liquid chromatograph comprises a degasser of G1322A type, a quaternary pump of G1311A type, an autosampler of G1313A type, a DAD diode array detector of G1315B type, a column incubator of G1316A type and a ChemStation chromatographic workstation;
electronic analytical balance: SHIMADZU AUW120D, Sartorius BSA 124S;
an ultrasonic cleaning machine: KQ-500DB ultrasonic instruments, Inc. of Kunshan;
electric jacket: DZTW type Bangxi Instrument science and technology (Shanghai) Co., Ltd;
an ultra-pure water machine: AXLK1820-2 Axiolou Chongqing Axiolou science and technology development, Inc.;
a chromatographic column: agilent5TC-C18(2)Column (column length 25cm, inner diameter 4.6mm, particle size 5 μm) batch number: 551725, 554265, 556323, 547698;
Phenomenex Kinetex C18a column (column length 25cm, inner diameter 4.6mm, particle size 5 μm);
Ultimate XB C18a column (column length 25cm, inner diameter 4.6mm, particle size 5 μm);
Hypersil GOLD C18a column (column length 25cm, inner diameter 4.6mm, particle size 5 μm);
Diamonsil C18a column (column length 25cm, inner diameter 4.6mm, particle size 5 μm);
Supersil ODS2 C18a column (column length 25cm, inner diameter 4.6mm, particle size 5 μm);
Agilent ZORBAX SB-C18(column length 25cm, inner diameter 4.6mm, particle size 5 μm);
reagent: acetonitrile, methanol, pure chromatogram; other reagents were analytically pure.
2. Selection of detection wavelength
Taking 10 μ l of each of the test solution and the control solution, detecting with a DAD detector by using acetonitrile (A) -0.1% phosphoric acid solution (B) as a mobile phase, and analyzing the spectra. Referring to detection wavelengths in Chinese pharmacopoeia angelica, liquorice, ginger, white paeony root and cassia twig content determination, comprehensively considering a 3D absorption diagram of a test solution and the absorption of a mixed reference solution.
The results show that glycyrrhizic acid and liquiritin have maximum absorption peaks at 247nm, multiple substances in the white peony root medicinal material have larger absorption at 210nm, paeoniflorin has a maximum absorption peak at 230nm, cinnamic acid and cinnamaldehyde have maximum absorption peaks at 290nm, ferulic acid has a maximum absorption peak at 327nm, and gallic acid has a maximum absorption peak at 210 nm.
In order to realize that each component of the traditional Chinese medicine compound can have an obvious absorption peak, the detection mode adopted by the application adopts different detection wavelengths in different time periods, as shown in the following table 1. By adopting the mode, the provided chemical composition information is large in amount, and an ideal chromatogram can be obtained.
Table 1 detection wavelength conversion table
3. Selection of mobile phase gradients
The acetonitrile (A) -0.1% phosphoric acid solution (B) system is used as a mobile phase, the detection wavelength is 250nm, the gradient of the mobile phase is changed, and the change of chromatographic peaks is compared.
Table 2 mobile phase gradient 1
The results show that when the mobile phase gradient elution 1 is used for gradient elution, peaks at the first end and the last end are too dense, the separation degree of paeoniflorin and adjacent peaks is poor, other peaks are clamped in the paeoniflorin peaks, and further optimization is needed, and the specific optimization gradient is shown in table 3.
TABLE 3 mobile phase gradient 2
The result shows that when the mobile phase gradient elution 2 is adopted for gradient elution, peak information is incomplete, peaks are lost, the peak separation degree is poor in 35-40 minutes, the paeoniflorin peak in the white paeony root is severely trailing, other peaks are mixed, further optimization is needed, and the specific optimization gradient is shown in table 4.
Table 4 mobile phase gradient 3
The result shows that when the mobile phase gradient elution 3 is adopted for gradient elution, the tailing condition of the paeoniflorin peak is improved, but the number of the peaks is small, the peaks are concentrated around 20 minutes, further optimization is needed, and the specific optimization gradient is shown in table 5.
TABLE 5 mobile phase gradient 4
The results show that when the mobile phase gradient elution 4 is adopted for gradient elution, the tailing of the paeoniflorin peak can be well solved, the separation degrees of the ferulic acid peak, the cinnamaldehyde peak, the glycyrrhizic acid peak, the 6-gingerol peak and the gallic acid peak are all more than 1.5, and the base line is stable, so the gradient 4 is selected as the follow-up research.
4. Selection of composition of mobile phase system
The mobile phase gradient change program was fixed, and when the mobile phases were compared with acetonitrile (a) -water (B) system, acetonitrile (a) -0.1% acetic acid solution (B) system, acetonitrile (a) -0.1% formic acid solution (B) system, and acetonitrile (a) -0.1% phosphoric acid solution (B) system, the detection wavelengths were switched as specified in table 6, and the change in the chromatographic peak was compared.
TABLE 6 examination of the composition of the mobile phase System
The results show that when the mobile phase system is acetonitrile (a) -0.1% formic acid solution (B) and acetonitrile (a) -0.1% acetic acid solution (B), the baseline shift of the chromatogram at a wavelength of 210nm is very severe; when the mobile phase system is acetonitrile (A) -water (B), each peak area in the chromatogram is obviously smaller than that of the mobile phase system of acetonitrile (A) -0.1% phosphoric acid solution (B), and the peak information is not as rich as that of the mobile phase system; when the mobile phase system is acetonitrile (A) -0.1% phosphoric acid solution (B), the separation condition of each main chromatographic peak is better, and chromatographic peak information is most abundant, so the mobile phase system is selected to be acetonitrile (A) -0.1% phosphoric acid solution (B). The elution is carried out by adopting a system with a mobile phase of acetonitrile (A) -0.1 percent phosphoric acid solution (B), the separation of various chromatographic peaks is good, the retention time is proper, the principle of maximizing fingerprint information is achieved, and the reproducibility and the stability are excellent.
5. Selection of different phosphoric acid concentrations
Fixing a mobile phase gradient change program, keeping the other chromatographic conditions unchanged for the same test solution, and inspecting the influence of three phosphoric acids with different concentrations on chromatographic peaks by using a 0.08 percent phosphoric acid solution, a 0.1 percent phosphoric acid solution and a 0.12 percent phosphoric acid solution flow. The results are shown in Table 7.
TABLE 7 examination results of mobile phase systems with different phosphoric acid concentrations
The results show that the influence difference of three phosphoric acid solutions with different concentrations, namely 0.08% phosphoric acid solution, 0.1% phosphoric acid solution and 0.12% phosphoric acid solution, on chromatographic peaks of gallic acid, ferulic acid and ammonium glycyrrhetate is not large, the separation base line is relatively stable when the concentration of the mobile phase phosphoric acid solution is 0.1%, and acetonitrile-0.1% phosphoric acid solution is selected for follow-up study by combining with a fingerprint.
6. Selection of column temperature
The traditional Chinese medicine compound is analyzed at column temperatures of 25 ℃, 30 ℃, 35 ℃ and 40 ℃ respectively, and compared with chromatograms at the column temperatures, experimental results show that chromatographic peak separation is better under the condition of 25 ℃ to 40 ℃, particularly under the condition of 30 ℃ column temperature, chromatographic peak separation is best, column pressure is lower, and therefore 30 ℃ is preferably the optimal column temperature condition.
7. Selection of chromatography columns
The invention screens different chromatographic columns through a large number of experiments, and adopts various chromatographic columns with different models for determination:
the chromatographic detection result shows that Agilent ZORBAX SB-C18The chromatographic column (the column length is 25cm, the inner diameter is 4.6mm, and the particle size is 5 mu m) can separate a sample to a base line, the separation degree of each chromatographic peak is good, the reproducibility and the stability are good, and a stable fingerprint spectrum can be established.
Through the screening of a large number of experiments, the optimal chromatographic conditions preferred by the invention are as follows: preferably, the material is Agilent ZORBAX SB-C18(column length 25cm, inner diameter 4.6mm, particle size 5 μm) chromatography column; mobile phase: phase A is acetonitrile, phase B is 0.1% phosphoric acid water solution, gradient elution, mobile phase flow rate is 1 ml/min; the change in the detection wavelength is shown in table 1; the column temperature is 30 ℃;
the gradient elution pattern is shown in table 8 below:
TABLE 8 gradient elution mode
Precision test
Taking a Chinese medicinal compound containing radix Angelicae sinensis of lot number DGJZT-1-1, preparing the sample solution according to the preparation method of the sample solution, continuously introducing sample for 6 times according to the chromatographic condition preferred by the fingerprint, and determining. And (3) inspecting the relative retention time of all the shared peaks and the shared peak area value with the relative peak area larger than 3%, wherein the RSD of the relative retention time of the 13 shared peaks is less than or equal to 0.15%, the RSD of the relative peak area of the shared peak with the relative peak area larger than 3% is less than or equal to 1.5%, calculating the similarity of the fingerprints obtained by 5 times of sample injection by taking the fingerprint obtained by the 1 st sample injection as a reference, and ensuring that the result of the similarity is larger than 0.99, thereby meeting the technical requirements of the fingerprints. The fingerprint spectrum detection method provided by the invention is good in precision.
Stability test
Taking a Chinese herbal medicine compound containing angelica in a batch number of DGJZT-1-1, preparing a test solution according to a preparation method of the test solution, respectively measuring the fingerprint for 6 times in 0, 3, 6, 9 and 12 hours according to the optimal chromatographic conditions of the fingerprint, investigating the relative retention time of 13 common peaks and the common peak area value of which the relative peak area is more than 3%, wherein the relative retention time RSD of each common peak is less than or equal to 1.5%, and the relative peak area RSD of the common peak of which the relative peak area is more than 3% is less than or equal to 2.0%, taking the fingerprint obtained by the 1 st sample injection as a reference, calculating the similarity of the fingerprints obtained by the 5 th sample injection, and the result similarity results are all more than 0.99, thereby showing that the fingerprint detection method provided by the invention is stable within 12 hours.
Repeatability test
Taking a Chinese herbal compound containing angelica in a batch number of DGJZT-1-1, preparing a test solution according to a preparation method of the test solution, respectively measuring the fingerprint for once in 0, 3, 6, 9 and 12 hours according to the preferable chromatographic conditions of the fingerprint, measuring for 6 times in total, investigating the relative retention time of all common peaks and the common peak area value of which the relative peak area is more than 3%, and taking the fingerprint obtained by the 1 st sample injection as a reference, wherein the relative retention time RSD of each common peak is less than or equal to 2.0%, and the relative peak area RSD of the common peak of which the relative peak area is more than 3% is less than or equal to 2.0%, and calculating the similarity of the fingerprints obtained by the 5 th sample injection, wherein the result similarity results are all more than 0.99, which indicates that the fingerprint detection method provided by the invention has good repeatability.
Consensus marking
A fingerprint method study of Chinese medicinal composition containing radix Angelicae sinensis on the basis of corresponding material by high performance liquid chromatography (0512 in 2015 pharmacopoeia of the four ministry of general regulations). Adopting fingerprint similarity evaluation software 'traditional Chinese medicine chromatogram fingerprint similarity evaluation system 2012 edition' compiled by pharmacopoeia committee, and adopting 26 batches of representative samples of the traditional Chinese medicine compound substance standard corresponding to object (lyophilized powder) to generate a reference fingerprint (shown in figure 1); and analyzing the chemical properties of the common peaks of the reference substance corresponding substance (lyophilized powder) of the Chinese medicinal compound, and identifying the peaks of ammonium glycyrrhizinate, ferulic acid, paeoniflorin, liquiritin, gallic acid and 6-gingerol.
The No. 1 peak is gallic acid, the No. 7 peak is paeoniflorin, the No. 8 peak is ferulic acid, the No. 9 peak is liquiritin, the No. 12 peak is ammonium glycyrrhizate, and the No. 13 peak is 6-gingerol. No. 8 peak is derived from radix Angelicae sinensis; 10. no. 11 peak is derived from ramulus Cinnamomi; 9. no. 12 Peak is derived from Glycyrrhiza uralensis Fisch; 1.2, 6 and 7 peaks are from radix paeoniae alba; peak 13 is derived from Zingiber officinale Roscoe. 3. Peak 5 is the common peak of Chinese angelica and white peony root; peak 4 is the common peak of licorice and white peony root.
The peak corresponding to the gallic acid reference peak is S peak, and according to the similarity evaluation system of traditional Chinese medicine chromatogram fingerprint, the similarity of the sample fingerprint and the reference physical object (lyophilized powder) corresponding to the traditional Chinese medicine compound material standard comparison fingerprint is greater than 0.90 by Mark peak similarity calculation.
Combining the verification result of the fingerprint methodology of the corresponding real object (lyophilized powder) of the Chinese medicinal compound material standard and the common peak condition of 26 batches of the corresponding real object (lyophilized powder) of the Chinese medicinal compound material standard, stipulating:
in the fingerprint of the test sample, chromatographic peaks with retention time same as that of the chromatographic peaks of ammonium glycyrrhizinate, ferulic acid, gallic acid, liquiritin, paeoniflorin, cinnamaldehyde and 6-gingerol should be respectively presented, 13 common peaks should appear, peaks 1(S), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13 are used as marks (as shown in figure 2), and the similarity of the fingerprint of the test sample and the reference fingerprint is calculated according to a similarity evaluation system of the traditional Chinese medicine chromatographic fingerprint, wherein the similarity is not lower than 0.90. Specifically, the results are shown in Table 9.
Table 926 lot material benchmark HPLC fingerprint matching similarity
Determination of content
The method is characterized in that the contents of ferulic acid and glycyrrhizic acid and the systematic adaptability of index components are taken as judgment indexes, the chromatographic condition and the systematic adaptability of the standard content determination of the Chinese medicinal compound substance containing Chinese angelica are verified, the pretreatment method of a sample is investigated, the preparation method of a test sample is determined, the methodology investigation of the determination method is carried out, the method for determining the contents of ferulic acid and glycyrrhizic acid in the standard of the Chinese medicinal compound substance is established, the contents of ferulic acid and glycyrrhizic acid in a plurality of batches of the standards of the Chinese medicinal compound substance are determined, and the result shows that the method is simple and easy to operate and has the advantages of good separation effect, high sensitivity, good repeatability and the like.
The actual measurement range of ferulic acid of a substance object corresponding to 26 batches of substance references is 0.024% -0.043%, the mean value is 0.033%, the fluctuation range of +/-30% of the mean value is 0.023% -0.043%, and the measured data of the ferulic acid content of the substance object corresponding to 26 batches of substance references all accord with the fluctuation range of +/-30% of the mean value (0.023% -0.043%).
The actual measurement range of the glycyrrhizic acid in the material objects of 26 batches is 0.11-0.20%, the mean value is 0.16%, the fluctuation range of the mean value +/-30% is 0.11-0.21%, and the measured data of the glycyrrhizic acid in the material objects of 26 batches all accord with the fluctuation range of the mean value +/-30% (0.11-0.21%).
According to the methodology examination results, the fingerprint spectrum detection method provided by the invention has good precision, repeatability and stability, can comprehensively, objectively and accurately detect and evaluate the quality of the Chinese herbal compound containing angelica, and has important significance for ensuring the clinical curative effect.
The foregoing is merely an example of the present invention and common general knowledge of known specific structures and features of the embodiments is not described herein in any greater detail. It should be noted that, for those skilled in the art, without departing from the present invention, several changes and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
The foregoing detailed description of the embodiments of the present application has been presented to illustrate the principles and implementations of the present application, and the description of the embodiments is only intended to facilitate the understanding of the methods and their core concepts of the present application. Meanwhile, a person skilled in the art should, according to the idea of the present application, change or modify the embodiments and applications of the present application based on the scope of the present application. In view of the above, the description should not be taken as limiting the application.
Claims (29)
1. A detection method of a Chinese herbal compound containing angelica is characterized by comprising the following steps:
detecting a test sample and a reference sample of a Chinese medicinal composition comprising radix Angelicae sinensis, ramulus Cinnamomi, Glycyrrhrizae radix, radix Paeoniae alba, rhizoma Zingiberis recens and fructus Jujubae, wherein the reference sample is gallic acid, paeoniflorin, ferulic acid, liquiritin, ammonium glycyrrhizate and 6-gingerol,
the chromatographic conditions for detection are as follows: by C18The chromatographic column takes acetonitrile as a mobile phase A and acid water solution as a mobile phase B, and the gradient elution procedure is as follows: 0-10 min, 5% -6% A; 10-12 min, 6% -12% A; 12-20 min, 12% A; 20-21 min, 12% -16% A; 21-38 min, 16% A; 38-49 min, 16% -25% A; 49-61 min, 25% -32% A; 61-79 min, 32% -56% A; 79-85 min, 56-72% A; 85-90 min, 72-85% A; 90-91 min, 85% -5% A; 91-100 min, 5% A; the flow rate is 0.8-1.2 mL/min; the column temperature is 25-40 ℃; the detection wavelength is 210-330 nm;
and obtaining the component information of the Chinese herbal compound according to the detection result.
2. The detection method according to claim 1, further comprising obtaining content information of the herbal compound.
3. The detection method according to claim 1, wherein the preparation process of the test article comprises: adding water or alcohol into the Chinese herbal compound to obtain a Chinese herbal compound solution; and filtering the traditional Chinese medicine compound solution after ultrasonic extraction or reflux extraction to obtain the test sample.
4. The detection method according to claim 1, wherein the preparation process of the reference substance comprises: weighing appropriate amount of gallic acid, paeoniflorin, ferulic acid, liquiritin, ammonium glycyrrhizate and 6-gingerol; and adding 30-70% methanol to obtain a mixture containing 0.02-0.2 mg/mL of each component-1The control of (1).
5. The detection method according to claim 1, wherein the flow rate is 1.0 to 1.2mL/min, and the column temperature is 30 to 40 ℃.
6. The detection method according to claim 1, wherein the number of theoretical plates is not less than 8000 in terms of gallic acid.
7. The detection method according to claim 1, wherein C is18The chromatographic column is Agilent Zorbax SB-C18Chromatographic column, Phenomenex Gemini C18Chromatography column, Thermo syncronis C18Chromatographic column, Agilent5TC-C18Chromatographic column, Kromasil 100-5C18Column, Waters symmetry C18Chromatographic column or Phenomenex Luna C18A chromatographic column.
8. The detection method according to any one of claims 1 to 7, wherein the aqueous acid solution is an aqueous weak acid solution having different concentrations.
9. The detection method according to any one of claims 1 to 7, wherein the aqueous acid solution is an aqueous solution of a weak polybasic acid having different concentrations.
10. The detection method according to any one of claims 1 to 7, wherein the acid is formic acid, acetic acid, phosphoric acid, trifluoroacetic acid, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid.
11. The detection method according to any one of claims 1 to 7, wherein the aqueous acid solution is a 0.08% -0.3% aqueous phosphoric acid solution.
12. The detection method according to any one of claims 1 to 7, wherein the Chinese herbal compound is prepared from the following components in parts by weight: 1-10 parts of angelica, 1-10 parts of cassia twig, 1-10 parts of liquorice, 1-10 parts of white paeony root, 1-10 parts of ginger and 1-10 parts of Chinese date.
13. The detection method according to any one of claims 1 to 7, wherein the Chinese herbal compound is prepared from the following components in parts by weight: 4 parts of angelica, 3 parts of cassia twig, 2 parts of liquorice, 6 parts of white paeony root, 3 parts of ginger and 4 parts of Chinese date.
14. The detection method according to claim 13, wherein the licorice is honey-fried licorice.
15. A method for constructing fingerprint of Chinese herbal compound containing angelica sinensis is characterized in that,
preparation of a test solution: adding water or alcohol into the Chinese medicinal compound to obtain Chinese medicinal compound solution; and the test solution is obtained by filtering after the ultrasonic extraction or reflux extraction of the traditional Chinese medicine compound solution, wherein the traditional Chinese medicine compound solution consists of angelica, cassia twig, liquorice, white paeony root, ginger and Chinese date;
preparation of control solutions: weighing gallic acid, paeoniflorin, ferulic acid, liquiritin, ammonium glycyrrhizate and 6-gingerol, adding 30% -70% methanol to obtain the final product with concentration of each component of 0.02-0.2 mg/mL-1The control solution of (a);
the chromatographic conditions of the high performance liquid detection are as follows: by C18The chromatographic column takes acetonitrile as a mobile phase A and acid water solution as a mobile phase B, and the gradient elution procedure is as follows: 0-10 min, 5% -6% A; 10-12 min, 6% -12% A; 12-20 min, 12% A; 20-21 min, 12% -16% A; 21-38 min, 16% A; 38-49 min, 16% -25% A; 49-61 min, 25% -32% A; 61-79 min, 32% -56% A; 79-85 min, 56-72% A; 85About 90min, 72% -85% A; 90-91 min, 85% -5% A; 91-100 min, 5% A; the column temperature is 25-40 ℃; the detection wavelength is 210-330 nm;
and obtaining the fingerprint of the Chinese herbal compound according to the high performance liquid detection result.
16. The construction method according to claim 15, wherein the flow rate of the HPLC is 1.0-1.2 mL/min, and the column temperature is 30-40 ℃.
17. The method of claim 15, wherein the number of theoretical plates is not less than 8000, calculated as gallic acid.
18. The method according to claim 15, wherein the fingerprint comprises peaks 1-13, wherein peak 1 is gallic acid as a reference peak, peak 7 is paeoniflorin, peak 8 is ferulic acid, peak 9 is liquiritin, peak 12 is glycinate, and peak 13 is 6-gingerol, and the retention times are 6.4-7.2 min, 28.4-29.2 min, 34.4-35.2 min, 36.4-37.2 min, 72.4-73.2 min, and 77.6-78.4 min, respectively.
19. The construction method of claim 15, wherein the Chinese herbal compound is prepared from the following components in parts by weight: 1-10 parts of angelica, 1-10 parts of cassia twig, 1-10 parts of liquorice, 1-10 parts of white paeony root, 1-10 parts of ginger and 1-10 parts of Chinese date.
20. The construction method of claim 15, wherein the Chinese herbal compound is prepared from the following components in parts by weight: 4 parts of angelica, 3 parts of cassia twig, 2 parts of liquorice, 6 parts of white paeony root, 3 parts of ginger and 4 parts of Chinese date.
21. The method of claim 20, wherein the licorice is radix Glycyrrhizae Preparata.
22. The method of any one of claims 15 to 21, wherein the aqueous acid solution is an aqueous solution of a weak acid at different concentrations.
23. The method of any one of claims 15 to 21, wherein the aqueous acid solution is an aqueous solution of a weak polybasic acid of varying concentration.
24. The method of any one of claims 15 to 21, wherein the acid is formic acid, acetic acid, phosphoric acid, trifluoroacetic acid, glycine, and hydrochloric acid, or phthalic acid and hydrochloric acid.
25. The method of any one of claims 15 to 21, wherein the aqueous acid solution is a 0.08% -0.3% aqueous phosphoric acid solution.
26. Construction method according to any one of claims 15 to 21, wherein C is18The chromatographic column is Agilent Zorbax SB-C18Chromatographic column, Agilent5TC-C18(2)Column, Phenomenex Kinetex C18Chromatographic column, Ultimate XB C18Chromatographic column, Hypersil GOLD C18Column chromatography, Diamonsil C18Chromatographic column or Supersil ODS 2C18A chromatographic column.
27. The method of claim 18, wherein peak 8 is from angelica; peak 10 and peak 11 are from cinnamomi; peak 9 and peak 12 are from licorice; peak 1, peak 2, peak 6, and peak 7 are from white peony; no. 13 is derived from rhizoma Zingiberis recens; the peak 3 and the peak 5 are common peaks of angelica and white peony root; peak 4 is the common peak of licorice and white peony root.
28. The construction method according to claim 18, wherein the retention time of peak 1-13 is 6.4-7.2 min, 7.6-8.4 min, 17.2-18.0 min, 19.6-20.4 min, 22.4-23.2 min, 26-26.8 min, 28.4-29.2 min, 34.4-35.2 min, 36.4-37.2 min, 46.4-47.2 min, 60-60.8 min, 72.4-73.2 min, 77.6-78.4 min.
29. Use of the detection method according to any one of claims 1 to 14 in constructing a fingerprint of a herbal compound comprising angelica.
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