Quality control method of volatile oil component of traditional Chinese medicine composition containing angelica and ligusticum and application thereof

Abstract

The application discloses a characteristic chromatogram construction method of volatile oil components of a traditional Chinese medicine composition containing angelica and ligusticum chuanxiong, and the method comprises the following steps: preparing a volatile oil component test sample solution; preparing a reference sample solution; obtaining the characteristic chromatogram of the volatile oil components according to the test sample solution and the reference sample solution. The detection method is simple, accurate and efficient, has good stability, high precision, good reproducibility, is convenient and easy to master, can effectively save energy and reduce analysis cost, and the technical content is not disclosed in the prior art.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a method for quality control of the volatile oil components of a traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong, and its application. Background Technology

[0002] In recent years, the integration of research on "Chinese herbal medicine chemistry" in the sense of phytochemistry with research on compound Chinese herbal medicine chemistry and life science research has enabled people to gain a deeper understanding of the in vivo processes of Chinese herbal medicine. Subsequently, the connotation and extension of drug metabolism have been expanded, not only referring to therapeutic drugs, but also including all exogenous chemical substances. The research content also includes not only the biotransformation process of chemical substances, but also the properties and quantitative laws of drug metabolism, involving processes such as absorption, distribution, metabolism and excretion. With the great progress of science and technology, there is also a richer content, such as predicting drug interactions and the impact of environmental factors on drug metabolism, and using these influences to improve therapeutic effects and drug safety. The research on the active and toxic components of Chinese herbal medicine has always occupied a pivotal position in the scientific research on complex systems of Chinese herbal medicine. It is a fundamental and core issue.

[0003] Traditional Chinese medicine (TCM) compound prescriptions, as a whole, require the combined action of all the individual medicinal materials to achieve therapeutic effects. As a primary form of clinical application, TCM compound prescriptions possess the advantages of multiple components, multiple targets, and multiple effects. However, the active substances in TCM compound prescriptions interact after entering the human body, producing a "natural compatibility" effect. Through multi-target binding, they achieve synergistic effects and reduce toxicity. These active substances may originate from the original components of the TCM, compounds generated during the preparation process, or metabolic products of the human body. These active substances do not exert their medicinal effects in a single medicinal material; they only exert their therapeutic effect when combined as a whole. To ensure safety and efficacy, reflect the inherent quality characteristics of this product, and embody its holistic nature, a series of studies have been conducted on the medicinal ingredients and preparation quality of this product, adhering to the principles of practicality, science, and standardization, based on the effects of each medicinal ingredient in the prescription.

[0004] Currently, no literature reports a comprehensive analysis and fingerprint study of the volatile oil components of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong claimed in this invention. In the prior art, only the quality analysis of a single volatile oil component in the traditional Chinese medicine compound is performed. There is no comprehensive and systematic quality control method to reflect the quality status of the volatile oil components in the main reference samples of the traditional Chinese medicine compound and finished product of this invention. It is impossible to effectively control its production process and product quality, and cannot better guarantee its clinical efficacy. Therefore, it is necessary to adopt a fingerprint quality control method that can comprehensively control the overall quality of the traditional Chinese medicine compound of this invention to control its key quality. Summary of the Invention

[0005] Based on this, the present invention provides a method for constructing a characteristic spectrum of volatile oil components in a traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong, the method comprising the following steps:

[0006] Preparation of the volatile oil component test solution: Take an appropriate amount of the extract of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong, heat and reflux to extract, and obtain volatile oil; take an appropriate amount of the volatile oil in a container, add the first solvent to dilute, make up to volume, shake well, and obtain the test solution of the volatile oil component; wherein, the traditional Chinese medicine composition includes Gentiana macrophylla, Glycyrrhiza uralensis, Ligusticum chuanxiong, Prunus persica, Carthamus tinctorius, Notopterygium incisum, Commiphora myrrha, Cyperus rotundus, Achyranthes bidentata, Pheretima aspergillum, Angelica sinensis and Trogopterus xanthipes;

[0007] Preparation of reference solution: Weigh an appropriate amount of ligustilide reference standard and add methanol to prepare a reference solution with a ligustilide concentration of 0.1~5 mg / ml;

[0008] Based on the results of high-performance liquid chromatography (HPLC) analysis of the test solution and the reference solution, characteristic spectra of volatile oil components were obtained.

[0009] The chromatographic conditions for this high-performance liquid chromatography (HPLC) detection are as follows: a column packed with octadecylsilane-bonded silica gel is used; mobile phase A is selected from one or more of acetonitrile, methanol, and tetrahydrofuran; mobile phase B is an aqueous acid solution, an aqueous alkaline solution, and / or an aqueous buffer solution; the gradient elution program is: 0–50 min, 56% B; 50–60 min, 56% B → 30% B; 60–65 min, 30% B; 65–70 min, 30% B → 20% B; 70–85 min, 20% B; 85–90 min, 20% B → 56% B; the flow rate is 0.62–0.78 ml / min; the column temperature is 25–35 °C; the detection wavelength is 190–400 nm; and the injection volume is 0.5–2 μl.

[0010] At a detection wavelength of 220 nm, the characteristic spectrum includes peaks 1-14, with peak 7 serving as a reference peak. The average relative retention times of peaks 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, and 14 are approximately 0.28, 0.60, 0.67, 0.70, 0.83, 0.88, 1.09, 1.15, 1.90, 2.18, 2.26, 3.46, and 3.75, respectively.

[0011] Furthermore, in the preparation method of the test solution, the heating and reflux extraction time is 4 to 6 hours, for example, about 5 hours.

[0012] Furthermore, the heating reflux extraction apparatus is a volatile oil extraction apparatus.

[0013] Furthermore, the sample amount of the extract is a 1- to 7-day dose, for example, a 1-day dose, a 5-day dose, or a 7-day dose.

[0014] Furthermore, the preparation method of the extract includes the following steps: weighing appropriate amounts of Gentiana macrophylla, Ligusticum chuanxiong, peach kernel, safflower, licorice, Notopterygium incisum, myrrh, Angelica sinensis, Trogopterus xanthipes, Cyperus rotundus, Achyranthes bidentata, and earthworm, adding appropriate amounts of water for extraction, filtering, and obtaining the first decoction; adding appropriate amounts of water to the residue for extraction, filtering, and obtaining the second decoction; combining the first decoction and the second decoction to obtain the extract.

[0015] Furthermore, this Wulingzhi is vinegar-processed Wulingzhi.

[0016] Furthermore, the mass ratio of the following herbs is (0.5~2):(1~3):(2~4):(2~4):(1~3):(0.5~2):(1~3):(2~4):(1~3):(0.5~2):(1~3):(2~4):(1~3):(0.5~2):(1~3):(2~4):(1~3):(0.5~2):(2~4):(1~3):(0.5~2):(2~4):(1~3).

[0017] Furthermore, the mass ratio of the following herbs is approximately 1:approximately 2:approximately 3:approximately 3:approximately 2:approximately 1:approximately 2:approximately 3:approximately 3:approximately 2:approximately 1:approximately 2:approximately 3:approximately 2 ...1:approximately 3:approximately 2:approximately 2:approximately 3:approximately 2:approximately 2:approximately 3:approximately 2.

[0018] Furthermore, the mass of the Gentiana macrophylla is 1-5 g, for example, about 3.73 g.

[0019] Furthermore, the mass of the chuanxiong is 5-10 g, for example, about 7.46 g.

[0020] Furthermore, the weight of the peach kernel is 10-15 g, for example, about 11.19 g.

[0021] Furthermore, the safflower weighs 10-15 g, for example, about 11.19 g.

[0022] Furthermore, the licorice has a mass of 5-10 g, for example, about 7.46 g.

[0023] Furthermore, the weight of the Notopterygium root is 1-5 g, for example, about 3.73 g.

[0024] Furthermore, the mass of the myrrh is 5 to 10 g, for example, about 7.46 g.

[0025] Furthermore, the weight of the angelica root is 10-15 g, for example, about 11.19 g.

[0026] Furthermore, the mass of this Wulingzhi is 5-10 g, for example, about 7.46 g.

[0027] Furthermore, the mass of the Cyperus rotundus is 1-5 g, for example, about 3.73 g.

[0028] Furthermore, the mass of the Achyranthes bidentata is 10-15 g, for example, about 11.19 g.

[0029] Furthermore, the earthworm's mass is 5-10 g, for example, about 7.46 g.

[0030] Furthermore, in the preparation of the first decoction, the volume-to-mass ratio of the water to the following herbs is 8 to 15, for example, about 12.

[0031] Furthermore, in the preparation of the second decoction, the volume-to-mass ratio of the water to the following herbs is 6 to 12, for example, about 10.

[0032] Furthermore, the extraction time is 30 to 60 minutes, for example, about 40 minutes or 45 minutes.

[0033] Furthermore, the mass of the volatile oil is 0.05~0.2 g, for example, about 0.1 g.

[0034] Furthermore, the container is a measuring bottle.

[0035] Furthermore, the volumetric flask has a volume of 10 ml.

[0036] Furthermore, the first solvent is methanol.

[0037] Furthermore, the mass / volume ratio of the volatile oil to the sample solution of the volatile oil component is 0.005 to 0.02, for example, about 0.01, in g / ml.

[0038] Furthermore, the method for preparing the test solution further includes the step of shaking and then filtering.

[0039] Furthermore, the filtration is membrane filtration.

[0040] Furthermore, the pore size of the filter membrane is 0.22 μm.

[0041] Furthermore, the concentration of ligustilide in the reference solution was approximately 1 mg / ml.

[0042] Furthermore, the flow rate for this high-performance liquid chromatography detection is 0.65~0.75 ml / min, for example, about 0.7 ml / min.

[0043] Furthermore, the column temperature is 27~33℃, for example, about 30℃.

[0044] Furthermore, the detection wavelength is 220~280 nm.

[0045] Furthermore, the detection wavelength is approximately 220 nm.

[0046] Furthermore, the injection volume is 0.8~1.2 μl, for example, about 1 μl.

[0047] Furthermore, the theoretical plate number of the chromatographic peak corresponding to this ligustilide is not less than 8000.

[0048] Furthermore, the chromatographic column is a HALO AQ C18 column.

[0049] Furthermore, the specifications of the chromatographic column are as follows: column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm.

[0050] Furthermore, mobile phase A is acetonitrile.

[0051] Furthermore, the aqueous acid solution, aqueous alkaline solution, and / or aqueous buffer salt solution are selected from one or more weak acids and their salts, weak bases and their salts of different concentrations.

[0052] Furthermore, the aqueous acid solution, aqueous alkaline solution, and / or aqueous buffer solution are selected from different concentrations of formic acid, glacial 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.

[0053] Furthermore, the acid aqueous solution is a 0.01% to 1% acid aqueous solution.

[0054] Furthermore, the acidic aqueous solution is a 0.01% to 1% aqueous solution of phosphoric acid.

[0055] Furthermore, the acidic aqueous solution is an aqueous solution of approximately 0.1% phosphoric acid.

[0056] Furthermore, the buffer salt solution is an acetate solution and / or an acetate solution.

[0057] Furthermore, the pH value of this buffer salt solution is no greater than 7.0.

[0058] Furthermore, at the detection wavelength of 220 nm, the characteristic spectrum includes peaks 1-14, with retention times of approximately 5.191 min, 11.256 min, 12.608 min, 13.196 min, 15.601 min, 16.592 min, 18.813 min, 20.488 min, 21.971 min, 36.260 min, 41.951 min, 43.627 min, 63.385 min, and 68.756 min, respectively.

[0059] Furthermore, at the detection wavelength of 220 nm, peak 7 is ligustilide.

[0060] Furthermore, at the detection wavelength of 220 nm, peak 1 originates from Ligusticum chuanxiong and Angelica sinensis, peak 2 originates from Ligusticum chuanxiong, peak 3 originates from Commiphora myrrha, peak 4 originates from Commiphora myrrha, peak 5 originates from Commiphora myrrha, peak 6 originates from Commiphora myrrha, peak 7 originates from Ligusticum chuanxiong and Angelica sinensis, peak 8 originates from Ligusticum chuanxiong and Angelica sinensis, peak 9 originates from Commiphora myrrha, peak 10 originates from Commiphora myrrha, peak 11 originates from Commiphora myrrha, peak 12 originates from Commiphora myrrha, peak 13 originates from Commiphora myrrha, and peak 14 originates from Commiphora myrrha.

[0061] According to another aspect of the present invention, a method for quality control of the volatile oil components of a traditional Chinese medicine composition comprising Angelica sinensis and Ligusticum chuanxiong is provided, the method comprising the following steps:

[0062] (1) Establish standard characteristic spectra of volatile oil components of traditional Chinese medicine compositions based on the above characteristic spectra construction method;

[0063] (2) Take the volatile oil component test solution and detect it according to the chromatographic conditions in the above-mentioned characteristic chromatogram construction method to obtain the characteristic chromatogram of the volatile oil component of the traditional Chinese medicine composition; and

[0064] (3) Compare the characteristic spectrum of the volatile oil component of the Chinese medicine composition obtained in step (2) with the standard characteristic spectrum of the volatile oil component of the Chinese medicine composition obtained in step (1). If the sample meets the requirements, it is a qualified product; if it does not meet the requirements, it is an unqualified product.

[0065] Furthermore, the compliance requirement includes one or more of the following:

[0066] (1) The volatile oil components of the Chinese herbal medicine composition showed 14 characteristic chromatographic peaks in the characteristic chromatographic spectrum of the sample to be tested. The retention time of each characteristic chromatographic peak was within ±10% of the retention time value of the corresponding reference peak in the standard characteristic chromatographic spectrum of the volatile oil components of the Chinese herbal medicine composition.

[0067] (2) Taking peak 7 as peak S, the relative retention times of each characteristic chromatographic peak in the characteristic chromatographic spectrum of the volatile oil component of the Chinese herbal medicine composition and peak S are within ±10% of the relative retention times of each characteristic chromatographic peak in the standard characteristic chromatographic spectrum of the volatile oil component of the Chinese herbal medicine composition reference sample; and

[0068] (3) According to the similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine, the similarity between the characteristic chromatogram of the volatile oil component of the traditional Chinese medicine composition and the standard characteristic chromatogram of the reference sample of the volatile oil component of the traditional Chinese medicine composition shall not be less than 0.90.

[0069] According to another aspect of the present invention, the above-described construction method or the above-described quality control method is provided for use in the quality detection and / or quality evaluation and / or quality control of volatile oil components in a traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong.

[0070] The beneficial effects of this invention are:

[0071] In summary, this invention provides a fingerprint spectral determination and quality control method for the volatile oil components of a traditional Chinese medicine composition reference sample containing Angelica sinensis and Ligusticum chuanxiong. This method identifies 14 common characteristic peaks, is simple to implement, and has a short analysis time. It solves the problems of difficult separation of fingerprint characteristic peaks and interference from impurity peaks, ensuring the chemical composition stability and safety of the reference sample. This provides an important reference for the quality control of subsequent preparations, ensuring product quality stability, guaranteeing the efficacy of the traditional Chinese medicine compound, and enabling the traditional Chinese medicine compound to better serve human health.

[0072] Specifically, compared with the prior art, the beneficial effects of the present invention are as follows: (1) The fingerprint spectrum of the volatile oil components of the traditional Chinese medicine compound reference sample of the present invention is established, which overcomes the defect that the content determination of a single component is difficult to reflect the overall content. It can control the intrinsic quality of the traditional Chinese medicine compound reference sample of the present invention from an overall and macroscopic perspective, ensuring the efficacy of the drug. It utilizes the main means of modern drug research to realize the inheritance of classics and make the classic prescriptions more formally controlled in terms of quality; (2) The chemical components in the traditional Chinese medicine compound reference sample of the present invention are complex, and it is difficult to separate its characteristic peaks. In the process of establishing the fingerprint spectrum, the present invention adopts the gradient elution method, which solves the problem of the fingerprint characteristic peaks being difficult to separate and the interference of impurity peaks; (3) In establishing the fingerprint spectrum of the traditional Chinese medicine compound reference sample of the present invention, the present invention has achieved the following advantages: During the process, 14 common characteristic peaks were identified, and their relative retention time, relative peak area and similarity were studied to ensure the chemical composition stability and safety of the reference sample, providing an important reference and quality reference for the subsequent quality control of compound preparations; (4) The fingerprint spectrum of each effective component in the traditional Chinese medicine compound reference sample of the present invention is regarded as a whole, and attention is paid to the order and interrelationship of each characteristic peak. This avoids the one-sidedness of judging the overall quality of the traditional Chinese medicine compound reference sample of the present invention by only measuring one or two chemical components, and reduces the possibility of artificial processing to meet the quality standards. This provides a new method and means for completely and accurately evaluating the quality of the traditional Chinese medicine compound reference sample of the present invention; (5) The method of the present invention has good stability, high precision, good reproducibility, convenience and easy mastery. Attached Figure Description

[0073] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0074] Figure 1 This is a characteristic spectrum of the volatile oil components of the reference sample of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention.

[0075] Figure 2 This is a common pattern diagram of the characteristic spectra of volatile oil components in 15 batches of reference samples of traditional Chinese medicine compositions containing Angelica sinensis and Ligusticum chuanxiong of the present invention. Detailed Implementation

[0076] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0077] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0078] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0079] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0080] In this invention, the term "comprising" and "including" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0081] As described in the background section, no literature reports a comprehensive analysis and fingerprint study of the volatile oil components of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong claimed in this invention. To address the above problem, this invention provides a method for constructing characteristic spectra of the volatile oil components of a traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong, the method comprising the following steps:

[0082] Preparation of the volatile oil component test solution: Take a one-day dose of the extract of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention, heat and reflux to extract, and obtain volatile oil; take an appropriate amount of the volatile oil and place it in a container, add the first solvent to dilute, make up to volume, shake well, and obtain the test solution of the volatile oil component; wherein, the traditional Chinese medicine composition includes Gentiana macrophylla, Glycyrrhiza uralensis, Ligusticum chuanxiong, Prunus persica, Carthamus tinctorius, Notopterygium incisum, Commiphora myrrha, Cyperus rotundus, Achyranthes bidentata, Pheretima aspergillum, Angelica sinensis and Trogopterus xanthipes;

[0083] Preparation of reference solution: Weigh an appropriate amount of ligustilide reference standard and add methanol to prepare a reference solution with a ligustilide concentration of 0.1~5 mg / ml;

[0084] Based on the results of high-performance liquid chromatography (HPLC) analysis of the test solution and the reference solution, characteristic spectra of volatile oil components were obtained.

[0085] The chromatographic conditions for this high-performance liquid chromatography (HPLC) detection are as follows: a column packed with octadecylsilane-bonded silica gel is used; mobile phase A is selected from one or more of acetonitrile, methanol, and tetrahydrofuran; mobile phase B is an aqueous acid solution, an aqueous alkaline solution, and / or an aqueous buffer solution; the gradient elution program is: 0–50 min, 56% B; 50–60 min, 56% B → 30% B; 60–65 min, 30% B; 65–70 min, 30% B → 20% B; 70–85 min, 20% B; 85–90 min, 20% B → 56% B; the flow rate is 0.62–0.78 ml / min; the column temperature is 25–35 °C; the detection wavelength is 190–400 nm; and the injection volume is 0.5–2 μl.

[0086] At a detection wavelength of 220 nm, the characteristic spectrum includes peaks 1-14, with peak 7 serving as a reference peak. The average relative retention times of peaks 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, and 14 are approximately 0.28, 0.60, 0.67, 0.70, 0.83, 0.88, 1.09, 1.15, 1.90, 2.18, 2.26, 3.46, and 3.75, respectively.

[0087] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.28" includes ±5% of 0.28, or from 0.266 to 0.294; "about 0.60" includes ±5% of 0.60, or from 0.57 to 0.63; "about 0.67" includes ±5% of 0.67, or from 0.6365 to 0.7035; "about 0.70" includes ±5% of 0.70, or from 0.665 to 0.735; "about 0.83" includes ±5% of 0.83, or from 0.7885 to 0.8715; "about 0.88" includes ±5% of 0.88, or from 0.836 to 0.924; "about 1.09" includes ±5% of 1.09. %, or from 1.0355 to 1.1445; “about 1.15” includes 1.15 ± 5%, or from 1.0925 to 1.2075; “about 1.90” includes 1.90 ± 5%, or from 1.805 to 1.995; “about 2.18” includes 2.18 ± 5%, or from 2.071 to 2.289; “about 2.26” includes 2.26 ± 5%, or from 2.147 to 2.373; “about 3.46” includes 3.46 ± 5%, or from 3.287 to 3.633; “about 3.75” includes 3.75 ± 5%, or from 3.5625 to 3.9375.

[0088] In this invention, when time, flow rate, column temperature, wavelength, injection volume, sample volume, mass, ratio, concentration, or other values ​​or parameters are expressed as ranges, preferred ranges, or a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “25~35” is disclosed, the described range should be interpreted as including ranges “25~35”, “25~33”, “25~31”, “25~29”, “25~27”, “27~35”, “27~33”, “27~31”, “27~29”, “29~35”, “29~33”, “29~31”, “31~35”, “31~33”, “33~35”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0089] In a preferred embodiment, the heating and reflux extraction time in the preparation method of the test solution is 4 to 6 hours, for example, about 5 hours.

[0090] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5" includes 5 ± 5%, or from 4.75 to 5.25.

[0091] In a preferred embodiment, the heating reflux extraction apparatus is a volatile oil extraction apparatus.

[0092] In a preferred embodiment, the amount of medicinal material sampled for the extract is a 1-7 day dose, for example, a 1-day dose, a 5-day dose, or a 7-day dose.

[0093] In a preferred embodiment, the preparation method of the extract includes the following steps: weighing appropriate amounts of Gentiana macrophylla, Ligusticum chuanxiong, peach kernel, safflower, licorice, Notopterygium incisum, myrrh, Angelica sinensis, Trogopterus xanthipes, Cyperus rotundus, Achyranthes bidentata, and earthworm, adding appropriate amounts of water for extraction, filtering, and obtaining a first decoction; adding appropriate amounts of water to the residue for extraction, filtering, and obtaining a second decoction; combining the first decoction and the second decoction to obtain the extract.

[0094] In a preferred embodiment, the five-spice powder is vinegar-processed five-spice powder.

[0095] In a preferred embodiment, the mass ratio of Gentiana macrophylla, Ligusticum chuanxiong, Prunus persica, Carthamus tinctorius, Glycyrrhiza uralensis, Notopterygium incisum, Commiphora myrrha, Angelica sinensis, Trogopterus xanthipes, Cyperus rotundus, Achyranthes bidentata, and Pheretima aspergillum is (0.5~2):(1~3):(2~4):(2~4):(1~3):(0.5~2):(1~3):(2~4):(1~3):(0.5~2):(2~4):(1~3).

[0096] In a preferred embodiment, the mass ratio of the following ingredients is approximately 1:approximately 2:approximately 3:approximately 3:approximately 2:approximately 1:approximately 2:approximately 3:approximately 3:approximately 2:approximately 1:approximately 2:approximately 3:approximately 2:approximately 2:approximately 2:approximately 3:approximately 2:approximately 1:approximately 3 ....

[0097] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05; "about 2" includes ±5% of 2, or from 1.9 to 2.1; and "about 3" includes ±5% of 3, or from 2.85 to 3.15.

[0098] In a preferred embodiment, the mass of the Gentiana macrophylla is 1 to 5 g, for example, about 3.73 g.

[0099] In a preferred embodiment, the mass of the Ligusticum chuanxiong is 5-10 g, for example, about 7.46 g.

[0100] In a preferred embodiment, the peach kernel weighs 10-15 g, for example, about 11.19 g.

[0101] In a preferred embodiment, the safflower weighs 10-15 g, for example, about 11.19 g.

[0102] In a preferred embodiment, the licorice has a mass of 5 to 10 g, for example, about 7.46 g.

[0103] In a preferred embodiment, the mass of the Notopterygium root is 1 to 5 g, for example, about 3.73 g.

[0104] In a preferred embodiment, the myrrh weighs 5 to 10 g, for example, about 7.46 g.

[0105] In a preferred embodiment, the mass of the angelica is 10-15 g, for example, about 11.19 g.

[0106] In a preferred embodiment, the mass of the five-spice powder is 5 to 10 g, for example, about 7.46 g.

[0107] In a preferred embodiment, the mass of the Cyperus rotundus is 1 to 5 g, for example, about 3.73 g.

[0108] In a preferred embodiment, the mass of the Achyranthes bidentata is 10-15 g, for example, about 11.19 g.

[0109] In a preferred embodiment, the earthworm has a mass of 5 to 10 g, for example, about 7.46 g.

[0110] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3.73" includes ±5% of 3.73, or from 3.5435 to 3.9165; "about 7.46" includes ±5% of 7.46, or from 7.087 to 7.833; "about 11.19" includes ±5% of 11.19, or from 10.6305 to 11.6445.

[0111] In a preferred embodiment, during the preparation of the first decoction, the volume-to-mass ratio of the water to the following herbs is 8 to 15, for example, about 12.

[0112] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 12" includes ±5% of 12, or from 11.4 to 12.6.

[0113] In a preferred embodiment, during the preparation of the second decoction, the volume-to-mass ratio of the water to the following herbs is 6 to 12, for example, about 10.

[0114] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0115] In a preferred embodiment, the extraction time is 30 to 60 minutes, for example, about 40 minutes or 45 minutes.

[0116] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 40" includes ±5% of 40, or from 38 to 42; "about 45" includes ±5% of 45, or from 42.75 to 47.25.

[0117] In a preferred embodiment, the volatile oil has a mass of 0.05 to 0.2 g, for example, about 0.1 g.

[0118] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.1" includes ±5% of 0.1, or from 0.095 to 0.105.

[0119] In a preferred embodiment, the container is a measuring bottle.

[0120] In a preferred embodiment, the volumetric flask has a volume of 10 ml.

[0121] In a preferred embodiment, the first solvent is methanol.

[0122] In a preferred embodiment, the mass / volume ratio of the volatile oil to the volatile oil component test solution is 0.005 to 0.02, for example, about 0.01, in g / ml.

[0123] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.01" includes ±5% of 0.01, or from 0.0095 to 0.0105.

[0124] In a preferred embodiment, the method for preparing the test solution further includes the step of shaking and then filtering.

[0125] In a preferred embodiment, the filtration is membrane filtration.

[0126] In a preferred embodiment, the filter membrane has a pore size of 0.22 μm.

[0127] In a preferred embodiment, the concentration of ligustilide in the reference solution is about 1 mg / ml.

[0128] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0129] In a preferred embodiment, the flow rate of the high-performance liquid chromatography (HPLC) detection is 0.65 to 0.75 ml / min, for example, about 0.7 ml / min.

[0130] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.7" includes ±5% of 0.7, or from 0.665 to 0.735.

[0131] In a preferred embodiment, the column temperature is 27~33°C, for example, about 30°C.

[0132] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes 30 ±5%, or from 28.5 to 31.5.

[0133] In a preferred embodiment, the detection wavelength is 220~280 nm.

[0134] In a preferred embodiment, the detection wavelength is approximately 220 nm.

[0135] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 220" includes ±5% of 220, or from 209 to 231.

[0136] In a preferred embodiment, the injection volume is 0.8 to 1.2 μl, for example, about 1 μl.

[0137] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0138] In a preferred embodiment, the theoretical plate number of the chromatographic peak corresponding to the ligustilide is not less than 8000.

[0139] In a preferred embodiment, the chromatographic column is a HALO AQ C18 column.

[0140] In a preferred embodiment, the chromatographic column has the following specifications: column length 150 mm, inner diameter 4.6 mm, and particle size 2.7 μm.

[0141] In a preferred embodiment, the mobile phase A is acetonitrile.

[0142] In a preferred embodiment, the acid aqueous solution, alkaline aqueous solution, and / or buffer salt aqueous solution are selected from one or more weak acids and their salts, and weak bases and their salts of different concentrations.

[0143] In a preferred embodiment, the acidic aqueous solution, alkaline aqueous solution, and / or buffer salt aqueous solution is selected from formic acid, glacial 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.

[0144] In a preferred embodiment, the acidic aqueous solution is a 0.01% to 1% acidic aqueous solution.

[0145] In a preferred embodiment, the acidic aqueous solution is a 0.01% to 1% aqueous solution of phosphoric acid.

[0146] In a preferred embodiment, the acidic aqueous solution is an aqueous solution of about 0.1% phosphoric acid.

[0147] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.1%" includes 0.1% ± 5%, or from 0.095% to 0.105%.

[0148] In a preferred embodiment, the buffer salt solution is an acetate aqueous solution and / or an acetate aqueous solution.

[0149] In a preferred embodiment, the pH value of the buffer saline solution is not greater than 7.0.

[0150] In a preferred embodiment, when the detection wavelength is 220 nm, the characteristic spectrum includes peaks 1-14, and the retention times of peaks 1-14 are approximately 5.191 min, approximately 11.256 min, approximately 12.608 min, approximately 13.196 min, approximately 15.601 min, approximately 16.592 min, approximately 18.813 min, approximately 20.488 min, approximately 21.971 min, approximately 36.260 min, approximately 41.951 min, approximately 43.627 min, approximately 63.385 min, and approximately 68.756 min, respectively.

[0151] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5.191" includes ±5% of 5.191, or from 4.93145 to 5.45055; "about 11.256" includes ±5% of 11.256, or from 10.6932 to 11.8188; "about 12.608" includes ±5% of 12.608, or from 11.9776 to 13.2384; "about 13.196" includes ±5% of 13.196. "Approximately 15.601" includes ±5% of 15.601, or from 14.82095 to 16.38105; "Approximately 16.592" includes ±5% of 16.592, or from 15.7624 to 17.4216; "Approximately 18.813" includes ±5% of 18.813, or from 17.87235 to 19.75365; "Approximately 20.488" includes ±5% of 20.488, or from 19.4636 to 21.5124; "Approximately 21.971" includes ±5% of 21.971, or from 20.87245 to 23.06955; "Approximately 36.260" includes ±5% of 36.260, or from 34.447 to 38.073; "Approximately 41.951" includes ±5% of 41.951, or From 39.85345 to 44.04855; “about 43.627” includes 43.627 ± 5%, or from 41.44565 to 45.80835; “about 63.385” includes 63.385 ± 5%, or from 60.21575 to 66.55425; “about 68.756” includes 68.756 ± 5%, or from 65.3182 to 72.1938.

[0152] In a preferred embodiment, peak 7 is ligustilide at a detection wavelength of 220 nm.

[0153] In a preferred embodiment, when the detection wavelength is 220 nm, peak 1 originates from Ligusticum chuanxiong and Angelica sinensis, peak 2 originates from Ligusticum chuanxiong, peak 3 originates from Commiphora myrrha, peak 4 originates from Commiphora myrrha, peak 5 originates from Commiphora myrrha, peak 6 originates from Commiphora myrrha, peak 7 originates from Ligusticum chuanxiong and Angelica sinensis, peak 8 originates from Ligusticum chuanxiong and Angelica sinensis, peak 9 originates from Commiphora myrrha, peak 10 originates from Commiphora myrrha, peak 11 originates from Commiphora myrrha, peak 12 originates from Commiphora myrrha, peak 13 originates from Commiphora myrrha, and peak 14 originates from Commiphora myrrha.

[0154] According to another aspect of the present invention, a method for quality control of the volatile oil components of a traditional Chinese medicine composition comprising Angelica sinensis and Ligusticum chuanxiong is provided, the method comprising the following steps:

[0155] (1) Establish standard characteristic spectra of volatile oil components of traditional Chinese medicine compositions based on the above characteristic spectra construction method;

[0156] (2) Take the volatile oil component test solution and detect it according to the chromatographic conditions in the above-mentioned characteristic chromatogram construction method to obtain the characteristic chromatogram of the volatile oil component of the traditional Chinese medicine composition; and

[0157] (3) Compare the characteristic spectrum of the volatile oil component of the Chinese medicine composition obtained in step (2) with the standard characteristic spectrum of the volatile oil component of the Chinese medicine composition obtained in step (1). If the sample meets the requirements, it is a qualified product; if it does not meet the requirements, it is an unqualified product.

[0158] In a preferred embodiment, the compliance requirement includes one or more of the following:

[0159] (1) The volatile oil components of the Chinese herbal medicine composition showed 14 characteristic chromatographic peaks in the characteristic chromatographic spectrum of the sample to be tested. The retention time of each characteristic chromatographic peak was within ±10% of the retention time value of the corresponding reference peak in the standard characteristic chromatographic spectrum of the volatile oil components of the Chinese herbal medicine composition.

[0160] (2) Taking peak 7 as peak S, the relative retention times of each characteristic chromatographic peak in the characteristic chromatographic spectrum of the volatile oil component of the Chinese herbal medicine composition and peak S are within ±10% of the relative retention times of each characteristic chromatographic peak in the standard characteristic chromatographic spectrum of the volatile oil component of the Chinese herbal medicine composition reference sample; and

[0161] (3) According to the similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine, the similarity between the characteristic chromatogram of the volatile oil component of the traditional Chinese medicine composition and the standard characteristic chromatogram of the reference sample of the volatile oil component of the traditional Chinese medicine composition shall not be less than 0.90.

[0162] According to another aspect of the present invention, the above-described construction method or the above-described quality control method is provided for use in the quality detection and / or quality evaluation and / or quality control of volatile oil components in a traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong.

[0163] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0164] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0165] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0166] Example

[0167] 1. Experimental Instruments and Materials

[0168] The instruments and materials are shown in Table 1.

[0169] Table 1. Instruments and Materials

[0170] name Model / Specification / Batch Number Manufacturer / Place of Origin High Performance Liquid Chromatography Agilent 1290 DAD Agilent Technologies chromatographic column HALO AQ C18 4.6mm×150mm, 2.7µm US AMT ligustilide 111737-201910 China National Institutes for Food and Drug Control Ceramic decoction pot 30B2 type Chaozhou City, Chaoan District Kangyashun Electric Appliance Co., Ltd. Electronic analytical balance AUW120D Shimadzu (Shanghai) Co., Ltd. Electronic analytical balance BSA124S Sartorius Group (Germany) peach kernel ZA01721100808 Jingbian County, Yulin, Shaanxi safflower ZA01821101104 Huocheng County, Ili, Xinjiang Myrrh ZA02021120803 Kenya Five Spirits Powder ZD02121101802 Danfeng County, Shangluo, Shaanxi Chuanxiong ZA01621101104 Pengshan County, Meishan City, Sichuan Province Angelica sinensis ZA00321031804-02 Zhanyi County, Qujing, Yunnan Notopterygium ZA01921121001 Xiaojin County, Aba Prefecture, Sichuan Province Gentiana macrophylla ZA01521111304 Gangu County, Tianshui City, Gansu Province Achyranthes bidentata ZA02321101803 Meng County, Jiaozuo, Henan earth dragon ZA02421101103 Gaozhou City, Maoming, Guangdong Cyperus rotundus ZA02221101104 Weishi County, Kaifeng, Henan licorice ZA00421031604 Yumen City, Jiuquan, Gansu

[0171] 2. Preparation method of extract of traditional Chinese medicine composition

[0172] Take a one-day dose of the traditional Chinese medicine composition of the present invention containing Angelica sinensis and Ligusticum chuanxiong (one-day dose: Gentiana macrophylla 3.73g, Ligusticum chuanxiong 7.46g, Prunus persica 11.19g, Carthamus tinctorius 11.19g, Glycyrrhiza uralensis 7.46g, Notopterygium incisum 3.73g, Commiphora myrrha 7.46g, Angelica sinensis 11.19g, Vinegar-processed Trogopterus xanthipes 7.46g, Cyperus rotundus 3.73g, Achyranthes bidentata 11.19g, Pheretima aspergillum 7.46g; total 93.25g), add 12 times the amount of water, extract for 45 minutes, filter, and obtain the first decoction; add 10 times the amount of water to the residue, extract for 40 minutes, filter, and obtain the second decoction; combine the first decoction and the second decoction to obtain the extract of the traditional Chinese medicine composition.

[0173] 3. Determination of volatile oil content

[0174] 3.1 Preliminary Experiments on Volatile Oil Research

[0175] The reference sample of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of this invention has a large number of medicinal ingredients, most of which contain volatile oils. Literature review shows that these volatile components are part of the active ingredients of the reference sample. Therefore, it is necessary to design an experiment to observe the volatile oil content of the reference sample. A method for determining volatile oil content is proposed to be used. Preliminary exploration of this detection method is needed, along with investigation of the sample size and other parameters in the testing process of multiple batches of reference samples.

[0176] The volatile oil content in the reference sample of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention was determined according to the method for determining volatile oil (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 2204). It was observed that the amount of volatile oil was about 0.1 ml, which was too small to be accurately read.

[0177] In summary, it has been determined that the quality standard for the reference sample of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of this invention needs to be supplemented with a "content determination" item for volatile oil content. Further investigation into the sampling amount will follow.

[0178] 3.2. Examination of the sampling quantity of the reference sample

[0179] Reference samples of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention, taken at 5-day and 7-day doses respectively, were determined according to the method for determination of volatile oil (General Chapter 2204, Part IV, Chinese Pharmacopoeia 2020).

[0180] The results show that when taking the 5-day dose, the volatile oil reading is about 0.5 ml, which is accurate. When taking the 7-day dose, the volatile oil is about 0.6 ml. Considering the batch-to-batch differences of multiple reference samples, it is tentatively proposed that when preparing and testing multiple batches of the reference sample of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of this invention, the 5-day dose reference sample shall be used as the sample amount for the determination of volatile oil content.

[0181] 3.3 Determination of methods for determining volatile oil content

[0182] In summary, the proposed method for determining the volatile oil content of the reference sample of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention is as follows:

[0183] A 5-day dose of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention was taken as a reference sample and its volatile oil content was determined according to the method for determining volatile oil content (General Chapter 2204, Part IV, Chinese Pharmacopoeia 2020).

[0184] 3.4 Determination of volatile oil content in multiple batches of reference samples

[0185] Fifteen batches of reference samples were taken, and the content of volatile oils in the reference samples was determined according to the above method. The results are shown in Table 2.

[0186] Table 2. Volatile oil content of 15 batches of reference samples

[0187] Reference sample batch Volatile oil (ml / g) Each dose contains volatile oil (ml) Reference Sample 1 0.086% 0.027 Reference Sample 2 0.089% 0.028 Reference Sample 3 0.099% 0.031 Reference Sample 4 0.090% 0.028 Reference Sample 5 0.120% 0.037 Reference Sample 6 0.109% 0.034 Reference Sample 7 0.118% 0.037 Reference Sample 8 0.124% 0.039 Reference Sample 9 0.120% 0.037 Reference Sample 10 0.094% 0.029 Reference Sample 11 0.073% 0.023 Reference Sample 12 0.090% 0.028 Reference Sample 13 0.086% 0.027 Reference Sample 14 0.124% 0.039 Reference Sample 15 0.086% 0.027

[0188] The results showed that the measured range of volatile oil content per dose in each batch of reference samples was 0.023 ml to 0.039 ml, with an average of 0.031 ml. The fluctuation range of the average ±30% was 0.022 ml to 0.041 ml, all within the range of ±30% of the average.

[0189] 4. Feature Map

[0190] 4.1 Preliminary Chromatographic Experiments and Selection of Detection Wavelength

[0191] According to the present invention, the medicinal composition of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong (containing volatile oils of Angelica sinensis, Ligusticum chuanxiong, Commiphora myrrha, Notopterygium incisum, and Cyperus rotundus), combined with chemical components and pharmacological effects, shows that ligustilide in Angelica sinensis (assistant) and Ligusticum chuanxiong (assistant) has anti-inflammatory and analgesic effects; the volatile oil in Commiphora myrrha (assistant) has blood-activating and stasis-removing effects; notopterygium in Notopterygium incisum (adjuvant) has the effects of relieving exterior cold and dispelling wind and dampness; and α-cyperone in Cyperus rotundus (adjuvant) has the effects of regulating qi and relieving pain. Therefore, it is proposed to characterize the volatile components in the reference sample of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention using liquid chromatography conditions. Based on the results of previous experimental studies, the volatile oil components in each decoction piece will be studied first to confirm the chromatographic peaks of the volatile components. The initial conditions are tentatively proposed as follows:

[0192] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512).

[0193] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel (CORTECS UPLC T3 2.1×100mm 1.6μm) as the stationary phase; acetonitrile:methanol (70:30) as mobile phase A and 0.05% phosphoric acid solution as mobile phase B, eluting according to elution gradient 1 in Table 3; column temperature 35℃, flow rate 0.3 ml / min, DAD wavelength scan 190–400 nm.

[0194] Table 3 Elution gradient 1

[0195] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～14 10 90 14～20 10→14 90→86 20～24 14→16 86→84 24～40 16→20 84→80 40～56 20→40 80→60 56～65 40→48 60→52 65～70 48 52 70～72 48→90 52→10

[0196] Preparation of test solution

[0197] Take a 3-day dose of the herbal composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention, add 2400 ml of water, connect the volatile oil extraction device, heat and reflux for 1 hour to extract the volatile oil, dissolve the extracted volatile oil in methanol and dilute it to a 10 ml volumetric flask, filter it through a 0.22 μm microporous membrane, and use it as the test solution.

[0198] According to the isolinear absorption chromatogram of the volatile oil test solution of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong according to the present invention, under the chromatographic conditions, most of the volatile oil chromatographic peaks have high response values ​​above 220 nm. In comparison, the chromatographic peak information of volatile components at the wavelength of 220 nm is relatively rich, and the baseline of the chromatogram is relatively stable. Therefore, the wavelength of 220 nm is temporarily selected for subsequent research.

[0199] However, under elution condition 1 in Table 3, the separation of each chromatographic peak was very poor, and the chromatographic peaks were basically concentrated after 60 min. Therefore, the chromatographic conditions were redeveloped.

[0200] 4.2 Investigation of the preparation method of the test solution 1

[0201] Based on the above research results, the method for preparing the reference sample of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention was examined, and the specific conditions are as follows:

[0202] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512).

[0203] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel (CORTECS UPLC T3 2.1×100mm 1.6μm) as the stationary phase; acetonitrile:methanol (70:30) as mobile phase A and 0.05% phosphoric acid solution as mobile phase B, eluting according to elution gradient 2 in Table 4; column temperature 35℃, flow rate 0.3 ml / min, and detection wavelength 220 nm.

[0204] Table 4 Elution gradient 2

[0205] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～14 10 90 14～20 10→14 90→86 20～24 14→16 86→84 24～34 16→20 84→80 34～52 20→40 80→60 52～60 40→48 60→52 60～63 48 52 63～72 48→58 52→42 72～78 58→70 42→30 78～80 70→90 30→10 80～90 90 10 90～93 90→10 10→90 93～100 10 90

[0206] Preparation of test solution

[0207] Test solution 1: Take the decoction of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention, filter it through a 0.22μm microporous membrane to obtain the solution.

[0208] Test solution 2: Take 20 ml of the decoction of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention, add 20 ml of cyclohexane and shake to extract, evaporate the cyclohexane solution to dryness, and filter the residue with methanol solution to 5 ml and filter through a 0.22 μm microporous membrane to obtain the test solution.

[0209] Test solution 3: Take 20 ml of the decoction of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention, add 20 ml of ether and shake to extract, evaporate the ether solution to dryness, and use methanol solution to make 5 ml of residue. Filter through a 0.22 μm microporous membrane to obtain the test solution.

[0210] Test solution 4: Take a one-day dose of the traditional Chinese medicine composition of the present invention containing Angelica sinensis and Ligusticum chuanxiong (Gentiana macrophylla 3.73g, Ligusticum chuanxiong 7.46g, Prunus persica 11.19g, Carthamus tinctorius 11.19g, Glycyrrhiza uralensis 7.46g, Notopterygium incisum 3.73g, Commiphora myrrha 7.46g, Angelica sinensis 11.19g, Vinegar-processed Trogopterus xanthipes 7.46g, Cyperus rotundus 3.73g, Achyranthes bidentata 11.19g, Pheretima aspergillum 7.46g; total 93.25g), add 12 times the amount of water, extract for 45 minutes, filter, add 10 times the amount of water to the residue, extract for 40 minutes, filter, combine the decoctions, connect to a volatile oil extraction device, heat under reflux for 5 hours, take 0.1g of the extracted volatile oil, dissolve and dilute with methanol to a 10ml volumetric flask, filter through a 0.22μm microporous membrane, and obtain the final product.

[0211] The assay involves precisely pipetting 1 μl of the test solution, injecting it into the liquid chromatograph, and measuring the sample.

[0212] The results showed that no volatile component peaks were detected in the chromatogram of test solution 1, indicating that the direct injection of the reference sample (decoction) of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention could not detect volatile component peaks. The chromatograms of test solutions 2 and 3 showed that the volatile component peaks could not be detected after the test solutions extracted with ether and cyclohexane were evaporated or dried, indicating poor extraction efficiency. The chromatogram of test solution 4 showed that after the extraction of volatile oil from the reference sample (decoction) of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention, there were many volatile component peaks, which was basically consistent with the chromatograms of the previously obtained traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong after the extraction of volatile oil.

[0213] Therefore, in order to better illustrate the quantitative and qualitative transfer relationship of volatile components, a characteristic spectral method was established to monitor the volatile components in the reference sample of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of this invention.

[0214] The tentatively proposed method for preparing the test sample of the reference sample containing Angelica sinensis and Ligusticum chuanxiong of the present invention is as follows: Take a one-day dose of the reference sample of the reference sample containing Angelica sinensis and Ligusticum chuanxiong of the present invention, connect it to a volatile oil extraction device, extract the volatile oil according to the volatile oil determination method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 2204), dissolve and dilute it with methanol to a 10ml volumetric flask, shake well, filter, and collect the filtrate to obtain the sample. However, under elution condition 2 in Table 4, the baseline is unstable, the separation of each chromatographic peak is poor, and the chromatographic peaks overlap significantly. Therefore, the chromatographic conditions need to be redeveloped.

[0215] 4.3 Elution gradient investigation

[0216] Table 5 Elution gradient 3

[0217] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～15 30→60 70→40 15～30 60 40 30～40 60→65 40→35 40～60 65→85 35→15 60～65 85 15 65～68 85→30 15→70 68～75 30 70

[0218] The results showed that under the elution gradient 3 conditions in Table 5, the separation of each chromatographic peak was poor, and they were mostly concentrated in the range of 20-30 min, which still did not meet the requirements of the characteristic chromatogram. Therefore, the chromatographic conditions were further optimized.

[0219] Table 6 Elution gradient 4

[0220] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～90 44 56

[0221] The results showed that under the elution gradient 4 conditions in Table 6, the separation of each chromatographic peak was improved. However, the chromatographic peaks between 30 and 40 min still could not meet the requirements for characteristic spectrum detection. Therefore, the chromatographic conditions were further optimized.

[0222] Table 7 Elution gradient 5

[0223] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～50 44 56 50～60 44→70 56→30 60～65 70 30 65～70 70→80 30→20 70～85 80 20 85～90 80→44 20→56

[0224] The results show that, under the elution gradient 5 conditions in Table 7, some volatile components still elute as the proportion of organic phase increases, and all chromatographic peaks are basically eluted by 85 minutes. Under these conditions, the chromatographic peaks have good resolution and symmetry, and basically meet the relevant requirements of the characteristic chromatograms.

[0225] 4.4 Determination of chromatographic conditions

[0226] After exploring and optimizing the chromatographic conditions for the reference sample characteristic chromatogram of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention, the final chromatographic conditions are as follows:

[0227] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512).

[0228] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel (HALO AQ C18 4.6×150mm, 2.7μm) was used as the stationary phase; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, eluting according to elution gradient 5 in Table 7; column temperature was 30℃, flow rate was 0.7 ml / min, and detection wavelength was 220 nm. The theoretical plate number, calculated based on the ligustilide peak, should be no less than 8000.

[0229] Preparation of reference solution: Take an appropriate amount of ligustilide reference standard, accurately weigh it, and add methanol to prepare a solution containing 1 mg per 1 ml, which is used as the reference solution.

[0230] Preparation of the test solution: Take a one-day dose of the traditional Chinese medicine composition of the present invention containing Angelica sinensis and Ligusticum chuanxiong (Gentiana macrophylla 3.73g, Ligusticum chuanxiong 7.46g, Prunus persica 11.19g, Carthamus tinctorius 11.19g, Glycyrrhiza uralensis 7.46g, Notopterygium incisum 3.73g, Commiphora myrrha 7.46g, Angelica sinensis 11.19g, Vinegar-processed Trogopterus xanthipes 7.46g, Cyperus rotundus 3.73g, Achyranthes bidentata 11.19g, Pheretima aspergillum 7.46g; total 93.25g), add 12 times the amount of water, extract for 45 minutes, filter, add 10 times the amount of water to the residue, extract for 40 minutes, filter, combine the decoctions, connect to a volatile oil extraction device, heat under reflux for 5 hours, take 0.1g of the extracted volatile oil, place it in a 10ml volumetric flask, dilute with methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0231] Determination method: Accurately pipette 1 μl of the test solution and the reference solution into the liquid chromatograph and determine the result.

[0232] 5. Methodological Validation

[0233] Based on the established method for preparing the test solution, the chromatographic conditions for the characteristic chromatograms of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of this invention were validated methodologically. Validation included: specificity, precision (instrument precision, intermediate precision, repeatability), and robustness (stability of the test solution, different flow rates, different column temperatures, different brands or batches of the same type of column, and mobile phase pH). The specific methods are as follows:

[0234] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512).

[0235] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel (HALO AQ C18 4.6×150mm 2.7μm) was used as the stationary phase; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, elution was performed according to elution gradient 5 in Table 7; column temperature was 30℃, flow rate was 0.7 ml per minute, and detection wavelength was 220 nm.

[0236] Preparation of reference solution: Take an appropriate amount of ligustilide reference standard, accurately weigh it, and add methanol to prepare a solution containing 1 mg per 1 ml, which is used as the reference solution.

[0237] Preparation of the test solution: Take a one-day dose of the traditional Chinese medicine composition of the present invention containing Angelica sinensis and Ligusticum chuanxiong (Gentiana macrophylla 3.73g, Ligusticum chuanxiong 7.46g, Prunus persica 11.19g, Carthamus tinctorius 11.19g, Glycyrrhiza uralensis 7.46g, Notopterygium incisum 3.73g, Commiphora myrrha 7.46g, Angelica sinensis 11.19g, Vinegar-processed Trogopterus xanthipes 7.46g, Cyperus rotundus 3.73g, Achyranthes bidentata 11.19g, Pheretima aspergillum 7.46g; total 93.25g), add 12 times the amount of water, extract for 45 minutes, filter, add 10 times the amount of water to the residue, extract for 40 minutes, filter, combine the decoctions, connect to a volatile oil extraction device, heat under reflux for 5 hours, take 0.1g of the extracted volatile oil, place it in a 10ml volumetric flask, dilute with methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0238] Determination method: Accurately pipette 1 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0239] 5.1 Specificity

[0240] The blank solvent, the volatile oil test solution of the reference sample of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention, the reference solution, the test solution prepared from the volatile oil of the single herb of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention, and the test solution prepared from the volatile oil of the negative control of the reference sample of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention without herb are injected together for analysis. The blank solvent should not interfere with the peak positions of each pseudo-characteristic peak.

[0241] Preparation of test solution

[0242] Take approximately 0.1g of the volatile oil (batch number: STZYKL2022120301-Y) collected from the reference sample of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of this invention, accurately weigh it, place it in a 10ml volumetric flask, dilute it to the mark with methanol, and shake well to obtain the product.

[0243] Preparation of reference solution

[0244] Take an appropriate amount of ligustilide reference standard, accurately weigh it, and add methanol to prepare a solution containing 1 mg per 1 ml, which is used as the reference solution.

[0245] Preparation of negative control solution

[0246] Twelve negative control solutions of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention, each lacking the corresponding medicinal flavor, were taken and connected to a volatile oil extraction device. The volatile oil was extracted according to the volatile oil determination method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 2204). The volatile oil was dissolved and diluted with methanol to a 10ml volumetric flask and filtered through a 0.22μm microporous membrane to obtain the final product.

[0247] blank solvent

[0248] The methanol is filtered through a 0.22μm microporous membrane to obtain the product.

[0249] The characteristic peak medicinal flavors of the reference sample of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention are assigned as shown in Table 8.

[0250] Table 8. Characteristic Peak Herbal Flavor Assignment Table of the Reference Sample of the Traditional Chinese Medicine Composition Containing Angelica sinensis and Ligusticum chuanxiong of the Present Invention

[0251] Gentiana macrophylla licorice Chuanxiong peach kernel safflower Notopterygium Myrrh Cyperus rotundus Achyranthes bidentata earth dragon Angelica sinensis Five Spirits Powder Peak 1 - - + - - - - - - - + - Peak 2 - - + - - - - - - - - - Peak 3 - - - - - - + - - - - - Peak 4 - - - - - - + - - - - - Peak 5 - - - - - - + - - - - - Peak 6 - - - - - - + - - - - - Peak 7 (ligustilide) - - + - - - - - - - + - Peak 8 - - + - - - - - - - + - Peak 9 - - - - - - + - - - - - Peak 10 - - - - - - + - - - - - Peak 11 - - - - - - + - - - - - Peak 12 - - - - - - + - - - - - Peak 13 - - - - - - + - - - - - Peak 14 - - - - - - + - - - - -

[0252] The results showed that the blank did not interfere with the elution positions of the 14 pseudo-characteristic peaks of the test sample; based on the comparison between the test sample and the reference solution, peak 7 could be identified as ligustilide.

[0253] Currently, peaks 3, 4, 5, 6, 9, 10, 11, 12, 13, and 14 are known to originate from myrrh decoction pieces and are negative with no interference; peak 7 is ligustilide, and along with peaks 1 and 8, it originates from angelica and chuanxiong decoction pieces, and is negative with no interference; peak 2 originates from chuanxiong decoction pieces and is negative with no interference.

[0254] Therefore, based on comprehensive judgment, this method has good specificity and meets the requirements of feature maps.

[0255] 5.2 Precision

[0256] 5.2.1 Instrument Precision

[0257] Prepare one test solution according to the established test solution preparation method, inject the sample six times consecutively, record the chromatogram, and calculate the RSD values ​​of the relative retention time and relative peak area of ​​each pseudo-characteristic peak.

[0258] The results showed that the relative retention times (RSDs) of the 14 pseudo-characteristic peaks of the volatile oil of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong in this invention were all less than 3.0%, and the relative peak area (RSD) of some chromatographic peaks was greater than 5.0%. However, since the peak area of ​​some chromatographic peaks accounted for a small proportion of the total peak area, less than 10%, no relevant requirements were imposed. Therefore, based on comprehensive judgment, this method has good instrument precision and meets the requirements of characteristic chromatograms.

[0259] 5.2.2 Intermediate Precision

[0260] Six test solutions were prepared by different personnel according to the established test solution preparation method. The solutions were then injected and detected on an Agilent ultra-high performance liquid chromatograph and a Waters ultra-high performance liquid chromatograph, respectively. Chromatograms were recorded, and the relative retention time and relative peak area of ​​each pseudo-characteristic peak were calculated.

[0261] The results showed that, in the intermediate precision test, the relative retention times (RSDs) of the 14 pseudo-characteristic peaks of the reference sample of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of this invention were all less than 3.0%. In summary, the intermediate precision test results of this method meet the relevant regulations.

[0262] 5.3 Repeatability

[0263] Take 6 portions of the volatile oil from the same batch of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of this invention, prepare 6 portions of test solution according to the established test solution preparation method, inject the samples for detection, record the chromatograms, and calculate the RSD values ​​of the relative retention time and relative peak area of ​​each pseudo-characteristic peak.

[0264] The results showed that the relative retention times (RSDs) of the 14 pseudo-characteristic peaks of the volatile oil of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong in this invention were all less than 3.0%, and the relative peak areas (RSDs) of the chromatographic peaks were all less than 5.0%. Therefore, based on comprehensive judgment, the method has good repeatability and meets the requirements of characteristic chromatograms.

[0265] 5.4 Durability

[0266] 5.4.1 Stability of the test solution

[0267] Prepare one test solution according to the established test solution preparation method, and inject the sample at 0h, 2h, 4h, 8h, 12h, 18h, 24h, 36h and 48h respectively. Record the chromatograms and calculate the RSD values ​​of the relative retention time and relative peak area of ​​each pseudo-characteristic peak.

[0268] The results showed that the peak areas of each pseudo-characteristic peak in the test solution decreased significantly at 48 hours. For the volatile oil test solution of the traditional Chinese medicine composition reference sample containing Angelica sinensis and Ligusticum chuanxiong, the relative retention times (RSDs) of all 14 pseudo-characteristic peaks were less than 3.0% within 36 hours, and the relative peak area (RSD) ranged from 0.3% to 6.1%. While some chromatographic peaks had RSDs greater than 5.0%, these peak areas accounted for a small proportion of the total peak area (less than 10%), so no specific requirements were imposed. Therefore, based on the overall assessment, the test solution exhibited good stability within 36 hours.

[0269] 5.5 Investigation of different flow velocities

[0270] Prepare one test solution according to the established test solution preparation method, and inject it at 0.6 ml / min, 0.7 ml / min, and 0.8 ml / min respectively. Record the chromatograms and calculate the relative average deviation of the relative retention time of each pseudo-characteristic peak.

[0271] The results showed that the relative average deviation of the relative retention times of the 14 pseudo-characteristic peaks of the volatile oil in the reference sample of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong was between 0.0% and 8.1%, indicating that the degree of separation of each chromatographic peak varied greatly under different flow rate conditions, resulting in significant variations in the retention times of each chromatographic peak. Based on the overall assessment, the flow rate robustness was poor. To ensure the reproducibility of the characteristic chromatograms, it is recommended to fix the flow rate at 0.7 ml / min as the detection flow rate for the characteristic chromatograms of the reference sample of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of this invention.

[0272] 5.6 Investigation at different column temperatures

[0273] Prepare one test solution according to the established test solution preparation method, and inject the sample at 27℃, 30℃, and 33℃ respectively. Record the chromatograms and calculate the relative average deviation of the relative retention time of each pseudo-characteristic peak.

[0274] The results showed that the relative average deviation of the relative retention times of the 14 pseudo-characteristic peaks of the reference sample of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong was less than 3.0%, indicating that the method has good durability at different column temperatures.

[0275] 5.7 Investigation of different acid concentrations

[0276] Prepare one test solution according to the established test solution preparation method, and inject it under the conditions of 0.08% phosphoric acid, 0.10% phosphoric acid, and 0.12% phosphoric acid, respectively. Record the chromatograms and calculate the relative average deviation of the relative retention time of each pseudo-characteristic peak.

[0277] The results showed that the relative average deviation of the relative retention times of the 14 pseudo-characteristic peaks of the reference sample of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong was less than 3.0%, indicating that the method has good robustness to phosphoric acid solutions of different concentrations.

[0278] 5.8 Investigation of different chromatographic columns

[0279] Prepare one test solution according to the established test solution preparation method. Inject the solution using chromatographic columns of the same specification but different brands or HALO AQ C18 (4.6×150mm 2.7μm) columns of the same type but different batches. Record the chromatograms and calculate the relative average deviation of the relative retention times of each pseudo-characteristic peak.

[0280] Column 1: HALO AQ C18 (4.6×150mm 2.7μm);

[0281] Column 2: XBridge Shield RP18 (4.6×150mm 3.5μm);

[0282] Column 2: Atlantis TMT3 (4.6×150mm 3μm).

[0283] The results showed that the relative average deviation of the relative retention times of the 14 pseudo-characteristic peaks of the volatile oil of the reference sample of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong in this invention ranged from 1.7% to 37.4%. The significant differences in results were due to variations in the packing materials of different brands and models of chromatographic columns, and the elution order of some peaks was also altered. Therefore, it is recommended to use a fixed chromatographic column model of HALO AQC18 (4.6 × 150 mm 2.7 μm).

[0284] 5.9 Summary of Methodological Validation

[0285] Based on the above methodological investigation results, it is known that the 14 pseudo-characteristic peaks in the reference chromatogram of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of this invention are affected to some extent by different chromatographic column and flow rate conditions, while the other chromatographic conditions have little effect. Taking all factors into consideration, it is tentatively determined that there are 14 characteristic peaks in the reference chromatogram of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of this invention.

[0286] In summary, this method has been used to determine the characteristic chromatograms of the reference samples of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention. Through methodological validation, it has demonstrated good precision, robustness, stability, and specificity. The established method is applicable to the detection of the characteristic chromatograms of the reference samples of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention.

[0287] 5.10 System Applicability Parameter Confirmation

[0288] Based on the methods established above, the system suitability parameters of each characteristic peak in the reference sample of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention were confirmed, and the results are shown in Table 9. The characteristic chromatograms of the reference sample of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention are as follows: Figure 1 As shown.

[0289] Table 9. Suitability parameters of the reference sample characteristic peak system for the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention.

[0290] Serial Number Retention time area high Resolution Symmetry factor USP Theoretical Plate Count 1 5.191 47376 9473 - 1.12 24788 2 11.256 109272 9488 27.71 0.89 23305 3 12.608 75143 6804 4.43 1.05 28129 4 13.196 111247 8132 1.79 0.82 24471 5 15.601 1350733 85314 6.22 1.13 22594 6 16.592 1511991 92614 2.30 0.96 24041 7 18.813 1915912 98140 4.66 1.03 22027 8 20.488 525440 24925 3.09 1.11 21664 9 21.971 3231040 151858 2.60 1.06 23898 10 36.260 271444 7592 9.41 1.02 22829 11 41.951 413482 9522 5.35 1.03 21213 12 43.627 458325 10221 1.38 1.03 20442 13 63.385 315375 24158 24.51 0.96 540185 14 68.756 1063178 78106 14.90 1.02 571202

[0291] The results showed that the chromatogram of the test solution exhibited 14 characteristic peaks, each with good peak shape, good separation, good symmetry, and a relatively stable baseline, meeting the relevant requirements for characteristic chromatogram detection. Taking all factors into consideration, the theoretical plate number of the reference peak, ligustilide, should be no less than 8000.

[0292] 6. Determination of characteristic spectra of multiple batches of reference samples

[0293] Fifteen batches of reference samples were taken for content determination. The volatile oil content under the volatile oil item was analyzed according to the established reference sample characteristic chromatogram determination method. Results are as follows: Figure 2 As shown.

[0294] The results showed that the volatile oil characteristic spectra of the reference samples of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention in 15 batches all exhibited 14 characteristic peaks, among which peak 7 should correspond to the retention time of the ligustilide reference peak. The peak corresponding to the ligustilide reference peak is peak S, and the calculated relative retention times of each characteristic peak and peak S are all within ±10% of the average value. By comparing the relative peak areas of the characteristic peaks of the volatile oil of the reference samples of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong of the present invention in 15 batches, it was found that the relative peak areas of each characteristic peak varied greatly among different batches and there was no discernible pattern. Therefore, no relevant regulations are made regarding the relative peak areas of the characteristic peaks.

[0295] The chromatogram of the test sample should show 14 characteristic peaks, among which peak 7 should correspond to the retention time of the ligustilide reference peak. The peak corresponding to the ligustilide reference peak is the S peak. Calculate the relative retention times of peaks 2-6 and 8-14 with peak S. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.60 (peak 2), 0.67 (peak 3), 0.70 (peak 4), 0.83 (peak 5), 0.88 (peak 6), 1.09 (peak 8), 1.15 (peak 9), 1.90 (peak 10), 2.18 (peak 11), 2.26 (peak 12), 3.46 (peak 13), and 3.75 (peak 14).

[0296] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, any changes or modifications made by those skilled in the art based on the ideas of the present invention, its specific implementation methods, and its application scope, are all within the scope of protection of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for constructing a characteristic spectrum of volatile oil components in a traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong, characterized in that, The construction method includes the following steps: Preparation of the volatile oil component test solution: Take an appropriate amount of the extract of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong, heat and reflux to extract, and obtain volatile oil; take an appropriate amount of the volatile oil in a container, add methanol to dilute, make up to volume, shake well, and obtain the test solution of the volatile oil component; wherein, the preparation method of the extract includes the following steps: weigh an appropriate amount of Gentiana macrophylla, Ligusticum chuanxiong, Prunus persica, Carthamus tinctorius, Glycyrrhiza uralensis, Notopterygium incisum, Commiphora myrrha, Angelica sinensis, Vinegar-processed Trogopterus xanthipes, Cyperus rotundus, Achyranthes bidentata, and Pheretima aspergillum, add an appropriate amount of water for extraction, filter, and obtain the first decoction; add an appropriate amount of water to the residue for extraction, filter, and obtain the second decoction; combine the first decoction and the second decoction to obtain the extract; Preparation of reference solution: Weigh an appropriate amount of ligustilide reference standard and add methanol to prepare the reference solution with a ligustilide concentration of 0.1~5 mg / ml; Based on the results of high-performance liquid chromatography (HPLC) analysis of the test solution and the reference solution, a characteristic spectrum of volatile oil components was obtained. The chromatographic conditions for high-performance liquid chromatography (HPLC) detection are as follows: a chromatographic column packed with octadecylsilane-bonded silica gel is used, wherein the column specifications are: column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm; mobile phase A is acetonitrile; mobile phase B is phosphoric acid aqueous solution; the gradient elution program is: 0–50 min, 56% B; 50–60 min, 56% B → 30% B; 60–65 min, 30% B; 65–70 min, 30% B → 20% B; 70–85 min, 20% B; 85–90 min, 20% B → 56% B; the flow rate is 0.62–0.78 ml / min; the column temperature is 25–35 °C; the detection wavelength is 190–400 nm; and the injection volume is 0.5–2 μl. When the detection wavelength is 220 nm, the characteristic spectrum includes peaks 1-14, with peak 7 serving as a reference peak. The average relative retention times of peaks 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, and 14 are approximately 0.28, 0.60, 0.67, 0.70, 0.83, 0.88, 1.09, 1.15, 1.90, 2.18, 2.26, 3.46, and 3.75, respectively.

2. The construction method according to claim 1, characterized in that, In the method for preparing the test solution, the heating and reflux extraction time is 4-6 h.

3. The construction method according to claim 2, characterized in that, In the method for preparing the test solution, the heating and reflux extraction time is approximately 5 hours.

4. The construction method according to claim 1, characterized in that, The heating reflux extraction device is a volatile oil extraction device.

5. The construction method according to claim 1, characterized in that, The amount of medicinal material sampled for the extract is a 1-7 day dose.

6. The construction method according to claim 5, characterized in that, The amount of medicinal material sampled for the extract is a 1-day dose, a 5-day dose, or a 7-day dose.

7. The construction method according to claim 1, characterized in that, The mass ratio of the following ingredients is (0.5~2):(1~3):(2~4):(2~4):(1~3):(0.5~2):(1~3):(2~4):(1~3):(0.5~2):(1~3):(2~4):(1~3):(0.5~2):(1~3):(2~4):(1~3):(0.5~2):(2~4):(1~3):(0.5~2):(2~4):(1~3):(0.5~2):(2~4):(1~3).

8. The construction method according to claim 7, characterized in that, The mass ratio of the following ingredients is (0.95~1.05):(1.9~2.1):(2.85~3.15):(2.85~3.15):(1.9~2.1):(0.95~1.05):(1.9~2.1):(2.85~3.15):(1.9~2.1):(0.95~1.05):(1.9~2.1):(2.85~3.15):(1.9~2.1):(0.95~1.05):(1.9~2.1):(2.85~3.15):(1.9~2.1):(0.95~1.05):(2.85~3.15):(1.9~2.1).

9. The construction method according to claim 1, characterized in that, The mass of the Gentiana macrophylla is 1~5 g.

10. The construction method according to claim 9, characterized in that, The mass of the Gentiana macrophylla is approximately 3.73 g.

11. The construction method according to claim 1, characterized in that, The mass of the chuanxiong rhizome is 5-10 g.

12. The construction method according to claim 11, characterized in that, The mass of the chuanxiong rhizome is approximately 7.46 g.

13. The construction method according to claim 1, characterized in that, The weight of the peach kernel is 10-15 g.

14. The construction method according to claim 13, characterized in that, The peach kernel weighs approximately 11.19 g.

15. The construction method according to claim 1, characterized in that, The weight of the safflower is 10-15 g.

16. The construction method according to claim 15, characterized in that, The safflower weighs approximately 11.19 g.

17. The construction method according to claim 1, characterized in that, The weight of the licorice is 5-10 g.

18. The construction method according to claim 17, characterized in that, The licorice weighs approximately 7.46 g.

19. The construction method according to claim 1, characterized in that, The mass of the notopterygium root is 1~5 g.

20. The construction method according to claim 19, characterized in that, The mass of the notopterygium root is approximately 3.73 g.

21. The construction method according to claim 1, characterized in that, The mass of the myrrh is 5-10 g.

22. The construction method according to claim 21, characterized in that, The mass of the myrrh is approximately 7.46 g.

23. The construction method according to claim 1, characterized in that, The weight of the Angelica sinensis is 10-15 g.

24. The construction method according to claim 23, characterized in that, The weight of the Angelica sinensis is approximately 11.19 g.

25. The construction method according to claim 1, characterized in that, The weight of the vinegar-processed five-spice powder is 5-10 g.

26. The construction method according to claim 25, characterized in that, The weight of the vinegar-processed five-spice powder is approximately 7.46 g.

27. The construction method according to claim 1, characterized in that, The mass of the Cyperus rotundus is 1~5 g.

28. The construction method according to claim 27, characterized in that, The mass of the Cyperus rotundus is approximately 3.73 g.

29. The construction method according to claim 1, characterized in that, The mass of the Achyranthes bidentata is 10-15 g.

30. The construction method according to claim 29, characterized in that, The mass of the Achyranthes bidentata is approximately 11.19 g.

31. The construction method according to claim 1, characterized in that, The mass of the earthworm is 5-10 g.

32. The construction method according to claim 31, characterized in that, The earthworm weighs approximately 7.46 g.

33. The construction method according to claim 1, characterized in that, In the preparation of the first decoction, the volume-to-mass ratio of water to the following ingredients is 8-15: Gentiana macrophylla, Ligusticum chuanxiong, Prunus persica, Carthamus tinctorius, Glycyrrhiza uralensis, Notopterygium incisum, Commiphora myrrha, Angelica sinensis, Vinegar-processed Trogopterus xanthipes, Cyperus rotundus, Achyranthes bidentata, and Pheretima aspergillum.

34. The construction method according to claim 33, characterized in that, In the preparation of the first decoction, the volume-to-mass ratio of water to the following ingredients is approximately 12: Gentiana macrophylla, Ligusticum chuanxiong, Prunus persica, Carthamus tinctorius, Glycyrrhiza uralensis, Notopterygium incisum, Commiphora myrrha, Angelica sinensis, Vinegar-processed Trogopterus xanthipes, Cyperus rotundus, Achyranthes bidentata, and Pheretima aspergillum.

35. The construction method according to claim 1, characterized in that, In the preparation of the second decoction, the volume-to-mass ratio of water to the following ingredients is 6-12: Gentiana macrophylla, Ligusticum chuanxiong, Prunus persica, Carthamus tinctorius, Glycyrrhiza uralensis, Notopterygium incisum, Commiphora myrrha, Angelica sinensis, Vinegar-processed Trogopterus xanthipes, Cyperus rotundus, Achyranthes bidentata, and Pheretima aspergillum.

36. The construction method according to claim 35, characterized in that, In the preparation of the second decoction, the volume-to-mass ratio of water to the following ingredients is approximately 10: Gentiana macrophylla, Ligusticum chuanxiong, Prunus persica, Carthamus tinctorius, Glycyrrhiza uralensis, Notopterygium incisum, Commiphora myrrha, Angelica sinensis, Vinegar-processed Trogopterus xanthipes, Cyperus rotundus, Achyranthes bidentata, and Pheretima aspergillum.

37. The construction method according to claim 1, characterized in that, The extraction time for the first or second decoction is 30-60 min.

38. The construction method according to claim 37, characterized in that, The extraction time for the first decoction or the second decoction is approximately 40 min or 45 min.

39. The construction method according to claim 1, characterized in that, The mass of the volatile oil is 0.05~0.2 g.

40. The construction method according to claim 39, characterized in that, The mass of the volatile oil is approximately 0.1 g.

41. The construction method according to claim 1, characterized in that, The container is a volumetric flask.

42. The construction method according to claim 41, characterized in that, The volumetric flask has a volume of 10 ml.

43. The construction method according to claim 1, characterized in that, The mass / volume ratio of the volatile oil to the volatile oil component test solution is 0.005~0.02, in g / ml.

44. The construction method according to claim 43, characterized in that, The mass / volume ratio of the volatile oil to the volatile oil component test solution is approximately 0.01, in g / ml.

45. The construction method according to claim 1, characterized in that, The method for preparing the test solution further includes the step of shaking and then filtering.

46. ​​The construction method according to claim 45, characterized in that, The filtration is membrane filtration.

47. The construction method according to claim 46, characterized in that, The filter membrane has a pore size of 0.22 μm.

48. The construction method according to claim 1, characterized in that, The concentration of ligustilide in the reference solution was approximately 1 mg / ml.

49. The construction method according to claim 1, characterized in that, The flow rate for the high-performance liquid chromatography (HPLC) detection is 0.65~0.75 ml / min.

50. The construction method according to claim 49, characterized in that, The flow rate for the high-performance liquid chromatography detection is approximately 0.7 ml / min.

51. The construction method according to claim 1, characterized in that, The column temperature is 27~33℃.

52. The construction method according to claim 51, characterized in that, The column temperature is approximately 30°C.

53. The construction method according to claim 1, characterized in that, The detection wavelength is 220~280 nm.

54. The construction method according to claim 1, characterized in that, The detection wavelength is approximately 220 nm.

55. The construction method according to claim 1, characterized in that, The injection volume is 0.8~1.2 μl.

56. The construction method according to claim 55, characterized in that, The injection volume is approximately 1 μl.

57. The construction method according to claim 1, characterized in that, The theoretical plate number of the chromatographic peak corresponding to the ligustilide is not less than 8000.

58. The construction method according to claim 1, characterized in that, The chromatographic column is a HALO AQ C18 column.

59. The construction method according to claim 1, characterized in that, The phosphoric acid aqueous solution is a 0.01% to 1% phosphoric acid aqueous solution.

60. The construction method according to claim 59, characterized in that, The phosphoric acid aqueous solution is approximately 0.1% phosphoric acid aqueous solution.

61. The construction method according to claim 1, characterized in that, When the detection wavelength is 220 nm, the characteristic spectrum includes peaks 1-14, and the retention times of peaks 1-14 are approximately 5.191 min, approximately 11.256 min, approximately 12.608 min, approximately 13.196 min, approximately 15.601 min, approximately 16.592 min, approximately 18.813 min, approximately 20.488 min, approximately 21.971 min, approximately 36.260 min, approximately 41.951 min, approximately 43.627 min, approximately 63.385 min, and approximately 68.756 min, respectively.

62. The construction method according to claim 1, characterized in that, At the detection wavelength of 220 nm, peak 7 is ligustilide.

63. The construction method according to claim 1, characterized in that, At the detection wavelength of 220 nm, peak 1 comes from Ligusticum chuanxiong and Angelica sinensis, peak 2 comes from Ligusticum chuanxiong, peak 3 comes from Commiphora myrrha, peak 4 comes from Commiphora myrrha, peak 5 comes from Commiphora myrrha, peak 6 comes from Commiphora myrrha, peak 7 comes from Ligusticum chuanxiong and Angelica sinensis, peak 8 comes from Ligusticum chuanxiong and Angelica sinensis, peak 9 comes from Commiphora myrrha, peak 10 comes from Commiphora myrrha, peak 11 comes from Commiphora myrrha, peak 12 comes from Commiphora myrrha, peak 13 comes from Commiphora myrrha, and peak 14 comes from Commiphora myrrha.

64. A method for quality control of the volatile oil components of a traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong, characterized in that, The quality control method includes the following steps: (1) Establish a standard characteristic spectrum of the volatile oil components of a traditional Chinese medicine composition reference sample using the characteristic spectrum construction method according to any one of claims 1 to 63; (2) Take the volatile oil component test solution and detect it under the chromatographic conditions in the characteristic spectrum construction method according to any one of claims 1 to 63 to obtain the characteristic spectrum of the volatile oil component of the traditional Chinese medicine composition to be tested, wherein the preparation of the volatile oil component test solution is as follows: take an appropriate amount of the extract of the traditional Chinese medicine composition containing Angelica sinensis and Ligusticum chuanxiong, heat and reflux to extract, and obtain volatile oil; take an appropriate amount of the volatile oil in a container, add methanol to dilute, make up to volume, shake well, and obtain the test solution of the volatile oil component; wherein the preparation method of the extract includes the following steps: weigh an appropriate amount of Gentiana macrophylla, Ligusticum chuanxiong, Prunus persica, Carthamus tinctorius, Glycyrrhiza uralensis, Notopterygium incisum, Commiphora myrrha, Angelica sinensis, Vinegar-processed Trogopterus xanthipes, Cyperus rotundus, Achyranthes bidentata and Pheretima aspergillum, add an appropriate amount of water to extract, filter, and obtain the first decoction; add an appropriate amount of water to the residue to extract, filter, and obtain the second decoction; combine the first decoction and the second decoction to obtain the extract; and (3) Compare the characteristic spectrum of the volatile oil component of the Chinese medicine composition obtained in step (2) with the standard characteristic spectrum of the volatile oil component of the Chinese medicine composition obtained in step (1). If the sample meets the requirements, it is a qualified product; if it does not meet the requirements, it is an unqualified product.

65. The quality control method according to claim 64, characterized in that, The compliance requirements include one or more of the following: (1) The volatile oil components of the Chinese herbal composition exhibit 14 characteristic chromatographic peaks in the characteristic chromatographic spectrum of the sample to be tested, and the retention time of each characteristic chromatographic peak is within ±10% of the retention time value of the corresponding reference peak in the standard characteristic chromatographic spectrum of the volatile oil components of the Chinese herbal composition. (2) Taking peak 7 as peak S, the relative retention time of each characteristic chromatographic peak in the characteristic chromatographic spectrum of the volatile oil component of the Chinese medicine composition and peak S is within ±10% of the relative retention time value of each characteristic chromatographic peak in the standard characteristic chromatographic spectrum of the volatile oil component of the Chinese medicine composition reference sample. as well as (3) According to the similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine, the similarity between the characteristic chromatogram of the volatile oil component of the traditional Chinese medicine composition and the standard characteristic chromatogram of the reference sample of the volatile oil component of the traditional Chinese medicine composition shall not be less than 0.

90.

66. The use of the construction method according to any one of claims 1 to 63 or the quality control method according to claim 64 or 65 in the quality detection and / or quality evaluation and / or quality control of the volatile oil components of a traditional Chinese medicine composition comprising Angelica sinensis and Ligusticum chuanxiong, wherein, The traditional Chinese medicine composition consists of Gentiana macrophylla, Glycyrrhiza uralensis, Ligusticum chuanxiong, Prunus persica, Carthamus tinctorius, Notopterygium incisum, Commiphora myrrha, Cyperus rotundus, Achyranthes bidentata, Pheretima aspergillum, Angelica sinensis, and Vinegar-processed Trogopterus xanthipes.

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